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 constituitive 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. PTHincreased 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 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.