interacts_with DB00136?
Down-regulation of lipoprotein lipase increases glucose uptake in Q9BTT4 muscle cells . Thiazolidinediones ( TZDs ) are synthetic hypoglycemic agents used to treat type 2 diabetes . TZDs target the peroxisome proliferator activated receptor-gamma ( P37231 ) and improve systemic insulin sensitivity . The contributions of specific tissues to TZD action , or the downstream effects of P37231 activation , are not very clear . We have used a rat skeletal muscle cell line ( Q9BTT4 cells ) to demonstrate that TZDs directly target P37231 in muscle cells . TZD treatment resulted in a significant repression of lipoprotein lipase ( P06858 ) expression in Q9BTT4 cells . This repression correlated with an increase in glucose uptake . Down-regulation of P06858 message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake . Thus , P06858 down-regulation improved insulin sensitivity independent of TZDs . This finding provides a novel method for the management of insulin resistance . Managing the underlying cause of cystic fibrosis : a future role for potentiators and correctors . Cystic fibrosis ( CF ) , a severe genetic disease , is caused by mutations that alter the structure and function of P13569 , a plasma membrane channel permeable to chloride and bicarbonate . Defective anion transport in CF irreversibly damages the lungs , pancreas , liver , and other organs . CF mutations cause loss of P13569 function in multiple ways . In particular , class 3 mutations such as p.Gly551Asp strongly decrease the time spent by P13569 in the open state ( gating defect ) . Instead , class 2 mutations impair the maturation of P13569 protein and its transport from the endoplasmic reticulum to the plasma membrane ( trafficking defect ) . The deletion of phenylalanine 508 ( p.Phe508del ) , the most frequent mutation among CF patients ( 70-90 % ) , destabilizes the P13569 protein , thus causing both a trafficking and a gating defect . These two defects can be overcome with drug-like molecules generically called correctors and potentiators , respectively . The potentiator Kalydeco™ ( also known as DB08820 or VX-770 ) , developed by Vertex Pharmaceuticals , has been recently approved by the US FDA and the European Medicines Agency ( P15941 ) for the treatment of CF patients carrying at least one P13569 allele with the p.Gly551Asp mutation ( 2-5 % of all patients ) . In contrast , the corrector VX-809 , which significantly improves p.Phe508del- P13569 trafficking in vitro , is still under study in clinical trials . Because of multiple defects caused by the p.Phe508del mutation , it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action . This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect . Vitamin D regulates the gut microbiome and protects mice from dextran sodium sulfate-induced colitis . The active form of vitamin D [ DB00136 , 1,25(OH)2D3 ] and the vitamin D receptor ( P11473 ) regulate susceptibility to experimental colitis . The effect of the bacterial microflora on the susceptibility of C57BL/6 mice to dextran sodium sulfate-induced colitis was determined . Mice that can not produce 1,25(OH)2D3 [ Cyp27b1 ( Cyp ) knockout ( KO ) ] , P11473 KO as well as their wild-type littermates were used . Cyp KO and P11473 KO mice had more bacteria from the Bacteroidetes and Proteobacteria phyla and fewer bacteria from the Firmicutes and Deferribacteres phyla in the feces compared with wild-type . In particular , there were more beneficial bacteria , including the Lactobacillaceae and Lachnospiraceae families , in feces from Cyp KO and P11473 KO mice than in feces from wild-type . Helicobacteraceae family member numbers were elevated in Cyp KO compared with wild-type mice . Depletion of the gut bacterial flora using antibiotics protected mice from colitis . 1,25(OH)2D3 treatment ( 1.25 μg/100 g diet ) of Cyp KO mice decreased colitis severity and reduced the numbers of Helicobacteraceae in the feces compared with the numbers in the feces of untreated Cyp KO mice . The mechanisms by which the dysbiosis occurs in P11473 KO and Cyp KO mice included lower expression of P12830 on gut epithelial and immune cells and fewer tolerogenic dendritic cells that resulted in more gut inflammation in P11473 and Cyp KO mice compared with wild-type mice . Increased host inflammation has been shown to provide pathogens with substrates to out-compete more beneficial bacterial species . Our data demonstrate that vitamin D regulates the gut microbiome and that 1,25(OH)2D3 or P11473 deficiency results in dysbiosis , leading to greater susceptibility to injury in the gut . Genetics of idiopathic disseminated bronchiectasis . Bronchiectasis is an abnormal dilation of bronchi , consequent to the destruction of their walls . It is included in the category of obstructive pulmonary diseases , along with chronic obstructive pulmonary disease ( P48444 ) , asthma , and cystic fibrosis . In approximately 50 % of cases , bronchiectasis is associated with underlying conditions ; in the remainder , known causes are not ascertainable ( idiopathic bronchiectasis ) . A search for genetic determinants of this phenotype , with the cystic fibrosis gene as a candidate , has been performed by three independent groups . The results of this search agreed on the association of bronchiectasis with cystic fibrosis gene mutations and polymorphisms . The cystic fibrosis gene is also associated with bronchiectasis due to rheumatoid arthritis and allergic bronchopulmonary aspergillosis . A few other genes have been investigated in idiopathic bronchiectasis , with negative results . Idiopathic bronchiectasis is , therefore , to be considered as an obstructive multifactorial disorder belonging to the category of cystic fibrosis monosymptomatic diseases ( or P13569 -opathies ) , whose pathogenesis is influenced by environmental factors and other undetermined genes . Distinct HDACs regulate the transcriptional response of human cyclin-dependent kinase inhibitor genes to Trichostatin A and 1alpha,25-dihydroxyvitamin D3 . The anti-proliferative effects of histone deacetylase ( HDAC ) inhibitors and 1alpha,25-dihydroxyvitamin D3 [ DB00136 ] converge via the interaction of un-liganded vitamin D receptor ( P11473 ) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor ( CDKI ) genes of the INK4 and Cip/Kip family . We investigated the effects of the HDAC inhibitor Trichostatin A ( P32119 ) and DB00136 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines . P32119 induced the INK4-family genes p18 and p19 , whereas the Cip/Kip family gene P38936 was stimulated by DB00136 . Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded P11473 and repressed the basal level of CDKI genes , but their role in regulating CDKI gene expression by P32119 and DB00136 were contrary . O15379 and HDAC7 attenuated DB00136 -dependent induction of the P38936 gene , for which NCoR1 is essential . In contrast , P32119 -mediated induction of the p18 gene was dependent on O15379 and P56524 , but was opposed by NCoR1 and un-liganded P11473 . This suggests that the attenuation of the response to P32119 by NCoR1 or that to DB00136 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes . 1alpha,25-dihydroxyvitamin D3 and a variety of its natural metabolites transcriptionally repress nuclear-factor-kappaB-mediated interleukin-8 gene expression . Regulation of interleukin-8 ( P10145 ) gene transcription occurs mainly through the sequences -94 to -71 of the 5'-flanking region of the P10145 gene , involving the transcription factors nuclear factor for interleukin-6 ( NF- P05231 ) and nuclear factor kappaB ( NF-kappaB ) . The human melanoma cell line A3 was derived from G-361 cells by stable transfection with an P10145 promoter-luciferase construct containing these sequences . DB00136 ( calcitriol ) repressed P10145 promoter activity induced by tumor necrosis factor-alpha ( P01375 ) by 50 % , compared to 30 % inhibition using dexamethasone , an effect consistent with its effect on P01375 -induced P10145 release and P10145 mRNA levels . A variety of vitamin D metabolites caused the same repressive effect on P10145 promoter activation as calcitriol . However , only those metabolites which were able to transactivate a classical vitamin D response element had the ability to repress P10145 promoter activation , suggesting that this repression is mediated via vitamin D receptor ( P11473 ) . Furthermore , overexpression of P11473 in the parental G-361 cell line enhanced the repressive effect of calcitriol on activation of the P10145 promoter by either P01375 stimulation or overexpression of the NF-kappaB subunit p65 . Electrophoretic mobility shift assays using nuclear extracts from A3 cells showed that calcitriol decreased the abundance of nuclear factors bound to the NF-kappaB binding site of the P10145 promoter and this reduced binding of NF-kappaB proteins presumably contributes to its inhibitory action . DB06643 for the treatment of bone metastases in advanced breast cancer . In women with advanced breast cancer , approximately three-quarters develop metastases to the bone , with a median survival after diagnosis of 2-3 years . Receptor activator of nuclear factor-κB ( Q9Y6Q6 ) and Q9Y6Q6 ligand ( O14788 ) belong to a signal pathway highly implicated in the development of bone metastases . DB06643 , a human monoclonal antibody with high affinity and specificity for O14788 , prevents the O14788 / Q9Y6Q6 interaction and inhibits osteoclast formation and function , thereby decreasing bone resorption and increasing bone mass . DB06643 compared with zoledronic acid showed superior efficacy in delaying time to first-on study SRE and time to first- and subsequent-on study SREs as well as reduction in bone turnover markers . These results led to the approval of denosumab by the European Medicines Agency ( P15941 ) and the US Food and Drug Administration ( FDA ) , for the prevention of SREs in adults with bone metastases from solid tumors , including breast cancer . Caveolae and caveolin-1 are implicated in 1alpha,25(OH)2-vitamin D3-dependent modulation of Src , MAPK cascades and P11473 localization in skeletal muscle cells . We previously reported that DB00136 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells . However , there is little information on the molecular mechanism underlying the initiation of DB00136 signaling through this pathway . Plasma membrane components have been involved in some non-genomic effects . In this work , we investigated the role of caveolae and caveolin-1 ( cav-1 ) in DB00136 -stimulation of c-Src and MAPKs . When proliferating cells were pretreated with methyl beta cyclodextrin ( MbetaCD ) , a caveolae disrupting agent , under conditions in which cell morphology is not affected and no signs of apoptosis are observed , DB00136 -dependent activation of P27361 /2 , p38 MAPK and c-Src was suppressed . Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone . By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions . Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus . Co-immunoprecipitation assays corroborated these observations . Changes in P11473 localization after DB00136 exposure were also investigated . Confocal microscopy images showed that the hormone induces P11473 translocation to the plasma membrane , and this effect is abolished by MbetaCD . Altogether , these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by DB00136 and that P11473 and cav-1 participate in the rapid signaling elicited by the hormone . Regulation of the human P38936 (waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor . The main regulator of the human tumor suppresser gene P38936 (waf1/cip1) is the transcription factor p53 , but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor P11473 in the presence of its ligand 1alpha,25-dihydroxyvitamin D3 ( DB00136 ) . To identify P11473 responding regions , we analyzed 20 overlapping regions covering the first 7.1 kb of the P38936 (waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays ( ChIP ) with antibodies against p53 and P11473 . We confirmed two known p53 binding regions at approximate positions -1400 and -2300 and identified a novel site at position -4500 . In addition , we found three P11473 -associated promoter regions at positions -2300 , -4500 and -6900 , i.e. two regions showed binding for both p53 and P11473 . In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five DB00136 response elements within the three P11473 -positive regions . Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and DB00136 . Moreover , re-ChIP assays confirmed the functionality of the three DB00136 -reponsive promoter regions by monitoring simultaneous occupancy of P11473 with the co-activator proteins CBP , Q15788 and Q15648 . Taken together , we demonstrated that the human P38936 ( ( waf1/cip1 ) ) gene is a primary DB00136 -responding gene with at least three P11473 binding promoter regions , in two of which also p53 co-localizes . The genes encoding cytokines P60568 , P22301 and P29460 are primary DB00136 target genes . A number of studies have described the effects of DB00136 in immune system . Most of the known effects of DB00136 are indirect since only two functional VDREs that regulate transcription of cytokine gene has been reported until today . In this study we have examined a possibility of direct transcriptional regulation of P60568 , P22301 and P29460 genes in activated Jurkat or THP-1 cells via liganded P11473 by using gene expression analysis and chromatin immunoprecipitation assays . According to our data the P60568 , P22301 and P29460 genes respond to DB00136 treatment by 3-6 h . In addition , all of these genes contain several genomic regions that recruit P11473 in a ligand dependent fashion . These data suggest that the above cytokines are under direct transcriptional regulation by DB00136 . Effect of canagliflozin on renal threshold for glucose , glycemia , and body weight in normal and diabetic animal models . BACKGROUND : DB08907 is a sodium glucose co-transporter ( SGLT ) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus ( T2DM ) . METHODS : (14)C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human , rat , or mouse SGLT2 or P13866 ; (3)H-2-deoxy-d-glucose uptake in Q9BTT4 myoblasts ; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed . Graded glucose infusions were performed to determine rate of urinary glucose excretion ( UGE ) at different blood glucose ( BG ) concentrations and the renal threshold for glucose excretion ( RT(G) ) in vehicle or canagliflozin-treated Zucker diabetic fatty ( ZDF ) rats . This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity . RESULTS : Treatment with canagliflozin 1 mg/kg lowered RT(G) from 415±12 mg/dl to 94±10 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RT(G) . DB08907 dose-dependently decreased BG concentrations in db/db mice treated acutely . In ZDF rats treated for 4 weeks , canagliflozin decreased glycated hemoglobin ( HbA1c ) and improved measures of insulin secretion . In obese animal models , canagliflozin increased UGE and decreased BG , body weight gain , epididymal fat , liver weight , and the respiratory exchange ratio . CONCLUSIONS : DB08907 lowered RT(G) and increased UGE , improved glycemic control and beta-cell function in rodent models of T2DM , and reduced body weight gain in rodent models of obesity . DB00136 inhibits transcriptional potential of nuclear factor kappa B in breast cancer cells . 1alpha,25-Dihydroxyvitamin D(3) ( VD(3) ) , the biologically active form of vitamin D , may have either pro- or anti-inflammatory activities because of its diverse actions on nuclear factor kappa B ( NF-kappaB ) . Previous studies indicated that VD(3) can either activate or inhibit NF-kappaB via Akt-induced I kappaB alpha phosphorylation and increase in I kappaB alpha synthesis respectively . At present , the relevant contribution of each mechanism has not been fully explored . We observed a VD(3)-mediated NF-kappaB inhibitory effect in vitamin D receptor ( P11473 ) -positive MCF-7 breast cancer cells . We showed that VD(3) induced P11473 -dependent I kappaB alpha expression but still able to lead on transient NF-kappaB p65 nuclear translocation through Akt-induced I kappaB alpha phosphorylation . Upon TNFalpha stimulation , VD(3) was not capable to inhibit I kappaB alpha degradation , p65 nuclear translocation and p65/p50-DNA binding . Here , we found that VD(3) strongly repressed p65 transactivation in MCF-7 cells using Gal4-p65 chimeras system . P11473 was required for the VD(3)-mediated transrepression and mutations in P11473 affected its suppressive ability . We also demonstrated that neither inhibition of p65 phosphorylation nor acetylation was responsible for the transrepression . In fact , we found that treatment of MCF-7 cells with histone deacetylase inhibitors abrogated VD(3)-induced p65 transrepression . In addition , knockdown of two nuclear corepressors O15379 and Q9Y618 relieved p65 transactivation and particular TNFalpha-triggered gene expression . In conclusion , the reduction of gene activation by VD(3) in breast cancer cells was caused by the interference of the transactivation potential of NF-kappaB p65 subunit . Our studies provide a scientific background for rational use of vitamin D in the prevention and treatment of inflammatory diseases . P01375 polymorphisms as a potential modifier gene in the cystic fibrosis . Modifier genes , as the P01375 -α gene , can modulate the cystic fibrosis ( CF ) severity . Thus , -238G > A and -308G > A polymorphisms of P01375 -α gene were analyzed as modifiers of CF . In this context , the present study enrolled 49 CF patients ( diagnosis performed by sweat test and complete P13569 screening ) . The -238G > A polymorphism analysis was performed by Q9ULH0 -PCR , and -308G > A , by PCR-RFLP . In our data , the -238G > A polymorphism was not associated with clinical variability . The AA genotype for -308G > A polymorphism was a risk factor for early gastrointestinal symptoms ( OR=5.98 , 95 % CI=1.06-49.68 ) and protection for the first Pseudomonas aeruginosa ( OR=0.05 , 95 % CI=0.0003-0.007 ) . For the first P. aeruginosa , GA genotype was a risk factor ( OR=10.2 , 95 % CI=1.86-84.09 ) ; for the same genotype , the diagnosis was made in minor time than the AA genotype ( p=0.031 ) . Considering the -308G > A polymorphism alleles , the G allele was a risk factor for early pulmonary symptoms ( OR=3.81 , 95 % CI=1.13-12.97 ) and P. aeruginosa ( OR=66.77 , 95 % CI=15.18-482.7 ) ; however , the same allele showed better transcutaneous oxygen saturation ( OR=9.24 , 95 % CI=1.53-206.1 ) . The A allele was a protective factor for early pulmonary symptoms ( OR=12.26 , 95 % CI=0.08-0.89 ) and P. aeruginosa ( OR=12.15 , 95 % CI=0002-0007 ) , however , the same allele was a risk factor for worst transcutaneous oxygen saturation ( OR=7.01 , 95 % CI=1.14-157.4 ) . As conclusion , the -308G > A polymorphism of the P01375 -α gene was associated with the CF severity . Synthesis and biological activities of 14-epi-MART-10 and 14-epi-MART-11 : implications for cancer and osteoporosis treatment . The 14-epimer of MART-10 , namely 14-epi-MART-10 ( 14-epi-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D3 ) and its 2-epimeric analog ( 14-epi-MART-11 ) were efficiently synthesized using the Julia coupling reaction to connect between the P01031 and P13671 positions ( steroid numbering ) . An A-ring precursor was prepared from (-)-quinic acid as shown in the previous MART-10 synthesis . The novel 14-epi-CD-ring coupling partner with an elongated two carbon unit as a sulfone was synthesized from 14-epi-25-hydroxy Grundmann 's ketone in good yield . The subsequent coupling reaction followed by a deprotection step afforded a mixture of 14-epi-MART-10 and 14-epi-MART-11 in 40 % yield . To separate 14-epi-MART-10 and 14-epi-MART-11 , each primary hydroxyl group was esterified with a pivaloyl group and the resulting pivalates 2alpha and 2beta were separated by high performance liquid chromatography . After the separation , the P06681 -stereochemistry of each ( 2alpha or 2beta ) was determined by 1H NMR ( nuclear magnetic resonance ) studies including NOE ( nuclear Overhauser effect ) experiments . The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11 , respectively . The P11473 ( vitamin D receptor ) -binding affinity , HL-60 ( human promyelocytic leukemia ) cell differentiation activity , antiproliferative activity in PZ-HPV-7 ( immortalized normal prostate ) cells and transactivation activity of the osteocalcin promoter in Q9UKB1 ( human osteoblast cell line ) cells ( serum-free conditions ) were investigated . In addition , the effects on bone mineral density ( BMD ) and the blood and urine calcium concentrations of ovariectomized ( OVX ) rats were examined . 14-epi-MART-10 has much greater antiproliferative and cell differentiation activities compared to 1alpha,25-dihydroxyvitamin D3 ( DB00136 ) . DB00136 induces nitric oxide production in cultured endothelial cells . BACKGROUND : Recently , DB00136 ( vitD ) has received increasing interest for its effects on many tissues and organs other than bone . A number of experimental studies have shown that vitD may have an important role in modifying risk for cardiovascular disease . AIMS : This study was planned to test the effects of vitD on endothelial nitric oxide ( NO ) production and to study the intracellular pathways leading to NO release . METHODS : In human umbilical vein endothelial cells ( HUVEC ) cultures the effects of vitD on NO production and p38 , Akt , P29323 and P29474 phosphorylations were examined in absence or in presence of the NO synthase inhibitor L-NAME and protein kinases specific inhibitors SB203580 , wortmannin and UO126 . RESULTS : VitD caused a concentration-dependent increase in NO production . The maximum effect was observed at a concentration of 1 nM and the optimal time of stimulation was 1 min . Effects induced by vitD were abolished by L-NAME and by pre-treatment with protein kinases inhibitors . To verify the effective involvement of vitD receptor ( P11473 ) in the action mechanism of vitD , experiments were repeated in presence of the specific P11473 ligands ZK159222 and ZK191784 . CONCLUSIONS : The results of this study demonstrate that vitD can induce a significant increase in endothelial NO production . VitD interaction with P11473 caused the phosphorylation of p38 , AKT and P29323 leading to P29474 activation . AP-1 transrepressing retinoic acid does not deplete coactivators or AP-1 monomers but may target specific Jun or Fos containing dimers . Retinoic acid ( RA ) inhibits tumor promotion in many models in vivo and in vitro , among them mouse epidermal JB6 cells . RA treatment suppresses 12-O-tetradecanoylphorbol-13-acetate ( TPA ) induced AP-1 activity , an activity that is required for transformation of JB6 P+ cells . The molecular mechanism of AP-1 transrepression by retinoids is unclear , especially as related to inhibition of transformation . Overexpression of AP-1 components did not rescue TPA induced AP-1 activation nor did a Q86UG4 pull down experiment implicate direct binding , thus rendering unlikely both a Jun/Fos-RA-RAR direct interaction and a Jun/Fos sequestration mechanism . Overexpression of p300 , Q15788 or pCAF did not abrogate AP-1 suppression by RA , thus arguing against coactivator competition . Overexpression of the corepressor silencing mediator for retinoic acid and thyroid hormone receptors ( Q9Y618 ) suppressed AP-1 activity . However , Q9Y618 but not RA inhibited cJun transactivation , suggesting Q9Y618 does not mediate RA transrepression . RA treatment also did not block TPA induced P29323 phosphorylation , Jun/Fos family protein expression except for cFos , or DNA binding of the AP-1 complex . The transcriptional activities of full-length JunB and full-length Fra-1 , but not the transactivation domain fusions , were increased by TPA treatment and suppressed by RA . Since these full-length fusions have bzip domains , the results suggest that JunB and/or Fra-1-containing dimers may constitute one target of RA for transrepression of AP-1 . Salacia oblonga extract increases glucose transporter 4-mediated glucose uptake in Q9BTT4 rat myotubes : role of mangiferin . BACKGROUND AND AIMS : To evaluate if the antidiabetic properties of Salacia oblonga extract are mediated not only by inhibiting intestinal alpha-glycosidases but also by enhancing glucose transport in muscle and adipose cells . METHODS : S. oblonga extract effects on 2-deoxy-D-glucose uptake were assayed in muscle Q9BTT4 -myotubes and 3T3-adipocytes . In Q9BTT4 -myotubes , the amount and translocation of glucose transporters were assayed . A fractionation of the extract was carried out to identify the active compounds . Furthermore , we analyzed the phosphorylation status of key components of signaling pathways that are involved in the molecular mechanisms regulating glucose uptake . RESULTS : S. oblonga extract increased 2-deoxy-D-glucose uptake by 50 % in Q9BTT4 -myotubes and 3T3-adipocytes . In Q9BTT4 -myotubes , the extract increased up to a 100 % the P14672 content , activating P14672 promoter transcription and its translocation to the plasma membrane . Mangiferin was identified as the bioactive compound . Furthermore , mangiferin effects were concomitant with the phosphorylation of DB00131 -activated protein kinase without the activation of P31749 /Akt . The effect of mangiferin on 2-deoxy-D-glucose uptake was blocked by GW9662 , an irreversible P37231 antagonist . CONCLUSIONS : S. oblonga extract and mangiferin may exert their antidiabetic effect by increasing P14672 expression and translocation in muscle cells . These effects are probably mediated through two independent pathways that are related to DB00131 -activated protein kinase and P37231 . Identification of Reverb(alpha) as a novel ROR(alpha) target gene . The nuclear receptor superfamily comprises a large number of ligand-activated transcription factors that are involved in numerous biological processes such as cell proliferation , differentiation , and homeostasis . ROR(alpha) ( P35398 ) and Reverb(alpha) ( P20393 ) are two members of this family whose biological functions are largely unknown . In addition , no ligand has been yet identified for these two receptors ; therefore , they are referred as orphan receptors . Here , we show that ROR(alpha) and Reverb(alpha) are expressed with a similar tissue distribution and are both induced during the differentiation of rat Q9BTT4 myoblastic cells . Ectopic expression of ROR(alpha)1 in Q9BTT4 cells significantly induces Reverb(alpha) expression as demonstrated by Northern blot analysis . Using reverse transcription-PCR to analyze Reverb(alpha) gene expression from staggerer mice , we found that there was a significant reduction of Reverb(alpha) mRNA in the skeletal muscle comparing it with the wild-type mice , which suggests that ROR(alpha) is involved in the regulation of Reverb(alpha) gene expression . Transient transfection assays using the Reverb(alpha) promoter demonstrate that ROR(alpha) regulates the Reverb(alpha) gene at the transcriptional level . Furthermore , mutagenesis experiments indicate that ROR(alpha) regulates Reverb(alpha) transcription via a monomeric ROR response element located in the Reverb(alpha) gene promoter . Electrophoretic mobility shift assays show that ROR(alpha) binds strongly to this site in a specific-manner . Finally , overexpression of Q9Y3R0 / Q06418 -2 , but not Q15788 , potentiates ROR(alpha)-stimulated Reverb(alpha) promoter activity in transient transfection experiments . Together , our results identify Reverb(alpha) as a novel target gene for ROR(alpha) . Integrated gene copy number and expression microarray analysis of gastric cancer highlights potential target genes . We performed an integrated array comparative genomic hybridization ( aCGH ) and expression microarray analysis of 8 normal gastric tissues and 38 primary tumors , including 25 intestinal and 13 diffuse gastric adenocarcinomas to identify genes whose expression is deregulated in association with copy number alteration . Our aim was also to identify molecular genetic alterations that are specific to particular clinicopathological characteristics of gastric cancer . Distinct molecular genetic profiles were identified for intestinal and diffuse gastric cancers and for tumors obtained from 2 different locations of the stomach . Interestingly , the P04626 amplification and gains at 20q13.12-q13.33 almost exclusively discriminated intestinal cancers from the diffuse type . In addition , the 17q12-q25 gain was characteristic to cancers located in corpus and the 20q13.12-q13.13 gain was more common in the antrum . Statistical analysis was performed using integrated copy number and expression data to identify genes showing differential expression associated with a copy number alteration . Genes with the highest statistical significance included P04626 , P15941 , Q14451 , Q9UD71 and Q15648 with concomitant changes in copy number and expression . Immunohistochemical analysis of P04626 and P15941 on a tissue microarray containing 78 independent gastric tissues showed statistically significant differences ( p < 0.05 and < 0.001 ) in immunopositivity in the intestinal ( 31 and 70 % ) and diffuse subtypes ( 14 and 41 % ) , respectively . In conclusion , our results demonstrate that intestinal and diffuse type gastric cancers as well as cancers located in different sites of the stomach have distinct molecular profiles which may have clinical value . The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation . Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors . We investigated the role of the mediator complex as a coactivator for vitamin D receptor ( P11473 ) in keratinocytes . Using P11473 affinity beads , the vitamin D receptor interacting protein ( DRIP ) /mediator complex was purified from primary keratinocytes , and its subunit composition was determined by mass spectrometry . The complex included core subunits , such as Q15648 /MED1 ( MED1 ) , that directly binds to P11473 . Additional subunits were identified that are components of the RNA polymerase II complex . The functions of different mediator components were investigated by silencing its subunits . The core subunit MED1 facilitates P11473 activity and regulating keratinocyte proliferation and differentiation . A newly described subunit Q13503 also has a role in promoting keratinocyte proliferation and differentiation , whereas Q9BTT4 has an inhibitory role . Blocking MED1/ Q13503 expression caused hyperproliferation of keratinocytes , accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog . Blocking MED1 or Q13503 expression also resulted in defects in calcium-induced keratinocyte differentiation , as indicated by decreased expression of differentiation markers and decreased translocation of P12830 to the membrane . These results show that keratinocytes use the transcriptional coactivator mediator to regulate P11473 functions and control keratinocyte proliferation and differentiation . Smoke exposure of human macrophages reduces O15379 activity , resulting in enhanced inflammatory cytokine production . Chronic obstructive pulmonary disease ( P48444 ) is a debilitating condition resulting from exposure to pollutants such as cigarette smoke . Pulmonary macrophages secrete a plethora of inflammatory mediators that are increased in the lungs of P48444 patients , but whether this phenotype results directly from smoke exposure remains unknown . Using an in vitro model for alveolar macrophages ( AM ) derived from human peripheral blood monocytes with granulocyte-macrophage stimulating factor ( GM-MØ ) , we analyzed the mechanistic connection between cigarette smoke exposure and histone deacetylase ( HDAC ) regulation , hypothesized to be a contributing factor in P48444 pathophysiology . Here we show that acute smoke exposure inhibits HDAC enzymatic activity in GM-MØ . Analysis of mRNA and total cellular proteins for expression of class I ( 1 , 2 , 3 and 8 ) , class II ( 4 , 5 , 6 , 7 , 9 , 10 ) , and class IV ( 11 ) HDAC revealed no effect of smoke exposure , whereas nuclear O15379 protein content was reduced . To better understand the physiological significance of reduced O15379 activity , we utilized siRNA to knockdown Q13547 , 2 and 3 individually . Interestingly , siRNA-mediated reduction of O15379 resulted in increased production of P10145 and IL1β in response to LPS stimulation , while Q92769 knockdown had no effect on either cytokine . Lower nuclear content of O15379 in the context of equivalent total HDAC protein levels following smoke exposure may reflect increased nuclear export of O15379 , allowing increased nuclear factor kappa b ( NF-κB ) driven cytokine expression that can contribute to inflammation . Increased epithelial permeability is the primary cause for bicarbonate loss in inflamed murine colon . BACKGROUND : Bicarbonate loss into the lumen occurs during intestinal inflammation in different species . However , candidate pathways like P13569 or P40879 are inhibited in the inflamed gut . This study addressed the question whether and how inflammation-associated increased intestinal permeability may result in epithelial HCO(3)(-) loss . METHODS : Murine proximal colon was studied because it does not express functional P40879 but is inflamed in the tumor necrosis factor α overexpressing mouse model ( P01375 (ΔARE) ) . DB01174 alkalization , (3)H-mannitol fluxes , impedance spectroscopy , and dilution potentials were measured in Ussing chambers , whereas expression and localization of tight junction-associated proteins were analyzed by Western blots and immunohistochemistry . RESULTS : DB01174 alkalization rates and (3)H-mannitol fluxes were increased in P01375 (+/ΔARE) proximal colon , whereas forskolin-stimulated I(sc) was not altered . Epithelial resistance was reduced , but subepithelial resistance increased . The epithelial lining was intact , and enterocyte apoptosis rate was not increased despite massively increased Th1 cytokine levels and lymphoplasmacellular infiltration . Measurement of dilution potentials suggested a loss of cation selectivity with increased anion permeability . Western analysis revealed a downregulation of occludin expression and an upregulation of both claudin-2 and claudin-5 , with no change in ZO-1 , P12830 , claudin-4 , and claudin-8 . Immunohistochemistry suggested correct occludin localization but reduced tight junction density in P01375 (+/ΔARE) surface epithelium . CONCLUSIONS : Inflammation during P01375 -α overexpression leads to increased epithelial permeability in murine proximal colon , decreased tight junctional cation selectivity , and increased HCO(3)(-) loss into the lumen . Inflammation-associated colonic HCO(3)(-) loss may occur through leaky tight junctions rather than through HCO(3)(-) secreting ion transporters . The genes of the coactivator Q15596 and the corepressor Q9Y618 are primary DB00136 targets . The complex of the receptor for the hormone 1alpha,25-dihydroxyvitamin D(3) ( 1alpha,25(OH)(2)D(3) ) , Vitamin D(3) receptor ( P11473 ) , the retinoid X receptor ( RXR ) and a 1alpha,25(OH)(2)D(3) response element ( VDRE ) is considered to be the molecular switch for nuclear 1alpha,25(OH)(2)D(3) signaling . In the presence of ligand the P11473 -RXR complex interacts with coactivator ( DB01992 ) proteins that in turn contact components of the basal transcriptional machinery resulting in an enhanced transcription of 1alpha,25(OH)(2)D(3) target genes . In the absence of ligand the P11473 remains bound to the DNA and interacts with corepressor ( CoR ) proteins that are involved in gene silencing activity . We treated MCF-7 breast cancer cells with 1alpha,25(OH)(2)D(3) for increasing amounts of time , extracted mRNA and screened by real-time PCR the members of the P52701 DB01992 and NCoR CoR families . We find that of the P52701 coactivators , only Q15596 was responsive to 1alpha,25(OH)(2)D(3) . Similarly Q9Y618 but not NCoR1 gene transcription was sensitive to 1alpha,25(OH)(2)D(3) treatment . In silico analysis revealed that both Q15596 and Q9Y618 promoters have substantial numbers of VDREs compared to the promoters of the other family members . These VDREs are formed by direct repeats of the core binding motif RGKTCA with a three nucleotide spacing ( Q93038 ) . We suggest that some or all of these Q93038 -type VDREs are responsible for the observed responsiveness of Q15596 and Q9Y618 to 1alpha,25(OH)(2)D(3) . Genotypes associated with lipid metabolism contribute to differences in serum lipid profile of GH-deficient adults before and after GH replacement therapy . OBJECTIVE : GH deficiency ( GHD ) in adults is associated with an altered serum lipid profile that responds to GH replacement therapy ( GHRT ) . This study evaluated the influence of polymorphisms in genes related to lipid metabolism on serum lipid profile before and after 1 year of GHRT in adults . DESIGN AND METHODS : In 318 GHD patients , total cholesterol ( TC ) serum concentrations , LDL-C , HDL-C , and triglycerides ( TG ) were assessed . Using a candidate gene approach , 20 single nucleotide polymorphisms ( SNPs ) were genotyped . GH dose was individually titrated to obtain normal serum IGF1 concentrations . RESULTS : At baseline , the minor alleles of cholesteryl ester transfer protein ( P11597 ) gene SNPs rs708272 and rs1800775 were associated with higher serum TC and apolipoprotein E ( P02649 ) gene SNP rs7412 with lower TC concentrations ; P11597 SNPs rs708272 , rs1800775 , and rs3764261 and apolipoprotein B ( P04114 ) gene SNP rs693 with higher serum HDL-C ; P02649 SNP rs7412 , peroxisome proliferator-activated receptor gamma ( P37231 ) gene SNP rs10865710 with lower LDL-C , and P11597 SNP rs1800775 with higher LDL-C ; and P02649 /C1/C4/ P06681 cluster SNP rs35136575 with lower serum TG . After treatment , P04114 SNP rs676210 GG genotype was associated with larger reductions in TC and LDL-C and P37231 SNP rs10865710 CC genotype with greater TC reduction . All associations remained significant when adjusted for age , sex , and BMI . CONCLUSIONS : In GHD adults , multiple SNPs in genes related to lipid metabolism contributed to individual differences in baseline serum lipid profile . The GH treatment response in TC and LDL-C was influenced by polymorphisms in the P04114 and P37231 genes . Rosiglitazone influences the expression of leukocyte adhesion molecules and P08571 receptor in type 2 diabetes mellitus patients . Diabetes mellitus is associated with increased inflammatory response , which may contribute to atherosclerosis progression . Experimental results demonstrated anti-inflammatory activity of glitazones ; their effect on leukocyte adhesion molecules has not been studied to date . We therefore studied the effect of rosiglitazone treatment on leukocyte surface expression of adhesion molecules in patients with type 2 diabetes mellitus and compared our results with findings in healthy subjects . 33 subjects with type 2 diabetes and 32 healthy controls were included ; patients were examined at baseline and after 5 months of rosiglitazone treatment ( 4 mg/d ) . Leukocyte expression of adhesion molecules LFA-1 , P05107 and P05362 was quantified using flow cytometry ; in addition , P08571 ( lipopolysaccharide receptor ) expression was analyzed as a marker of nonspecific immunity . The expression of examined molecules at baseline was higher in patients compared to controls . Despite only mild decrease in blood glucose , rosiglitazone treatment induced substantial decrease of P05107 and P08571 expression and borderline decrease of LFA-1 and P05362 expression ( on monocytes only ) . We thus observed improvement in the expression of leukocyte inflammatory markers after rosiglitazone treatment . This effect is supposed to be mediated by direct effect of rosiglitazone on P37231 receptors on leukocytes . Effects of alpha/beta- DB01524 immune regulating hormones on bone remodeling and apoptosis in osteoblasts . A large body of evidence suggests that the immune system directly impacts bone physiology . We tested whether immune regulating hormones ( P48061 ) , 17beta- DB01524 ( beta-AED ) , 7beta,17beta-androstenetriol ( beta-AET ) or the 17alpha- DB01524 ( alpha-AED ) , and 7alpha,17beta-androstenetriol ( alpha-AET ) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts ( FOB-9 ) . The impact on bone remodeling was examined by comparing the ratio of O14788 / O00300 gene expression in response to AED and AET compounds . The alpha-AED was found to significantly increase in the ratio of O14788 / O00300 gene expression and altering the morphology of O14788 stained FOB-9 cells . Cell viability was assessed using a Live/Dead assay . Again alpha-AED was unique in its ability to reduce the proportion of viable cells , and to induce mild apoptosis of FOB-9 cells . Treatment of FOB-9 cells with WY14643 , an activator of Q07869 and -gamma , also significantly elevated the percentage of dead cells . This increase was abolished by co-treatment with GW9962 , a specific inhibitor of P37231 . Analysis of P37231 mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased P37231 activation by 19 % , while beta-AED conferred a 37 % decrease in P37231 activation . In conclusion , alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells . The increase in O14788 / O00300 is modulated by an activation of P37231 that in turn caused mild apoptosis of FOB-9 cells . 1,25-Dihydroxycholecalciferol enhances butyrate-induced P38936 (Waf1/Cip1) expression . Butyrate , a short-chain fatty acid produced in the colon , as well as its prodrug tributyrin , reduce proliferation and increase differentiation of colon cancer cells . P38936 (Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines . We studied the effects of butyrate on differentiation , P11473 expression , as well as on P38936 (Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells ( Caco-2 ) . Butyrate induced cell differentiation , which was further enhanced after addition of DB00136 . Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of P11473 . While butyrate as well as dihydroxycholecalciferol increased P38936 (Waf1/Cip1) and p27(Kip1) expression , in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of P38936 (Waf1/Cip1) , but not of p27(Kip1) expression . These data imply that butyrate selectively increases P38936 (Waf1/Cip1) expression via upregulation of P11473 in Caco-2 cells . P04150 interacting protein-1 restores glucocorticoid responsiveness in steroid-resistant airway structural cells . Glucocorticoid ( GC ) insensitivity represents a profound challenge in managing patients with asthma . The mutual inhibition of transcriptional activity between GC receptor ( GR ) and other regulators is one of the mechanisms contributing to GC resistance in asthma . We recently reported that interferon regulatory factor ( Q969Q1 ) -1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle ( P17405 ) cells by interfering with GR signaling ( Tliba et al. , Am J Respir Cell Mol Biol 2008;38:463-472 ) . Here , we sought to determine whether the inhibition of GR function by P10914 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 ( Q9Y3R0 ) , a known GR transcriptional co-activator . We here found that siRNA-mediated Q9Y3R0 depletion attenuated P10914 -dependent transcription of the luciferase reporter construct and the mRNA expression of an P10914 -dependent gene , P28907 . In parallel experiments , Q9Y3R0 silencing significantly reduced GR-mediated transactivation activities . Co-immunoprecipitation and Q86UG4 pull-down assays showed that Q9Y3R0 , through its repression domain , physically interacts with P10914 identifying Q9Y3R0 as a bona fide transcriptional co-activator for P10914 . Interestingly , the previously reported inhibition of GR-mediated transactivation activities by either P01375 and P01579 treatment or P10914 overexpression was fully reversed by increasing cellular levels of Q9Y3R0 . Together , these data suggest that the cellular accumulation of P10914 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of Q9Y3R0 from the GR transcriptional regulatory complexes . The anti-proliferative effects of DB00136 on breast and prostate cancer cells are associated with induction of P38398 gene expression . The anti-proliferative action of the seco-steroid hormone 1alpha , 25-dihydroxyvitamin D3 [ DB00136 ] extends to some , but not all breast and prostate cancer cell lines . By elucidating the molecular mechanisms mediating the sensitivity of these cells , we can identify critical target genes regulated directly or indirectly by DB00136 and pathways potentially disrupted during transformation . In this study , we demonstrated the induction of expression of P38398 mRNA and protein as well as transcriptional activation from the P38398 -promoter by DB00136 in the sensitive breast cancer cell line MCF-7 . This was not observed in the DB00136 -resistant breast cancer cell line MDA-MB-436 . The induction of P38398 mRNA was blocked by cyclohexamide . This indicated that transcriptional activation was mediated indirectly by the vitamin D receptor ( P11473 ) . Inhibition of P11473 protein levels by stable transformation of the anti-sense P11473 in MCF-7 reduced the sensitivity of MCF-7 to DB00136 by 50-fold . In addition , the induction of P38398 protein and transcriptional activation of a P38398 promoter-luciferase reporter construct was abrogated in the stable transformant with the greatest reduction of P11473 levels . Examination of other breast and prostate cancer cell lines revealed that sensitivity to the anti-proliferative effects of 1alpha , 25(OH)2D3 was strongly associated with an ability to modulate P38398 protein . Furthermore , the expression of the estrogen receptor in these cell lines strongly correlated with their sensitivity to DB00136 and their ability to modulate P38398 expression . Taken together , our data support a model whereby the anti-proliferative effects of DB00136 are mediated , in part , by the induction of P38398 gene expression via transcriptional activation by factors induced by the P11473 and that this pathway is disrupted during the development of prostate and breast cancers . c-Fos protein as a target of anti-osteoclastogenic action of vitamin D , and synthesis of new analogs . Although active vitamin D drugs have been used for the treatment of osteoporosis , how the vitamin D receptor ( P11473 ) regulates bone cell function remains largely unknown . Using osteoprotegerin-deficient mice , which exhibit severe osteoporosis due to excessive receptor activator of NF-kappaB ligand/receptor activator of NF-kappaB ( O14788 / Q9Y6Q6 ) stimulation , we show herein that oral treatment of these mice with 1alpha,25-dihydroxyvitamin D3 [ DB00136 ] inhibited bone resorption and prevented bone loss , suggesting that P11473 counters O14788 / Q9Y6Q6 signaling . In P09603 -dependent osteoclast precursor cells isolated from mouse bone marrow , DB00136 potently and dose-dependently inhibited their differentiation into multinucleate osteoclasts induced by O14788 . Among signaling molecules downstream of Q9Y6Q6 , DB00136 inhibited the induction of c-Fos protein after O14788 stimulation , and retroviral expression of c-Fos protein abrogated the suppressive effect of DB00136 on osteoclast development . By screening vitamin D analogs based on their c-Fos-suppressing activity , we identified a new analog , named DD281 , that inhibited bone resorption and prevented bone loss in ovariectomized mice , more potently than DB00136 , with similar levels of calcium absorption . Thus , c-Fos protein is an important target of the skeletal action of P11473 -based drugs , and DD281 is a bone-selective analog that may be useful for the treatment of bone diseases with excessive osteoclastic activity . 20-Epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers of DRIP coactivator complex binding to the vitamin D3 receptor . 1,25-Dihydroxyvitamin D3 ( 1,25(OH)2D3 ) plays a major role in the stimulation of bone growth , mineralization , and intestinal calcium and phosphate absorption ; it also acts as a general inhibitor of cellular proliferation . Several new , clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH)2D3 , but the molecular basis for this has not been clearly elucidated . Here , we tested whether the potency of one class of compounds , 20-epi analogues , to induce myeloid cell differentiation , is because of direct molecular effects on vitamin D receptor ( P11473 ) . We report that two 20-epi analogues , MC1627 and MC1288 , induced differentiation and transcription of P38936 (Waf1,Cip1) , a key P11473 target gene involved in growth inhibition , at a concentration 100-fold lower than that of 1,25(OH)2D3 . We compared this sensitivity to analogue effects on P11473 interacting proteins : RXR , Q9Y3R0 , and Q15648 , a subunit of the DRIP coactivator complex . Compared with the interaction of P11473 with RXR or Q9Y3R0 , the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to P11473 and to the ability of the receptor to activate transcription in a cell-free system . These results provide compelling links between the efficiency of the 20-epi analogue in inducing P11473 /DRIP interactions , transactivation in vitro , and its enhanced ability to induce cellular differentiation . Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage . The vitamin D receptor knockout ( P11473 -KO ) mouse presents with a skeletal phenotype typical for complete lack of genomic DB00136 effects . Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression . In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon , we characterized the amount and site-specific distribution of the P11473 along the large intestine in wild-type ( WT ) , heterozygote ( HT ) and KO mice . We also evaluated expression of proliferating cell nuclear antigen ( P12004 ) , of cyclin D1 and the levels of 8-hydroxy-2'-deoxyguanosine ( 8-OHdG ) , a marker of oxidative stress . In colon ascendens , proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice . A decrease of P11473 expression did not affect expression significantly . In colon descendens , however , fewer proliferative cells were solely located in the lower third of the crypt , and an inverse relationship between P11473 reduction , P12004 positivity and cyclin D1 expression was found in HT and KO mice . In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred . Therefore , the sigmoid colon of P11473 -KO mice , fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes , is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence . SUMOylation of tissue transglutaminase as link between oxidative stress and inflammation . Cystic fibrosis ( CF ) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator ( P13569 ) gene . CF is characterized by chronic bacterial lung infections and inflammation , and we have previously reported that tissue transglutaminase ( TG2 ) , a multifunctional enzyme critical to several diseases , is constitutively up-regulated in CF airways and drives chronic inflammation . Here , we demonstrate that the generation of an oxidative stress induced by P13569 -defective function leads to protein inhibitor of activated P35610 (PIAS)y-mediated TG2 SUMOylation and inhibits TG2 ubiquitination and proteasome degradation , leading to sustained TG2 activation . This prevents peroxisome proliferator-activated receptor ( Q07869 )gamma and IkBalpha SUMOylation , leading to NF-kappaB activation and to an uncontrolled inflammatory response . Cellular homeostasis can be restored by small ubiquitin-like modifier ( SUMO ) -1 or Q8N2W9 gene silencing , which induce TG2 ubiquitination and proteasome degradation , restore PPARgamma SUMOylation , and prevent IkBalpha cross-linking and degradation , thus switching off inflammation . DB06757 superoxide dismutase overexpression as well as the treatment with the synthetic superoxide dismutase mimetic EUK-134 control Q8N2W9 -TG2 interaction and TG2 SUMOylation . TG2 inhibition switches off inflammation in vitro as well as in vivo in a homozygous F508del- P13569 mouse model . Thus , TG2 may function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or degradation . Targeting TG2-SUMO interactions might represent a new option to control disease evolution in CF patients as well as in other chronic inflammatory diseases , neurodegenerative pathologies , and cancer . Loss of coordinated androgen regulation in nonmalignant ovarian epithelial cells with P38398 /2 mutations and ovarian cancer cells . Epidemiological studies have implicated androgens in the etiology/progression of epithelial ovarian cancer . Because normal and malignant ovarian epithelial cells are growth inhibited by transforming growth factor ( TGF ) beta , we tested the ability of 5alpha-dihydrotestosterone ( DB02901 ) to modulate this response and the expression of TGF-beta receptor types I and II . Cells derived from the ovarian surface epithelium of women undergoing oophorectomy ( n = 7 ) for nonovarian indications or with a germ-line P38398 or 2 mutation ( n = 9 ) , and from the ascitic fluid of patients with primary ovarian cancer ( n = 8 ) were cultured with and without DB02901 . Cell proliferation after TGF-beta1 or vehicle treatment was determined , and transcripts for TGF-beta receptors were measured by quantitative reverse transcription-PCR . As low levels of androgen receptor were observed in the cultures , we also measured transcript levels for steroid receptor coactivators Q15788 , Q13772 , and Q9Y6Q9 . TGF-beta1 inhibited growth in 12 of 13 cultures tested , and DB02901 generally reversed this effect , demonstrating that androgens can block TGF-beta-induced growth inhibition in both malignant and nonmalignant ovarian epithelial cells . Transcripts for TGF-beta receptors , Q15788 , and Q13772 were found to be coordinately regulated by androgen in control cells , but not in either malignant or P38398 /2-positive cell cultures . These findings raise the possibility that by modulating steroid receptor coactivator expression , androgen might affect other hormonal responses and contribute to the initiation of ovarian cancer . Evidence that Q13507 is a molecular component of the DB00136 -activated capacitative calcium entry ( CCE ) in muscle and osteoblast cells . In chick skeletal muscle and in rat osteoblast-like cells ( ROS 17/2.8 ) , 1alpha,25-dihydroxy-Vitamin-D(3) [ 1alpha,25(OH)(2)D(3) ] stimulates release of Ca(2+) from inner stores and extracellular cation influx through both voltage-dependent and capacitative Ca(2+) entry ( CCE ) channels . We investigated the involvement of TRPC proteins in CCE induced by 1alpha,25(OH)(2)D(3) . Two fragments were amplified by RT-PCR , exhibiting > 85 % sequence homology with human Q13507 . Northern and Western blots employing Q13507 -probes and anti- Q13507 antibodies , respectively , confirmed endogenous expression of a Q13507 -like protein . Both cell types transfected with anti- Q13507 antisense oligodeoxynucleotides showed reduced CCE and Mn(2+) entry induced by either thapsigargin or 1alpha,25(OH)(2)D(3) . In muscle cells , anti- P11473 antisense inhibited steroid-induced Ca(2+) and Mn(2+) influx and co-immunoprecipitation of Q13507 and P11473 was observed , suggesting an association between both proteins and a functional role of the receptor in 1alpha,25(OH)(2)D(3) activation of CCE . In osteoblasts , two PCR fragments showing high homology with human INAD-like sequences were obtained . Northern blot and antisense functional assays suggested the involvement of the INAD-like protein in CCE regulation by the hormone . Therefore , we propose that an endogenous Q13507 protein mediates 1alpha,25(OH)(2)D(3) modulation of CCE in muscle and osteoblastic cells , which seems to implicate P11473 - Q13507 association and the participation of a INAD-like scaffold protein . Expression of transient receptor potential P13671 channels in human lung macrophages . Chronic obstructive pulmonary disease ( P48444 ) is associated with pulmonary inflammation with increased numbers of macrophages located in the parenchyma . These macrophages have the capacity to mediate the underlying pathophysiology of P48444 ; therefore , a better understanding of their function in chronic inflammation associated with this disease is vital . Ion channels regulate many cellular functions ; however , their role in macrophages is unclear . This study examined the expression and function of transient receptor potential ( TRP ) channels in human macrophages . Human alveolar macrophages and lung tissue macrophages expressed increased mRNA and protein for Q9Y210 when compared with monocytes and monocyte-derived macrophages . Moreover , Q9Y210 mRNA expression was significantly elevated in alveolar macrophages from patients with P48444 compared with control subjects . There were no differences in mRNA for Q13507 or TRPC7 . Although mRNA for O94759 and Q8NER1 was detected in these cells , protein expression could not be determined . Fractionation of lung-derived macrophages demonstrated that Q9Y210 protein was more highly expressed by smaller macrophages compared with larger macrophages . Using whole-cell patch clamp electrophysiology , Q9Y210 -like currents were measured in both macrophage subpopulations with appropriate biophysical and basic pharmacological profiles . These currents were active under basal conditions in the small macrophages . These data suggest that Q9Y210 -like channels are functional on human lung macrophages , and may be associated with P48444 . Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells . The active vitamin D3 metabolite DB00136 ( 1,25(OH)2D3 ) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediator implicated particularly in the function of cells belonging to the monocyte/macrophage lineage . These effects are controlled by the vitamin D receptor ( P11473 ) , a member of the steroid hormone receptor family . The objective of this study was to develop U937 transfectants expressing antisense P11473 mRNA , and to use these to examine the role of 1,25(OH)2D3- P11473 interaction in this lineage . A 2-kb P11473 cDNA insert ( including the complete P11473 coding region ) was cloned in an antisense orientation into the EBV episomal vector pMEP4 under the control of an inducible promoter and transfected into U937 . The resultant cell line , DH42 , was hygromycin resistant , contained P11473 cDNA , expressed fewer VDRs than controls , and showed a substantial decrease in antiproliferative response to 1,25(OH)2D3 . However , 1,25(OH)2D3 increased the number of cells expressing macrophage cell surface Ags , including P08571 and CD11b . A subpopulation of smaller cells did not express the differentiation markers after cadmium stimulation . Cell cycle analysis showed shifts in the distribution of cells from P55008 to S phase , which were more pronounced after cadmium treatment . A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis . Thus , the functional outcome of the P11473 antisense transfection suggests that in the myelomonocytic lineage , P11473 expression may act as a protective mechanism against programmed cell death . Maxi-anion channel as a candidate pathway for osmosensitive DB00171 release from mouse astrocytes in primary culture . In the present study , we aimed to evaluate the pathways contributing to DB00171 release from mouse astrocytes during hypoosmotic stress . We first examined the expression of mRNAs for proteins constituting possible DB00171 -releasing pathways that have been suggested over the past several years . In RT-PCR analysis using both control and osmotically swollen astrocytes , amplification of cDNA fragments of expected size was seen for connexins ( P08034 , P35212 , P17302 ) , pannexin 1 ( Px1 ) , the Q99572 receptor , MRP1 and P08183 , but not P13569 . Inhibitors of exocytotic vesicular release , gap junction hemi-channels , P13569 , MRP1 , P08183 , the Q99572 receptor , and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive DB00171 release from astrocytes . In contrast , the hypotonicity-induced DB00171 release from astrocytes was most effectively inhibited by gadolinium ( 50 muM ) , an inhibitor of the maxi-anion channel , which has recently been shown to serve as a pathway for DB00171 release from several other cell types . Thus , we propose that the maxi-anion channel constitutes a major pathway for swelling-induced DB00171 release from cultured mouse astrocytes as well . A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology . The Mediator is a multiprotein transcriptional coactivator that is expressed ubiquitously in eukaryotes from yeast to mammals and is required for induction of RNA polymerase II ( pol II ) transcription by DNA binding transcription factors . In the work described here , we exploit multidimensional protein identification technology ( MudPIT ) to carry out a proteomic analysis of the subunit composition of the mammalian Mediator complex . By comparing MudPIT data sets obtained from six independent Mediator preparations immunoaffinity purified through their Nut2 ( Q9BTT4 ) , Med25 ( Q9NWA0 ) , Intersex ( Q9NX70 ) , A0JLT2 ( A0JLT2 ) , AK007855 ( Q9H204 ) , or CRSP70 ( O95402 ) subunits , we identify a set of consensus mammalian Mediator subunits . In addition , we identify as Mediator-associated proteins the P49336 -like cyclin-dependent kinase CDK11 and the Q9UHV7 -like Q71F56 protein ( Q71F56 ) , which is mutated in patients with the congenital heart defect transposition of the great arteries ( TGA ) . P12004 D-cdk4 activity modulates the subnuclear localization and interaction of Q02078 with P12931 -family coactivators during skeletal muscle differentiation . Prior work has indicated that D-type cyclin-cdk4 complexes , which are only active in proliferating cells , can suppress the skeletal muscle differentiation program in proliferating myoblasts . In this study , we show that cyclin D-cdk activity can block the activity of the Q02078 family of transcriptional regulators , which are crucial regulators of skeletal muscle gene expression . We have found that cyclin D-cdk activity blocks the association of Q06413 with the coactivator protein Q9Y3R0 and thereby inhibits the activity of Q02078 . During skeletal muscle differentiation , Q9Y3R0 is localized to punctate nuclear structures and can apparently tether Q02078 to such structures . Cotransfection of Q9Y3R0 can both potentiate the transcriptional activity of a Gal4- Q06413 construct and induce Q06413 localization to punctate nuclear structures . Consistent with the absence of punctate nuclear Q9Y3R0 in proliferating myoblasts , we have found that ectopic cyclin D-cdk4 expression disrupts the localization of both Q9Y3R0 and Q06413 to these punctate subnuclear structures . Our findings indicate that cyclin D-cdk4 activity represses skeletal muscle differentiation in proliferating cells by blocking the association of Q02078 with the coactivator Q9Y3R0 and concomitantly disrupts the association of these factors with punctate nuclear subdomains within the cell . Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells . OBJECTIVE : This study was performed to assess adult human bone marrow mesenchymal stem/progenitor cells at a single-cell level and to determine a hierarchy based on proliferative potential . MATERIALS AND METHODS : Adult bone marrow mesenchymal cells expressing the enhanced green fluorescent protein ( EGFP ) were sorted as single cells into 24-well plates , each well confirmed with single EGFP-positive cells by fluorescence microscopy , and counted every 3 days . Colonies derived from single cells were expanded then sorted and evaluated using established differentiation protocols for adipogenic , chondrogenic , and osteogenic lineages . Cells were further analyzed by real-time reverse transcription polymerase chain reaction ( RT-PCR ) ( peroxisome proliferator-activated receptor[ Q07869 ]-gamma2 , P41159 , P06858 , P51884 , P49747 , BIG , Q13950 , P21815 , P02818 ) and immunocytochemistry ( Q07869 -gamma1/2 , collagen II , bone sialoprotein II ) specific for trilineage differentiation . RESULTS : Bone marrow mesenchymal cells were found to contain high proliferative potential ( HPP ) mesenchymal colony-forming cells ( MCFC ) ( 7 % ) , low proliferative potential ( Q93052 ) MCFC ( 29 % ) , mesenchymal cell clusters ( MCC , 26 % ) , and mature mesenchymal cells ( DB00305 , 38 % ) . All Q93052 -MCFC , MCC , and DB00305 colonies reached senescence at the end of the evaluation period . However , HPP-MCFC continued to grow , showed differentiation toward all three lineages , and demonstrated the capacity to give rise to secondary HPP-MCFC upon replating at a clonal level . CONCLUSION : These findings suggest that there is a low frequency of bone marrow-derived HPP-MCFC that can both self-renew at a single-cell level and differentiate toward multiple lineages of mesenchymal origin . Dynamic genetic linkage of intermediate blood pressure phenotypes during postural adaptations in a founder population . Blood pressure ( BP ) is a dynamic phenotype that varies rapidly to adjust to changing environmental conditions . Standing upright is a recent evolutionary trait , and genetic factors that influence postural adaptations may contribute to BP variability . We studied the effect of posture on the genetics of BP and intermediate BP phenotypes . We included 384 sib-pairs in 64 sib-ships from families ascertained by early-onset hypertension and dyslipidemia . Blood pressure , three hemodynamic and seven neuroendocrine intermediate BP phenotypes were measured with subjects lying supine and standing upright . The effect of posture on estimates of heritability and genetic covariance was investigated in full pedigrees . Linkage was conducted on 196 candidate genes by sib-pair analyses , and empirical estimates of significance were obtained . A permutation algorithm was implemented to study the postural effect on linkage . ADRA1A , APO , CAST , Q9Y5Q5 , P34998 , P24530 , P09038 , GC , P17302 , Q92953 , P08254 , P01303 , P08235 , P26678 , P37173 , P25445 , and P34981 showed evidence of linkage with any phenotype in the supine position and not upon standing , whereas P15121 , P16671 , P25101 , P12259 , P14780 , PKD2 , P27169 , P37231 , Q9UBK2 , P17252 , and P07949 were specifically linked to standing phenotypes . Genetic profiling was undertaken to show genetic interactions among intermediate BP phenotypes and genes specific to each posture . When investigators perform genetic studies exclusively on a single posture , important genetic components of BP are missed . Supine and standing BPs have distinct genetic signatures . Standardized maneuvers influence the results of genetic investigations into BP , thus reflecting its dynamic regulation . Recruitment and subnuclear distribution of the regulatory machinery during 1alpha,25-dihydroxy vitamin D3-mediated transcriptional upregulation in osteoblasts . The architectural organization of the genome and regulatory proteins within the nucleus supports gene expression in a physiologically regulated manner . In osteoblastic cells ligand activation induces a nuclear punctate distribution of the 1alpha,25-dihydroxy vitamin D3 ( DB00136 ) receptor ( P11473 ) and promotes its interaction with transcriptional coactivators such as Q15788 , NCoA-62/Skip , and Q15648 . Here , we discuss evidence demonstrating that in osteoblastic cells P11473 binds to the nuclear matrix fraction in a DB00136 -dependent manner . This interaction occurs rapidly after exposure to DB00136 and does not require a functional P11473 DNA binding domain . The nuclear matrix-bound P11473 molecules colocalize with the also nuclear matrix-associated coactivator Q15648 . We propose a model where the rapid association of P11473 with the nuclear matrix fraction represents an event that follows DB00136 -dependent nuclear localization of P11473 , but that precedes DB00136 -dependent transcriptional upregulation at target genes . An essential role of the CAAT/enhancer binding protein-alpha in the vitamin D-induced expression of the human steroid/bile acid-sulfotransferase ( Q06520 ) . The vitamin D receptor ( P11473 ) regulates steroid and drug metabolism by inducing the genes encoding phase I and phase II enzymes . Q06520 is a liver- and intestine-expressed sulfo-conjugating enzyme that converts the alcohol-OH of neutral steroids , bile acids , and drugs to water-soluble sulfated metabolites . DB00136 [ 1,25-(OH)2D3 ] induces Q06520 gene transcription after the recruitment of P11473 to the vitamin D-responsive chromatin region of Q06520 . A composite element in human Q06520 directs the 1,25-(OH)2D3-mediated induction of natural and heterologous promoters . This element combines a P11473 /retinoid X receptor-alpha-binding site [ vitamin D response element ( VDRE ) ] , which is an imperfect inverted repeat 2 of AGCTCA , and a CAAT/enhancer binding protein ( C/EBP ) -binding site located 9 bp downstream to VDRE . The binding sites were identified by EMSA , antibody supershift , and deoxyribonuclease I footprinting . C/EBP-alpha at the composite element plays an essential role in the P11473 regulation of Q06520 , because 1 ) induction was lost for promoters with inactivating mutations at the VDRE or C/EBP element ; 2 ) Q06520 induction by 1,25-(OH)2D3 in C/EBP-alpha-deficient cells required the expression of cotransfected C/EBP-alpha ; and 3 ) C/EBP-beta did not substitute for C/EBP-alpha in this regulation . P11473 and C/EBP-alpha were recruited concurrently to the composite element along with the coactivators p300 , steroid receptor coactivator 1 ( Q15788 ) , and P12931 -2 , but not Q9Y6Q9 . P11473 and C/EBP-alpha associated endogenously as a DNA-dependent , coimmunoprecipitable complex , which was detected at a markedly higher level in 1,25-(OH)2D3-treated cells . These results provide the first example of the essential role of the interaction in cis between C/EBP-alpha and P11473 in directing 1,25-(OH)2D3-induced expression of a P11473 target gene . Osteoblast and osteocyte-specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing . Connexin43 ( P17302 ) plays an important role in osteoblastic differentiation in vitro , and bone formation in vivo . Mice with osteoblast/osteocyte-specific loss of P17302 display decreased gap junctional intercellular communication ( GJIC ) , bone density , and cortical thickness . To determine the role of P17302 in fracture healing , a closed femur fracture was induced in P02818 -Cre+ ; P17302 (flox/flox) ( Cx43cKO ) and Cre- ; P17302 (flox/flox) ( WT ) mice . We tested the hypothesis that loss of P17302 results in decreased bone formation and impaired healing following fracture . Here , we show that osteoblast and osteocyte-specific deletion of P17302 results in decreased bone formation , bone remodeling , and mechanical properties during fracture healing . Cx43cKO mice display decreased bone volume , total volume , and fewer TRAP+ osteoclasts . Furthermore , loss of P17302 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post-fracture , compared to WT mice . These studies identify a novel role for the gap junction protein P17302 during fracture healing , suggesting that loss of P17302 can result in both decreased bone formation and bone resorption . Therefore , enhancing P17302 expression or GJIC may provide a novel means to enhance bone formation during fracture healing . Dynamic and ligand-selective interactions of vitamin D receptor with retinoid X receptor and cofactors in living cells . The vitamin D receptor ( P11473 ) mediates vitamin D signaling in numerous physiological and pharmacological processes , including bone and calcium metabolism , cellular growth and differentiation , immunity , and cardiovascular function . Although transcriptional regulation by P11473 has been investigated intensively , an understanding of ligand-selective dynamic P11473 conformations remains elusive . Here , we examined ligand-dependent dynamic interactions of P11473 with retinoid X receptor ( RXR ) , steroid receptor coactivator 1 ( Q15788 ) , and silencing mediator of retinoic acid and thyroid hormone receptor ( Q9Y618 ) in cells using fluorescence resonance energy transfer ( FRET ) and chromatin immunoprecipitation ( ChIP ) assays . We compared the effects of 1α,25-dihydroxyvitamin D(3) [ 1,25(OH)(2)D(3) ] , lithocholic acid ( LCA ) , and ( 25R ) -25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-20-epivitamin D(3) ( ADTT ) , a partial agonist/antagonist vitamin D derivative . In the absence of ligand , P11473 homodimers were preferred to RXR heterodimers and were associated with Q9Y618 . 1,25(OH)(2)D(3) induced heterodimerization with RXR , dissociation of Q9Y618 , and association of Q15788 . LCA and ADTT induced those effects to a lesser extent at concentrations that did not induce expression of the P11473 target gene Q07973 in human embryonic kidney ( P29320 ) 293 cells . Unlike in HEK293 cells , ADTT increased Q07973 expression in HCT116 cells and increased the association of P11473 and Q9Y618 on the Q07973 promoter . The results indicate that ligand-selective conformation may lead to unique cofactor complex formation in a cell context-dependent manner . The combination of FRET and ChIP assays is a powerful tool useful in understanding ligand-selective dynamic P11473 conformations and the development of selective P11473 modulators . A concise and efficient route to 2alpha-(omega-hydroxyalkoxy)-1alpha,25-dihydroxyvi tam in D3 : remarkably high affinity to vitamin D receptor . [ reaction : see text ] A convenient and potentially valuable synthetic approach to the novel 2alpha-functionalized 1alpha,25-dihydroxyvitamin D3 [ DB00136 ] derivatives ( 1a-c ) , which are the P06681 -epimer of ED-71 and its analogues , has been developed . The C2alpha-modified ring A precursors ( 1,7-enynes 16 , n = 0 , 1 , and 2 ) were constructed stereoselectively starting from D-glucose in high yield . In the synthesized 2alpha-(omega-hydroxyalkoxy)- DB00136 derivatives , 1a and 1b showed a greater binding affinity to vitamin D receptor ( P11473 ) , up to 1.8 times that of the native hormone . Inhibition of serum-stimulated mitogen activated protein kinase by 1alpha,25(OH)2-vitamin D3 in MCF-7 breast cancer cells . DB00136 [ DB00136 ] , the hormonally active form of vitamin D3 , has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo . DB00136 acts through two different mechanisms . In addition to regulating gene transcription via its specific intracellular receptor ( vitamin D receptor , P11473 ) , DB00136 induces rapid , non-transcriptional responses involving activation of transmembrane signal transduction pathways , like growth factors and peptide hormones . The mechanisms that mediate the antiproliferative effects of DB00136 in breast cancer cells are not fully understood . Particularly , there is no information about the early non-genomic signal transduction effectors modulated by the hormone . The present study shows that DB00136 rapidly inhibits serum induced activation of P27361 and P28482 Q96HU1 kinases . The tyrosine kinase Src is involved in the pathway leading to activation of P29323 1/2 by serum . Furthermore , DB00136 increases the tyrosine-phosphorylated state of Src and inhibits its kinase activity , while induces the association of the P11473 with Src , either in the presence or absence of serum . In parallel , the hormone rapidly increases the amounts of P11473 associated to plasma membranes ( PM ) . Pretreatment with the tyrosine phosphatase inhibitors orthovanadate or bpV ( phen ) prevented mitogen-activated protein kinase ( MAPK ) inhibition by DB00136 . These data altogether suggest that DB00136 inhibits the MAPK cascade by inactivating Src tyrosine kinase through a mechanism mediated by the P11473 and tyrosine phosphatases . Nongenotropic , anti-apoptotic signaling of 1alpha,25(OH)2-vitamin D3 and analogs through the ligand binding domain of the vitamin D receptor in osteoblasts and osteocytes . Mediation by Src , phosphatidylinositol 3- , and JNK kinases . Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors , we have explored the possibility that the vitamin D nuclear receptor ( P11473 ) might exhibit similar nongenotropic actions . We report that the conformationally flexible full P11473 agonist , 1alpha,25(OH)2-vitamin D3 ( DB00136 ) , and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 ( JN ) analog , also acting through the P11473 but with poor transcriptional activity , protected murine osteoblastic or osteocytic cells from apoptosis . This effect was reproduced in HeLa cells transiently transfected with either wild type P11473 or a mutant consisting of only the P11473 ligand binding domain . The P11473 ligand binding domain bound [3H] DB00136 as effectively as wild type P11473 but did not induce vitamin D response element-mediated transcription . The anti-apoptotic effects of DB00136 and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors : Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine ( P50391 ) , phosphatidylinositol 3 kinase inhibitor Wortmannin , and the JNK kinase inhibitor SP600125 . However , inhibition of p38 with SB203580 or P29323 with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions . Further , DB00136 induced rapid ( 5 min ) association of P11473 with Src kinase in OB-6 cells . Finally , actinomycin D or cycloheximide prevented the anti-apoptotic effect of DB00136 , indicating that transcriptional events are also required . These findings suggest that nongenotropic modulation of kinase activity is also a general property of the P11473 and that ligands that activate nongenotropic signals , but lack transcriptional activity , display different biological profiles from the steroid hormone DB00136 . Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells . P13569 ( P13569 ) is a P13569 in epithelial cells ; recently , we identified it in mast cells . Previous work that we confirmed showed that interferon gamma ( IFNgamma ) down-regulated P13569 expression in epithelial cells ( T84 ) , but by contrast , we found that IFNgamma up-regulated P13569 mRNA and protein expression in rat and human mast cells . IFNgamma up-regulation of P13569 in mast cells was inhibited by p38 and extracellular signal-regulated kinase ( P29323 ) kinase inhibitors but not a Janus tyrosine kinase (JAK)2 inhibitor , whereas in T84 cells IFNgamma-mediated down-regulation of P13569 was O60674 -dependent and P29323 - and p38-independent . Furthermore , IFNgamma down-regulation of P13569 in T84 epithelial cells was P42224 -dependent , but up-regulation of P13569 in mast cells was P42224 -independent . Thus , differential regulatory pathways of P13569 expression in mast cells and epithelial cells exist that depend upon either p38/ P29323 or JAK/ P35610 pathways , respectively . Surprisingly , IFNgamma treatment of mast cells inhibited Cl(-) efflux , in contrast to up-regulation of P13569 /mRNA and protein expression . However , down-regulation of Cl(-) flux correlated with IFNgamma-mediated inhibition of mediator secretion . This and other work suggests that the effect of IFNgamma on P13569 expression in mast cells is important for their function . DB09280 - DB08820 in Patients with Cystic Fibrosis Homozygous for Phe508del P13569 . Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells : Potential mechanism for excessive P10145 expression . Cystic fibrosis ( CF ) is a lethal disease caused by defective function of the cftr gene product , the CF transmembrane conductance regulator ( P13569 ) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients . We here report the effects of oxidative stress ( hyperoxia , 95 % O(2) ) on the expression of pro-inflammatory interleukin ( IL ) -8 and P25024 /2 receptors in two human CF lung epithelial cell lines ( IB3-1 , with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation ) and two control non-CF lung epithelial cell lines ( S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o- ) . Under oxidative stress , the expression of P10145 and P25024 /2 receptors was increased in CF , corrected and normal lung cell lines . The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB ( NF-kappaB ) and activator protein-1 ( AP-1 ) activities . Under oxidative stress , no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells . The signalling of mitogen-activated protein ( Q96HU1 ) kinases was further studied . We demonstrated that extracellular signal-regulated kinase ( P27361 /2 ) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress . Consistently , inhibition of P27361 /2 in oxidative stress-exposed CF lung cells strongly decreased both the P10145 production and P25024 /2 expression . Therefore , targeting of P27361 /2 Q96HU1 kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients . Telomere shortening is associated with reduced duodenal HCOFormula secretory but normal gastric acid secretory capacity in aging mice . The incidence of duodenal ulcer , especially Helicobacter pylori-negative duodenal ulcer , strongly increases with age . In humans , telomere length shortening is considered to be one critical factor in cellular senescence and organ survival . In this study , we compared basal and stimulated gastric acid and duodenal HCO(3)(-) secretory rates in aged late-generation ( G(3) ) telomerase-deficient ( mTERC(-/-) ) mice , which are characterized by severe telomere dysfunction due to the inability to elongate telomeres during cell division . We found that basal and forskolin-stimulated HCO(3)(-) secretion and short-circuit current ( I(sc) ) in isolated duodenal mucosa of G(3) mTERC(-/-) mice were markedly reduced compared with age-matched wild-type mice . In contrast , basal and forskolin-stimulated acid secretory rates in isolated G(3) mTERC(-/-) gastric mucosa were not significantly altered . Correspondingly , duodenal mucosa of G(3) mTERC(-/-) mice showed slimming and shortening of villi , whereas gastric mucosal histology was not significantly altered . However , the ratios of cystic fibrosis transmembrane conductance regulator ( P13569 ) and solute-linked carrier 26 gene family ( Slc26a6 ) mRNA expression in relation to cytokeratin-18 were not altered in duodenal mucosa . The further knockout of P38936 , which is a downstream effector of telomere shortening-induced senescence , rescued villus atrophy of duodenal mucosa , and basal and forskolin-stimulated duodenal HCO(3)(-) secretion and I(sc) in mTERC(-/-) P38936 (-/-) double-knockout mice were not different from wild-type controls . In conclusion , genetic ablation of telomerase resulted in P38936 -dependent duodenal mucosal atrophy and reduced duodenal HCO(3)(-) secretory capacity , whereas gastric morphology and acid secretory function were preserved . This suggests that telomere shortening during aging may result in an imbalance between aggressive and protective secretions against duodenal mucosa and thus predispose to ulcer formation . Isoform-specific transcriptional regulation by thyroid hormone receptors : hormone-independent activation operates through a steroid receptor mode of co-activator interaction . Thyroid hormone receptors ( T3Rs ) are hormone-regulated transcription factors that play important roles in vertebrate homeostasis , differentiation , and development . T3Rs are synthesized as multiple isoforms that display tissue-specific expression patterns and distinct transcriptional properties . Most T3R isoforms associate with co-activator proteins and mediate transcriptional activation only in the presence of thyroid hormone . The pituitary-specific T3Rbeta-2 isoform departs from this general rule and is able to interact with P52701 co-activators , and to mediate transcriptional activation in both the absence and presence of hormone . We report here that this hormone-independent activation is mediated by contacts between the unique N terminus of T3Rbeta-2 and an internal interaction domain in the Q15788 ( steroid receptor co-activator-1 ) and Q9Y3R0 ( glucocorticoid receptor interacting protein 1 ) co-activators . These hormone-independent contacts between T3Rbeta-2 and the P52701 co-activators are distinct in sequence and function from the LXXLL motifs that mediate hormone-dependent transcriptional activation and resemble instead a mode of co-activator recruitment previously observed only for the steroid hormone receptors and only in the presence of steroid hormone . Our results suggest that the transcriptional properties of the different T3R isoforms represent a combinatorial mixture of repression , antirepression , and hormone-independent and hormone-dependent activation functions that operate in conjunction to determine the ultimate transcriptional outcome . Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells . During reprogramming of porcine mesenchymal cells with a four-factor ( Q01860 / P48431 / O43474 /MYC ) mixture of vectors , a fraction of the colonies had an atypical phenotype and arose earlier than the recognizable porcine induced pluripotent stem ( iPS ) cell colonies . Within days after each passage , patches of cells with an epithelial phenotype formed raised domes , particularly under 20 % O(2) conditions . Relative to gene expression of the iPS cells , there was up-regulation of genes for transcription factors associated with trophoblast ( TR ) lineage emergence , e.g. , P23769 , P37231 , P35548 , O60479 , O96004 , Q9NP62 , Q99626 , Q02363 , Q9UKW6 , TCFAP2C , and Q15561 and for genes required for synthesis of products more typical of differentiated TR , such as steroids ( P14061 , P05108 , and STAR ) , pregnancy-associated glycoproteins ( PAG6 ) , and select cytokines ( IFND , P01579 , and P01584 ) . Although Q01860 was down-regulated relative to that in iPS cells , it was not silenced in the induced TR ( iTR ) cells over continued passage . Like iPS cells , iTR cells did not senesce on extended passage and displayed high telomerase activity . Upon xenografting into immunodeficient mice , iTR cells formed nonhemorrhagic teratomas composed largely of layers of epithelium expressing TR markers . When cultured under conditions that promoted embryoid body formation , iTR cells formed floating spheres consisting of a single epithelial sheet whose cells were tethered laterally by desmosome-like structures . In conclusion , reprogramming of porcine fibroblasts to iPS cells generates , as a by-product , colonies composed of self-renewing populations of TR cells , possibly containing TR stem cells . Down-regulated P13569 During Aging Contributes to Benign Prostatic Hyperplasia . Benign prostatic hyperplasia ( BPH ) is a hyper-proliferative disease of the aging prostate ; however , the exact mechanism underlying the development of BPH remains incompletely understood . The present study investigated the possible involvement of the cystic fibrosis transmembrane conductance regulator ( P13569 ) , which has been previously shown to negatively regulate nuclear factor-κB ( NF-κB ) /cyclooxygenase 2 ( P35354 ) /prostaglandin E2 ( DB00917 ) pathway , in the pathogenesis of BPH . Our results showed decreasing P13569 and increasing P35354 expression in rat prostate tissues with aging . Furthermore , suppression of P13569 led to increased expression of P35354 and over-production of DB00917 in a normal human prostate epithelial cell line ( PNT1A ) with elevated NF-κB activity . DB00917 stimulated the proliferation of primary rat prostate stromal cells but not epithelial cells , with increased P12004 expression . In addition , the condition medium from PNT1A cells after inhibition or knockdown of P13569 promoted cell proliferation of prostate stromal cells which could be reversed by P35354 or NF-κB inhibitor . More importantly , the involvement of P13569 in BPH was further demonstrated by the down-regulation of P13569 and up-regulation of P35354 /NF-κB in human BPH samples . The present results suggest that P13569 may be involved in regulating DB00917 production through its negative regulation on NF-κB/ P35354 pathway in prostate epithelial cells , which consequently stimulates cell growth of prostate stromal cells . The overstimulation of prostate stromal cell proliferation by down-regulation of P13569 -enhanced DB00917 production and release during aging may contribute to the development of BPH . The human peroxisome proliferator-activated receptor delta gene is a primary target of 1alpha,25-dihydroxyvitamin D3 and its nuclear receptor . Peroxisome proliferator-activated receptor ( Q07869 ) delta is the most widely expressed member of the Q07869 family of nuclear receptor fatty acid sensors . Real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARdelta expression was increased 1.5 to 3.2-fold after three hours stimulation with the natural vitamin D receptor ( P11473 ) agonist , 1alpha,25-dihydroxyvitamin D3 ( DB00136 ) . In silico analysis of the 20 kb of the human PPARdelta promoter revealed a Q93038 -type DB00136 response element approximately 350 bp upstream of the transcription start site , which was able to bind P11473 -retinoid X receptor ( RXR ) heterodimers and mediate a DB00136 -dependent upregulation of reporter gene activity . Chromatin immuno-precipitation assays demonstrated that a number of proteins representative for DB00136 -mediated gene activation , such as P11473 , RXR and RNA polymerase II , displayed a DB00136 -dependent association with a region of the proximal PPARdelta promoter that contained the putative Q93038 -type VDRE . This was also true for other proteins that are involved in or are the subject of chromatin modification , such as the histone acetyltransferase CBP and histone 4 , which displayed ligand-dependent association and acetylation , respectively . Finally , real-time PCR analysis demonstrated that DB00136 and the synthetic PPARdelta ligand L783483 show a cell and time-dependent interference in each other 's effects on P11473 mRNA expression , so that their combined application shows complex effects on the induction of P11473 target genes , such as Q07973 . Taken together , we conclude that PPARdelta is a primary DB00136 -responding gene and that P11473 and PPARdelta signaling pathways are interconnected at the level of cross-regulation of their respective transcription factor mRNA levels . Selective use of multiple vitamin D response elements underlies the 1 alpha,25-dihydroxyvitamin D3-mediated negative regulation of the human O15528 gene . The human 25-hydroxyvitamin D3 ( DB00146 ) 1alpha-hydroxylase , which is encoded by the O15528 gene , catalyzes the metabolic activation of the DB00146 into 1alpha,25-dihydroxyvitamin D3 ( DB00136 ) , the most biologically potent vitamin D3 metabolite . The most important regulator of O15528 gene activity is DB00136 itself , which down-regulates the gene . The down-regulation of the O15528 gene has been proposed to involve a negative vitamin D response element ( nVDRE ) that is located approximately 500 bp upstream from transcription start site ( TSS ) . In this study , we reveal the existence of two new P11473 -binding regions in the distal promoter , 2.6 and 3.2 kb upstream from the TSS , that bind vitamin D receptor-retinoid X receptor complexes . Since the down regulation of the O15528 gene is tissue- and cell-type selective , a comparative study was done for the new DB00136 -responsive regions in P29320 -293 human embryonic kidney and MCF-7 human breast cancer cells that reflect tissues that , respectively , are permissive and non-permissive to the phenomenon of DB00136 -mediated down-regulation of this gene . We found significant differences in the composition of protein complexes associated with these O15528 promoter regions in the different cell lines , some of which reflect the capability of transcriptional repression of the O15528 gene in these different cells . In addition , chromatin architecture differed with respect to chromatin looping in the two cell lines , as the new distal regions were differentially connected with the proximal promoter . This data explains , in part , why the human O15528 gene is repressed in P29320 -293 but not in MCF-7 cells . 20-Hydroxycholecalciferol , product of vitamin D3 hydroxylation by P450scc , decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes . The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc ( P05108 ) to form 20-hydroxycholecalciferol , which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes . Since nuclear factor-kappaB ( NF-kappaB ) plays a pivotal role in the regulation of cell proliferation , differentiation and apoptosis , we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB , using DB00136 ( calcitriol ) as a positive control . 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes . Also , 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein , IkappaB alpha , in a time dependent manner , while no changes in total NF-kappaB-p65 mRNA or protein levels were observed . Another measure of NF-kappaB activity , p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes . Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes , as determined by immunoblotting and immunofluorescent staining . In keratinocytes lacking vitamin D receptor ( P11473 ) , 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels , indicating that it requires P11473 for its action on NF-kappaB activity . Comparison of the effects of calcitrol , hormonally active form of vitamin D3 , with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB . Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators , our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases . P13569 mutations impart elevated immune reactivity in a murine model of cystic fibrosis related diabetes . Increased life expectancy in cystic fibrosis ( CF ) is accompanied by an increasing incidence of CF related diabetes ( CFRD ) . Altered immune reactivity occurs in CF , which we hypothesize , is exacerbated by hyperglycemia . P13569 deficient ( P13569 -/- ) mice were rendered hyperglycemic by streptozotocin ( Q11206 ) to test this hypothesis . P13569 -/- , C57BL/6J , and FVB/NJ mice received either Q11206 or lactated ringers ( LR ) ( n=5-10 ) . Four weeks later , splenocytes were harvested , mitogen stimulated , and analyzed for cytokine production ( P60568 , P05112 , and P22301 ) along with stimulation indices ( SI ) . SI of Q11206 -treated P13569 -/- were elevated compared to LR-treated mice , although both were greater than C57BL/6J and FVB/NJ ( p < 0.05 ) . Fasting glucose levels of Q11206 -treated P13569 -/- mice correlated with SI ( p < 0.003 ) . Stimulated P22301 concentrations were elevated in Q11206 -treated P13569 -/- compared to LR-treated animals and controls ( p < 0.05 ) . P60568 levels were greater in P13569 -/- mice compared to controls ( p < 0.05 ) , but unrelated to Q11206 . Reinforcing generalized cytokine up-regulation in P13569 -/- , P05112 levels were greater in P13569 -/- mice compared to C57BL/6J , but FVB/NJ mice demonstrated greatest concentrations following Q11206 . These results suggest that , hyperglycemia may exacerbate the clinical course in CF by impacting immune reactivity . There is clear need to maximize metabolic management in CFRD . Role of P49336 and beta-catenin in colorectal adenocarcinoma . Colorectal adenocarcinoma is a major cause of morbidity and mortality . The Wnt/beta-catenin pathway plays an important role in colon cancers . However , relatively little is known about the regulatory mechanism of beta-catenin in colon cancers . P49336 is a cyclin-dependent kinase ( CDK ) member of the mediator complex that couples transcriptional regulators to the basal transcriptional machinery , and is implicated in the transcriptional regulation of key pathways involved in colon cancers . To determine the relationship between P49336 and beta-catenin expressions , a population-based study was conducted for immunohistochemical staining analysis of tumor tissues , and Western blot analysis and P49336 interference studies of colon cancer cell lines . The hypothesis that colorectal cancers with P49336 expression have distinct clinical , prognostic and molecular attributes was tested . Among 127 colorectal cancers , P49336 expression was detected in 96 ( 76 % ) tumors by immunohistochemistry . P49336 and beta-catenin expression had significant positive correlation with carcinogenesis , tumor progression and patient survival . Immunohistochemically , P49336 expression in colorectal cancer was independently associated with beta-catenin activation ( P=0.0002 ) . However , beta-catenin expression was not completely suppressed by P49336 interference in the colon cancer cell lines HCT-116 , HT-29 and SNU- P01031 . These data support a potential link between P49336 and beta-catenin , and suggest that P49336 may identify a subset of colon cancer patients with a poor prognosis . However , control of P49336 is not an effective therapeutic strategy through beta-catenin regulation of general colon cancer . DB00136 induces capacitative calcium entry involving a Q13507 protein in skeletal muscle and osteoblastic cells . This work describes the involvement of TRPC proteins in capacitative calcium entry ( CCE ) induced by 1alpha,25-dihydroxy-vitamin-D3 [ DB00136 ] in chick skeletal muscle and in rat osteoblast-like cells ( ROS 17/2.8 ) and the role of the vitamin D receptor ( P11473 ) in this non-genomic rapid response mediated by the hormone . We propose that an endogenous Q13507 protein mediates DB00136 modulation of CCE in these cells , which seems to implicate P11473 - Q13507 association and the participation of an INAD-like scaffold protein . Inhibition by 1alpha,25-dihydroxyvitamin D3 of activin A-induced differentiation of murine erythroleukemic P12259 -5 cells . DB00136 ( 1alpha,25-(OH)2D3 ) and other vitamin D3 ( VD3 ) analogs enhanced the inhibitory effect of Activin A on murine erythroleukemia ( P61006 ) cell proliferation and differentiation in a dose-dependent manner . 1alpha,25-(OH)2D3 inhibited differentiation more potently than proliferation by one order of magnitude . The VD3 analog study demonstrated either effect of VD3 on P61006 cells via vitamin D receptor ( P11473 ) , as evidenced from the close relationship with the reported affinities for P11473 . The effects of 1alpha,25-(OH)2D3 were preceded by the suppression of ornithine decarboxylase ( ODC ) activity , a rate-limiting enzyme in polyamine metabolism . Difluoromethylornithine ( DB06243 ) , an inhibitor of ODC , inhibited P61006 cell proliferation , which was reversed by the simultaneous addition of putrescine , a product of ODC , but did not affect differentiation . 1alpha,25-(OH)2D3 inhibited cell differentiation during the phenotype-expression stage as reflected by the inhibition of beta-globin gene expression , while it inhibited proliferation in the commitment stage . Furthermore , it seems unlikely that the different effects of VD3 on proliferation and differentiation may be a result of upregulation of P11473 or nongenomic action . In summary , it was suggested that 1alpha,25-(OH)2D3 inhibited Activin A-induced P61006 cell proliferation and differentiation by distinct mechanisms and inhibited the proliferation by inhibiting ODC activity . We demonstrated the presence of 1alpha,25-(OH)2D3 action on leukemic cells at physiological concentration , which was distinct from the pharmacological effect of VD3 reported thus far . The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation . We compared the effects of 1alpha,25-dihydroxyvitamin D3 [ DB00136 ] and its analog , 1alpha,25-dihydroxy-16-ene-vitamin D3 [ 1alpha,25(OH)2-16-ene-D3 ] , as well as their interactions with 17-beta estradiol ( E2 ) on osteoblastic function in our human normal ( HOB ) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation , the more differentiated HOB+DEX cells and SaOS+DEX cells , and the corresponding less differentiated HOB-DEX and SaOS-DEX cells . The differential effects of DB00136 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant . The most significant effects were seen in SaOS+DEX cells , in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than DB00136 , the maximal enhancement being exerted at 0.1 nM and 10 nM , respectively . E2 enhanced the stimulatory effects of both compounds , with ALP being increased 2-fold at 0.1 nM ( p < 0.001 ) . P02818 ( OC ) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by DB00136 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM , with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3 . In SaOS-DEX and SaOS+DEX cells , DB00136 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of DB00136 . Western blot analysis of DB00136 receptor ( P11473 ) levels showed that in SaOS+DEX cells , the effect of DB00136 was larger than that of 1alpha,25(OH)2-16-ene-D3 . These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts .