| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 101 === Mesenchymal stem cells (MSC) can differentiate into a variety of tissues including bone, cartilage, fat, muscle, nerves. Osteogenic differentiation of MSCs can be divided into various stages depending on the cells (osteoprogenitors, preosteoblasts, osteoblasts and then become mature osteocytes) they become. Mouse pre-osteoblastic cell line, MC3T3-E1, is an unipotency stem cell that has been used frequently for study bone differentiation, because when cultured with the osteogenic differentiation-inducing factors, MC3T3-E1 cells can differentiate into osteoblasts with matrix mineralization.
c-Cbl is an E3 ubiquitin ligase that ubiquitinates a variety of growth factor receptor or non-receptor tyrosine kinases, resulting in their degradation to block signal transduction. c-Cbl plays regulatory roles in the signalings of FGFR2, Src, PI3K and αvβ１ integrin which either promote or block osteoblastic differentiation. In order to understand the role of c-Cbl in osteogenic differentiation of preosteoblasts, I used lentiviruses expressing the shRNAs targeting c-Cbl or green fluorescent protein (GFP) to infect MC3T3-E1 cells and established the stable clones shGFP, shCbl-1 and shCbl-3. After confirming significant decreases of c-Cbl expression in shCbl-1 and shCbl-3 cells, they together with shGFP cells were cultured in an osteogenic differentiation medium (OS) containing ascorbic cid and KH2PO4 for 4 and 7 days, respectively. I found that c-Cbl mRNA and protein levels induced by OS in shCbl-1 and shCbl-3 stable clones were much lower than those of the shGFP cells. Moreover, I also found that alkaline phosphatase (ALP) activity induced by OS in two c-Cbl-knockdowned clones were drastically decreased. In addition, the mRNA levels of ALP and Type Ⅰcollagen, two early osteogenic differentiation marker genes, osteonectin and bone sialoprotein, two middle differentiation marker genes, osteonectin and bone sialoprotein, two late differentiation marker genes induced by OS in shCbl-1 and shCbl-3 cells were drastically reduced. In agreement, OS-induced mineralization of these c-Cbl-knockdowned clones was greatly diminished. To understand the effects of c-Cbl knockdown on reduced osteogenic differentiation of MC3T3-E1 cells, I next examined Runx2, a master transcription factor for osteogenic differentiation of MSC, in the aforementioned stable clones. Interestingly, both basal of OS-induced levels and Runx2 mRNA and protein levels were drastically decreased in shCbl-1 and shCbl-3 cells. However, no significant difference in the stability of Runx2 protein was found among three stable clones. In the meantime, protein levels of Snail, a known repressor of Runx2 expression, were found to be elevated in two c-Cbl-knockdowned clones, which was due to an increase of it stability. The presence of c-Cbl and Snail in a similar complex was demonstracted by the result of co-immunoprecipitation, implying that Snail might be a substrate for c-Cbl. Based on the assumption, I also established Snail-knockdowned clones respectively from shGFP and shCbl-1 cells, and showed good enhancement of ALP activity and mineralization induced by OS in those clones. Taken together, my results suggest that c-Cbl may ubiquitinate Snail in MC3T3-E1 cells. When c-Cbl expression is downregulated in these cells, Snail protein levels will be drastically elevated, due to an increase of its stability. Consequently, Runx2 expression will be repressed more severely, leading to a decreased osteogenic differentiation of these preosteoblasts.
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