Functional involvements of heterogeneous nuclear ribonucleoprotein A1 in smooth muscle differentiation from stem cells in vitro and in vivo.

To investigate the functional involvements of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in smooth muscle cell (SMC) differentiation from stem cells, embryonic stem cells were cultivated on collagen IV-coated plates to allow for SMC differentiation. We found that hnRNPA1 gene and protein expression was upregulated significantly during differentiation and coexpressed with SMC differentiation markers in the stem cell-derived SMCs as well as embryonic SMCs of 12.5 days of mouse embryos. hnRNPA1 knockdown resulted in downregulation of smooth muscle markers and transcription factors, while enforced expression of hnRNPA1 enhanced the expression of these genes. Importantly, knockdown of hnRNPA1 also resulted in impairment of SMC differentiation in vivo. Moreover, we demonstrated that hnRNPA1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Acta2 and Tagln genes using luciferase and chromatin immunoprecipitation assays. We further demonstrated that the binding sites for serum response factor (SRF), a well-investigated SMC transcription factor, within the promoter region of the Acta2 and Tagln genes were responsible for hnRNPA1-mediated Acta2 and Tagln gene expression using in vitro site-specific mutagenesis and luciferase activity analyses. Finally, we also demonstrated that hnRNPA1 upregulated the expression of SRF, myocyte-specific enhancer factor 2c (MEF2c), and myocardin through transcriptional activation and direct binding to promoters of the SRF, MEF2c, and Myocd genes. Our findings demonstrated that hnRNPA1 plays a functional role in SMC differentiation from stem cells in vitro and in vivo. This indicates that hnRNPA1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine. STEM CELLS 2013;31:906-917