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 sub(2) production, enhances interleukin-1-stimulated PGE sub(2) production, and can stimulate PG-mediated bone resorption. We found that TGF beta induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE sub(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/ml) in MC3T3-E1 cells; mRNA levels peaked at 4-8 h and were still elevated at 24 h. Induction of PGHS-2 protein and PGE sub(2) production correlated with PGHS-2 mRNA levels. In contrast, TGF beta had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGF beta was not enhanced by cycloheximide pretreatment, suggesting a requirement for new protein synthesis. To study transcriptional regulation, cells were stably transfected with a PGHS-2 promoter-luciferase reporter construct containing 371 bp of the 5'-flanking region and 70 bp of untranslated DNA from the PGHS-2 gene. TGF beta -stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGF alpha , forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGF beta . A 90% drop in luciferase 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 areas between -371 and -213 bp in the 5'-flanking region of the PGHS-2 gene.