Cellular and molecular effects of growth hormone and estrogen on human bone cells.

The aim of the present thesis is to examine some aspects of the biological effects of growth hormone (GH) and estrogen on bone cells in vitro. The first part of the thesis describes characterization of model systems to study normal human osteoblasts and osteoclasts in vitro. Three culture systems for human osteoblasts have been characterized, representing different stages of osteoblasts differentiation/maturation: (1) mature osteoblasts cultured from trabecular bone explants (trabecular osteoblasts), (2) less mature osteoblasts (stromal osteoblasts) cultured from bone marrow and (3) osteoblast precursor cells cultured also from bone marrow. This classification is based on quantitative and qualitative differences in the expression of osteoblast phenotypic markers by these cells. These systems are useful in studying the regulation of hormones and growth factors of different stages of osteoblast differentiation. Further characterization of the osteoblast differentiation pathway is still needed, especially the identification of surface markers that can definitively identify intermediate stages of osteoblast differentiation. Normal human osteoclasts were cultured from bone marrow mononuclear cells and exhibited the main characteristics of osteoclast phenotype: production of tartrate-resistant acid phosphatase, presence of a ruffled border and the ability to resorb mineralized matrix. This model is useful for studying factors regulating osteoclast commitment and differentiation. The second part of the thesis deals with the effects of GH on proliferation and differentiation of trabecular and stromal osteoblasts. Effects of GH on human osteoblasts were dependent on their degree of maturation. GH stimulated cell proliferation in both trabecular and stromal osteoblasts. While it increased the functional activity of trabecular osteoblasts, these effects were absent in stromal osteoblast cultures. Human trabecular osteoblasts produce mainly IGF-II, IGFBP-3 and minute quantities of IGF-I in culture. GH does not seem to regulate the local production of IGF-II or IGFBP-3. However, IGFs and their binding proteins may exert important regulatory effects on the biological effects of GH on human osteoblasts, and this role needs to be studied. Sincer GH exerted profound effects on the biological functions of human osteoblasts in vitro, the hypothesis that either decreased production of GH or decreased sensitivity of bone cells to its action leads to bone loss and osteoporosis was examined. No differences in the basal or GH-stimulated production of IGF-I, IGF-II or IGFBP-3 were found between osteoporotic patients and age-matched normals. Similarly, The response of bone cells to in vivo and in vitro stimulation by GH was similar in the two groups. These studies do not support the hypothesis of the presence of major defects in production of GH or the presence of resistance to its effects in bone cells in patients with osteoporosis. The last part of the thesis deals with the cellular mechanisms mediating estrogen actions on bone cells. Thus, the potential role of estrogen regulation of bone-resorbing cytokines in the pathogenesis of bone loss in postmenopausal women was examined. Estrogen was found to inhibit IL-6 production and gene expression in an immortalized human osteoblastic cell line expressing high levels of estrogen receptors (hFOB/ER9 cell line). To further examine the relationship between estrogen deficiency and early postmenopausal bone loss, levels of IL-6, IL-6sR, IL-1 alpha, IL-1 beta and IL-1ra were measured in bone marrow plasma and bone marrow cultures obtained from 40 postmenopausal women, half of whom were on estrogen replacement therapy. No difference was found between the groups in any of these parameters. This suggests that neither IL-1 nor IL-6 by itself is the major mediator for increased bone loss due to estrogen deficiency in the early postmenopausal period. (ABSTRACT TRUNCATED)