Functional Involvement Of Heterogeneous Nuclear Ribonucleoprotein A1in Smooth Muscle Differentiation From Stem Cells In Vitro And In Vivo

Atherosclerosis is a chronic, medium and large size vessel related disease. Atherosclerosis is a progressive disease in which lipids, inflammatory cells and vascular smooth muscle cells (VSMCs) accumulate in the neointimal layer of the artery and thus form a plaque with fibrous cap. Under normal physical condition, media smooth muscle cells have less proliferation with the formation called’hills and valleys’. Those smooth muscle cells express some special differentiation gene, such as SM a-actin, SM-MHC, hl-calponin, SM22a and smoothelin. But, under pathological situation, smooth muscle cells migrate from media to intima and start to proliferate with cobblestone-like structure. There is always controversy about where are these two different type cells coming from. Most recently, growing evidence suggests that stem/progenitor cells exsited in the blood vessel wall or surrounding tissues is one of the major cellular sources of the smooth muscle cells in the atherosclerosis intima.Three major types of stem cells-embryonic stem cells, adult or somatic stem cells and induced pluripotent stem cells have been investigated intensively in the stem cell field. In terms of the disadvantages and advantages of all three types of stem cells, although embryonic stem cell has been consistently challenged because of its immunological rejection and ethics issues, such problems can be easily resolved by setting-up the HLA and ABO antigen matching human embryonic stem cell bank from which150-200human embryonic stem cell (hESC) lines with various HLA genotypes will suffice to provide HLA-matches for populations in UK, Japan or China. Moreover, since embryonic stem cell posseses full-potential to generate every single cell type or tissue inside human body which is not naturally achived by other stem cells, it will become an excellent model for in vitro cell differentiation.RNA binding protein HnrnpAlis a member of conserve heterogeneous nuclear ribonucleoprotein family. Heterogeneous As reported, hnrnpAl has four major functions:transcription, mRNA splicing, nucleus-cytoplasm shuttle and effecting micro-RNA biosynthesis. It has been reported that changes on hnrnpA1expression levels and/or activiries will impact post-transcription for diverse RNAs, and such changes have been observed in cancer, neuro-degenerate and Alzheimer diseases.Gene transcriptional regulation is a delicate process, which plays an important role in many diseases including cancer, neuro-degeneration and smooth muscle cells de/differentiation related atherosclerosis. Our project, based on the emerging evidence in this field that the smooth muscle cells in the atherosclerotic intima is possiblely differentiated from stem cells haboring in the vessel wall or surrounding tissues, by using the the well-established mouse smooth muscle cell differentiation model in Dr Xiao’s group, aims to investigate the molecular mechanisms by which hnrnpAl regulates smooth muscle cell differentiation.In our previous nuclear proteomics analysis, we found hnRNPAl protein expression increased in the early stage of mouse smooth muscle cell differentiation, suggesting that hnRNPA1plays a role in smooth muscle cell differentiation from stem cells. Indeed, PCR and western demonstrated that hnRNPAl was positively associated with smooth muscle cell differentiation genes, like SM22a, and transcription factors, like SRF. Moreover, double immunohistochemical stainings confirmed the expression patterns of hnRNPAl in stem cell-derived smooth muscle cells, during embryonic cardiovascular development, and in adult aorta and heart tissues. Importantly, function-loss and function-gain experiment showed that hnRNPA1is indispensable in smooth muscle cell differentiation from stem cells, and it is especially for smooth muscle cell differentiation. Furthermore, in vivo Matrigel plug double confirmed the in vitro results. Data from our actinomycin D inhibiton, Luciferase reporter analysis and chromatin immunoprecipitation (ChIP) assay proved hnRNPAl regulates smooth muscle specific differentiation gene by directly binding with the promoter of these genes. Our data also reveal that hnRNPA1and SRF interactions is involved in hnRNPAl-mediated smooth muscle cell differentiation, and hnRNPAl regulates smooth muscle specific differentiation gene expressiojn through SRF binding site within gene promoter as demonstrated by phnRNPAl/SRF siRNAs cotrasfection, protein complex immunoprecipitation (Co-P), promoter gene mutation analyses, respectively.Finally, we also discovered that hnRNPAl regulates transcription factor SRF, MEF2and Myocardin at transcription level.