Abstract 10202: Sp1-Dependent Activation of HDAC7 is Required for PDGF-BB-Induced Smooth Muscle Cell Differentiation From Stem Cells
Background: Embryonic stem (ES) cells possess the potential to differentiate into specific cell lineages, such as vascular smooth muscle cells (SMCs). We have previously demonstrated that histone deacetylases 7 (HDAC7) plays an important role in mediating a PDGF-BB-induced ES cell differentiation into SMCs but the molecular mechanisms are currently unknown. Therefore, we determined whether PDGF-BB upregulates HDAC7 expression during ES cell differentiation towards SMCs through transcription activation and the underlying molecular mechanisms.
Methods and results: Mouse ES cells were seeded on collagen IV-coated flasks and cultured in the absence of Leukemia Inhibitory Factor (LIF) in differentiation medium for 3 to 9 days in order to induce SMCs differentiation. We found that PDGF-BB induced a 3-fold increase in the transcripts of HDAC7 in differentiated ES cells as determined by real-time PCR. To characterize the molecular mechanism underlying PDGF-BB-induced HDAC7 expression, we employed the luciferase reporter system with a series of deletion mutants of the HDAC7 5′-flanking region. The PDGF-BB responsive region is located between −343 and −292bp in the 5′-flanking region of HDAC7 gene. Analysis of the sequence of this region identified one consensus binding site for the transcription factor, Sp1. Mutation of the Sp1 site within the HDAC7 promoter abolished PDGF-BB-induced activity and the results of Chromatin immunoprecipitation (ChIP) assay demonstrated enhanced Sp1 binding to the HDAC7 promoter in SMCs treated with PDGF-BB in vivo. To further investigate the effects of altered expression of Sp1 on HDAC7 transcription, siRNA knockdown experiments were performed. Our data revealed that PDGF-BB-induced HDAC7 expression was abrogated in differentiated ES cells with Sp1 siRNA. In addition, enforced expression of Sp1 was sufficient to increase the activity of the HDAC7 promoter. Finally, we found that the Sp1-dependent activation of HDAC7 is involved in PDGF-BB-induced multiple SMC-specific marker gene expression in differentiated ES cells.
Conclusion: We have demonstrated that PDGF-BB induces the transcription activation of HDAC7 during ES cell differentiation towards SMCs, which is regulated by the transcription factor Sp1.
- © 2010 by American Heart Association, Inc.