Work leading to this application indicates that cells of the stromal/osteoblastic lineage collectively display all the known alpha and Beta subunits of the receptors for the interleukin-6 family of cytokines that includes IL-6, IL-II, LIF, CNTF, and OSM; different representatives of the lineage, however, express different receptor repertoire. Furthermore, acting via their specific receptors IL-6 type cytokines increase alkaline phosphatase and inhibit cell growth, suggesting their ability to induce differentiation. In addition, the level of expression of both the alpha and Beta subunits of these receptors is modulated in vitro by systemic hormones, such as sex steroids, parathyroid hormone and Vitamin D3. Based on these lines of evidence, the following hypotheses will be tested: IL-6 type cytokines act on cells of the osteoblastic lineage to promote their differentiation. Different members of the group act at specific stages of the differentiation process and such stage- dependent responsiveness to a particular cytokine is achieved by expression or repression of the respective receptor. Systemic hormones and other locally produced growth factors influence the prodifferentiating effects of these cytokines by modulating the level of cytokine receptor expression. To test these interrelated hypotheses, the specific aims of this application are to determine the effects of IL-6, IL-II, LIF, CNTF, and OSM on the expression of collagen type I, alkaline phosphatase and osteocalcin, osteoblast phenotype-related genes representative of proliferation, maturation and mineralization, respectively. In these experiments established osteoblast-like cell lines from animals and humans as well as primary cultures of murine calvaria cells and Northern blot analysis will be employed. Further, the effects of sex steroids, PTH, 1,25(OH)2D3, glucocorticoids, and retinoids on the expression of the ligand-binding subunit of IL-II and CNTF, and LIFRBeta and OSMRBeta, in the above cell models will be determined: and whether these changes alter cellular responsiveness to these cytokines. Finally, the pattern of distribution of cytokine receptors will be determined in cells of the osteoblastic lineage in vivo; from immature osteoblast precursors of the marrow, to the earliest morphologically recognizable elliptical preosteoblasts (type I cells) to cuboidal mature osteoblasts (type II cells) to older flattened and more elongated lining cells (types III and IV cells) and osteocytes. These studies will be performed in: a) bone marrow cell aspirates and b) sections of bone tissue from mice, and in situ RT-PCR in combination with histostaining and dynamic bone histomorphometry.
