Abstract 9127: C-myb-regulated Expression Of Mir-143/145 In Embryonic Stem Cell-derived And Adult Vascular Smooth Muscle Cells
Background: The c-myb gene encodes for the transcription factor c-Myb, which has been implicated in the differentiation of vascular smooth muscle cells (VSMCs) from neural crest and embryonic stem cells (ESCs). Mouse ESCs lacking c-myb (KO) failed to express SMC-specific genes and did not differentiate into contractile SMCs in embryoid bodies (EB), with c-Myb regulating the formation of a specific Flk-1+/PDGFRα- (F+/P-) progenitor of VSMCs. Others showed that expression of the miR-143/145 cluster is restricted to adult SMC lineages and promotes the expression of contractile genes. Hypothesis: c-Myb mediates the transcriptional regulation of miR-143/145.
Methods &Results: Flow-sorting of cardiovascular-directed d3.75 EBs showed high levels of miR-143/145 expression in F+/P- (c-myb high) cells, with low levels in other (c-myb low: F+/P+ and F-/P+) cell populations. Importantly, no differences in the expression levels of miR-143/145 could be detected in F+/P-, F-/P+ and F+/P+ cells derived from c-myb KO EBs. Multiple putative c-Myb consensus-binding sites (conserved in human and mouse) were identified in 3kb and 20kb enhancers upstream of the miR-143/145 cluster. Chromatin immunoprecipitation assays in both cardiovascular-directed d3.75 EBs and immortalized mouse adult aortic SMCs (MOVAS) revealed c-Myb binding to multiple specific sites. Next, c-Myb responsiveness of wild-type and deletion mutant enhancer-reporter constructs was assayed in HEK293 cells. These studies confirmed two c-Myb-dependent miR-143/145 enhancer activities.
Conclusion: Our data suggest that c-Myb-regulated VSMC differentiation and contractile gene expression may be mediated by direct binding to specific transcription enhancer elements in the miR-143/145 cluster. *Authors have contributed equally to this work.
- © 2012 by American Heart Association, Inc.