TRANSFORMING GROWTH-FACTOR-BETA-1 REGULATION OF PROSTAGLANDIN G H SYNTHASE-2 EXPRESSION IN OSTEOBLASTIC MC3T3-E1 CELLS/

Transforming growth factor-beta (TGF beta) plays an important role in bone development and remodeling TGF beta stimulates PGE(2) production, enhances interleukin-1-stimulated PGE(2) production, and can stimulat e PG-mediated bone resorption. Me found that TGF beta induced prostagl andin G/H synthase(PGHS-2) messenger RNA (mRNA) and PGE(2) production in neonatal mouse calvarial cultures and in primary cells derived from these calvariae. We used MC3T3-E1 cells, an immortalized osteoblastic cell. line derived from mouse calvariae, to examine the mechanism of PGHS-2 induction. PGHS-2 mRNA was rapidly induced by TGF beta (10 ng/m l) in MC3T3-E1 cells; mRNA levels peaked at 4-8 h and were still eleva ted at 24 h. Induction of PGHS-2 protein and PGE(2), production correl ated with PGHS-2 mRNA levels. In contrast, TGF beta had much less effe ct on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS- 2 mRNA induction by TGF beta was not enhanced by cycloheximide pretrea tment, suggesting a requirement for new protein synthesis, To study tr anscriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-fla nking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGF b eta-stimulated luciferase activity paralleled PGHS-2 mRNA induction. S timulation of luciferase activity and PGHS-2 mRNA. levels by other ago nists, including interleukin-1, TGF alpha, forskolin, and phorbol 12-m yristate 12-acetate, were enhanced by TGF beta. A 90% drop in lucifera se activity occurred with deletion of the region from -371 to -213 bp of the PGHS-2 promoter. The PG response to TGF beta in MC3T3-E1 cells appears to be mediated primarily by transcriptional regulation of PGHS -2 expression through one or more cis-acting elements located between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.