Prostaglandins (PGs) are local factors produced by bone cells which can mediate cellular responses to systemic hormones, cytokines, growth factors and mechanical forces. They are potent stimulators of bone resorption and can both stimulate and inhibit bone formation. Prostaglandin G/H synthase (pGHS), the major enzyme in the conversion of arachidonic acid (AA) to PGs, has two isoforms encoded by separate genes, the """"""""constitutive"""""""" PGHS (pGHS-1) and the newly identified """"""""inducible"""""""" PGHS (pGHS-2). PGHS-2 mRNA is abundantly expressed in osteoblasts, and PGHS-2 may be the major synthase mediating such physiologic processes as skeletal growth and repair and the response to mechanical loading, as well as pathologic inflammatory responses. Our preliminary data suggest that PGHS-2 is the primary isoform mediating PG responses to cytokines, growth factors and glucocorticoids in osteoblasts. Studies in osteoblastic MC3T3-E1 cells stably transfected with PGHS-2 promoter-luciferase reporter (p2-Luc) constructs indicate that this regulation of PGHS-2 expression is at least in part transcriptional. Our goal is to determine mechanisms of this regulation. Specifically, we will examine transcriptional regulation by the cytokines, interleukin-1 and tumor necrosis factor-alpha, and by transforming growth factors-alpha and -beta using MC3T3-E1 cells stably transfected with P2-Luc deletion constructs. Specific DNA binding regions mediating responses will be characterized by mobility shift analysis and their function confirmed by site-directed mutagenesis. We will assess the glucocorticoid inhibition of agonist-induced PGHS-2 expression using MC3T3-E1 cells transfected with deletion constructs and with DNA motifs, identified as capable of mediating agonist induction, linked to a heterologous promoter. mRNA stability will be assessed by steady state mRNA decay studies and by comparison of accumulation of spliced and unspliced PGHS-2 mRNA transcripts. Because the levels of PGHS-1 and PGHS-2 mRNA expression do not always correlate with PG production, we will also examine other aspects of the PG pathway, including AA release and PGHS-1 and PGHS-2 protein levels and activity. Similar protocols for determining mechanisms of regulation of PG production, as well as mechanisms of transcriptional regulation, will be used to examine the induction of PGHS- 2 expression by fluid shear stress in neonatal primary calvarial digest cells and in MC3T3-E1 cells. We will also measure collagen protein synthesis and mRNA levels in the presence and absence of nonsteroidal anti-inflammatory drugs to assess the role of PGs in mediating the effects of fluid shear stress on bone formation.
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