Regulation of the prostaglandin G/H synthase gene by parathyroid hormone in osteoblastic cells
Date of Completion
Parathyroid hormone (PTH), the major regulator of serum calcium levels, has important effects on bone metabolism. Binding of PTH to its receptor initiates protein kinase A (PKA), protein kinase C (PKC) and calcium pathways. PTH induces release of prostaglandins (PGs) by osteoblastic cells. Prostaglandin G/H synthase (PGHS), a central enzyme in the synthesis of PGs, is found in the constitutive PGHS-1 and the inducible PGHS-2 forms. The objective of this thesis was to study the mechanisms by which PTH induces PGHS-2 expression in the osteoblastic MC3T3-E1 cells.^ PTH rapidly and transiently induced PGHS-2 mRNA levels that peaked at one and returned to baseline by six h. PTH increased PGHS-2 transcription suggesting that PTH regulates the PGHS-2 mRNA levels, at least in part, through transcriptional activation. Experiments with inhibitors and mimetics showed that PKA is the main pathway mediating PTH induction of PGHS-2 expression. PTH superinduced PGHS-2 mRNA in the presence of cycloheximide, suggesting that PGHS-2 is a primary response gene. While cycloheximide did not affect PTH induction of PGHS-2 transcription at 0.5 h, cycloheximide did block the subsequent decline of PGHS-2 transcription at 2 h. Therefore, PTH induces the synthesis of a factor(s) that attenuates PGHS-2 transcription.^ The inducible cAMP early repressor (ICER) is a transcriptional inhibitor induced by the cAMP/PKA pathway and transcribed from an intronic promoter of the CREM gene. PTH rapidly induced ICER mRNA levels in MC3T3-E1 cells and calvarial organ cultures suggesting that ICER could mediate the attenuation of the PTH-induced PGHS-2 transcription.^ To identify promoter elements that mediate the PTH regulation, MC3T3-E1 cells were stably transfected with PGHS-2 promoter constructs driving a luciferase reporter. Deletion analysis revealed that elements downstream of $-$150 bp mediate baseline and PTH-mediated promoter activity. Electrophoretic mobility shift assays showed that the strongest binding to the $-$150/ + 70 bp promoter probe was competed by a $-$63/$-$42 bp fragment that contains overlapping CRE and E-box response elements. However, mutations of the CRE and/or E-box sites did not affect PTH stimulation of luciferase activity, suggesting that neither the consensus CRE at $-$57/$-$51 bp, nor the E-box at $-$52/$-$47 bp mediate the PTH and FSK effects. ^
Tetradis, Sotirios, "Regulation of the prostaglandin G/H synthase gene by parathyroid hormone in osteoblastic cells" (1996). Doctoral Dissertations. AAI9707848.