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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR043627-02
Application #
2442840
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Project Start
1996-07-05
Project End
1999-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Hefferan, T E; Reinholz, G G; Rickard, D J et al. (2000) Overexpression of a nuclear protein, TIEG, mimics transforming growth factor-beta action in human osteoblast cells. J Biol Chem 275:20255-9
Hefferan, T E; Subramaniam, M; Khosla, S et al. (2000) Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family. J Cell Biochem 78:380-90
Spelsberg, T C; Subramaniam, M; Riggs, B L et al. (1999) The actions and interactions of sex steroids and growth factors/cytokines on the skeleton. Mol Endocrinol 13:819-28
Fautsch, M P; Vrabel, A; Subramaniam, M et al. (1998) TGFbeta-inducible early gene (TIEG) also codes for early growth response alpha (EGRalpha): evidence of multiple transcripts from alternate promoters. Genomics 51:408-16
Subramaniam, M; Hefferan, T E; Tau, K et al. (1998) Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 68:226-36
Tau, K R; Hefferan, T E; Waters, K M et al. (1998) Estrogen regulation of a transforming growth factor-beta inducible early gene that inhibits deoxyribonucleic acid synthesis in human osteoblasts. Endocrinology 139:1346-53
Fautsch, M P; Vrabel, A; Rickard, D et al. (1998) Characterization of the mouse TGFbeta-inducible early gene (TIEG): conservation of exon and transcriptional regulatory sequences with evidence of additional transcripts. Mamm Genome 9:838-42
Tachibana, I; Imoto, M; Adjei, P N et al. (1997) Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 99:2365-74