Functional Involvements of Heterogeneous Nuclear Ribonucleoprotein A1 in Smooth Muscle Differentiation from Stem Cells In Vitro and In Vivo

Abstract

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 Disclosure of potential conflicts of interest is found at the end of this article.

Upregulated sirtuin 1 by miRNA-34a is required for smooth muscle cell differentiation from pluripotent stem cells

“…Data shown in Figure 7a revealed that gene expression levels of all three factors were significantly upregulated by SirT1, suggesting SirT1 may have a direct role in regulation of these transcription factors during SMC differentiation. Indeed, luciferase activity assays with SRF, MEF2c and Myocd gene reporters 11 showed that the SirT1 overexpression significantly increased SRF, MEF2c or Myocd gene-promoter activities (Figure 7b), indicating that SirT1 upregulates transcriptional activity of these three genes. Importantly, data SirT1 promotes SMC gene expression through inhibiting H3K9 methylation around SMC gene promoters.…”

“…Murine ESCs were induced to differentiate towards SMCs as described previously (Supplementary Figure S1). [11][12][13][14] miRNA microarrays analysis was conducted to identify potential miRNA candidates for SMC differentiation, and the data revealed that miR-290 family members, the reported ESC-specific miRNA cluster, 15 were dramatically downregulated upon differentiation (Supplementary Table S1). Conversely, muscle differentiation-related miRNAs (miR-143/145/133) were increased in our SMC differentiation model, while SMC proliferation-related miR-21 was undetectable at early stage but dramatically increased at late stage of SMC differentiation, indicating that some miRNAs may initiate SMC differentiation, whereas others may have important roles in the late stage of SMC differentiation.…”

“…Expectedly, data summarised in Supplementary Table S2 showed that a panel of SMC-specific genes (highlighted in yellow) were upregulated by miR-34a overexpression. Importantly, several SMCspecific transcription factors, including serum response factor (SRF), myocardin (Myocd) and myocyte enhancer factor 2C (MEF2c), that were reported to be activated in our SMC differentiation system 11,17 were also significantly upregulated by miR-34a, alongside SirT1 (Supplementary Table S2). Furthermore, overexpression of miR-34a upregulated SRF, myocardin and MEF2c, whereas knockdown of miR-34a downregulated these genes (Supplementary Figure S2), suggesting that miR-34a works in concert with these SMC transcription factors during SMC differentiation.…”

“…*Po0.05 miR-34a and SirT1 in smooth muscle differentiation X Yu et al miR-34a in SMC differentiation in vivo, pLL3.7-GFP and pLL3.7-GFP-miR-34a ESCs were subcutaneously injected into mice with 100 ng/ml of PDGF-BB to promote in vivo SMC differentiation as described in our previous study. 11,12 Data showed that more smooth muscle myosin heavy chain (SM-MHC)-positive cells were presented in the implants of miR-34a-overexpressing ESCs (Figures 2a and b) and that the majority of cells in the Matrigel implants were GFPpositive, indicating its exogenous origins (Figure 2a). Importantly, the expression levels of miR-34a, smooth muscle alpha-actin (SMαA), SM-MHC and SirT1 in the Matrigel implants of pLL3.7-GFP-miR-34a ESCs were significantly higher than that of control pLL3.7-GFP ESCs as demonstrated by immunofluorescence staining (Figure 2a), RTqPCR ( Figure 2c) and immunoblotting (Figure 2d) analyses, respectively, further confirming the efficiency of miR-34a overexpression and the importance of miR-34a in SMC differentiation in vivo.…”

“…33 have nicely demonstrated that RNAbinding protein HuR attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA. Moreover, our previous studies have suggested an important role for another two RBPs, hnRNPA1 11 and hnRNPA2B1, 34 in SMC differentiation. These evidence together with our observation that much less miR-34a and SirT1 3′UTR were loaded into Ago2-RISC complex upon SMC differentiation prompts us to speculate that miR-34a upregulates SirT1 during SMC differentiation through a similar mechanism as reported by Kundu et al 33 In other words, the upregulation of SirT1 by miR-34a may well be a result of RBPs, such as HuR, hnRNPA1 and hnRNPA2B1, interacting with miR-34a-binding sites and/or other elements within the 3′UTR of SirT1 to disengage it from Ago2-RISC complex during differentiation.…”