Title

Inducible cAMP early repressor expression and function in parathyroid hormone-treated osteoblasts

Date of Completion

January 2000

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Parathyroid hormone (PTH) induces expression of several early response genes in osteoblasts, including prostaglandin G/H synthase-2 (PGHS-2). Early response gene expression is rapid, transient and does not require new protein synthesis. While pretreatment of osteoblastic MC3T3-E1 cells with the protein synthesis inhibitor cycloheximide does not prevent PTH-induced PGHS-2 expression, it does inhibit attenuation of PGHS-2 transcription. This suggests that PTH simultaneously induces expression of both PGHS-2 and a transcriptional inhibitor. One likely candidate is inducible CAMP early repressor (ICER), a member of the CAMP response element modulator family of basic leucine zipper transcription factors. We hypothesized that PTH induces ICER in osteoblasts and that ICER represses PTH-mediated gene expression. Our specific aim was to determine if ICER is an inducible, cAMP-dependent, early response gene that acts as a transcriptional repressor in PTH-treated osteoblasts. ^ PTH induced ICER mRNA and protein in osteoblastic MC3T3-E1 cells and cultured neonatal mouse calvariae in both a time- and dose-dependent manner. PTH also induced ICER mRNA expression in ROS 17/2.8, UMR-106, and Py1a osteoblastic cell lines. In all cells tested, ICER induction was rapid, transient, and did not require new protein synthesis, thus classifying ICER as an early response gene in PTH-treated cells. PTH also induced ICER mRNA in neonatal mouse calvariae in vivo. Unexpectedly, ICER mRNA was detectable in control-treated animals. Handling stress did not appear to affect this response. ^ PTH's cellular effects are transduced by the PTH/PTHrP receptor, which is coupled to the cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. To determine which pathways mediate PTH-induced ICER expression, MC3T3-E1 cells were treated with selective signaling agonists and antagonists. ICER expression was significantly induced only after cAMP-PKA activation. ^ Dose-dependent overexpression of ICER produced biphasic effects on a PGHS-1(−368/+6)-luciferase reporter vector. Low ICER overexpression enhanced, while high ICER overexpression repressed, basal and induced promoter activity. Mutation of the AP-1 site mimicked ICER's effect on the wild type. ^ Based on these studies, we conclude that ICER is a PTH-inducible, cAMP-dependent early response gene that represses PTH-mediated gene transcription in osteoblasts. However, low concentrations of ICER may also enhance PGHS-1 promoter activity, suggesting that ICER is not strictly a transcriptional repressor. ^