Transforming growth factor-beta1 regulation of prostaglandin G/H synthase-2 expression in osteoblastic MC3T3-E1 cells.
Transforming growth factor-beta (TGFbeta) plays an important role in bone development and remodeling. TGFbeta stimulates PGE2 production, enhances interleukin-1-stimulated PGE2 production, and can stimulate PG-mediated bone resorption. We found that TGFbeta 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 TGFbeta (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, TGFbeta had much less effect on PGHS-1 mRNA levels. Unlike the response to other agonists, PGHS-2 mRNA induction by TGFbeta 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. TGFbeta-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 TGFbeta. 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 TGFbeta 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.
Pilbeam
C
C
Department of Medicine, University of Connecticut Health Center, Farmington 06030, USA. pilbeam@nso.uchc.edu
Rao
Y
Y
Voznesensky
O
O
Kawaguchi
H
H
Alander
C
C
Raisz
L
L
Herschman
H
H
D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis; D005784:Gene Amplification; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D010006:Osteoblasts / Q000201:enzymology* / Q000502:physiology; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*
D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D003513:Cycloheximide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone