NOVEL TGF-BETA INDUCIBLE GENE IN HUMAN OSTEOBLASTS

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DESCRIPTION: (Adapted from investigator's abstract) Transforming Growth Factor-beta (TGF-beta) is thought to play a major role in human bone cell physiology. It is highly concentrated in human bone, produced by both normal bone forming osteoblasts (hOB) and bone resorbing osteoclasts (OC), and has major effects on activities of these cells. Its production and activation in hOB cells are regulated by steroids, parathyroid hormone, TGF-beta itself, and other agents. It is possible that TGF-beta plays a role in the coupling between hOB and OC, and in aging and osteoporosis. The applicant laboratories have identified a new TGF-beta inducible early gene (TIEG) using differential display polymerase chain reaction. When this differentially expressed cDNA was used as a probe in hOB cDNA library screening, a 2.9 kb cDNA of this gene was isolated. Northern analyses using this cDNA has shown that TIEG mRNA is 3.5 kb long. Its levels are increased within 30 minutes of TGF-beta or BMP-2 treatment, and reaches a maximum of 10-fold above control values at 2 hours in human cells from both fetal sources (hF0Bs) and adults (hOBs). A dozen other growth factors and cytokines do not regulate TIEG expression. This 2.9 kb cDNA has been sequenced and the computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino acid protein. The C-terminal end of TIEG protein shows significant homology to a zinc finger-containing transcription factor family of genes, whereas the N-terminal region seems to be unique. Recently, the applicants have: 1) prepared polyclonal antibodies to the TIEG protein; 2) shown that the TIEG is expressed in both hOB and MCF-7 breast cancer cells; as well as in select cell types in a variety of other tissues. The latter shows hOB cells (but not other bone cells) and epithelial cells of the breast (but not stromal cells) contain/express TIEG using immunohistochemistry. 3) shown that its regulation is highly growth factor/cytokine specific, (restricted to TGF- beta family and EGF); 4) identified the TIEG protein by Western blotting; 5) determined that the TIEG protein has src homology-3 binding domains and appears to be tyrosine phosphorylated; and 6) expressed the TIEG protein in bacteria. They propose to utilize newly developed human fetal osteoblast cell lines, immortalized with a temperature-sensitive SV-40 T antigen, to further determine: 1) the tissue/cell type specificity of the TIEG expression and the TGF-beta regulation; 2) the growth factor/cytokine specificity of its regulation; 3) the regulation of the TIEG protein using PAb and MAb via Western blotting, intracellular localization, half-life analyses, etc.; 4) whether the protein is phosphorylated and whether the src-tyrosine kinase pathway is involved; 5) the function of TIEG protein via its action on the promoter activities of TGF-beta regulated genes, as well as its action on the zinc finger and SP-1 consensus sequences, and the effects of TIEG antisense oligonucleotides and overexpression in stable cell transfectants on hFOB cell proliferation and bone forming functions; and finally 6) the TIEG protein DNA binding element in gene promoters and determine the effect of TIEG protein DNA binding element in gene promoters and determine the effect of TIEG protein phosphorylation on this binding. Because TIEG is an early induced putative transcription factor gene, its protein products might play an important role as a signaling molecule in osteoblastic cells.