ROLE OF A TGF-BETA REGULATED GENE IN HUMAN OSTEOBLASTS

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Periodontitis is a chronic inflammatory disease associated with destruction of connective tissue and alveolar bone loss and is the primary cause of tooth loss in adults. The cytokine, TGF-beta, is released at inflammatory sites and plays a role in periodontal tissue and bone remodeling by regulating the synthesis of matrix proteins and matrix metalloprotease inhibitors. Not only do bone forming osteoblasts synthesize and activate latent complex TGF-beta, they also respond to TGF-beta in many functions, including increased bone matrix protein production. TGF-beta also plays an important role in osteoblast and osteoclast coupling. This laboratory discovered a TGF-beta inducible early gene (TIEG) in normal human osteoblasts (hOB) and characterized it as an immediate response gene for TGF-beta. TIEG encodes a 72kDa, 3-zinc finger, transcription factor-like, phosphoprotein. Early studies in this and other laboratories have correlated the levels of TIEG expression with TGF-beta responses, including the inhibition of cell proliferation and apoptosis, in several different cell types, including hOB cells. We have recently reported that TIEG overexpression in MG-63 cells mimics the TGF-beta effects on these cells by inhibiting cell proliferation and enhancing bone matrix protein gene expression in a TIEG dose-dependent pattern. Now we show evidence that TIEG expression enhances Smad binding element-reporter gene activity by down regulating the inhibitory Smad 7 gene expression at the level of its promoter. To elucidate the biological role of TIEG in human osteoblasts, we plan to determine: 1) the mechanism by which TIEG down-regulates Smad 7 promoter; 2) the effects of TIEG knockout mice in both embryo development and adult skeleton; and identify 3) proteins that interact with TIEG protein using the yeast-two hybrid systems; and 4) the DNA consensus binding element for TIEG by using random sequence oligonucleotide approach. We will compare this element to the sequences identified above in the Smad 7 promoter-reporter gene experiments. The outcome of these studies should provide new insights into the mechanisms of TGF-beta action and the role of TIEG in human osteoblast cells and bone disease such as periodontitis.