doi: 10.1161/CIRCRESAHA.108.172114
© 2008 American Heart Association, Inc.
Editorials |
Vascular Endothelial Growth Factor Gene Regulation by HEXIM1 in Heart
From the Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis.
Correspondence to Atsushi Asakura, Stem Cell Institute, University of Minnesota Medical School, McGuire Translational Research Facility, 2001 6th St. SE, Mail Code 2873, Minneapolis, MN 55455. E-mail asakura{at}umn.edu
See related article, pages 415–422
Key Words: angiogenesis • VEGF • heart • HEXIM1 • C/EBP
Hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) was cloned as an upregulated gene in vascular smooth muscle cells after treatment with the differentiating agent HMBA.1 A recent report has shown that HEXIM1 is an inhibitor of positive transcription elongation factor (P-TEF)b, which plays an important role in regulation of RNA polymerase II elongation.2–4 HEXIM1 was previously cloned as a potential cardiac transcriptional regulatory factor suppressing the cardiac myosin light chain-2v promoter and termed cardiac lineage protein (CLP)-1.5 HEXIM1 was also cloned as the novel inhibitor of breast cell growth estrogen-downregulated gene (EDG)1.6 HEXIM1 protein level is downregulated by estrogens. HEXIM1 also suppresses estrogen receptor- transcriptional activity.6,7 Gene and protein expression indicate the broad expression of HEXIM1 during postnatal development, with highest levels in heart, skeletal muscle, and brain. Protein localization of endogenous HEXIM1 in vascular smooth muscle cells and primary cardiomyocytes suggests that HEXIM1 is a nuclear protein. Furthermore, HEXIM1 has been shown to be involved in many biological processes, including cancers, AIDS, cardiac hypertrophy, and inflammation, through transcriptional repression.4
In vitro experiments demonstrate that HEXIM1 functions as a transcriptional repressor by inhibiting P-TEFb, a protein complex composed of cyclin-dependent kinase 9 and a cyclin partner that regulates RNA polymerase II elongation.2–4 HEXIM1 interacts with 7SK small nuclear RNA (snRNA), a component of P-TEFb complex containing cyclin-dependent kinase 9 and cyclin T1. This interaction occurs through the KHRR 7SK snRNA binding motif of HEXIM1 (Figure). HEXIM1 also associates with cyclin T1 through the PYNT motif to suppress P-TEFb activity (Figure). Recently, HEXIM2 was identified as a HEXIM1 homolog in the DNA database. HEXIM2 also contains 7SK snRNA and cyclin T1 binding motifs.8,9 HEXIM1 functions as a homodimer or heterodimer with HEXIM2 via their C-terminal regions. Based on these homologies, HEXIM2 can functionally compensate for the loss of HEXIM1 on regulation of P-TEFb activity in vitro. Gene knockout mouse experiments demonstrate that CLP-1/HEXIM1 is essential for heart development10: CLP-1 gene–knockout (CLP-1–/–) mice display lethality in late fetal stages. CLP-1–/– hearts show a reduced left ventricular chamber with thickened myocardial walls, indicating the characteristics of cardiac hypertrophy. The HAND1 gene, a basic helix–loop–helix (bHLH) transcription factor involved in early heart development, is significantly reduced in CLP-1–/– hearts. Therefore, CLP-1/HEXIM1, together with HAND1, plays a critical role in heart development, and loss of the CLP-1/HEXIM1 gene can lead to cardiac hypertrophy. Similarly, elevated P-TEFb activity by overexpression of cyclin T1 is observed in cardiac hypertrophy. Taken together, these data strongly suggest the involvement of HEXIM1 and P-TEFb in cardiac hypertrophy. However, little is known about HEXIM1 function on other transcription factors.
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Vascular endothelial growth factor (VEGF), which belongs to the VEGF family containing several different isoforms, is a key growth factor during vascular development throughout the body by binding to its receptors, VEGFR1 (Flt1) and VEGFR2 (Flk1/KDR).11 VEGF is among the most important growth factors to stimulate angiogenesis during embryogenesis, postnatal development, and tissue repair and regeneration. The loss of this gene results in reduced angiogenesis and embryonic lethality, suggesting that it plays essential roles in the development and differentiation of the vascular system. The transcriptional regulation of the VEGF gene is critical to adjust to the VEGF expression level. Therefore, the VEGF expression level is strictly controlled by transcription factors that bind to several response elements within the VEGF promoter region.11 These transcription factors include hypoxia-induced factor (HIF)-1, HIF-2, nuclear factor (NF)-
B, activator protein (AP)-1, specificity protein (SP)1, STAT, and CCAAT/enhancer binding protein (C/EBP). HIF-1 and -2 are well-characterized bHLH-PAS (bHLH-Per Arnt Sim)–type transcription factors of the VEGF gene. Hypoxia induced by ischemia upregulates both HIF-1 and -2 gene expression, which can activate VEGF gene transcription through binding to hypoxia-response elements located at regulatory regions of VEGF gene. NF-B is a transcription factor for the VEGF gene, and the activity of NF-B is regulated by intracellular localization change from cytoplasm to the nucleus by inductions of stresses and cytokines. AP-1 is a transcription factor that binds to TPA-response element (TRE) and is highly activated by hypoxia. SP1 is involved in a variety of gene regulation. SP1 seems to regulate differential overexpression of VEGF. Expression of the VEGF gene is also regulated by leukemia inhibitory factor through a STAT transcription factor that binds to the promoter region of the VEGF gene. C/EBPs are a family of 6 proteins, the expression of which is detected in a wide variety of cell types.12 These proteins contain the BR-LZ (basic region leucine zipper) DNA-binding domain. C/EBPs bind to DNA elements as homo- or heterodimers. The N-terminal region of the proteins contains transcriptional activation, repression, and autoregulatory functions. In general, C/EBP proteins function as transcriptional activators. However, recent data demonstrate that C/EBPs also repress transcription of certain target genes. Several studies reported that C/EBP blocks cell cycle progression at the G1/S boundary. Montano et al first demonstrate the negative transcriptional regulation of VEGF gene by C/EBP in the heart (Figure).13 Interestingly, HEXIM1 can attenuate the inhibitory effect of C/EBP on VEGF gene expression. Montano et al established new HEXIM1 gene–knockout mice to examine involvement of this gene in heart development. This group created mice carrying insertional mutation in the HEXIM1 gene that disrupted its C-terminal region. These homozygous HEXIM1 (1-312) mutant mice display perinatal death and several heart defects, including abnormal coronary patterning, thinner ventricular wall, and decreased vascularization. These defects were attributable to decreased expression of VEGF and fibroblast growth factor-9, and increased apoptosis. They found that HEXIM1 can activate VEGF gene expression through interaction between the HEXIM1 C-terminal region and C/EBP, which is a suppressor for VEGF gene expression through binding to the VEGF promoter region (–2079/–1252). Therefore, the authors suggest that VEGF is a direct transcriptional target of HEXIM1 that attenuates a repression of C/EBP on VEGF gene transcriptional control. Taken together, these results suggest that HEXIM1 plays critical roles in coronary vessel development and myocardial growth through direct interaction with C/EBP. Recent work has revealed that HEXIM1 can also bind to estrogen receptor-,7 glucocorticoid receptor,14 and NF-B.15 Clearly, HEXIM1 may interact with more transcription factors. Thus, HEXIM1 regulates wide varieties of developmental programs and diseases through transcriptional regulation.
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Acknowledgments |
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The author thanks Mayank Verma for critical reading.
Sources of Funding
This work was supported by grants from the Korea Institute of Science and Technology.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2008 102: 415-422.
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