Identification of Molecular Mechanisms Mediating TWIST-1 Regulation of Mesenchymal Stem Cell Proliferation and Differentiation

Abstract

Bone marrow-derived mesenchymal stem/ stromal cells (BMSC) are self-renewing, multipotent cells that can give rise to multiple lineages including osteoblasts (bone), chondrocytes (cartilage) and adipocytes (fat). Interestingly, various pathways that promote BMSC osteogenesis/chondrogenesis simultaneously suppress adipogenesis and vice versa. The basic Helix-Loop-Helix (bHLH) transcription factor, TWIST-1 is highly expressed by BMSC and plays an important role in BMSC proliferation, lifespan, differentiation and commitment. Enforced expression of TWIST-1 enhances proliferation potential and lifespan of BMSC. It also enhances the adipogenic potential of BMSC yet inhibits chondrogenesis and osteogenesis. However, the underlying mechanisms mediating TWIST-1 regulation of BMSC growth and differentiation are not fully understood. In order to identify novel TWIST-1 gene targets involved in BMSC proliferation and osteogenic differentiation, previous studies from our laboratory have compared the gene expression profile of BMSC, which express either endogenous or enforced expression of TWIST-1 during either normal growth conditions or osteogenic inductive conditions, using microarray analysis. Two novel differentially expressed genes were identified, HOPX and CMTM8, as being suppressed by TWIST-1. The aim of this thesis is to determine whether HOPX and CMTM8 are novel targets of TWIST-1 in BMSC and whether they are involved in mediating the effects of TWIST-1 on cell proliferation and lineage commitment. To assess the functional role of HOPX and CMTM8 in the context of BMSC biology, expression of HOPX and CMTM8 were overexpressed using retroviral transduction and supressed using siRNA. The present thesis demonstrated that HOPX counteracts TWIST-1/EZH2 regulation of BMSC cell fate determination via suppression of adipogenic genes such as C/EBPα, ADIPOQ, FABP4, PLIN1 and PLIN4, while HOPX is also a promoter of BMSC proliferation. This thesis also reported that CMTM8 is a suppressor of BMSC osteogenic differentiation and promoter of proliferation and cell migration via the EGFR signalling pathway. This thesis provides better understanding of the downstream molecular mechanisms of TWIST-1 in bone development and post-natal homeostasis and therefore, provides insight into possible future therapeutic strategies that will alter the function of TWIST-1 targets.