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Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat
J Fujimoto1, R E Straub, M C Gershengorn
1Department of Medicine, New York Hospital, Cornell University Medical College, New York 10021.
Abstract:
In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Thyrotropin-releasing hormone (TRH) transiently decreases thyrotropin-releasing hormone receptor (TRH-R) mRNA levels in GH3 cells. Protein kinase C activation by phorbol myristate acetate (PMA) mimics this effect, which is blocked by H-7.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Thyrotropin-releasing hormone (TRH) down-regulates its own receptors (TRH-Rs) on pituitary tumor cells.
- TRH-R mRNA levels decrease before TRH-R density reduction.
- TRH signaling involves calcium and diacylglycerol pathways, activating protein kinases.
Purpose of the Study:
- Investigate the mechanism of TRH-induced TRH-R downregulation.
- Determine the role of protein kinase C (PKC) in TRH-R mRNA regulation.
- Examine the effects of TRH, calcium, PMA, and H-7 on TRH-R mRNA levels.
Main Methods:
- GH3 cells were treated with TRH, elevated cytoplasmic Ca2+, PMA, or H-7.
- TRH-R mRNA levels were quantified using Northern analysis and nuclease protection assays.
- Probes were generated from mouse pituitary TRH-R cDNA.
Main Results:
- TRH (1 μM) caused a transient decrease in TRH-R mRNA (20-45% of control by 3-6 h).
- Elevated cytoplasmic Ca2+ had no effect on TRH-R mRNA levels.
- PMA (1 μM) mimicked TRH's effect on TRH-R mRNA, while H-7 blocked both TRH and PMA effects.
Conclusions:
- TRH-induced TRH-R downregulation involves a decrease in TRH-R mRNA.
- Protein kinase C activation, not calcium elevation, mediates TRH's effect on TRH-R mRNA.
- PKC plays a crucial role in regulating TRH-R mRNA levels in GH3 cells.