| Connective tissue growth factor (CTGF/CCN2) regulates osteoblast differentiation and acts as an effector of transforming growth factor beta1 (TGF-beta1) in specific cell types. We examined the role of CTGF as a mediator of TGF-beta1 induced extracellular matrix (ECM) production and cell growth in osteoblasts and characterized the molecular mechanisms of CTGF induction through TGF-beta1 signaling. We show that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts. TGF-beta1 treatment of osteoblasts increased collagen and fibronectin expression. When CTGF specific siRNA was used to prevent CTGF induction by TGF-beta1, it also inhibited collagen and fibronectin production, demonstrating the requirement of CTGF for their up-regulation. When osteoblast cultures were treated with TGF-beta1, cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression consistent with TGFbeta-1 induced cell cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1 induced growth arrest is independent of CTGF. To investigate the involvement of the TGF-beta1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction we cloned the rat CTGF proximal promoter containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using promoter deletion and site-directed mutagenesis approach we demonstrated the requirement of both the TRE and SBE for CTGF induction by TGF-beta1 in osteoblasts. Western blot analysis revealed that treatment of osteoblast cultures with TGF-beta1 activated ERK, JNK and p38. MAPK inhibitors PD98059, SB203580 and SP600125 inhibited activation of ERK, p38 and JNK respectively and impaired the TGF-beta1 stimulation of CTGF promoter activity. The expression of a dominant-negative (DN) mutant of ERK also showed significant inhibition (∼8 fold) of CTGF promoter activity. In contrast, the expression of DN-p38 or DN-JNK failed to inhibit activation of CTGF promoter activity. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1 induced ECM production, and that these two growth factors function independently regarding their opposing effects on osteoblast proliferation. Further, CTGF induction by TGF-beta1 requires both the TRE and SBE as well as the ERK signaling cascade in primary osteoblasts. |
