roles of TGF-beta-targeted genes in bone development.
Published: 2006
Language: eng
Number of pages: 247
ISBN-13: 9780494219089
Description: Bone matrix contains growth factors belonging to the TGF-beta super family, which regulate growth, differentiation, and matrix synthesis. To understand the consequences of TGF-beta signaling during osteogenesis, cDNA microarray technology was used to identify TGF-beta responsive target genes during osteoblast development. From the 120 differentially expressed genes in the clonal osteoblastic cell line MC3T3-E1 treated with TGF-beta1, collagen, type V, alpha1 (COL5A1) {Differential expression = + 4.9} and nephronectin {Differential expression = - 5.2} were selected for further studies since they represented previously uncharacterized components of the bone matrix. Northern blotting confirmed the microarray results, and immunostaining showed that treatment with TGF-beta1 dramatically down-regulates nephronectin expression. Furthermore, COL5A1 and nephronectin mRNA and protein expression were detected in the developing bone of the E15.5 and E17.5 mouse embryos, in which nephronectin mRNA was found to be highly expressed in proliferating osteoblasts, thereby suggesting that nephronectin could be a potential marker for distinguishing proliferating from mature osteoblasts.To determine the function of nephronectin in osteogenesis, various nephronectin constructs were generated to produce stable cell lines of MC3T3-E1, expressing and secreting nephronectin protein, including full-length (NN), nephronectin lacking EGF-like repeats (NN-MAM), nephronectin lacking RGD and MAM domains (NN-EGF), and full-length with 3'-untranslated region (3'-UTR, named NN-3'). I demonstrated for the first time that nephronectin promotes differentiation during osteoblast development. This result was confirmed by expression of an siRNA targeting nephronectin, which resulted in down-regulation of osteoblast differentiation. The motif responsible for enhanced osteoblast differentiation was identified as that comprising multiple EGF-like repeats. Expression of nephronectin lacking these repeats inhibited the morphological transition from fibroblastic to cuboidal that accompanies the onset of differentiation in osteoblasts. In comparison, over-expression of full-length nephronectin resulted in earlier formation of bone nodules in MC3T3-El cells. It was also found that the nephronectin 3'-UTR contains a binding site for microRNA-378. Inclusion of the 3'-UTR repressed nephronectin translation, resulting in the delay in differentiation exhibited by the NN-3'-transfected cells compared to the NN-transfected cells. Lastly, analysis of second messenger signaling revealed that the effects of nephronectin are mediated by ERK and that ERK activation is essential for osteoblast differentiation.
Notes: Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0092.

Thesis (Ph.D.)--University of Toronto, 2006.

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