Transforming Growth Factor-β1 Regulation of Prostaglandin G/H Synthase-2 Expression in Osteoblastic MC3T3-E1 Cells

• Published in: Endocrinology (Philadelphia) 1997-11, Vol.138 (11), p.4672-4682
• Main Authors: Pilbeam, C., Rao, Y., Voznesensky, O., Kawaguchi, H., Alander, C., Raisz, L., Herschman, H.
• Format: Article
• Language: English
• Subjects: Acetic acid; Agonists; Bone growth; Bone remodeling; Bone resorption; Cycloheximide; Cytokines; Forskolin; Gene deletion; Gene regulation; Growth factors; Interleukin 1; Interleukins; Neonates; Osteoblasts; Prostaglandin E2; Protein biosynthesis; Protein synthesis; Proteins; Transcription; Transforming growth factor-a
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.138.11.5495

Abstract Transforming growth factor-β (TGFβ) plays an important role in bone development and remodeling. TGFβ stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFβ induced prostaglandin G/H synthase (PGHS-2) messenger RNA (mRNA) and PGE2 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 TGFβ (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 PGE2 production correlated with PGHS-2 mRNA levels. In contrast, TGFβ had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFβ 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. TGFβ-stimulated luciferase activity paralleled PGHS-2 mRNA induction. Stimulation of luciferase activity and PGHS-2 mRNA levels by other agonists, including interleukin-1, TGFα, forskolin, and phorbol 13-myristate 12-acetate, were enhanced by TGFβ. 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 TGFβ 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.