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The hexosamine pathway regulates the plasminogen activator inhibitor-1 gene promoter and Sp1 transcriptional activation through protein kinase C-βI and -δ

Increased flux through the hexosamine biosynthesis pathway (HBP) has been shown to stimulate the expression of a number of genes. We previously demonstrated in glomerular mesangial and endothelial cells that both high glucose concentrations and glucosamine activated the plasminogen activator inhibitor-1 (PAI-1) gene promoter through the transcription factor, Sp1; and that the glutamine:fructose-6-phosphate amidotransferase inhibitor, 6-diazo-5-oxonorleucine, inhibited the effect of high glucose, but not that of glucosamine. Here, we examined the role of protein kinase C (PKC) isoforms in the regulation of the PAI-1 promoter and Sp1 transcriptional activity by the HBP. In transient transfections, exposure to 2 mm glucosamine or 20 mmglucose for 4 days increased the activities of a PAI-1 promoter-luciferase reporter gene as well as the Sp1 transcriptional activation domain fused to the GAL4 DNA-binding domain cotransfected with a GAL4 promoter-luciferase reporter. Cotransfected dominant negative PKC-βI and -δ completely blocked the induction of PAI-1 promoter transcription by both sugars, whereas only dominant negative PKC-βI interfered with Sp1-GAL4 activation. Both glucosamine and high glucose stimulated the in vitro kinase activity of immunoprecipitated PKC-βI and -δ. Furthermore, 6-diazo-5-oxonorleucine suppressed high glucose-induced PKC kinase activity and Sp1-GAL4 transcriptional activation. These findings demonstrate a requirement for the PKC-βI and -δ signal transduction pathways in HBP-induced transcription.

Prolonged exposure to high glucose concentrations (high glucose) promotes the development of microvascular disease associated with diabetes mellitus (1,2). The pathways by which high glucose triggers changes in cellular behavior are incompletely understood. Proposed mechanisms include nonenzymatic protein glycation and formation of advanced glycation end products (3), enhanced flux of glucose through the polyol pathway (4), generation of oxidative stress (5), and the activation of the diacylglycerol-protein kinase C (PKC) 1 signaling pathways (6 -14). Recently, we (15)(16)(17) and others (18 -27) have provided evidence for a potential contribution by an additional pathway of glucose metabolism, the hexosamine biosynthesis pathway (HBP).
We have used cultured rat glomerular mesangial cells to study hexosamine-induced gene expression. These cells are largely responsible for the extracellular matrix accumulation that occurs in diabetic nephropathy and provide a well established in vitro model for this condition (6 -10, 15, 27, 45). High glucose-mediated flux through the HBP in mesangial cells may be related not only to increased cellular entry of glucose, but also to up-regulation of mesangial cell GFA (as noted in rats with experimental diabetes) (24) and to the increased production of reactive oxygen species by mitochondrial glucose metabolism (5,16). Using the PAI-1 gene promoter as a model, we demonstrated that both glucose and glucosamine up-regulated PAI-1 promoter activity and that the GFA inhibitor, 6-diazo-5oxonorleucine (DON), inhibited the action of glucose but not that of glucosamine (15). Overexpression of GFA also stimulated the PAI-1 promoter (17). Furthermore, we found that the transcription factor, Sp1, was required for activation of the PAI-1 promoter by high glucose or glucosamine (15).
The mechanisms by which the HBP activates Sp1 and the PAI-1 promoter are unclear. However, based on some previous data, it has been hypothesized that O-GlcNAcylation of Sp1 increases gene expression. There are at least nine O-GlcNAc moieties on Sp1 that are located preferentially in the aminoterminal transcriptional activation domain (39,44,46). It was reported earlier that Sp1 produced in Escherichia coli, which lacks O-GlcNAc, activates in vitro transcription to a lesser extent than mammalian Sp1, which contains O-GlcNAc (44). Recently, high glucose has been found to activate the PAI-1 promoter via the HBP and Sp1 in endothelial cells and to increase overall Sp1 O-GlcNAcylation (16). In contrast, Kudlow and colleagues (46,47) have provided evidence suggesting that O-GlcNAcylation of the Sp1 transcriptional activation domain suppresses, rather than stimulates, Sp1 transcriptional activation. They mapped one of the Sp1 O-GlcNAcylation sites to serine 484 in the B transcriptional activation domain (46). O-GlcNAcylation of this site reduced interactions between Sp1 and the transcriptional cofactor, dTAF II 110 (46). Mutation of serine 484 to alanine enhanced the activity of a GAL4 fusion protein containing part of the Sp1 B domain in an in vitro transcription assay (47). This mutant construct was also more active than wild-type in transiently transfected HIT-T15 cells, which contain high levels of OGT, but not in HepG2 or HeLa cells, containing low OGT levels (47).
These data led us to consider an alternate explanation for hexosamine-induced transcription, namely that O-GlcNAcylation of cytoplasmic proteins may result in perturbations in intracellular signaling, thus impacting indirectly on transcription factors. The Sp1 site in the PAI-1 promoter could be regulated in this way. Although Sp1 is ubiquitous, constitutively expressed, and binds to GC-rich sites that are abundant in housekeeping genes (48,49), its activity is modulated by a variety of stimuli, including high glucose, PKC, cAMP, p38 mitogen-activated protein kinase (MAPK), growth factors, lipopolysaccharide, and TGF-␤ (15, 16, 48 -55).
Members of the PKC family of phospholipid-dependent serine/threonine kinases are particularly good candidates to be effectors of the hexosamine pathway. PKC isoforms are classified according to their sensitivity to diacylglycerol and calcium (56 -61). Classical PKC isoforms (␣, ␤I, ␤II, ␥) are activated by calcium and diacylglycerol, novel PKC isoforms (␦, ⑀, , ) depend only on diacylglycerol, and atypical PKC isoforms (/, ) respond to products of the phosphatidylinositol 3-kinase pathway. PKC isoforms differ in their patterns of expression, subcellular localization, and activation in response to external stimuli, conferring different functions upon them (56 -61). This is illustrated by the ability of PKC-⑀ to protect against ischemic injury, by the participation of PKC-␦ in apoptosis, and by the requirement of PKC-␤II for cellular proliferation (58). High glucose results in de novo synthesis of diacylglycerol and membrane translocation of various PKC isoforms, including PKC-␣, -␤I, -␦, -⑀, and -in mesangial and other cells in vitro and in experimental animals (6 -13). In addition, there is good evidence that these activated PKC isoforms contribute to high glucose-induced increases in gene transcription and participate in the pathogenesis of diabetes complications (6,9,11,12,14,26,27,62).
In this study we tested the hypothesis that alteration in PKC signaling is involved in the modulation of gene expression by increased flux through the HBP. We found that both high glucose and glucosamine increased the kinase activity of three PKC isoforms, ␤I, ␦, and ⑀, and interestingly, that this activation by high glucose was attenuated by inhibition of GFA. Specific inhibition of PKC-␤I and -␦, by cotransfection of dominant negative (DN) PKC mutants, blocked PAI-1 promoter activation. High glucose and glucosamine strongly stimulated the activity of a Sp1 transcriptional activation domain-GAL4 DNA binding domain fusion protein cotransfected with a GAL4-luciferase reporter and this was specifically blocked by DN-PKC-␤I. These data indicate that HBP-mediated gene expression involves both PKC-␤I and -␦, and that the required Sp1 transcriptional activation is dependent on altered cell signaling by PKC-␤I.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatments-Rat glomerular mesangial cells were grown in Dulbecco's modified Eagle's medium supplemented with 17.5% fetal bovine serum, as previously described (15). To increase flux through the hexosamine pathway, mesangial cells were exposed for 4 days to 2 mM glucosamine, prepared fresh and adjusted to pH 7.4. One day prior to harvest, the cells were washed three times with phosphatebuffered saline, without calcium or magnesium (PBS), and switched to serum free medium (minimal essential medium, 20 mM HEPES, 0.1% bovine serum albumin) containing 2 mM glutamine. For high glucose experiments, the cells were grown in Dulbecco's modified Eagle's medium containing 2 mM glutamine and either 1 mM glucose or 20 mM glucose, and changed to serum-free medium for the last 24 h of the experiment.
Plasmids-Sp1 (holo)-GAL4, which contains most of the human Sp1 cDNA (amino acids 83-778), fused to the DNA-binding domain of the yeast transcription factor GAL4 (amino acids 1-147), was a gift from S. Smale (UCLA, Los Angeles, CA) (63). The Sp1 transcriptional activation domain (TAD) (amino acids 91-604) fused to GAL4, (Sp1 (TAD)-GAL4) was generated by polymerase chain reaction (PCR) using Sp1 (holo)-GAL4 as a template and cloned into the vector, pFA-CMV (Stratagene) between EcoRI and BglII. The construct, AP-2-GAL4, containing a region (amino acids 31-76) from the proline-rich domain of the human AP-2 transcription factor fused to GAL4, was previously described by Alevizopoulos et al. (64) as a negative control. The cDNA for this AP-2 fragment was prepared by PCR and inserted between the BamHI and HindIII sites of pFA-CMV. Expression vectors for kinase-dead, DN-PKC-␣, -␦, -⑀, and -, which have a critical lysine in their ATP binding domains mutated to arginine, were kindly provided by J.-W. Soh and I. B. Weinstein (Columbia University, New York, NY) (59). The equivalent expression vectors for DN-PKC-␤I and -␤II, which have the critical lysine in their ATP binding domains mutated to methionine, were gifts from R. V. Farese (University of South Florida, Tampa, FL) (61). These vectors are all under control of a cytomegalovirus (CMV) promoter, except for DN-PKC-␤I, which was in the vector, pTB107, that is regulated by an SV40 promoter and a viral long terminal repeat (61). An empty pTB107 vector was created by PCR by deleting the DN-PKC-␤I cDNA insert. An expression vector for DN-stress-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (SEK), (also called MAPK kinase 4) was supplied by J. Woodgett (University of Toronto, Toronto, Canada). A luciferase reporter gene driven by a CMV promoter (CMV-luciferase) was a gift from N. C. W. Wong (University of Calgary, Calgary, Canada). All PCRs were performed with the high fidelity DNA polymerases, Pfu or Pfu turbo (Stratagene), and verified by dideoxy sequencing.
Transient Transfections-Semi-confluent mesangial cells, seeded in 24-well plates, were transiently transfected with the lipid reagent, FuGENE-6 (Roche Molecular Biochemicals), according to the manufacturer's instructions. Each well received 0.5 g of DNA and 0.83 l of FuGENE-6. Luciferase activity was measured in serum-starved cells and normalized for protein, as we have previously described (15).
Lysates were passed three times through a 26 G syringe and centrifuged at 100,000 ϫ g for 60 min at 4°C. The pellet was washed with buffer A and then solubilized with buffer A and 1% Triton X-100 on ice for 20 min. After centrifugation at 100,000 ϫ g for 60 min, the supernatant was saved as the membrane fraction. Samples containing equal amounts of proteins (5 g) (determined by a Bio-Rad DC kit) were separated by SDS-PAGE on a 10% polyacrylamide gel and then transferred overnight to nitrocellulose (Schleicher & Schuell). Ponceau S staining was used to confirm equal protein loading. The blots were blocked with 7% milk, 1% bovine serum albumin, 0.1% Tween 20, 20 mM Tris-HCl, pH 7.6, and 137 mM NaCl, and probed overnight in the same buffer with 1/5000 anti-PKC-␣, -␦, or -⑀ (Sigma), 1/2000 anti-PKC-, or 1/1000 anti-PKC-␤I or -␤II antibodies (Santa Cruz). Blots were developed with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1/5000, Invitrogen), Lumiglo ECL reagent (KPL, Gaithersburg, MD), and Biomax x-ray film (Eastman Kodak Co.). All blots were scanned and quantification was performed with Scion Image software (Scion Corp., Frederick, MD). Whole cell extracts were obtained by washing mesangial cells seeded in 10-cm dishes with ice-cold PBS and scraping into lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 10 mM ␤-glycerophosphate, 1 mM sodium vanadate, 5 mM NaF, and Complete protease inhibitor mixture. After incubation on ice for 20 min, lysates were extracted by centrifugation at 16,000 ϫ g for 5 min, boiled in SDS sample buffer, and 10 g of protein were separated by SDS-PAGE and immunoblotted with PKC-␤I, -␦, or -⑀ antibodies.
To assess tyrosine phosphorylation of PKC isoforms, mesangial cells were washed with PBS and scraped into lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 10 mM NaF, 1% Triton X-100, 1 mM sodium vanadate, and Complete protease inhibitor mixture. After incubation on ice for 20 min, the extracts were cleared by centrifuging at 16,000 ϫ g for 5 min at 4°C. For immunoprecipitation, 500 g of protein lysate were incubated with 2 g of PKC antibodies (Santa Cruz) for 3 h and the immune complexes recovered by rotating with Protein G-agarose (50 l) (Santa Cruz) for a further 60 min. The immunoprecipitates were washed four times with lysis buffer, and the pellet was then resuspended in 100 l, separated by SDS-PAGE on a 10% polyacrylamide gel, and then transferred overnight to nitrocellulose (Schleicher & Schuell). Immunoblotting was performed with anti-phosphotyrosine antibodies, PY99 (1/2000, Santa Cruz), or with PKC-␤1 or -␦ antibodies.
In Vitro PKC Kinase Activity-PKC isoform in vitro kinase activities were determined by modifications of previously reported assays (65,66). Serum-starved mesangial cells in 10-cm dishes were washed three times with ice-cold PBS and scraped into lysis buffer consisting of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 10 mM NaF, 50 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 100 nM Microcystin LR, and Complete protease inhibitor mixture. After incubation on ice for 20 min, the extracts were cleared by centrifuging at 16,000 ϫ g for 5 min at 4°C. For immunoprecipitation, aliquots containing 200 g of protein were incubated with 2 g of isoform-specific PKC antibodies (Santa Cruz) for 3 h and the immune complexes recovered by rotating with Protein G-agarose (40 l) (Santa Cruz) for an additional 60 min. PKC-␤I assays used 400 g of protein.
The immunoprecipitates were washed three times with 0.5 ml of lysis buffer and three times with 0.5 ml of kinase buffer consisting of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 0.1 mM sodium orthovanadate, 1 mM NaF, 0.1 mM sodium pyrophosphate, 100 nM Microcystin LR, and complete protease inhibitor mixture. The pellet was then resuspended in 100 l of kinase buffer containing 5 Ci of [␥- 32 (66) or from the myristoylated alanine-rich C kinase substrate protein (amino acids 151-175) (BIOMOL, Plymouth Meeting, PA) were substituted, respectively. The reaction was stopped with 25 l of 100 mM EDTA, pH 8.0, 0.1 mM ATP, and 60 l spotted onto Whatman P-81 ion exchange paper. These were washed four times for 5 min each with 75 mM orthophosphoric acid, washed once with 80% ethanol, and subjected to liquid scintillation counting. Specific counts were calcu-lated by subtracting from total radioactivity the nonspecific counts obtained when antibody was excluded from or nonspecific IgG used in the immunoprecipitation procedure, both yielding similar results. Nonspecific counts were less than 10%, except for PKC-␤I, which had relatively low basal activity (see below). PKC kinase activity was confirmed by the ability of added PKC inhibitor, GF109203X (100 nM), to block the reaction.
Statistics-Results are expressed as means Ϯ S.E. Student's t tests for unpaired samples were performed with Statistica software (Statsoft, Tulsa, OK). Values of p Ͻ 0.05 were considered significant. All experiments were repeated at least three times.

RESULTS
Glucosamine-induced PAI-1 Promoter Transcription Requires PKC-Regulation of transcription by the HBP was assessed by incubating glomerular mesangial cells with exogenous glucosamine (2 mM) in the presence of 5.6 mM glucose for 4 days. This approach has the advantage of potently inducing outcomes caused by the hexosamine pathway, while avoiding stimuli from other high glucose-activated pathways. Glucosamine is taken up by the glucose transporter, GLUT1, and phosphorylated to glucosamine 6-phosphate, thereby bypassing GFA (36). Elevated levels (13-fold increase) of hexosamine pathway end products, UDP-N-acetylhexosamines, were documented in our previous experiments and by others in glucosamine-treated mesangial cells (15,18,26).
To define a functional requirement for PKC isoforms in hexosamine-induced PAI-1 gene expression, we performed transient transfections with a 740-bp PAI-1 promoter linked to a luciferase reporter gene (PAI-LUC), as previously described (15). A 4.8 Ϯ 0.6-fold increase in PAI-1 promoter activity was observed in mesangial cells incubated with 2 mM glucosamine for 4 days. Pretreatment with the general PKC inhibitor, GF109203X (5 M) (9,12), diminished this to 1.5 Ϯ 0.3-fold (p Ͻ 0.01), but many chemical kinase inhibitors lack specificity (67). To confirm these results and address the roles of individual PKC isoforms, we cotransfected expression vectors for kinaseinactive, DN-PKC mutants with PAI-LUC. Previous reports have documented the ability of this type of construct to interfere with signal transduction mediated by specific PKC isoforms, presumably by competing with endogenous PKC isoforms for binding to substrates or to PKC-binding proteins (59 -61). Expression of the transiently transfected DN-PKC mutants was verified by immunoblotting and by immunofluorescence imaging with antibodies against their HA epitope tags in studies reported elsewhere (62). Glucosamine caused an 8-fold increase in PAI-LUC transcription in cells transfected with PAI-LUC and the empty vector, pcDNA3 (Fig. 1). Cotransfected DN-PKC-␤I, -␤II, or -␦ completely blocked glucosaminemediated PAI-LUC transcription (0.3 -, 0.7-, and 1.1-fold stimulation, respectively, versus untreated cells) whereas DN-PKC-␣ and -⑀ were only partially inhibitory (4.3-and 3-fold stimulation, respectively), and DN-PKC-had no effect (10-fold stimulation) (Fig. 1). The pTB107 vector, which was the backbone for DN-PKC-␤I, also had no effect (data not shown). Toxic effects of the DN-PKC isoforms in the presence of glucosamine were ruled out by transfecting a luciferase reporter gene driven by a CMV promoter (CMV-LUC). Glucosamine did not significantly alter expression from CMV-LUC cotransfected with the empty vector, pcDNA3 (1.09 Ϯ 0.23-fold stimulation). Cotransfection of DN-PKC-␤I or -␦ diminished this only slightly or not at all (0.83 Ϯ 0.1-and 1.45 Ϯ 0.33-fold, respectively, p ϭ NS). These results suggest that several PKC isoforms participate in glucosamine-stimulated transcription, with PKC-␤ and -␦ being the most critical.
Glucosamine Stimulates Mesangial Cell PKC Activity-The above data indicated that basal or stimulated PKC activity was required for glucosamine to induce PAI-1 expression. It has been well documented that high glucose causes PKC isoforms to translocate to membranes, the hallmark of PKC activation (6 -13). To determine whether the membrane content of PKC was affected by glucosamine, membrane fractions were prepared from mesangial cells and analyzed by immunoblotting with antibodies, as previously described (7,13). PMA (100 nM for 30 min) was used as a positive control. As expected, PMA caused PKC-␣, -␤I, -␦, and -⑀ to translocate to membranes (Fig.  2). PKC-␤II was not detected. Conversely, incubation with 2 mM glucosamine did not result in any increase in membranebound PKC isoforms. Furthermore, glucosamine did not affect membrane PKC content at shorter time points (0 -4 days). To be certain that this did not result from variations in membrane preparation techniques, the experiments were repeated by the protocol of Kolm-Litty et al. (25) and yielded identical results (data not shown).
PKC isoforms may be regulated by tyrosine and serine/threonine phosphorylation as well as by the allosteric effects of diacylglycerol (57, 68 -71). This prompted us to test the possibility that hexosamines increase intrinsic PKC kinase activity in the absence of membrane translocation. Different PKC isoforms were immunoprecipitated from whole cell lysates and used in immune complex kinase assays that included [␥-32 P]ATP, PMA, and peptide substrates, as previously reported (26, 57, 65, 66, 68 -73). Fig. 3A shows that exposure to 2 mM glucosamine augmented PKC-␤I, -␦, and -⑀ kinase activity 3-, 1.8-, and 1.4-fold, respectively, compared with untreated cells, but did not affect PKC-␣ or -activity. Increased cellular PKC content did not account for these results, because PKC-␤I, -␦, and -⑀ protein levels, determined by immunoblotting, were not increased by glucosamine (Fig. 3B). Basal PKC-␤I activity was much weaker than that of the other isoforms (ϳ1/30 of PKC-␦). This is consistent with PKC-␤I protein expression being much lower than that of the other isoforms and with other published reports (73). The 42-kDa PKC-␦ catalytic fragment, which is produced by caspase-dependent proteolysis in cells undergoing apoptosis (65), was not detected in either glucosamine-treated or control cells (data not shown).
The regulation of intrinsic PKC kinase activity is not fully understood at present. Nevertheless, there is evidence that reactive oxygen species enhance PKC kinase activity (69, 70, 74 -76). Interestingly, flux through the hexosamine pathway has been associated with oxidative stress (77). To begin to explore the role of reactive oxygen species in glucosaminestimulated PKC kinase activity, mesangial cells were exposed to glucosamine and treated for the final 24 h with, or without, the antioxidant, probucol (78,79). In these experiments, glucosamine stimulated PKC-␤I kinase activity, 2.1 Ϯ 0.1-fold (p Ͻ 0.01) and incubating the cells with 50 M probucol reduced activation by glucosamine to 0.6 Ϯ 0.1-fold (p Ͻ 0.01 versus glucosamine). Oxidative stress and other stimuli induce tyrosine phosphorylation of PKC isoforms (68 -70, 80 -82). Therefore, we assessed the tyrosine phosphorylation of PKC-␤I or -␦ immunoprecipitated from mesangial cells treated with glucosamine. These immunoblots did not reveal phosphotyrosine on PKC-␤I or -␦ in the basal state or in response to glucosamine, although both 100 nM PMA and 5 mM H 2 O 2 (used as positive controls) increased PKC-␦ tyrosine phosphorylation as previously reported (69, 70) (data not shown).
Glucosamine Enhances the Transactivation Function of Sp1-We previously showed the transcription factor, Sp1, to be a target of hexosamines in stimulating the PAI-1 promoter (15). In these earlier experiments, the increase in Sp1 DNA-binding in glucosamine-treated cells (1.3-fold) appeared to be insufficient to explain the marked induction of PAI-1 promoter activity (15). We, therefore, hypothesized that the hexosamine pathway regulates the Sp1 transcriptional activation domain. To test this, an expression vector encoding the Sp1 transcriptional activation domain fused to the yeast GAL4 DNA-binding domain (Sp1(TAD)-GAL4) was cotransfected with a GAL4-dependent luciferase reporter gene (GAL4-LUC). In these transfections, GAL4-LUC alone was inactive, with or without cotransfected GAL4 DNA-binding domain or glucosamine. Treatment of mesangial cells with glucosamine enhanced transactivation by the Sp1(TAD)-GAL4 construct 6.3-fold and caused a similar 5.9-fold increase in the activity of a vector expressing all the domains of Sp1 fused to GAL4 (Sp1(holo)-GAL4) (Fig. 4A). In contrast, a construct containing part of the proline-rich domain of the transcription factor AP-2, fused to GAL4 (AP2-GAL4) was resistant to activation by glucosamine. Sp1-GAL4 protein levels, determined by Western blotting, were not increased by glucosamine in these transfections (data not shown).

PKC-␤I Is Required for Glucosamine-induced Sp1
Transcriptional Activation-Because several PKC isoforms were activated by glucosamine and required for stimulation of the PAI-1 promoter by glucosamine, the role of these PKCs in Sp1 transcriptional activation was tested. To this end, the Sp1(holo)-GAL4 and GAL4-LUC containing vectors were cotransfected with DN-PKC isoform mutants. As shown in Fig. 4B, DN-PKC-␤I significantly diminished activation of Spl-GAL4 by glucosamine from 5-to 2-fold (p Ͻ 0.01). DN-PKC-␦ and DN-SEK, used as a control, had no significant inhibitory effects. These data indicate a requirement of PKC-␤I for the regulation of Sp1 transcriptional activity by glucosamine.
High Glucose-induced PAI-1 Promoter Activation Requires PKC-In our earlier study, high glucose activated the PAI-1 promoter and this was blocked by inhibition of the HBP with DON (15). Thus, it was important to determine whether this effect of high glucose required PKC, as we found for glucosamine. Because the kinase activities of PKC-␤I, -␦, and -⑀ were increased by glucosamine, we first assessed the effect of high glucose on PKC isoform kinase activity. In these experiments, mesangial cells were cultured in either 20 mM glucose (high glucose) or 1 mM glucose for 4 days. A relatively low base-line glucose concentration was used because we previously found that the glucose dose-response curve for PAI-1 promoter acti-vation was shifted to the left (15). Others have reported a similar phenomenon (30,83), which likely reflects elevated glucose uptake associated with high level GLUT1 expression in these cultured cells. Exposure of mesangial cells to 20 mM glucose increased PKC-␤I and PKC-␦ activity 5.2-and 1.6-fold, respectively (Fig. 5A). In contrast, PKC-⑀ kinase activity was only slightly enhanced by high glucose (1.18 Ϯ 0.08 of control; p Ͻ 0.05). There were small, but not statistically significant, increases in total cellular PKC-␤I (1.38 Ϯ 0.24-fold) and PKC-␦ (1.12 Ϯ 0.08-fold), which were insufficient to account for the elevated kinase activities (Fig. 5B). Interestingly, the activation of PKC -␤I and -␦ by high glucose was blocked by coincubation with DON (1.6-fold for PKC-␤I and 0.9-fold for PKC-␦), indicating involvement of the HBP (Fig. 5).
To determine the role of these isoforms in PAI-1 promoter activation, mesangial cells were cotransfected with PAI-LUC and either DN-PKC isoform mutants or empty vector. High glucose (20 mM) up-regulated PAI-1 promoter activity 3.8-fold compared with 1 mM glucose. Both DN-PKC-␤I and -␦ mutants inhibited this effect (1.5-fold increase for both isoforms) (Fig. 6).

PKC-␤I Is Required for High Glucose-induced Sp1
Transcriptional Activation-We postulated that, similar to glucosamine, the action of high glucose on the PAI-1 promoter was mediated by PKC-dependent Sp1 transcriptional activation. In experiments carried out as described above, high glucose augmented the activity of Sp1(holo)-GAL4 cotransfected with GAL4-LUC 4.6-fold, and this effect of high glucose was blocked by coincubation with DON (1.1-fold) (Fig. 7A). Cotransfection of DN-PKC-␤I reduced the activation of Spl-GAL4 by high glucose from 3.7-to 0.9-fold, whereas DN-PKC-␦ and DN-SEK did not inhibit activation by high glucose (5.7-and 3.6-fold stimulation, respectively) (Fig. 7B). The compound LY333531 and its analogue, LY379196, are relatively specific inhibitors of the PKC-␤ isoforms (6,11,84,85), and LY 333531 has been shown to inhibit high glucose-induced extracellular matrix synthesis (6,11,84). The inhibitor LY379196 has an IC 50 of 50 nM for PKC-␤I and 700 nM for PKC-␦ (85). Coincubating mesangial cells with 60 -240 nM LY379196 (Eli Lilly, Indianapolis, IN) abolished high glucose-induced PAI-LUC transcription (Fig. 8). DISCUSSION We previously found that enhanced flux through the HBP activated the PAI-1 promoter via Sp1 (15). In this study, we demonstrate that this process requires PKC activation and is mediated by increases in the transcriptional activity of Sp1. Exposure of mesangial cells to glucosamine resulted in in- creased PKC-␤I and -␦ and, to a lesser extent, PKC-⑀ kinase activity. Furthermore, cotransfection of DN-PKC isoforms (DN-PKC-␤I and -␦) almost completely inhibited PAI-1 promoter induction by glucosamine. Although PKC-⑀ and -␣ may have minor or permissive roles, the combination of strong stimulation of PKC-␤I and -␦ kinase activity and marked inhibition of glucosamine-induced PAI-1 promoter transcription by DN-PKC-␤I and -␦ mutants implicates these isoforms in the effects of glucosamine. This conclusion is further supported by findings that high glucose significantly increased the kinase activity of these same PKC isoforms, and that high glucose-induced PAI-1 promoter activity was also inhibited by DN-PKC-␤I and -␦. Our earlier study detected only a small increase in Sp1 DNA binding (ϳ30%) following treatment of mesangial cells with glucosamine (15), suggesting that Sp1 transcriptional activation could play a role in PAI-1 promoter activation by glucosamine. Here, we obtained direct evidence for glucosamine and high glucose- and 7). Although not directly comparable, the induction was not less than that of the transfected PAI-1 promoter, suggesting that transcriptional activation of Sp1 was relevant. The role of PKC in Sp1 transcriptional activation was probed with the DN-PKC isoforms, and PKC-␤I activation was found to be essential. Thus, stimulation of Sp1(TAD)-GAL4 by glucosamine or high glucose was inhibited by DN-PKC-␤I, but not by DN-PKC-␦ or DN-SEK (Figs. 4 and 7). PAI-1 promoter activation by high glucose was also blocked by the PKC-␤I specific inhibitor, LY 379196 (Fig. 8). The effects of high glucose on both PKC kinase activity and Sp1 transcriptional activation were inhibited by DON, which blocks glucose flux through the HBP (Figs. 5 and 7). Taken together, the data indicate that activation of PKC-␤I and -␦ is mediated partly by increased flux through the HBP and that these PKC isoforms play important, but distinct roles in PAI-1 promoter activation. In particular, the role of PKC-␤I appears to be to enhance the transcriptional activity of Sp1.
Glucosamine-stimulated PKC kinase activity was not associated with PKC membrane translocation. This implies that enhanced HBP flux does not result in increased synthesis of diacylglycerol, a well known effect of high glucose (see above). Rather, this regulation of PKC kinase activity by the HBP may serve to enhance the effects of diacylglycerol synthesized in response to high glucose. Further investigation will be required to determine the precise impact of this increased PKC activity on in vivo PKC function. Increased PKC activity in whole cell lysates has been previously documented in response to high glucose, glycated albumin, hydrogen peroxide, serum, insulin, and cellular adhesion (26, 57, 68 -71, 86). Apart from membrane translocation, the mechanisms may involve altered PKC serine/threonine or tyrosine phosphorylation (57, 68 -71). It is possible that HBP-mediated O-GlcNAcylation of kinases, phosphatases, or their regulatory proteins could indirectly increase PKC kinase activity. In preliminary studies, immunoblotting of immunoprecipitated PKC-␤I and -␦ with a monoclonal antibody directed against O-GlcNAc, CTD 110.6 (87) (kindly provided by G. Hart, Johns Hopkins University, Baltimore, MD), did not reveal O-GlcNAc on these PKCs (data not shown). The experiments with the antioxidant, probucol, suggest the involvement of oxidative stress in hexosamine-stimulated PKC kinase activity, but the exact role of reactive oxygen species in the actions of hexosamines remains to be defined.
Other studies have also suggested that flux through the HBP regulates PKCs. Our findings are generally consistent with a report by Singh et al. (26) showing high glucose and glucosamine increased PKC kinase activity in SV-40 transformed mesangial cells. Similar to our observations, PKC membrane translocation did not occur in response to glucosamine in that study (26). Moreover, Singh et al. (26,27) have shown that pharmacological PKC inhibitors blocked laminin and fibronectin synthesis stimulated by the hexosamine pathway. Filippis et al. (70) noted that high glucose or glucosamine increased PKC kinase activity in adipocyte membranes, but did not examine PKC membrane translocation by immunoblotting. In contrast, Kolm-Litty (25) reported membrane translocation of PKCs in response to a higher concentration of glucosamine, 12 mM, than used in the current study or by Singh et al., and that azaserine blocked high glucose-induced membrane translocation of PKC isoforms. Differences in glucosamine concentrations, experimental protocols, or cell-specific responses may account for the precise nature of PKC activation observed in the different studies. The mechanism by which O-GlcNAcylation alters Sp1 transcriptional activation is incompletely defined at present. One possibility is that direct O-GlcNAcylation of Sp1 or one of its transcription cofactors up-regulates Sp1 transcriptional activity. Consistent with this, increased Sp1 O-GlcNAcylation is observed in endothelial cells exposed to high glucose (16). On the other hand, O-GlcNAcylation of serine 484 in Sp1 results in decreased Sp1 transcriptional activation (46,47). Overexpression of OGT suppressed a promoter containing multiple Sp1 sites (47), but the outcome of overexpressing OGT may differ from that of high glucose or glucosamine. For example, overexpressed OGT may bind to and inhibit the function of its substrates or cause excessive O-GlcNAcylation that is inhibitory. Reconciliation of these results will require a detailed study to evaluate the functional effects of high glucose-induced O-GlcNAcylation of specific sites in Sp1 and Sp1 transcription cofactors in mesangial cells. It is established that O-GlcNAcylation of residues in the Sp1 amino terminus increases Sp1 protein stability (41,42). This effect, discovered in the context of glucose deprivation, does not appear to account for our findings because we added glucosamine to a medium containing normal (5.6 mM) glucose and the Sp1(TAD)-GAL4 and Sp1(holo)-GAL4 constructs lacked the amino-terminal region required for this mechanism of regulation of Sp1 degradation (42).
Instead, our data suggest that the HBP indirectly activates Sp1 or its transcription cofactors through PKC-␤I. Although overall Sp1 serine phosphorylation decreases reciprocally with increased O-GlcNAcylation in high glucose-treated endothelial cells (16), the regulation of individual Sp1 and Sp1 transcription cofactor phosphorylation sites by the HBP has not been defined. Indeed, the requirement for the transcription cofactor, p300, in PMA-induced Sp1 activation (50) raises the possibility that PKC-␤ may target Sp1 transcription cofactors. In this respect, the effects of PKC-␤ on Sp1 transcriptional activation appear to differ from that of a number of other kinases, including PKA, PKC-, ERK, a MEK-dependent kinase distinct from ERK, and p38 MAPK (48,49,53,54,88,89) and protein phosphatase-1, all of which predominately augment Sp1 DNA binding rather than transcriptional activation.
It has been reported that PKC-␤ is required for lipoproteinstimulated PAI-1 protein synthesis (85) and that PKC regulates endogenous Sp1 (50,52,53) as well as a transfected Spl-GAL4 construct (50). Our data link a specific PKC isoform, PKC-␤I, for the first time with stimulation of the Sp1 transcriptional activation domain. This relationship could be important in the pathogenesis of diabetic nephropathy, where PKC-␤ plays an important role, as exemplified by the ability of the specific PKC-␤ inhibitor, LY333531, to inhibit the increased TGF-␤1 and extracellular matrix protein expression in rats with streptozotocin-induced diabetes and db/db mice (6,11,84). Beyond diabetic nephropathy, the link between PKC-␤I and Sp1 activation may have broader implication for diseases in which fibrosis is uncontrolled, given that Sp1 is involved in the regulation of many extracellular matrix genes (90).
Although O-GlcNAcylation is a potential mediator of HBPstimulated gene expression, it remains possible that high glucose and glucosamine-induced PAI-1 promoter activation occurs through other mechanisms. Interestingly, glucosamineinduced insulin resistance correlates poorly with UDP-GlcNAc concentrations in adipocytes (91). Inhibition of glucose-6-phosphate dehydrogenase by glucosamine or glutamine was explained by the glucosamine metabolite, glucosamine 6-phosphate (92). Furthermore, impaired pancreatic ␤-cell function induced by glucosamine or GFA overexpression was attributed to oxidative stress rather than O-GlcNAcylation (77). It will be necessary to identify the targets of O-GlcNAcylation to definitively link this post-translational modification to the apparent signaling function of the HBP.
In summary, this study demonstrates that increased flux through the HBP promoted by glucosamine or high glucose results in increased PKC-␤I and -␦ kinase activity, and that these isoforms play key roles in PAI-1 promoter activation. Furthermore, the HBP stimulates Sp1 transcriptional activity via activation of PKC-␤I.