Abstract 5444: Cbx3 Identified by Nuclear Proteomics Plays a Key Role in Stem Cell Differentiation Into Smooth Muscle Cells in vitro and in vivo
Our previous studies demonstrated that Sca-1+ progenitors derived from embryonic stem cells (ESCs) and adventitial tissues of adult apoE−/− mice can differentiate into smooth muscle cells (SMCs) that participated in the neointimal formation. However, the molecular mechanism of stem cell differentiation into SMCs is not fully elucidated. To identify potential signal transducers mediating SMC differentiation, nuclear proteins were harvested from undifferentiated mouse ESCs and differentiating ESC at different time points, and subjected to proteomic analysis by nanoflow liquid chromatography tandem mass spectrometry. We found that the majority of upregulated nuclear proteins during SMC differentiation were involved in chromatin remodeling, cell proliferation, and RNA processing processes. Among those upregulated nuclear proteins, we further focused on chromobox protein homolog 3 (Cbx3) because Cbx3 functions as activator and/or repressor within both heterochromatin and euchromatin. Knockdown of Cbx3 in the differentiating stem cells resulted in down-regulation of smooth muscle markers including smooth muscle actin, SM22, calponin and smooth muscle myosin heavy chain, while enforced expression of this gene enhanced SMC differentiation in a dose-dependent manner. CHIP assay suggests that Cbx3 mediated SMC differentiation through regulation of smooth muscle specific transcription factor, serum response factor (SRF), and coactivatior myocardin. Furthermore, we also demonstrated that the transcription factor, Dia1, functioned as a bridge protein between Cbx3 and SRF, through which Cbx3 modulates SRF activation, mediates ultimately SMC differentiation. Interestingly, in situ hybridization and immunohistochemical staining indicates that Cbx3 localized at smooth muscle system during development in different stages of chicken embryos. Importantly, in vivo knockdown of Cbx3 in the vessel wall significant increased wire-injury-induced neointima formation in mice. Our findings demonstrated for the first time that Cbx3 has a crucial role in SMC differentiation and possesses a protective function in vessel injury-induced neointima formation, providing novel information on potential therapeutic targets for vascular diseases.