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Abstract
Backgrounds
GH3 cells lack growth hormone (GH)-releasing hormone (GHRH) receptors. In this study, GH3 cells permanently transfected with human GHRH receptor cDNA (GH3-GHRHR cells), were established in order to examine the effects of GHRH and G protein mutation (gsp oncogene) on the levels of somatostatin receptor mRNA.
Methods
GH3 cells were permanently transfected with a plasmid expressing human GHRH receptor cDNA. The GHRH receptor mRNA was detected by RT-PCR. The responsiveness to GHRH was evaluated using a GHRH binding assay, Western blot analysis, Northern blot analysis, and measurements of the intracellular cAMP levels and GH release. Cells were transiently transfected with the gsp oncogene, and then treated with GHRH or octreotide for 4h. The sst1 and sst2 mRNA levels were measured using real-time RT-PCR analyses.
Results
GHRH receptor mRNA was detected in the GH3 cells permanently transfected with human GHRH receptor cDNA. The GHRH binding assay showed that GHRH was bound to the GH3-GHRHR cells. The GHRH treatment increased the intracellular cAMP levels, GH release, GH mRNA levels, and MAPK activity, as well as the levels of sst1 and sst2 mRNA. Transient expression of the gsp oncogene for 48h increased the cAMP, GH release, and levels of sst1 and sst2 mRNA. In the gsp-transfected GH3-GHRHR cells, GHRH stimulation resulted in decreases in the magnitude of the increase in the levels of sst1 and sst2 mRNA compared to those transfected with a control vector. Octreotide treatment did not alter the levels of sst1 and sst2 mRNA in either the control or gsp-transfected cells.
Conclusion
These results suggest that GH3 cells permanently transfected with the GHRH receptor are useful in the in vitro studies on the actions of GHRH. The gsp oncogene was shown to increases the levels of sst1 and sst2 mRNA in GH3 cells, but these findings are unlikely to be the major mechanism by which gsp-positive pituitary tumors show a greater response to somatostatin. The discrepancy between the in vivo and these in vitro results should be examined further.
Figures and Tables
Fig. 1
Characteristics of GH3 cells permanently transfected with human GHRH receptor cDNA (GH3-GHRHR cells).
(A) RT-PCR analysis of GHRH receptor mRNA in wild-type GH3 and GH3-GHRHR cells. GHRH receptor mRNA was only detected in GH3-GHRHR cells. (B) Binding assay of GHRH to GHRH receptors in wild-type GH3, GH3-GHRHR, and primary cultured rat anterior pituitary cells. 125I-Labeled and increasing concentrations of unlabeled hGHRH (1-44) amide were added to cells. Results are expressed as a percentage of the maximum specific binding. (C) Intracellular cAMP levels in response to GHRH (10, 100 nM) and forskolin (10 µM). (D) GH response to GHRH (10 nM) in wild-type GH3, GH3-GHRHR, and primary pituitary cells. GH levels were measured 15, 30, and 60 min after adding GHRH. (E) Effects of GHRH (10 nM) and dexamethasone (10 µM) on GH mRNA levels in GH3-GHRHR cells. Cells were treated with GHRH and dexamethasone for 72h. GH mRNA levels were measured by Northern blot assay. (F) MAPK activation in GH3-GHRHR cells. Western blot analysis was performed using rabbit anti-phospho-p44/p42 MAPK. TRH was used as a positive control. (G) Effects of GHRH (10 nM) and forskolin (10 µM) on sst1 and sst2 mRNA levels in GH3-GHRHR cells. Cells were incubated with GHRH and forskolin for 4h. Sst1 and sst2 mRNA levels were measured by real-time RT-PCR. *, P < 0.05, **, P < 0.01.
Fig. 2
Effect of transient transfection of gsp oncogenes into GH3-GHRHR cells on intracellular cAMP levels. Cells were transfected with pSV2, pSV2-wt (wt), pSV2-αs-R201H (201), and pSV2-αs-Q227L (227) and incubated for 24h, 48h, and 72h. Intracellular cAMP levels were measured by RIA. Results are expressed as a percent of pSV2 controls (Mean±S.E.; n=4 wells from two independent experiments). *, P < 0.05, **, P < 0.01.
Fig. 3
Effect of transient transfection of gsp oncogenes into GH3-GHRHR cells on GH release. Cells were transfected with pSV2, pSV2-wt (wt), pSV2-αs-R201H (201), and pSV2-αs-Q227L (227) and incubated for 48h. GH levels were measured by RIA. Data are expressed as Mean±S.E. (n=4 wells from two independent experiments). *, P < 0.05.
Fig. 4
Effect of transient transfection of gsp oncogenes into GH3-GHRHR cells on sst1 and sst2 mRNA levels. Cells were transfected with pSV2, pSV2-wt (wt), pSV2-αs-R201H (201), and pSV2-αs-Q227L (227) and incubated for 48h. Sst1 and sst2 mRNA levels were measured by real-time RT-PCR assay. Receptor mRNA levels were adjusted by β-actin and expressed as a percent of pSV2 controls (Mean±S.E.; n=4 wells from two independent experiments). *, P < 0.05.
Fig. 5
Effects of GHRH and octreotide stimulation on sst1 and sst2 mRNA levels in GH3-GHRHR cells transfected with pSV2, pSV2-wt (wt), pSV2-αs-R201H (201), and pSV2-αs-Q227L (227). Transfected cells were incubated for 48h and stimulated with GHRH (10 nM) and octreotide (10 nM) for 4h. Sst1 and sst2 mRNA levels were measured by real-time RT-PCR assay. Receptor mRNA levels were adjusted by β-actin and expressed as a percent of pSV2 controls (Mean±S.E.; n=4 wells from two independent experiments). *, P < 0.05, **, P < 0.01.
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