text:
Sneha Ramakrishna and Il-Man Kim contributed equally to this work.
Research Article
Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box m1 transcription factor
Version of Record online: 15 MAR 2007
DOI: 10.1002/dvdy.21113
Copyright © 2007 Wiley-Liss, Inc.
Developmental Dynamics
Volume 236, Issue 4, pages 1000–1013, April 2007
Additional Information
How to Cite
Ramakrishna, S., Kim, I.-M., Petrovic, V., Malin, D., Wang, I.-C., Kalin, T. V., Meliton, L., Zhao, Y.-Y., Ackerson, T., Qin, Y., Malik, A. B., Costa, R. H. and Kalinichenko, V. V. (2007), Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box m1 transcription factor. Dev. Dyn., 236: 1000–1013. doi: 10.1002/dvdy.21113
Publication History
- Issue online: 15 MAR 2007
- Version of Record online: 15 MAR 2007
- Manuscript Accepted: 9 FEB 2007
Funded by
- National Institute of Health. Grant Number: HL 84151-01
- American Heart Association. Grant Number: 0335036N
- March of Dimes Birth Defects Foundation. Grant Number: 6-FY2005-325
- Abstract
- Article
- References
- Cited By
- winged helix DNA binding domain;
- Forkhead transcription factor;
- heart development;
- Trident;
- cardiomyocyte;
- p21;
- knockout mice;
- NFATc3
The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 −/− mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 −/− hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21Cip1 protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the −9,259/−9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development. Developmental Dynamics 236:1000–1013, 2007. © 2007 Wiley-Liss, Inc.
Development of mouse hearts initiates at 7.0 days post coitum (dpc) when the cardiac lineage becomes specified from cells of the primitive streak (Zaffran and Frasch, 2002). The cardiac mesoderm migrates anterolaterally and fuses at the ventral aspect of the embryo to form a linear heart tube, which subsequently undergoes complex looping and septation resulting in the formation of the four-chambered heart (Zaffran and Frasch, 2002; Eisenberg and Markwald, 2004). The transcriptional network, which controls the development of mammalian hearts, has been extensively studied. Disruption of this transcriptional network results in a variety of human diseases including congenital heart disease, dilated cardiomyopathy, and cardiac hypertrophy (Frey and Olson, 2003; Olson, 2004; Clark et al., 2006). Central to the regulation of these developmental processes are the cardiac transcription factors GATA4 (Watt et al., 2004), NKX2.5 (Cripps and Olson, 2002), Hand (McFadden et al., 2005), nuclear factors of activated T-cell (NFAT) c3 and c4 (Molkentin, 2000; Graef et al., 2001; Bushdid et al., 2003), Foxp4 (Li et al., 2004) and FOG2 (Svensson et al., 2000; Lin et al., 2004), and T-box transcription factors TBX1 and TBX5 (Plageman and Yutzey, 2005), all of which have been shown to regulate cardiomyocyte proliferation, cardiac-specific gene expression, and proper patterning of the developing heart.
The Forkhead Box (Fox) proteins are an extensive family of transcription factors, which share homology in the Winged Helix/Forkhead DNA binding domain (Clark et al., 1993; Clevidence et al., 1993; Kaestner et al., 1993). Expression of the Foxm1 transcription factor (previously known as HFH-11B, Trident, Win, or MPP2) is induced during cellular proliferation in a variety of different cell types and extinguished in terminally differentiated cells (Korver et al., 1997; Ye et al., 1997). Liver regeneration studies demonstrated that mice with postnatal hepatocyte-specific deletion of the Foxm1 fl/fl (LoxP targeted) allele exhibit a significant reduction in hepatocyte DNA replication and mitosis, which is associated with altered expression of proteins that limit Cdk1 and Cdk2 activity required for normal cell cycle progression (Wang et al., 2002). Recently, we demonstrated that Foxm1 is required for aberrant proliferation of tumor cells during progression of liver, lung, and prostate cancers (Kalinichenko et al., 2004; Kalin et al., 2006; Kim et al., 2006).
Previous studies described the generation of Foxm1−/−neo mouse line that contained a targeted insertion of PGK-neomycin cassette into the third exon of Foxm1 gene without deleting any of the coding sequences (Korver et al., 1998). Although the Foxm1−/−neo mice died postnatally, displaying accumulation of polyploid cardiomyocytes and hepatoblasts (Korver et al., 1998), the authors did not provide any molecular mechanisms of this cardiovascular phenotype and, therefore, the precise role of Foxm1 during heart development remains uncharacterized. We generated a distinct Foxm1 −/− mouse line, which contains a targeted deletion of exons 4 to 7 encoding the Foxm1 winged helix DNA binding and the C-terminal transcriptional activation domains (Krupczak-Hollis et al., 2004). Most of the Foxm1 −/− embryos died in utero between 13.5 and 16.5 dpc due to severe defects in development of the embryonic liver, lung, and heart (Krupczak-Hollis et al., 2004; Kim et al., 2005a). Foxm1 −/− livers displayed abnormal accumulation of polyploid hepatoblasts resulting from diminished DNA replication and a failure to enter mitosis (Krupczak-Hollis et al., 2004). Mouse embryonic fibroblasts (MEF) from our Foxm1 −/− embryos were unable to grow in culture due to a block in mitotic progression and undergo cellular senescence at passage three with high expression levels of senescence-associated β-galactosidase and cell cycle inhibitors p21Cip1, p27Kip1, p16Ink4A, and p19ARF proteins (Wang et al., 2005). This mitotic block was also associated with diminished protein levels of the Polo-like kinase 1 and Aurora B kinase (Krupczak-Hollis et al., 2004; Kim et al., 2005a; Wang et al., 2005), both of which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis (Glover et al., 1998; Adams et al., 2001). Foxm1 is required for differentiation of hepatoblast precursor cells toward billiary epithelial cell lineage because Foxm1 −/− livers fail to develop intrahepatic bile ducts (Krupczak-Hollis et al., 2004). Likewise, Foxm1 −/− embryos exhibit defects in differentiation of pulmonary mesenchyme into mature capillary endothelial cells during the canalicular stage of lung development (Kim et al., 2005a).
In this report, we used our Foxm1 −/− mouse line to investigate the role of Foxm1 in cardiac morphogenesis. We demonstrate that Foxm1 deletion does not affect looping heart morphogenesis or cardiomyocyte differentiation, but reduces both DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in embryonic Foxm1 −/− hearts were associated with reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21Cip1 protein. Likewise, HL-1 cardiomyocytes depleted in Foxm1 levels by siRNA transfection exhibited altered expression of these genes, indicating a direct influence of Foxm1 on cardiomyocyte proliferation. We also showed that Foxm1 binds to the −9,259/−9,288-bp region of endogenous mouse NFATc3 promoter in the developing heart, suggesting that the mouse NFATc3 gene is a direct transcriptional target of Foxm1 during heart development. In contrast to other embryonic tissues, Foxm1 −/− hearts displayed normal expression of many known Foxm1-target genes including Polo-like kinase 1, Aurora B kinase, Skp2, Cks1, and Laminin α4. We also demonstrated that endothelial-specific deletion of the Foxm1 fl/fl allele does not affect heart development or cardiomyocyte proliferation, suggesting that Foxm1 deficiency in endothelial cells does not contribute to proliferation defects in embryonic Foxm1 −/− hearts. These results suggest that the Foxm1 transcription factor is essential for mouse embryonic heart development through regulation of distinct cellular pathways essential for proliferation of cardiomyocytes.
Foxm1 −/− Embryonic Hearts Exhibited Ventricular Hypoplasia
Paraffin sections from mouse embryos that contained a targeted deletion of exons 4 to 7 of the Foxm1 gene (Foxm1 −/−; Krupczak-Hollis et al., 2004) were stained with hematoxylin and eosin (H&E) and compared with embryonic hearts isolated from aged-matched wild type (WT) embryos to examine for gross morphological defects in heart development. Although no obvious heart abnormalities were observed in Foxm1 −/− 9.5 dpc embryos (Fig. 1A and E), Foxm1 −/− 13.5–18.5-dpc hearts exhibited thinning of ventricular myocardium (Fig. 1B–D, F–H), which is consistent with development of ventricular hypoplasia. Morphometric analysis of embryonic hearts demonstrated that Foxm1 −/− 13.5-dpc hearts displayed a 43% reduction in the thickness of left ventricle compared to WT hearts, whereas a 57% reduction in the thickness of left ventricle was found in Foxm1 −/− 15.5-dpc hearts (Fig. 1R). Although no significant differences in heart size were observed in Foxm1 −/− 13.5-dpc embryos, Foxm1 −/− 15.5-dpc hearts exhibited a 25% reduction in the heart size when compared to WT embryos (Fig. 1Q). Interestingly, Foxm1 −/− 13.5-dpc hearts displayed a significant 1.3-fold increase in thickness of heart valve leaflets compared to WT littermates (Fig. 1J, N; and data not shown).
Although enlarged cardiomyocytes were found in the Foxm1 −/− atrium and ventricle as early as 9.5 dpc (Fig. 1I and M; and data not shown), severe accumulation of polyploid cardiomyocytes was observed in Foxm1 −/− 13.5–18.5-dpc heart tissues, including ventricular myocardium, interventricular septum (IVS), atrium, and heart trabeculae (Fig. 1D,H, K,L,O,P). No significant differences were found in either length or thickness of heart trabeculae by morphometric analysis of Foxm1 −/− and WT 13.5-dpc hearts (data not shown). These results suggest that Foxm1 is dispensable for trabeculogenesis in the heart. Despite a reduced number of cardiomyocytes in Foxm1 −/− hearts (Fig. 1O,P), they still expressed desmin (Fig. 2E–F), laminin α4 extra-cellular matrix protein (Lama4; Fig. 2I,J), and GATA-4 transcription factor (Fig. 2G,H), all of which are essential for maintaining a differentiated phenotype in cardiomyocyte lineage (Watt et al., 2004; Wang et al., 2006).
To determine Foxm1 expression pattern in embryonic hearts, paraffin sections from WT hearts were stained with antibodies specific to Foxm1 protein. Foxm1 was expressed in highly proliferative cells of compact layer and trabecular cardiomyocytes of 9.5-dpc WT hearts (Fig. 2A). Abundant Foxm1 nuclear staining was also detected in cardiomyocytes and endothelial cells of 15.5-dpc WT embryos, whereas Foxm1 staining was significantly decreased in 18.5-dpc WT hearts (Fig. 2B–D), correlating with reduced proliferation rates. Foxm1 protein was not detected in adult WT hearts (data not shown).
Foxm1 −/− Hearts Exhibited Diminished DNA Replication and Mitosis
Foxm1 −/− 15.5-dpc embryos displayed significantly reduced numbers of cardiomyocytes (Fig. 3G), suggesting that Foxm1 stimulates cardiomyocyte proliferation during heart development. In order to determine the mechanism(s) of the proliferation defect in Foxm1 −/− cardiomyocytes, we measured DNA replication rates in Foxm1 −/− and WT 15.5-dpc hearts by injecting 5-bromo-2′-deoxyuridine (BrdU) into pregnant female mice 2 hr prior to harvesting the embryos. Immunohistochemistry with BrdU antibody demonstrated that Foxm1 −/− 15.5-dpc hearts exhibited a significant reduction in the number of BrdU-positive cardiomyocytes compared to age-matched WT embryos (Fig. 3A,B,H), whereas DNA replication rates were not affected in Foxm1 −/− 9.5-dpc hearts (Fig. 3H). To determine whether Foxm1 deficiency would result in decreased entry of cardiomyocytes into mitosis, we performed immunofluorescent staining of heart sections with an antibody specific to the phosphorylated form of Histone 3 (PH3). Consistent with proliferation defects in Foxm1 −/− livers (Krupczak-Hollis et al., 2004), a 75% reduction in the number of cardiomyocytes undergoing mitosis was observed in Foxm1 −/− 15.5-dpc hearts (Fig. 3C,D,I). Interestingly, cell death did not contribute to reduced cardiomyocyte number in Foxm1 −/− 15.5-dpc hearts, as was determined by immunostaining with antibody against cleaved caspase 3 and TUNEL assay (Fig. 3E,F; and data not shown). These results demonstrated that Foxm1 deficiency reduces both DNA replication and mitosis in developing cardiomyocytes.
Gene Expression Profile in Foxm1 −/− Heart
In order to determine potential target genes regulated by Foxm1 in the developing heart, we performed RT-PCR analysis of total heart RNA prepared from WT or Foxm1 −/− 14.5-dpc embryos. Although we confirmed undetectable levels of Foxm1 mRNA in Foxm1 −/− hearts (Fig. 4), Foxm1 −/− embryos displayed normal cardiac levels of Polo-like kinase 1, Aurora B kinase, Cyclin B1, Skp2, Cks1, and Lama4 (Fig. 4), all of which are known transcriptional targets for Foxm1 protein in hepatocytes and fibroblasts (Krupczak-Hollis et al., 2004; Wang et al., 2005). These results suggest that the Foxm1 deficiency does not influence expression of these genes in the developing heart. However, consistent with decreased entry into mitosis in Foxm1 −/− cardiomyocytes (Fig. 3I), we observed an 80% reduction in the expression of Cdc25B phosphatase (Fig. 4), a known transcriptional target for Foxm1 protein (Wang et al., 2001). Because Cdc25B is essential for activation of Cdk1/ cyclin B complex during M-phase progression (Borgne and Meijer, 1996), diminished Cdc25B levels may cause delayed cardiomyocyte entry into mitosis in Foxm1 −/− hearts, causing polyploid phenotype. Furthermore, numbers of cells expressing Cdk inhibitor p21Cip1 (p21) protein were increased in Foxm1 −/− hearts (Fig. 5A–E), a finding consistent with elevated p21 protein levels in Foxm1 −/− MEFs (Wang et al., 2005). Because p21 reduces Cdk2 activity, causing diminished DNA replication (Sherr and Roberts, 1999), induced cardiac p21 levels may contribute to the reduction of cell cycle progression in Foxm1 −/− hearts.
Foxm1 −/− hearts displayed a 70% reduction in the expression levels of the nuclear factor of activated T-cell c3 (NFATc3), whereas cardiac expression of related NFATc4 protein was not changed in the absence of Foxm1 (Fig. 4). Since NFATc3 protein regulates genes essential for cardiac hypertrophy and cardiomyocyte proliferation (Wilkins et al., 2002; Sanna et al., 2005), diminished NFATc3 levels may contribute to the proliferation defects seen in Foxm1 −/− cardiomyocytes. Interestingly, we observed normal cardiac expression of calmodulin, H11 kinase, serotonin 5-HT2B receptor, and FoxO3a transcription factor (Fig. 4), all of which are essential for the development of hypertrophy in cardiomyocytes (Nebigil et al., 2000; Depre et al., 2002; Colomer et al., 2004; Skurk et al., 2005).
Endothelial-Specific Deletion of Foxm1 fl/fl Allele Does Not Alter Proliferation Rates in the Heart
Previous reports demonstrated that Foxm1 −/− embryos displayed diminished number of blood vessels in the liver and lung (Krupczak-Hollis et al., 2004; Kim et al., 2005a), suggesting vascular defects in these organs. Furthermore, endothelial-specific disruption of Foxm1 diminished proliferation of endothelial cells following LPC-induced vascular injury (Zhao et al., 2006). Since Foxm1 is expressed in both cardiomyocytes and cardiac endothelial cells in the developing heart (Fig. 2A–D), we sought to determine whether Foxm1 −/− embryos exhibit defects in development of cardiac endothelial cells. Immunohistochemical staining and RT-PCR analysis of 14.5 dpc Foxm1 −/− hearts displayed normal cardiac expression of the Platelet-endothelial cell adhesion molecule-1 (Pecam-1), suggesting no gross defects in vascular patterning (Fig. 4 and data not shown).
To determine whether the Foxm1 −/− cardiomyocyte defects were indirect due to defects in development of Foxm1-deficient endothelial cells, we used a Tie2-Cre recombinase transgene to mediate endothelial-specific deletion of the Foxm1 fl/fl allele in mouse embryos. Although the majority of Tie2-Cre Foxm1 fl/fl mice survived at birth, approximately 20% of Tie2-Cre Foxm1 fl/fl embryos died in late gestation-postnatally. These Tie2-Cre Foxm1 fl/fl embryos with the most severe phenotype exhibited vascular abnormalities in the lung as demonstrated by diminished Pecam-1 staining (Fig. 6I–J), a result consistent with reduced Pecam-1 levels in Foxm1 −/− lungs (Kim et al., 2005a). These same Tie2-Cre Foxm1 fl/fl embryos were then examined for heart defects. The Tie2-Cre Foxm1 fl/fl 17.5-dpc hearts displayed undetectable Foxm1 staining in cardiac endothelial cells (Fig. 6G,H), indicating efficient endothelial-specific deletion of Foxm1 fl/fl allele in the developing heart. However, no morphological defects were observed in the structure of coronary vessels and cardiac microcirculation in Tie2-Cre Foxm1 fl/fl heart (Fig. 6A,B and data not shown). Furthermore, the Tie-2 Foxm1 fl/fl hearts displayed normal structure of atria-ventricular valves and the ventricular outflow tract (Fig. 6C,D and data not shown), suggesting that Foxm1 is not essential for the formation of endocardial cushion during heart development.
The Tie2-Cre Foxm1 fl/fl embryos displayed similar numbers of ventricular cardiomyocytes compared to control Foxm1 fl/fl embryos (Fig. 6K). Furthermore, cardiomyocyte proliferation was similar in Tie2-Cre Foxm1 fl/fl and Foxm1 fl/fl hearts (Fig. 6E,F,L), as demonstrated by immunohistochemical staining for Proliferation Cell Nuclear Antigen (PCNA). These results suggest that the endothelial Foxm1 deficiency does not contribute to proliferation defects in embryonic Foxm1 −/− heart.
Foxm1 Depletion in HL-1 Cardiomyocytes Causes Diminished DNA Replication, Delayed Entry Into Mitosis, and Reduced Expression of NFATc3 and Cdc25B
To determine whether Foxm1 deficiency directly reduces the cell cycle progression in cardiomyocytes, HL-1 mouse cardiomyocytes were transfected with short interfering RNA (siRNA) duplex specific to the mouse Foxm1 cDNA (siFoxm1) or with mutant siFoxm1 duplex (mutFoxm1) containing two mutations in the siRNA structure (Fig. 7D). Seventy-two hours later, cells were labeled for 2 hr with BrdU and then used for immunostaining with BrdU antibody. Depletion of Foxm1 caused a 50% reduction in the number of HL-1 cells undergoing DNA replication compared to either untransfected HL-1 cells or cells transfected with mutFoxm1 duplex (Fig. 7A,B). Furthermore, we performed time-course experiments in serum-starved HL-1 cells to determine whether Foxm1-deficiency influences mitotic progression in cardiomyocytes. BrdU and DAPI double staining demonstrated that although mitotic figures in control HL-1 cultures were detected at 16 hr following BrdU labeling, no mitotic figures were observed in siFoxm1-transfected HL-1 cells until 24 hr after BrdU labeling (Fig. 8), suggesting that Foxm1 deficiency causes an 8-hr delay in the entry of HL-1 cells into mitosis. These results suggest that Foxm1 is critical for both DNA replication and mitotic progression in HL-1 cardiomyocytes.
Since Foxm1 −/− hearts displayed reduced expression of NFATc3 and Cdc25B phosphatase (Fig. 4), and increased nuclear levels of the Cdk-inhibitor p21cip1 protein (Fig. 5C,D), we examined the expression levels of these genes in siFoxm1-transfected HL-1 cardiomyocytes by Western blot and quantitative real-time RT-PCR analysis. These transfection studies revealed that siFoxm1 efficiently reduces the expression of mouse Foxm1 in HL-1 cells, whereas transfection of mutant siFoxm1 duplex has a minimal effect on Foxm1 levels (Fig. 7C). In comparison with either untransfected cells or cells transfected with mutant siFoxm1, depletion of Foxm1 levels by siFoxm1 caused a significant decrease in the expression of M-phase promoting Cdc25B phosphatase and NFATc3 transcription factor (Fig. 7C). Furthermore, Western blot demonstrated that transfection of siFoxm1 caused increased nuclear levels of Cdk inhibitor p21cip1 protein (Fig. 7D), a result consistent with increased cardiac staining for p21cip1 protein in Foxm1 −/− hearts (Fig. 5C,D). Interestingly, expression levels of Cyclin B1 and Cdk-inhibitor p27Kip1 were not affected by siFoxm1 transfection (Fig. 7D), suggesting that Foxm1-deficiency does not influence the expression of these genes in cultured HL-1 cells.
Foxm1 Binds to the −9,259/−9,288-bp Region of Endogenous Mouse NFATc3 Promoter
Previous studies demonstrated that Foxm1 directly regulates Cdc25B and p21Cip1 genes in hepatocytes and mouse embryonic fibroblasts (Wang et al., 2001; Krupczak-Hollis et al., 2004; Wang et al., 2005). However, little is known about cardiomyocyte-specific Foxm1 targets. Since NFATc3 mRNA levels were reduced in both Foxm1 −/− hearts and Foxm1-depleted HL-1 cardiomyocytes, we used Chromatin Immunoprecipitation (ChIP) assays to determine whether Foxm1 protein directly binds to the NFATc3 promoter in context of embryonic mouse DNA and to identify specific Foxm1-binding regions. Four potential Foxm1 DNA binding sites were found in the −10-Kb promoter region of the mouse NFATc3 gene: −4,263/−4,277, −4,989/−5,000, −5,533/−5,547, and −9,259/−9,288 bp, the latter of which contains eight overlapping Foxm1-binding motifs (Fig. 7E). The cross-linked and sonicated chromatin from mouse WT 17.5-dpc embryonic hearts was immunoprecipitated (IP) with antibodies specific to either Foxm1 or IgG control antibody. Binding of NFATc3 promoter DNA associated with the IP chromatin was determined by PCR with primers specific to potential Foxm1-binding sites in mouse NFATc3 promoter. Although Foxm1 did not recognize the first three NFATc3 promoter regions (data not shown), Foxm1 specifically binds to the −9,259/−9,288-bp region of endogenous mouse NFATc3 promoter (Fig. 7F, left panel). Furthermore, mouse HL-1 cardiomyocytes transfected with CMV-Foxm1 expression vector displayed efficient binding of the −9,259/−9,288 bp NFATc3 promoter region and Foxm1 protein (Fig. 7F, right panel). These results suggest that the mouse NFATc3 gene is a direct transcriptional target of Foxm1 in cardiomyocytes, a result consistent with reduced NFATc3 levels in Foxm1 −/− heart (Fig. 4) and Foxm1-depleted HL-1 cardiomyocytes (Fig. 7C).
The requirement for Foxm1 activity in cardiac development was examined in Foxm1 −/− mouse embryos that contain a targeted deletion of exons 4 through 7, encoding the Foxm1 DNA binding domain and C-terminal transcriptional activation domains, both of which are essential for Foxm1 transcriptional activity (Krupczak-Hollis et al., 2004). Most of the Foxm1 −/− embryos exhibited an embryonic lethal phenotype between 13.5 and 16.5 dpc because of severe abnormalities in liver morphogenesis, hypertrophy of blood vessels, and inability of lung mesenchyme to form peripheral pulmonary capillaries (Krupczak-Hollis et al., 2004; Kim et al., 2005a). Overall developmental defects in our Foxm1 −/− embryos were more severe compared to the previously reported Foxm1−/−neo mouse line, which contains a targeted insertion of PGK-Neo into Foxm1 gene locus without deleting any of the coding sequences (Korver et al., 1998). This includes a complete embryonic lethality, earlier thinning of myocardium, and dilation of the heart chambers (Krupczak-Hollis et al., 2004; this report). One explanation for these differences between Foxm1 −/− and Foxm1−/−neo mouse lines is that the Foxm1−/−neo mice may contain hypomorphic levels of Foxm1 protein, providing residual Foxm1 activity during embryonic development. This conclusion is supported by the fact that Foxm1 −/− mouse embryonic fibroblasts (MEF) were unable to grow in culture due to severe mitotic defects and premature senescence (Wang et al., 2005), whereas Foxm1−/−neo MEFs grew at passage 4 in culture, displaying reduced G2/M progression and abnormal mitosis due to defective chromosomal segregation (Laoukili et al., 2005).
Proliferation defects in Foxm1 −/− cardiomyocytes were also associated with increased nuclear expression of p21Cip1 protein (p21), which inactivates the Cdk2 protein required for DNA replication (Sherr and Roberts, 1999). Cdk2 complexes with either Cyclin E or Cyclin A cooperate with Cyclin D-Cdk4/6 to phosphorylate the Retinoblastoma (RB) protein, which releases bound E2F transcription factor and allows it to stimulate expression of genes required for DNA replication (Harbour and Dean, 2000; Ishida et al., 2001). Therefore, defects in DNA replication in Foxm1 −/− hearts may be a direct consequence of increased cardiac levels of p21 protein. This result is consistent with previously published data demonstrating that transgenic over-expression of Foxm1 in Rosa26-Foxm1 transgenic mice is associated with decreased p21 protein levels and premature proliferation of different lung cell types following Butylated Hydroxytoluene (BHT) lung injury (Kalinichenko et al., 2003). We previously demonstrated that Foxm1 regulates transcription of Skp2 and Cks1 proteins, which are specificity subunits of the Skp1-Cullin-F-Box (SCF) ubiquitin ligase complex that targets both p21 and p27 proteins for degradation during the G1/S transition (Wang et al., 2005). Unchanged expression levels of Skp2 and Cks1 in Foxm1 −/− hearts suggest that degradation of p21 protein by SCF ubiquitin ligase is not involved in abnormal nuclear accumulation of p21 protein in the heart. Interestingly, accumulation of polyploid cells was found in livers and lungs of Skp2 −/− mice, but not in Skp2 −/− hearts (Kossatz et al., 2004), suggesting that the Skp2 protein is not essential for cardiomyocyte proliferation. Although the mechanism of nuclear p21 accumulation in Foxm1 −/− cardiomyocytes remains unknown, cardiac p21 mRNA levels remained unchanged in Foxm1 −/− hearts (data not shown), excluding a transcriptional regulation of p21 gene by the Foxm1.
Proliferation defects in Foxm1 −/− hepatoblasts and vascular smooth muscle cells were associated with reduced expression of Polo-like kinase 1 (PLK1) and Aurora B kinase (Krupczak-Hollis et al., 2004; Kim et al., 2005a), which contribute to a failure to complete mitosis and cytokinesis. (Glover et al., 1998; Adams et al., 2001). Furthermore, a recent study demonstrated that these genes are direct transcriptional targets for the Foxm1 transcription factor in MEFs and U2OS human osteosarcoma cell line (Wang et al., 2005). Interestingly, even though cardiac expression of PLK1 and Aurora B kinase was unchanged in embryonic Foxm1 −/− hearts, Foxm1 −/− cardiomyocytes still exhibited reduced mitosis causing the accumulation of polyploid cells. These results suggest that Foxm1 may regulate other genes essential for cardiomyocyte entry into mitosis during heart morphogenesis. In this study, we found that Foxm1 −/− hearts display reduced expression of Cdc25B phosphatase, a result consistent with diminished levels of Cdc25B in proliferating Foxm1 −/− hepatocytes following a partial hepatoctomy (Wang et al., 2001). In addition, increased nuclear levels of Cdk inhibitor p21 may not only prevent activation of Cdk2, but p21 can also complex with and inhibit activity of M-phase promoting Cdk1 protein (Op De Beeck et al., 2001; Patel et al., 2002; Dash and El-Deiry, 2005). Furthermore, reduced Cdc25B expression and increased nuclear p21 protein levels were found in cultured HL-1 cardiomyocytes after siRNA-mediated Foxm1 depletion. Progression into mitosis requires the activation of Cdk1 through assembly with cyclin B1 regulatory subunit and the removal of Cdk1 inhibitory phosphates at Thr 14 and Tyr 15 by the Cdc25B and Cdc25C phosphatases (Borgne and Meijer, 1996; Wells et al., 1999; Nilsson and Hoffmann, 2000). Therefore, diminished cardiac levels of Cdc25B and increased nuclear levels of p21 protein can decrease Cdk1 activation and M-phase progression causing delayed entry into mitosis in Foxm1 −/− cardiomyocytes.
The nuclear factor of the activated T-cell (NFAT) family of transcription factors regulates genes essential for morphogenesis of the heart, skeletal muscle, cartilage, bone, skin, neuronal, and immune systems (reviewed in Graef et al., 2001). NFATc3 activity is critical for the generation of primary fibers in skeletal muscle (Crabtree and Olson, 2002), whereas over-expression of NFATc4 induces cardiac hypertrophy in adult mice (Molkentin, 2000). In this study, we demonstrated that Foxm1 −/− embryos display a significant reduction in the expression of NFATc3 in the heart, whereas NFATc4 levels remained unchanged. Since a combinatorial disruption of NFATc3 and NFATc4 genes caused embryonic lethality and diminished cardiomyocyte proliferation (Bushdid et al., 2003), it is unlikely that NFATc3 deficiency is solely responsible for the proliferation defects in Foxm1 −/− cardiomyocytes. However, in combination with either reduced Cdc25B or elevated p21 levels, diminished NFATc3 expression may contribute to the cell cycle defects in developing Foxm1 −/− heart. Interestingly, quantitative real-time RT-PCR demonstrated that siRNA-mediated Foxm1 depletion in cultured HL-1 cardiomyocytes causes a significant decrease in the expression of NFATc3, suggesting that the mouse NFATc3 gene is a direct transcriptional target of Foxm1 in cardiomyocytes. Furthermore, we used Chromatin Immunoprecipitation (ChIP) assay to demonstrate that Foxm1 protein directly binds to the −9,259/−9,288-bp region of endogenous mouse NFATc3 promoter in the developing heart and in cultured HL-1 cells, providing a further support for this concept.
Previous studies have demonstrated that endothelial-specific disruption of either neurofibromin (NF1) or platelet-derived growth factor-B (PDGFB) caused abnormal development of myocardium (Gitler et al., 2003; Bjarnegard et al., 2004). These studies suggest that signaling from cardiac endothelial cells is required for heart development. Since Foxm1 is expressed in both cardiomyocytes and cardiac endothelial cells (Ye et al., 1997; and this report), we used Tie2-Cre Foxm1 fl/fl mice to investigate the consequences of endothelial Foxm1 deficiency for heart development. Surprisingly, the Tie2-Cre Foxm1 fl/fl embryos displayed normal heart morphology and similar cardiomyocyte proliferation compared to control Foxm1 fl/fl embryos. These results suggest that the endothelial Foxm1 deficiency does not contribute to proliferation defects in embryonic Foxm1 −/− heart. Although the Tie2-Cre Foxm1 fl/fl embryos exhibited defects in development of pulmonary vasculature, no gross morphological defects in vascular patterning were observed in Tie2-Cre Foxm1 −/− hearts. This indicates that Foxm1 promotes the lung vascular development, but is dispensable for the development of cardiac endothelium. This is consistent with previous reports demonstrating that Foxm1 −/− embryos displayed reduced numbers of endothelial cells in developing lung (Krupczak-Hollis et al., 2004; Kim et al., 2005a), suggesting different requirements for Foxm1 activity in distinct populations of endothelial cells.
In summary, Foxm1 −/− embryos displayed ventricular hypoplasia and severe reduction in cardiomyocyte DNA replication and mitosis. Proliferation defects in Foxm1 −/− cardiomyocytes were associated with reduced expression of NFATc3, Cdc25B phosphatase, and abnormal nuclear accumulation of the Cdk-inhibitor p21cip1 protein. Depletion of Foxm1 expression by short interfering RNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in mouse embryos did not affect heart development and cardiomyocyte proliferation. Foxm1 binds to the −9,259/−9,288-bp region of endogenous mouse NFATc3 promoter in the developing heart, suggesting that the mouse NFATc3 gene is a direct transcriptional target of Foxm1 during heart development. The identification of critical regulators of cardiomyocyte proliferation, such as Foxm1, may provide novel strategies for diagnosis and treatment of congenital heart defects and dilated cardiomyopathy.
Foxm1 −/− Mice
We previously described the generation of Foxm1 +/− mice, in which the targeted allele lacked the DNA binding and transcriptional activation domains (Krupczak-Hollis et al., 2004). These Foxm1 +/− mice were bred for eight generations into the C57BL/6 mouse genetic background. Pregnant females were killed by carbon dioxide asphyxiation to dissect Foxm1 −/− embryos at various days of gestation. Internal cartilage tissue samples were used for genotyping the Foxm1 null allele by PCR analysis as described previously (Krupczak-Hollis et al., 2004). Two hours before sacrifice, an i.p. injection of PBS containing 10 mg/ml 5-bromo-2′-deoxyuridine (BrdU, Sigma; 50 μg/g body weight) was administered to pregnant females.
Conditional knockout mice containing an endothelial-specific deletion of the Foxm1 LoxP allele were generated by breeding Foxm1 LoxP/LoxP (fl/fl) mice with transgenic Tie2-Cre C57BL/6 mice (Zhao et al., 2006). Although 20% of Tie2-Cre Foxm1 fl/fl embryos died in late gestation/postnatally, the majority of Tie2-Cre Foxm1 fl/fl mice survived through birth and were capable of mating (Zhao et al., 2006).
Immunohistochemical Staining
Wild type (WT), Foxm1 −/−, Tie2-Cre Foxm1 fl/fl, and Foxm1 fl/fl embryos were harvested, fixed overnight with 10% buffered formalin, and then embedded into paraffin blocks. Paraffin in 5-μm sections was stained with hematoxylin and eosin (H&E) for morphological examination and morphometric analysis. The thickness of myocardial walls and heart trabeculae, and the length of trabeculae were measured on photomicrographs using Axiovision 3.0 software. Means ± SD were calculated from the average measurements from three distinct Foxm1 −/− and WT embryos, in which 10 randomized sections of left ventricle were analyzed for each genotype. The heart size was determined as the largest size between apex and the tip of right atrium using 10 heart sections for each embryo (five embryos per group). The thickness of atrioventricular (A/V) valve leaflets was determined from an average of measurements taken at the base, middle, and tip of the tricuspid and mitral valve leaflets. An overall average was calculated from 7 random heart sections from three embryos for each genotype. Statistical significance was determined using a two-tailed Student's t-test of values for either Foxm1 −/− vs. WT hearts or Tie2-Cre Foxm1 fl/fl vs. Foxm1 fl/fl hearts (P < 0.05).
Paraffin sections were used for immunostaining with mouse monoclonal antibodies against desmin (1:1,000; A8; Sigma, St Louis, MO), BrdU (1:100; Bu 20A; Dako), and PCNA (1:1,000; PC-10; Roche), rabbit polyclonal antibodies against Foxm1 (1:100; (Kalinichenko et al., 2004)), and cleaved caspase 3 (1:200; 5A1; Cell Signaling) or goat polyclonal antibodies against laminin α4 (1:100; V-20; Santa Cruz). Antibody-antigen complexes were detected using either secondary antibody directly conjugated with alkaline phosphatase (AP) or biotinylated secondary antibody followed by avidin-AP complex (Vector Labs, Burlingame, CA). BCIP/NBT was used as AP-substrate as described (Kalinichenko et al., 2001b). Sections were counterstained with nuclear fast red (Vector Labs). We also used mouse monoclonal antibodies against p21Cip1 (1:200; SXM30; BD Biosciences), goat polyclonal antibodies against GATA-4 (1:100; C-20; Santa Cruz), and rat monoclonal antibodies against Pecam-1 (1:50; MEC 13.3, Pharmingen). Antibody-antigen complexes were detected using biotinylated secondary antibody, avidin-horseradish peroxidase (HRP) complex, and DAB substrate (all from Vector Labs) as described (Kalinichenko et al., 2002; Kim et al., 2005b). For immunofluorescent detection of phosphohistone H3 (PH3), we used rabbit polyclonal PH3 antibody (1:200; MC463; Upstate) followed by anti-rabbit antibody conjugated with FITC (Vector Labs) as described previously (Kalinichenko et al., 2003).
Semi-Quantitative RT-PCR and Quantitative Real-Time RT-PCR
Total mouse heart RNA was prepared from 14.5-dpc hearts of WT or Foxm1 −/− embryos using RNA-STAT-60 (Tel-Test "B" Inc. Friendswood, TX) and then used for reverse transcriptase (RT)-PCR analysis as described (Kalinichenko et al., 2002). The following sense and antisense primers were used for amplification: laminin α4 (Lama4), 5′- ggatcccgactggtcattgatggtctacgag and 5′-gtcgaccgccttctgtggaaaaataagttc; Foxm1, 5′-ggatcctgccaccccagaccttgttc and 5′- gtcgactccct- gatgcttttcgctgtc; Cdc25B, 5′-attccag- ctctgcccaagctttggc and 5′-tccacaaatccgtcatcttctca; Aurora B, 5′-ttgacaactttgagattggg and 5′-gctggtcgtagaagtagttgt; Plk-1, 5′- ctcctggagctgcacaaga- ggaggaa and 5′- tctgtctgaagcatcttctggatgag; Cyclin B1, 5′-atcggggaacctctgatttt and 5′-tcacacacaggcaccttctc; Skp2, 5′- gtatgttagggaaccatttgcgag and 5′-ttagaagggcacttggaagagtt; Cks1, 5′-gacctcaaagccctcgtgt and 5′- tgaaacataaatccataagtcatca; laminin α2 (Lama2), 5′-tgtcgtggggattctgtatgtc and 5′-caagaaggtccaatccaacttt; integrin β1, 5′-attggctttggctcatttgtgg and 5′-ccagcagtcgtgttacattcc; NFATc3, 5′-tggatctcagtatc- ctttaa and 5′-cacacgaaatacaagtcgga; NFATc4, 5′-cattggcactgcagatgag and 5′- cgtagctcaatgtctgaat; H11 Kinase, 5′-cttgcttggttgcgttaggt and 5′-tggtggtgatggtttgagag; 5-HT2B receptor, 5′-ctcttttcaactgcctccatc and 5′-ccagcattgcc- accttttc; calmodulin, 5′-tccgttcttccttcttcgctcgcaccatggc and 5′-gtgtgtggacagaggggcttctgacatcag; FoxO3a, 5′-gtcacactacggcaaccaga and 5′-cctgagagaga- gtccgagagg; cyclophilin, 5′-agctctgagcactggagagaaa and 5′-tcctgagctacagaaggaatgg. Two different cDNA concentrations were used for RT-PCR reactions to ensure that RT-PCR conditions were in the linear range. Quantitation of expression levels was determined with Tiff files of ethidium bromide-stained gels by using the BioMax 1D program (Kodak) as described (Kalinichenko et al., 2002).
For the real-time RT-PCR, the BioRAD cDNA Synthesis Kit containing both oligo-dT and random hexamer primers was used to synthesize cDNA from 10 μg of total RNA prepared from mouse HL-1 cells as described (Wang et al., 2005; Kim et al., 2006). The following sense and anti-sense primers and annealing temperature (Ta) were used to amplify and measure the amount of mRNA by Real-Time RT-PCR: Foxm1, 5′-cacttggattgaggaccactt and 5′-gtcgtttctgctgtgattcc (Ta: 57.5°C); Cdc25B, 5′-ccctt- ccctgttttcctttc and 5′-acacacactcctgccatagg (Ta: 61.7°C), NFATc3, 5′-actgcctcatcaccatctcc and 5′-tcccaataatctcgttcacatc (Ta: 58.0°C), and cyclophilin, 5′-ggcaaatgctggaccaaacac and 5′- ttcctggacccaaaacgctc (Ta: 57.5°C). Reactions were amplified and analyzed in triplicate using a MyiQ Single Color Real-Time PCR Detection System (Bio-Rad, CA).
siRNA Transfection
Mouse cardiomyocyte HL-1 cells were obtained from Dr. Claycomb and were cultured in Claycomb medium (JRH Bioscience). In order to inhibit Foxm1 expression in HL-1 cells, we used a 19-nucleotide short interfering RNA (siRNA) duplex specific to mouse Foxm1 cDNA (siFoxm1, 5′-gga cca cuu ccc uua cuu u-3′). Foxm1 siRNA containing symmetric 2-Uracil (U) 3′ overhangs was designed and synthesized using Dharmacon Research algorithm. We transfected 100 nM of either siFoxm1 or mutant siFoxm1 (mutFoxm1; mutated nucleotides are indicated by capital letters, 5′-gga cca cuu Ucc Cua cuu u-3′) duplexes into mouse HL-1 cells using Lipofectamine™ 2000 reagent (Invitrogen) in serum free tissue culture media as described previously (Wang et al., 2005; Kim et al., 2006). HL-1 cells were harvested at 72 hr after transfection for total RNA preparation or immunostaining. HL-1 cells were labeled with BrdU for 2 hr, and then fixed with ethanol and immunostained for BrdU incorporation using the BrdU-Labeling and Detection kit II (Roche Diagnostics) according to manufacturer's recommendations. For time-course experiments, HL-1 cells were transfected with siRNA. Twenty-four hours after transfection, the cells were serum starved for 48 hr and then re-stimulated and labeled with BrdU for 2 hr as described (Major et al., 2004).
Western Blot Analysis
Nuclear protein extracts were prepared from HL-1 cells, and then subjected to Western Blot analysis (Kalinichenko et al., 2001a) using mouse monoclonal antibody against cyclin B1 (1:500; GNS-11; BD Biosciences), p27Kip1 (1:3,000; G173-524; BD Biosciences), or p21Cip1 (1:200; SXM30; BD Biosciences). We also used rabbit polyclonal antibody against Cdk2 (1:1,500; M2; Santa Cruz) for loading controls. Detection of the immune complex was accomplished by using secondary antibodies directly conjugated with HRP followed by chemi-illuminescence (Supersignal, Pierce, Rockford, IL).
Chromatin Immunoprecipitation (ChIP) Assays
Embryonic heart tissue from WT 17.5-dpc mouse embryos was cross-linked by addition of formaldehyde and used to prepare protein extract as described (Wells and Farnham, 2002). Cross-linked extracts were also prepared from HL-1 cells that were transiently transfected with CMV-Foxm1 expression vector for 24 hr. Extracts were subsequently sonicated and used for the immunoprecipitation (IP) with Foxm1 rabbit antiserum as described previously (Rausa et al., 2003). IP with P-selectin rabbit antiserum (BD Biosciences, Palo Alto, CA) was used as a control. Crosslinks were reversed on all samples by addition of TE buffer containing 10 μg of RNase A and then incubated for 15 min at 25°C. Proteinase K (10 μg) was then added and samples were digested for 16 hr at 65°C. DNA was extracted from the digested samples using PCR purification columns according to the manufacturer's instructions (Qiagen, Maryland). We then used these ChIP DNA samples for PCR reaction using primers specific to the −9,259/−9,288-bp region of endogenous mouse NFATc3 promoter: sense 5′-ttgatgacctgaactccttgcc (−9,331/−9,310) and antisense 5′-tcttagaaaagtatgcccccacc (−9,230/−9,252).
Statistical Analysis
The Student's t-test was used to determine statistical significance. P values ≤ 0.05 were considered significant. Values for all measurements were expressed as the mean ± standard deviation (SD).
We thank H. M. Yoder and A.V. Kalin for excellent technical assistance, and Eric Svensson for critically reviewing the manuscript. This work was supported by grants from National Institute of Health (HL 84151-01 to V.V.K.), American Heart Association ((0335036N to V.V.K.), and March of Dimes Birth Defects Foundation (6-FY2005-325 to V.V.K.).
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Ducommun Incorporated (NYSE: DCO) today announced that its Ducommun AeroStructures, Inc. (DAS) subsidiary has been awarded an indefinite delivery, indefinite quantity (IDIQ) contract for the V-22 Osprey titanium longeron assemblies. The V-22 Osprey is a multi-mission, military tiltrotor aircraft with both a vertical takeoff and landing (VTOL) and short takeoff and landing (STOL) capabilities. It is designed to perform conventional helicopter missions but with the long-range, high-speed cruise performance of a turboprop aircraft. The V-22 was developed by Bell Helicopter, which manufactures it in partnership with Boeing Rotorcraft Systems.  Final assembly of both the left and right longerons will be delivered from DAS' Coxsackie, NY (DAS–NY) facility.  Additionally, other Ducommun manufacturing facilities will support DAS-NY with critical build components.
Tony Reardon, president and  chief executive officer of Ducommun, stated, "This is one of the most critical outsourced assemblies that Bell Helicopter has awarded to date on the V-22 program.  The contract is indicative of the scope of work we anticipate being awarded with the addition of DAS-NY to the Ducommun family.  We are clearly leveraging the synergies in customer base, programs, products and processes in the expanded DAS family to take on more substantial and complex statements of work for our key customers.  We believe this award is an indication of the confidence that Bell Helicopter has in Ducommun and our ability to support the V-22 Osprey program.â€
About Ducommun, Incorporated
Founded in 1849, Ducommun Incorporated provides engineering and manufacturing services to the aerospace and defense industry.  The company is a supplier of critical components and assemblies for commercial aircraft, military aircraft, and missile and space programs through its three business units: Ducommun AeroStructures (DAS), Ducommun Technologies (DTI), and Miltec.  Additional information can be found at www.ducommun.com.
The statements made in this press release include forward-looking statements that involve risks and uncertainties. The Company's future financial results could differ materially from those anticipated due to the Company's dependence on conditions in the airline industry, the level of new commercial aircraft orders, production rates for Boeing commercial aircraft, the C-17 and Apache helicopter rotor blade programs, the level of defense spending, competitive pricing pressures, manufacturing inefficiencies, start-up costs and possible overruns on new contracts, technology and product development risks and uncertainties, product performance, risks associated with acquisitions and dispositions of businesses by the Company, increasing consolidation of customers and suppliers in the aerospace industry, possible goodwill impairment, availability of raw materials and components from suppliers, and other factors beyond the Company's control.  See the Company's Form 10-K for the year ended December 31, 2008 and Form 10-Q for the quarter ended October 3, 2009 for a more detailed discussion of these and other risk factors and contingencies.
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- Bell-Boeing awarded $151M to develop the CMV-22B Osprey variant
- Bell-Boeing awarded $14M for CV-22 Integrated Aircraft Survivability Equipment
- Tiltrotor documentary re-released at lower price
- Bell-Boeing awarded $8.3M order for MV-22 TCAS
- Rolls-Royce awarded $117M order for 50 AE1107C engines for V-22
- MV-22 proves firefighting capability in Cal Fire exercise
- Bell-Boeing awarded $15M order for V-22 survivability equipment
- Bell-Boeing awarded $20M more on V-22 flight test contract
- Bell-Boeing awarded $11M more on V-22 training device contract
- M-1 Support Services win $12M contract option for helicopter support at Kirtland AFB
- Special Ops V-22 maintainers win 2015 Maintenance Excellence award


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THE NIGHT; The Dance Of the Images
By PHOEBE HOBAN
Published: October 12, 1997
THE disco song ''Bad Girls'' boomed throughout the marble-floored duplex penthouse, which was a study in shocking pink: candelabras wreathed in magenta feather boas sat on pink-velvet-draped tables. On the balcony over Fifth Avenue, a row of brightly lighted mannequins looked like high-fashion gargoyles. A small army of caterers dressed like Las Vegas showgirls wore red dynel wigs.
But the glitzy decorations paled in comparison to the guest list for the party given on Monday night by the songwriter Denise Rich and Helen Gurley Brown, the former Cosmopolitan editor, to celebrate Francesco Scavullo's latest photo album, ''Scavullo'' (Harry N. Abrams), a collection of 50 years of glamorous celebrity shots -- from a juvenile Brooke Shields to Sting posed as Jesus.
Ms. Rich's gargantuan apartment was filled with enough celebrities for another Scavullo book: Martha Stewart (photographed by Mr. Scavullo for Cosmopolitan when she was just 17); Gloria Vanderbilt; Geoffrey Holder and his wife, Carmen de Lavallade; Donald and Ivana Trump (attending separately); Geraldo Rivera; Kathy Sledge; Ahmet and Mica Ertegun; Earle Mack; RuPaul; Joel Grey, and Tommy Tune.
Then there was the eye candy provided by the models Joel West and Marcus Schenkenberg, who has just come out with his own book, ''New Rules''; among other photos, it features a Cosmopolitan cover Mr. Scavullo shot of Mr. Schenkenberg paired with Cindy Crawford that never ran -- he was airbrushed out, and Ms. Crawford appeared solo.
Mr. Scavullo later pronounced his party, ''very 70's.''
''Book parties in the 1990's can be so pretentious and uptight, with everybody air-kissing each other,'' he said. ''But this was wild, everybody dancing and drinking Champagne.''
As Ashford and Simpson crooned ''Ain't No Mountain High Enough'' to Mr. Scavullo, Ms. Brown danced with RuPaul. ''That was my big thrill,'' she said. ''Why should this divine person dance by himself? I had tons of fun. I didn't know there still were parties like this in New York.''
Ms. Rich, Ms. Sledge and Deborah Cooper sang a rousing ''We Are Family.'' ''We were trying to create the whole 1970's Studio 54 environment,'' Ms. Rich said. ''That was really a Scavullo era. He's the icon.''
Mr. Scavullo may be an icon, but he also created one: the Cosmo girl, whose cleavage-plus-career image has been an enduring influence. ''Helen told me the cover had to be sexy, bosomy, lush and colorful,'' he said. ''At first I wasn't crazy about doing it. The truth is, I don't like anything that's vulgar. But the Cosmo girl is a Scavullo creation. She was always changing, but she never became Barbie.''
How does Mr. Scavullo, 68, feel about being one of fashion photography's Old Masters? ''It's hard to believe it's been half a century,'' he said. ''It seems like it took 10 minutes. I feel like a kid.''


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Beaufort County Council last week whittled down a list of more than 100 topics into 26 action items and seven top priorities for 2016 at its annual three-day strategic planning retreat.
The Lowcountry Civil War Round Table is offering its second Civil War History Scholarship to a local high school senior in an effort to promote interest and education about the American Civil War.
As part of Black History Month during February, the Osher Lifelong Learning Institute at the University of South Carolina Beaufort (USCB) will present a two-hour educational discussion of The Charleston Nine and the history and culture of the African Methodist Episcopal Church.
A new set of officers will serve on Coastal Carolina Hospital's Governing Board.
Author Pat Conroy says he has pancreatic cancer, but promises he will fight the disease hard and finish a novel he owes his fans.
Republican presidential candidate Donald Trump is lashing out against his rivals, and again floating a potential third-party run in the lead-up to South Carolina's first-in-the-South primary.
Authorities say a restaurant owner whose bribes to keep his workers in the county illegally has been charged with murder.
Tourism is an $18 billion industry in South Carolina.
The body of U.S. Supreme Court Justice Antonin Scalia was taken to a West Texas airport Sunday afternoon and was being flown to Virginia
Proposals to raise the minimum wage in South Carolina and repeal an anti-union law have virtually no chance of passing in the Republican-dominated Legislature
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15 Parks near Charlotte, North Carolina
Harrisburg, North Carolina 13.38 miles NE
Waxhaw, North Carolina 18.96 miles SE
Concord, North Carolina 19.84 miles NE
Monroe, North Carolina 20 miles SE
Denver, North Carolina 24.41 miles NW
Concord, North Carolina (map) | (704) 455-4445
Sites: 462 | Latest Rate: $27.00 | 53 Reviews
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Mar 2016 - $27.00
We have stayed here several times in the past. This park is a facility associated with Charlotte Motor Speedway and is available for camping throughout the year with some dates blacked out for events in the racing complex. The area is basically a large gravel lot with pull through full hook up sites, however during events they become back in sites. The shower house and laundry room are both clean and well taken care of. This park caters to fully self-contained RVs, no tents or pop-ups have ever been seen by us here. The secured shower house is quite away from the main parking area. The sites are easy to level up on and have easy central access to hookups. - Teacher's Pet
Nov 2015 - $21.00
This is a great park with open, graveled sites, very close to the Charlotte Motor Speedway on Bruton Smith Blvd. It is actually located on the speedway property next to Tom Johnson Camping Center sales and service center. They are wide long pull-throughs. The only downside to staying there is you have to vacate the property every time the is an event at the speedway, unless you are willing to pay $100.00 per night for the blackout period (assuming there is a site available). - Dngreen51
Oct 2015 - $27.00
A a full sun gravel parking lot with full hookups next to Charlotte Motor Speedway. Level sites with excellent full hookups. No restrooms or showers, however there is a laundry on site. We had to move the picnic table, very heavy. - Teacher's Pet
Charlotte, North Carolina (map) | (704) 588-2600
Sites: 199 | Latest Rate: $57.00 | 49 Reviews
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Jul 2016 - $57.00
We had made reservations for Carowinds so we could stop on our way home for Florida. The staff upon arrival was very friendly and courteous. We pulled into our campsite got hooked up and went to the park. We were waiting on friends. They called and said they were hooked as well. The next morning they woke up with their camper completely loaded with ants. And I mean loaded. Now, I know you can get them anywhere camping but we found out this was a known problem. That came straight from the management. They thought it was under control. The ants were so bad that they got into their food and laundry. So he went back to the manager and they put them up in a cabin which I thought was very nice. But the problem I had was that they actually put another camper in the same spot within the hour. Unacceptable. Probably will not stay again, as we found out we had ants as well. - pacevette
Jul 2016 - $60.00
A nice park. New paved roads and pads. Our site had some dirt where a patio should be, but overall well cared for with a very nice and helpful staff. We stayed here for Carowinds so being right next to the park was great! They have a new shower facility in the campground that was clean and private for users. Each shower has its own room. Roads are narrow, but definitely fine for one way traffic. The only issue would be if someone were going the opposite direction. Our visit was peaceful and pleasant. Our site was a back-in that was fairly easy to get in to, but the pull through sites are definitely the easiest in the park. All sites looked level while walking except the ones near the store/pool used mainly by tents. - IBpraisingGod
Jun 2016 - $65.00
Other than as a base camp for Carowinds and Carolina Harbor, I do not understand the lure of this campground. We stayed multiple weeks on and off while waiting to move into our house, and it's not great. The laundry room contains 2 washers and 2 dryers for the entire campground which is completely unacceptable for the size of it. Not to mention the laundry room and bathrooms are dirty most of the time with sand and dirt all over the floor. It's obvious with the trash around that employees do not groom the sites after visitors leave and prior to the next guest's arrival. The campers were rude and walked through occupied campsites to get to the pool, office, and the front of the park. Watch out if you're in site 195 because it becomes the short cut for all those rude people. And those guests who live here tend to park on the road making it difficult to get a rv in and out. Only if we need to camp close to Charlotte again will we stay here. I would be remiss not to say that the staff are very friendly and helpful. In a pinch, they helped us find a place to stay. It's not the staff's fault that management does not require cleanliness. And it's not their fault that the campers here don't know camping etiquette 101. - Roadschool
Fort Mill, South Carolina (map) | (803) 548-1148
Sites: 146 | Latest Rate: $40.00 | 87 Reviews
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Jul 2016 - $40.00
As many campers have said the entrance is confusing at best. If you miss it and have a large rig it's really a hassle. Registration office is shut down so you have to go to the gas station/convenience store to check in. This requires parking at the KOA (plenty of room) and walking to the gas station. Employee did not seem to like his job. Site was level and easy to get in and out. We asked for pull thru so were give two sites since they don't have any real pull-thrus. There's a road through the camp which is pretty busy. The street names are creepy. Scary Lane, Scream Street, etc. The gas station/office has a liquor store on one end and a bar on the other (people sit outside). There's a Publix market nearby which is great. The restrooms/showers were closed. We weren't told this when making the reservation. Wifi & cable was OK. Wouldn't stay more than overnight or stay again. Just too weird. I wouldn't recommend for folks with kids. - Catsinatrailer
Jun 2016 - $43.00
Stayed overnight found everything to be fine, check in was a little tight in the lot since you check in at the convenience store out front but other than that it was great. Gas station and convenience store out front with a liquor store on one side and a bar on the other that are part of the campground. - fil564
Jun 2016 - $43.00
Signage is confusing. Don't turn where the sign is, its the next turn. Sign in at the Valero after hours (1930). No yellow shirts here. Given a map to go find our site. Directly on the flight path to nearby Charlotte airport. Mostly uneven gravel sites except a few concrete pads. The Wi-Fi is nonexistent. - Cardiak1
4490 Boatshore Rd Rock Hill, South Carolina 29732 (map) | (803) 366-6620
Sites: 69 | Latest Rate: $26.00 | 26 Reviews
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Apr 2016 - $26.00
Really nice campground near Rock Hill and Ft Mill. Our site had a great view of the water, overlooking the picnic pavilion, play areas, boat docks, etc., but from a far distance. There was 1 tree. Other sites had a few more trees, and the other side was heavily wooded. Sites 23 and 25 are the best, as they view the lake nicely. All are spacious. All in all, a great location! - Autigerinnc70
Feb 2016 - $26.00
Camped at this York County Park, on the shores of Lake Wylie near Rock Hill, SC in late February. The Sites were well spaced, and very well priced with full hookup (water, electric, sewage) and even internet. Park Ranger was able to accommodate a move to a new site when 30+mph winds worked against us. Staff was friendly, efficient and professional. The restrooms were clean and heated and convenient trash bins with small solar night light and daily pickup completed the picture. Run like a good private campground but with the open space, boat dock and launch of a public park. - Hikers
Dec 2014 - $28.00
Ebenezer State Park is a terrific campground. The price for out of state residents is only $28 a night and they have some beautiful waterfront sites. In addition they have excellent boat access, free wifi, and the TV signal through antenna is very good for the major networks. WE stayed for six weeks and although their rule is you have to move every two weeks, we enjoyed all of the sites we stayed in. They do book fast however, so reserving in advance on the web or over the phone is a good idea. Off season there was plenty of spaces but they are pretty crowded in season and do require a day off property every two weeks to give other campers a chance. About half their sites are first come first serve and the others are reserveable, but once you get used to the system staying for an extended period is not a problem. They have many 50 amp sites and the water is from the city and tastes as good as bottled. Overall we were very very happy staying here and I give it my highest recommendation. - Lee and Trace
Rock Hill, South Carolina (map) |
Sites: 40 | Latest Rate: $16.00 | 1 Reviews
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Dec 2007 - $16.00
This is an attractive park on a lovely lake. However the sites were difficult to navigate, We were in a 38' fifth wheel and ended up parking in the large lakefront lot and walking around trying to find a site that we could back into. They claim to have pull throughs, but I didn't see any. If you have a smaller RV and can get here early in the afternoon ( no reservations), this would be a nice place to spend some time. - eimac
Charlotte, North Carolina (map) | (704) 399-3521
Sites: 94 | Latest Rate: $30.00 | 15 Reviews
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Mar 2015 - $30.00
The price here is very low, which might be a plus. The bathroom, which is basically inaccessible to most of the RV spots, is also the worst we've seen on our entire trip around the country. It looked like a bombed-out tool shed and wasn't stocked with any TP, soap, etc.
As reported in several other reviews, the husband is a very difficult and unhappy person. Unfortunately, his wife runs everything and is often gone, so you very likely will have to deal with him. Everything we communicated with the office was lost, confused, or not passed on to the appropriate person.
Others report an OK, stay, and I can see that happening if you can avoid dealing with the office and don't mind using your own RV's bathroom/shower. If so, might be worth a look. Otherwise, no way. We certainly won't be back. - 5thWheelAroundTheCountry
May 2014 - $25.00
They take cash only, hmmm that was very interesting. Also, good luck trying to level. I have a 40 ft motorhome and I got nowhere near level after 30 minutes of trying. Watch out for the speed bumps on the east side of the property. They are impossible to hit straight so everything in your refrigerator and cabinets will be thrown around big time. No use in returning to this one. - Johnnyboy21
Dec 2012 - $25.00
This is more of a trailer park with some transient sites. Owner was helpful in backing us in and giving directions needed for morning travel. Small area for dog walking. Cash only. - dotr
Charlotte, North Carolina (map) | (704) 588-5224
Sites: 56 | Latest Rate: $28.00 | 22 Reviews
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Aug 2015 - $28.00
We found a site without a reservation in August and stayed here for about a week. The tent sites do not have grass and you are required to set up in a specific gravel area so bring plenty of padding. The areas are level and will allow at least two tents, depending on which site you choose. There are individual, lockable bathroom/showers which is convenient and they are right next to a small laundry which is open only 8a to 4p. Definitely a good and secluded place to camp, with easy access to Charlotte. No Alcohol which is bad but no out of state fees, which is good. All in all a good place to be. - CaliDiscGolf
Jun 2015 - $38.00
The park itself is very beautiful and sits on Lake Wylie. We arrived late and had to knock on the door of another camper to find out about the gate code since there was a big sign that stated the gate locked at dark (which could be open to interpretation). Had trouble finding the site in the dark. Sites are gravel, not level and most are on an incline. Trees are low and the access roads have sharp curves. It would be a great place for smaller campers (less than 30 feet) and not the 40 foot one we were driving, which we were assured would not be a problem. The next day when we checked at the office, we did not receive any information, receipts, gate code, etc. Staff seemed to be more interested in their private conversation than helping. Since it is a state park, local residents receive a considerable discount, but there appeared to be several "permanent" camps which did not add to the overall appearance of the campground. We had to clean up the trash that had been left by a previous guest to make the site nice. - h2oskier
Apr 2015 - $26.00
Of the few sites that they have for RVs, make sure that you get a level one. Site R13 in particular has a 8" side to side fall that makes it impossible to level your rig on. We moved to site R2 and it was very level and long so we could remain hooked up if wanted. There are many interesting short hike trails and you are just outside of a very busy city with lots of things to do. No wifi but our AT&T worked good. - VtLee
Fort Mill, South Carolina (map) | (803) 547-3500
Sites: 138 | Latest Rate: $34.00 | 24 Reviews
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Jan 2016 - $34.00
This park is convenient to Charlotte, NC. The office staff is very friendly and helpful. I agree with the previous reviews, not much to add. I understand if you are here for several months, the office will give you a wi-fi modem which is very helpful in such a large park. - lateblumer
Dec 2015 - $39.00
We stayed here three nights to attend the Belk Bowl in Charlotte, NC. This was a convenient location for visiting Charlotte. Sites were asphalt. As happens with many asphalt sites, the sites were sloping across the site. For us that meant having to level by putting downslope wheels on about 4 inches of wooden blocks. Almost all the campers around us had to do the same. This is a nice campground, but most sites were occupied by long-term occupants. Our site was a pull-through, but we had to back out of it. There was no way to pull forward out of the site and make the turn on the camp road in front. It was too tight with trees in the way. We would camp here again if in the Charlotte area. - jwindham
Aug 2015 - $34.00
Stayed here 4 nights in a 40 foot MH, Friendly check in service, good 50 amp, water sewer, wifi very slow, could not get cable to work, BUT that was probably my fault, haven't used cable in years. Too many trees for Satellite dish. I was parked in site 42, long enough site for coach and car although it was a bit slanted. It appears that a lot of the sites are for seasonal or permanents, that's ok as they were all clean and pretty well kept. This place is in an area that is still being developed housing wise, lots of construction and lots of traffic, BUT the park was a quite place.
We would return. - walt66
Harrisburg, North Carolina (map) | (704) 773-9349
Sites: 44 | Latest Rate: $20.00 | 10 Reviews
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Jun 2015 - $20.00
Gravel roads and sites with some green space between sites. Most back-in sites are level. Pull-through sites are not. All sites are open. There is no shade. 4 bars on Verizon. Satellite TV was no problem. Turns were tight for our 45 footer. Owners were very nice. - eaglessoc
Sep 2014 - $20.00
This "park's" not family friendly, only a few sites have sewer, and the park is in someone's backyard. No showers or toilets available. That being said, it's near the highway (I-85), the owner is nice, and Charlotte Speedway is right there. We like it for an overnight and would stay again. - bbnh2000
May 2013 - $25.00
I would rate this campground higher, but there are zero amenities (no shower house, laundry facilities, pool). Charlotte just is not an RV destination town. That said, the owners are very friendly and helpful and the hookups were good. Most sites face each other so if you're there with friends, that can be a good thing. If not, it's a bad thing. We stayed on the "deluxe" RV sites which put us back to back with another unit. Easy access and about 14 miles from downtown Charlotte and directly across the street from the Speedway. There are also RV parks across the street, but you have to search the city Concord. - sixtires58
Concord, North Carolina (map) | (704) 455-9282
Sites: 50 | Latest Rate: $60.00 | 12 Reviews
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May 2016 - $60.00
Owner was very accommodating, he helped set trailer in camp site under very wet conditions. Park is kept very neat and clean by employees continually picking up any trash. We stayed there for 10 days during race week. This park has a very safe, family type atmosphere. This was our second 10 day stay. We will definitely stay again. Best location for races in the area and cleanest by a long shot. On site store and gas station.. Very level lots with excellent drainage. Full hook ups. Owner constantly checks to see if you need anything. Porta-potties cleaned/pumped daily, very clean and this was during race week. This is by far the cleanest camp ground near track. There were full sun or shaded lots, many options. - Gary Ohio Camper
May 2016 - $25.00
This is a smaller RV Park, but it has huge overflow fields used for race weekends. It's RIGHT next to Charlotte Motor Speedway...walking distance. So, race weekends are crazy crowded. But the rest of the time it is awesome. The regular park has full hookups, pull through sites. The sites are fairly good size and level. It is very convenient to 85 and 485. Near Concord Mills, lots of shopping.
If you come during race time, the overflow fields are mostly dry camping...just a field to park in with porta potties...but it is really convenient. - GranNan06
May 2015 - $30.00
In 40ft MH towing site and roads plenty of room. - Dan Kleine


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Click here to list ALL Makes
This is a Radiomodile Model 100 which were fitted to quality cars in the late 1940s including the Bristol 400.This particular set has just been serviced and is now in good working order.
Any accuratly .....
When the Bristol 630 was introduced by Bristol Cars to replace the 411 in 1976 two variants were available a 5.2 litre "E" (Economy) version and the "S" (Sport) version with the 5.9 litre Chrysler V8 .....
The Bristol 401 was the first 4-seater coupe produced by the Company and one of the most outstanding cars of the immediate post-war period. This car was renovated to show condition in the 1990's and is .....
Chassis no: 411-7507
First registered: 19 January 1970
Current (and original) registration: CGN 490H
One of the first 10 of the first series of 411.
Wind up windows and slightly different interior .....
First registered: 22nd May 2008
Indicated mileage: 29,000 (probably correct).
Dark blue paint with cream leather, piped in dark blue.
SUMMARY: very low .....
Car that had a restoration in England, excellent condition, repainted, metallic color, engine still running completely redone, original interior very healthy, rubberized leather, homologated ASI, registered .....
1983 Bristol Brigand. A two owner car with interesting history and 79,000 miles. Recent bare metal respray in Rolls Royce Caribbean Blue, with new headlining and carpets.
Mechanically very good with .....
1972 � Bristol 411 � Series III
Chassis: 4117614128
Eng: F3834038
Finished in Metallic Midnight Blue.
With a newly re upholstered St James Red Connolly Hide Interior.
It has been fitted with .....
chassis number 405/4538
Engine no: 100B/3701
First registered: 9th November 1956
Carries its original (and only) registration of XUM 620.
One family ownership since 1965
Colour: Arabian red with .....
A rare and exclusive "Gentleman's Express" in fine order.
To be sold by Silverstone Auctions at �The Silverstone Classic� auction on the 30th/31st July 2016 at The Wing Building, Silverstone Circuit, .....
Very rare 409 Bristol, only 76 built, V8 engine. One of the last cars equipped with pushing buttons automatic gear box.
Car in excellent condition.
First registered: 31 October 1974
Taxation class: Historic Vehicle, ie no �road tax�.
Chassis no: 7727.467
Current owner has owned and loved this car since 16th October 1993.
For sale due .....
Bristol 400 first registered: 4 May 1949 ; sold by AFN Ltd, Middlesex
Chassis no: 400/1/614 ; Car no: 400/A/387
Engine no: 85C/1026
Current owner from July 1986
Good history file from 1986 .....
1949 Bristol 401 Coupe blue with tan interior. This is an extremely rare car. An excellent original car for restoration for only $39,500.
Finished in mediterranean blue with blue hide trim. Restored by time served Bristol Employee some years ago and has been in a private collection ever since. Would sell outright or listen to interesting .....
The Bristol 411 was in production for eight years from 1969 and early example likes this are becoming quite scarce. This is the first Bristol 411 we have had the pleasure of selling in many years and .....
A true gentleman�s express, this Bristol Blenheim III is believed to be one of only 8 manufactured and is a 2002 car with only two owners from new and 85,000 miles on the clock.
Serviced regularly, .....
In 1949 Jean Simmons and Stewart Granger were two of the worlds biggest film stars. So close were the British couple (who married a year later in America in a ceremony organised by Howard Hughes) that .....
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Children's Health
Infant Ear Infections Becoming Less Common
By Amy Norton
HealthDay Reporter
MONDAY, March 28, 2016 (HealthDay News) -- Painful ear infections remain a scourge of childhood, but fewer American babies are getting them now compared with 20 years ago, new research shows.
The study didn't dig into the reasons for the decline. But experts say the credit likely goes to certain childhood vaccines, rising rates of breast-feeding and the drop in Americans' smoking rate.
The new research found that 46 percent of babies followed during 2008 to 2014 had a middle ear infection by the time they were 1 year old.
But while the infections were common, those rates were lower when compared against U.S. studies from the 1980s and '90s, the researchers added. Back then, around 60 percent of babies had suffered an ear infection by their first birthday, the study authors said.
The decline is not surprising, according to lead researcher Dr. Tasnee Chonmaitree, a professor of pediatrics at the University of Texas Medical Branch, in Galveston.
"This is what we anticipated," she said.
That's in large part because of a vaccine that's been available in recent years: the pneumococcal conjugate vaccine, Chonmaitree said. The pneumococcal conjugate vaccine protects against several strains of pneumococcal bacteria, which can cause serious diseases like pneumonia, meningitis and bloodstream infections.
Those bacteria are also one of the major causes of children's middle ear infections, Chonmaitree said.
She added that yearly flu shots, which are now recommended for children starting at the age of 6 months, are probably helping, too: Ear infections often arise after a viral infection like the flu or common cold, the study authors said.
Vaccinations "could very well be one of the drivers" behind the decline in infant ear infections, agreed Dr. Joseph Bernstein, a pediatric otolaryngologist who wasn't involved in the study.
But there are other important factors, too, both he and Chonmaitree said -- namely, rising rates of breast-feeding and a decrease in babies' exposure to secondhand smoke.
"The data really do suggest that breast-feeding -- particularly exclusive breast-feeding in the first six months of life -- helps lower the risk of ear infections," said Bernstein, who is director of pediatric otolaryngology at the New York Eye and Ear Infirmary of Mount Sinai, in New York City.


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It was {A href="http://bothell.komonews.com/content/does-billy-idol-do-birthdays"}two years ago that Michael Henrichsen launched his relentless campaign to convince Idol to come to the Emerald City to play his birthday.
All that work paid off Friday night when Idol and his band took the stage at Seattle's Showbox at the Market.
Idol said he had been aware of Henrichsen's campaign for some time.
"Two or three years ago I started to see some of the Youtube videos he'd made with some of the different musicians, sports people and celebrities who were asking me to play his birthday party," Idol said. "He also drove Steve Stevens, my guitar player's wife, nuts by tweeting her all the time."


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Burwell Jobs
Looking for jobs in Burwell? Snagajob has great opportunities with the area's leading companies. And applying for Burwell jobs has never been easier. Look at the job listings below, click on a job title that interests you and hit the "apply now" button. Part-time jobs, teen jobs, student jobs and summer jobs are now just a click away. Start your job search now.
Burwell, NE Job Openings
Bosselman Administrative Services, Inc.517 S 1St
Burwell, NE