REGULATION OF THE PROSTAGLANDIN G/H SYNTHASES IN BONE

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

We will study the regulation and function in bone of prostaglandin G/H synthase-2 (PGHS-2), the inducible form of the rate-limiting enzyme that converts arachidonic acid to prostaglandins (PG). PGHS-2 is an early response gene that is highly expressed in osteoblasts and is responsible for most acute PG responses in bone. We will study transcriptional regulation by the proinflammatory agents, IL-1 ,TNFalpha and LPS, potent inducers of PGHS-2, whose effects may be mediated in part via PGs. We hypothesize that stimulation of PGHS-2 promoter activity by these agents will involve an activating protein-I motif, which we have recently identified, and a nearby cAMP response element. In osteoblastic MC3T3- E1 cells transfected with PGHS-2 promoter-luciferase reporter (Pluc) constructs, we will examine effects on luciferase activity of mutating these sites, look for interactions with other cis-acting sites, identify transacting factors on electrophoretic mobility shift assay with supershifting antibodies, and co-transfect with Gal4-Jun or Gal4-CREB fusion proteins to assess ability to activate transcription. To determine if controls of PGHS-2 gene expression identified in vitro also function in vivo, we will develop mice transgenic for Pluc mutated and unmutated constructs. We will compare Pluc responses in tissues and isolated cells with Pluc responses in MC3T3-E1 cells. Despite their abundance in bone, the role of PGs in bone turnover is not clear. To address this question, we will study mice with disruption of one (P2+/-) or both (P2-/-) alleles for the PGHS-2 gene. We hypothesize that PGs produced by PGHS-2 stimulate formation of both osteoclasts and osteoblasts. Preliminary studies suggest formation may be more reduced than resorption by diminished PGHS-2 in adult mice. We will compare bone histomorphometry after double calcein labeling in vivo in aging P2+/+, +/-,and -/- mice. We will examine osteoclastogenesis in marrow cultures and in spleen-osteoblast co-cultures and examine in vivo resorption after local PTH injection above calvariae. We will study osteoblastic proliferation and differentiation in long-term cultures of primary osteoblasts and marrow stromal cells. To examine compensatory effects of constitutively expressed PGHS-1, we will do selected experiments on PGHS-1 knockout (PI-/-) mice and breed P2+/-, P1-/- mice. A better understanding of the regulation and function of PGHS-2 may lead to novel therapies for increasing bone mass.