Functional Impact of Heterogeneous Nuclear Ribonucleoprotein A2/B1 in Smooth Muscle Differentiation from Stem Cells and Embryonic Arteriogenesis

Abstract

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play various roles in transcriptional and post-transcriptional modulation of gene expression. However, it remains unclear if hnRNPs are associated with smooth muscle cell (SMC) differentiation from stem cells and embryonic arteriogenesis. In this study, mouse embryonic stem (ES) cells were cultivated on collagen IV-coated plates and smooth muscle differentiation medium. We found that hnRNPA2/B1 gene and protein expression was significantly up-regulated following 3-7 days of cell differentiation. hnRNPA2/B1 knockdown resulted in down-regulation of specific smooth muscle markers and transcription factors, whereas enforced expression of hnRNPA2/B1 enhanced the expression of these genes. Moreover, we demonstrated by using luciferase and chromatin immunoprecipitation assays that hnRNPA2/B1 could transcriptionally regulate SMC gene expression through direct binding to promoters of Smαa and Sm22α genes. We further demonstrated that chromobox protein homolog gene 3, a previously identified SMC differentiation regulatory nuclear protein, is required for hnRNPA2/B1-mediated SMC differentiation gene expression. Importantly, specifically designed Hnrnpa2/b1 morpholinos for in vivo knockdown could inhibit the migration and differentiation of neural crest cells into SMCs in chick embryos. This resulted in the maldevelopment of branchial arch arteries and increased embryo lethality at a later developmental stage. Our findings demonstrated that hnRNPA2/B1 plays a functional role in SMC differentiation from stem cells in vitro and embryonic branchial arch artery development. This indicates that hnRNPA2/B1 is a potential modulating target for deriving SMCs from stem cells and cardiovascular regenerative medicine.