Recent advances have now implicated both osteoblasts and osteoclasts as target cells for steroid hormones including estrogen (E2) It is the hypothesis of this laboratory that estrogen (E2) plays a major role in the coupling of bone resorbing osteoclasts and bone forming osteoblasts and is involved in osteoporosis. Our laboratories discovered estrogen receptors (as well as progesterone and androgen receptors) in normal human osteoblast-like (hOB) cells and steroid receptors in avian osteoclasts. However, we have identified no E2 effects on cell proliferation or matrix protein production in normal human osteoblast cells, but have identified a significant E2 induction of TGF-beta in these cultured (hOB) cells. TGF-beta appears to play a key role in E2 action on bone. Since the TGF-beta gene was reported to be regulated by Fos/Jun, we then investigated and have preliminary results which demonstrate that E2 rapidly and markedly regulates the steady state mRNA levels of the c-fos and c-jun nuclear proto-oncogenes in the hOB cells. The goal of this application is to further characterize the rapid E2 regulation of these protooncogenes in the hOB cells by identifying the transcriptional regulatory elements for E2. We will continue studies on the regulation of c-fos and c-jun mRNA steady state levels in hOB cells in response to E2, l7alphaE2 (inactive) or 4-hydroxytamoxifen (anti-E2). We have succeeded in (and will continue) transfection of the c-fos/CAT and cjun/CAT constructs into hOB cells to demonstrate the E2 regulation of the transfected genes. Using deletion mutants of the c-fos and c-jun 5' domains, it is hoped that all sequences required for E2 response will be identified. Verification of these putative E2 functional elements will be achieved using oligonucleotides containing these elements in tk/CAT constructs. We will assess both normal hOB cells, other bone cells, and reproductive cells for differences in the regulation of these elements. Final studies will focus on the analyses of nuclear protein factors from hOB cells, other bone cells, and reproductive cells which bind specifically to these elements. The characterization of the E2 regulation of these regulatory proto-oncogenes in cells of the osteoblast lineage should help elucidate the role of E2 in regulating these genes in normal and pathological states.
