Cyclooxygenase-2 induction by parathyroid hormone in osteoblasts and its role in parathyroid hormone-induced osteoclast formation

Date of Completion

January 2009

Keywords

Biology, Molecular|Biology, Cell

Degree

Ph.D.

Abstract

Parathyroid hormone (PTH) strongly induces the expression of cyclooxygenase (COX)-2, the enzyme responsible for most prostaglandin (PG) production in osteoblasts. PTH and PGs are potent regulators of bone remodeling. The goals of this work were to study the transcriptional mechanism of COX-2 induction by PTH in osteoblasts and to investigate the role of COX-2 in PTH-induced receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) expression and osteoclast formation.^ First, we analyzed the transcriptional mechanism and signaling pathway leading to COX-2 induction by PTH in osteoblasts. Single mutation of the cAMP response element (CRE), the activating protein-1 (AP-1), or the nuclear factor of activated T-cells (NFAT) sites in COX-2 promoter sequence decreased PTH-stimulated COX-2 promoter activity 40-60% in stably transfected MC3T3-E1 or MC-4 cells, while joint mutation of NFAT and AP-1 abrogated the induction. PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos and c-Jun to an oligonucleotide spanning the composite NFAT/AP-1 site. The cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than AP-1. Both PTH and forskolin stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 60-80% by calcium chelation or calcineurin inhibitors and 60-98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium–calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.^ Next, we examined osteoclast formation and gene expression in marrow cultures from COX-2 wild type (WT) and knockout (KO) mice. Osteoclast formation was reduced 55-75% in KO cultures compared to WT cultures and in WT cultures treated with NS-398, an inhibitor of COX-2 activity, compared to vehicle-treated cultures. The PTH-induced RANKL/OPG ratio, the determining factor regulating osteoclastogenesis, was decreased 56-70% in marrow cultures and 60-90% in primary calvarial osteoblast cultures lacking COX-2 expression or activity. We conclude that PTH-stimulated COX-2 expression and PG production increase the effect of PTH on osteoclastogenesis in marrow cultures through enhancing the RANKL/OPG ratio.^

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