Recent evidence suggests that the inflammatory cytokine interleukin-J (IL-I) plays a key role in the pathogenesis of osteoporosis and in the bone loss which accompanies chronic inflammatory disease. We propose to determine the molecular mechanism by which this cytokine regulates transcription of three physiologically important target genes in osteoblastic cells, namely prostaglandin G/H synthase-2 (PGHS-2) and interleukin-6 (IL-6), which are up- regulated, and type I collagen (COL1Al), which is downregulated by IL-I. PGHS-2 is the key regulated enzyme in the conversion of arachidonic acid to biologically active prostanoids. IL-6 is a cytokine produced by osteoblasts which stimulates osteoclast differentiation and may play a role in post-menopausal osteoporosis. Type I collagen is the major structural component of bone matrix. The following specific aims are proposed: l)To examine the basal expression and regulation of NF-IL6, NF-kB and related transcription factors by IL-I in osteoblastic MC3T3-EI cells. Northern analysis will be performed in MC3T3-El cells and neonatal mouse calvariae to determine basal expression of individual members of the NF-IL6 and NF-kB multigene families, and the effects of IL-l on transcription factor mRNA levels will be determined. Protein levels will be assessed by Western blotting, Electrophoretic mobility shift analysis will be used to examine DNA- protein interactions at NF-IL6 and NP-kB sites in the PGHS-2, IL-6 and COLIAI promoters, 2) The function of NF-IL6 and NP-kB elements in the regulation of the PGHS-2, IL-6 and COLIAI genes will be analyzed by 5' deletion mapping, site directed mutagenesis of cis-acting elements, overexpression of trans-acting factors and dominant-negative regulators, and antisense approaches to inhibit the expression of specific transcription factors. 3) The mechanisms of COLIAI gene regulation by cytokines will be examined in the Co1CAT transgenic mouse model. Mapping of negative cytokine response elements in the COLIAI promoter will be performed by 5' deletion analysis, and by analysis of heterologous promoter constructs in which the osteoblast basal enhancer element is placed upstream of a basal enhancerless promoter. Elucidation of the mechanisms by which IL-I stimulates IL-6 and PGHS-2 expression while inhibiting type I collagen synthesis m osteoblasts should lead to a better understanding of the molecular basis for the bone loss which occurs in postmenopausal osteoporosis and chronic inflammatory disease states. In addition, these studies will contribute to our basic understanding of the molecular mechanisms involved in the transcriptional regulation of gene expression by cytokines via the NF-kB and NF-IL6 transcription factor families.
