Endocrinology and Metabolism

Original Articles

Changes in Somatostatin Receptor mRNA Levels by G Protein Mutation in GH3 Cells Which Show Responsiveness to Growth Hormone-Releasing Hormone.: Eun Hee Kim, Sook Jin Sohn, Min A Lee, Sang Hee Seo, Sung Hee Ju, Dahm Lee, Hyun Ju Chung, Jee Chang Jung, Seung Joon Park; J Korean Endocr Soc. 2005;20(4):323-333. Published online August 1, 2005; DOI: https://doi.org/10.3803/jkes.2005.20.4.323

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S: 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.

Effect of TRH on Phospholipase D Activity in GH3 Cell.: Dong Sun Kim, Chang Beom Lee, You Hern Ahn, Tae Wha Kim, Mee Sup Yoon, Joong Soo Han; J Korean Endocr Soc. 2002;17(4):465-472. Published online August 1, 2002

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GH3 cells are a well characterized and widely used model used for the in vitro study of growth hormone (GH) secretion. Thyrotropin releasing hormone (TRH) binds to receptors belonging to the family of G protein-coupled receptors, and secrets both GH & prolactin. Phospholipase D (PLD) is an enzyme that hydrolyses phosphatidylcholine to yield phosphatidic acid and choline, and plays important roles in cellular proliferation and hormonal secretion. To elucidate the pathway of the action of TRH in GH3 cells, we investigated the activities of PLC and PLD in GH3 cells treated with TRH or phorbor 12-myristate 13-acetate (PMA). METHODS: GH3 cells were labeled with [3H] myristate, followed by incubation of with 0.3% ethanol, prior to before the addition of the agonists. The total lipids were extracted from the harvested cells following treatment with the agonists. The PLD activity was assessed by measuring [3H] phosphatidylethanol from the [3H] phospholipid using thin layer chromatography. RESULTS: TRH (1 muM) stimulated the PLC activity by 44-fold over that of the control values. TRH (1 microM), mastoparan (5 muM), and PMA (500 muM) for 30 minutes increased PLD activity by 1.9, 1.5 and 2.2 fold, respectively, in comparison to the controls. The PLD activities after 15, 30, 60, 120 and 240 min treatments of TRH (1 microM) were 142%, 170%, 172%, 160% and 115%, respectively. CONCLUSION: These results suggest that TRH stimulates not only the PLC activity, but also the PLD activity in GH3 cells.

Gene Expression of the Somatostatin Receptors, Gi2 alpha and Pit-1 alpha in GH3 Cells Permanently Transfected with a Mutant Gs alpha Gene.: Cheol young Park, In myung Yang, Eun hee Kim, Sook jin Sohn, Mee sook Ryu, Jeong taek Woo, Sung woon Kim, Jin woo Kim, Young seol Kim, Young kil Choi, Seung joon Park; J Korean Endocr Soc. 2002;17(2):170-182. Published online April 1, 2002

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Cyclic AMP stimulates the expression of the somatostatin (SRIF) receptor (sst1-5) and human growth hormone (GH)-secreting pituitary tumors with the gsp oncogene which increases intracellular cAMP levels, and shows a good inhibitory response of the GH to SRIF. Taken together, we hypothesized that the gsp oncogene may increase the SRIF receptor expression or and factors related to the postreceptor signal transduction of the SRIF, in order to enhance its responsiveness to SRIF. To test this hypothesis, we investigated if the gsp oncogene could increase the sst1, sst2, Gi2 alpha, and pit-1 alpha gene expression in GH3 cells. METHODS: GH3 cells were permanently transfected with the plasmid expressing Gs alpha gene, where the arginine of codon 201 was replaced with histidine. Intracellular cAMP levels and GH concentrations were measured by radioimmunoassays. Gene expressions of the sst1, sst2, Gi2 alpha, and pit-1 alpha were determined by RT-PCR. RESULTS: Intracellular cAMP levels and medium GH release were increased by 1.7 and 2.7-fold in GH3 cells expressing the gsp oncogene, respectively. In GH3 cells expressing the gsp oncogene, the sst1 mRNA levels were decreased, whereas those of the sst2, Gi2 alpha and pit-1 alpha mRNA were increased. A 4-h forskolin (10 M) stimulation remarkably increased the sst1 and sst2 mRNA levels in GH3 cells expressing wild and mutant Gs alpha . However, forskolin did not affect the Gi2 alpha and pit-1 alpha mRNA levels. In contrast, SRIF (1 M, 2 h) decreased the sst2 mRNA levels only in GH3 cells expressing the gsp oncogene. CONCLUSION: These results suggest that higher expressions of sst2, Gi2 alpha, and pit-1 alpha, induced by the gsp oncogene may be a mechanism by which gsp-positive pituitary tumors show a greater response to SRIF. The discrepancy between these and in vivo results should be explored further.

Thyrotropin-releasing Hormone(TRH) Receptor Gene Expression in GH3 Cells Permanently Transfected with a Mutant Gs alpha Gene.: Seung Joon Park, In Myung Yang, Sung Vin Yim, Joo Ho Chung, Jee Chang Jung, Kye Chang Ko, Young Seol Kim, Young Kil Choi; J Korean Endocr Soc. 2000;15(1):46-54. Published online January 1, 2001

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Gs alpha gene mutation, that constitutively increases intracellular cAMP, is found in some acromegalic patients. It was demonstrated that increased intracellular cAMP levels suppress the expression of rat TRH receptor (TRH-R) mRNA. We previously demonstrated that transient expression of a mutant Gs alpha gene suppress the rat TRH-R gene expression in the cultured rat growth hormone-secreting tumor cell line (GH3), whereas TRH-R gene expression in adenomas with Gs alpha gene mutation (gsp oncogene) did not differ from that in tumors without the mutation. The discrepancy suggests the possibilities that the effect of permanent expression of mutant Gs alpha gene on TRH-R gene expression is different from that of transient expression of the mutant gene and hypothalamic hormones including TRH regulate the gene expression. METHODS: We investigated whether permanent expression of the mutant-type Gs alpha does not suppress the TRH receptor gene expression in GH3 cells, and whether TRH suppresses the gene expression by using reverse transcription-polymerase chain reaction (RT-PCR) and in vitro transcription. RESULTS: Permanent expression of a mutant-type Gs alpha increased basal cAMP levels up to 1.7-fold relative to the controls, whereas the wild-type cell line did not show increased cAMP levels. Permanent expression of a mutant-type Gs alpha increased TRH receptor mRNA level up to 2.8 fold compared with the controls. Treatment of the permanently transfected GH3 cells with TRH suppressed TRH-R gene expression more prominently compared to the wild type GH3 cells. CONCLUSION: These results suggest that permanent expression of mutant Gs alpha enhances the expression of TRH-R in GH-secreting pituitary tumors with gsp oncogene, but the gene expression may also be regulated by other factors including TRH.

Effect of Gs alpha Gene Mutation on the Expression of the Rat Somatostatin Receptor Gene.: I M Yang, S J Park, H G Jhang, G J Lee, S J Oh, S W Kim, J W Kim, Y S Kim, Y K Choi; J Korean Endocr Soc. 1999;14(4):657-666. Published online January 1, 2001

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Abstract PDF: BACKGROUND
The growth hormone (GH)-secreting pituitary adenoma with mutation of Gs alpha gene (gsp oncogene) is known to show a higher response to somatostatin. The mechanism for the higher response is unclear. We previously demonstrated that transcription of the rat somatostatin receptor (SSTR) gene was increased by cAMP. Therefore, we investigated whether the mutation of Gs alpha gene can increase SSTR gene expression. METHODS: The mutant Gs alpha expressing plasmid of which arginine of codon 201was replaced with histidine was transfected transiently and permanently into GH3 cells. cAMP and rat GH were measured by radioimmunoassay. mRNA expression of the rat SSTR2 was determined by a semiquantitative RT-PCR. RESULTS: The intracellular cAMP production was increased by 1.8 folds in the transiently transfected cells and by 1.5 folds in permanently transfected cells compared to those in the cells transfected with the vehicle plasmid, plasmid expressing the wild type or the plasmid expressing the silent mutant of codon 226. The transcriptional activation by cAMP response element of somatostatin.gene was also increased by 2 folds 24 hours after transient transfection and by 3.5 folds in the permanently transfected cells. The transcriptional activation was not enhanced by forskolin in the transiently transfected cells, whereas it was more remarkably inhibited by somatostatin compared to that in the other transfected cells. The expression of SSTR2 was increased by 1.8 folds 24 hours after transfection in the transiently transfected cells and by 3 folds in the permanently transfected cells, and it was not enhanced by forskolin and isobutylmethylxanthine. The secretion of GH from the transiently transfected cells was not significantly higher than that from the wild type-expressing cells, but it was higher in the permanently transfected cells. The suppression of GH by somatostatin was more prominent in the permanently transfected cells compared to the other transfected cells. CONCLUSION: This study suggests that a higher expression of SSTR induced by the mutant Gs alpha may be a mechanism by which gsp-positive adenomas showed a higher response to somatostatin.

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