Endocrinology and Metabolism

Original Articles

Identification of TPA - Response Element (TRE) in the Rat Thyrotropin - Releasing Hormone (TRH) Gene.: Woon Won Jung, Young Kil Choi, In Myung Yang, Kwang Sik Seo, Jeong Taek Woo, Sung Woon Kim, Jin Woo Kim, Young Seol Kim, Young Kil Choi, Seung Joon Park; J Korean Endocr Soc. 1994;10(3):200-213. Published online November 6, 2019

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Abstract PDF: There are two potential imperfect copies of the TRE consensus sequence between -47 and -113bp position on 5' upstream of the rat TRH gene. The upstream element(5'-TGcCgTCA-3') is located between -101 bp and -94 bp, and the downstream element(5'-TGAcCTCA-3') is positioned between -59bp and -52bp relative to the stranscription start site. The downstream variant differs from the consensus sequence of TRE(AP-1)(5'-TGACTCA-3'), by addition of one nucleotide. As there is no direct evidence that TPA stimulates the transcription of rat TRH gene, and there is no study to define TRE of the rat TRH gene, we performed Northern blot assay, transient gene expression study and gel shift assay to identify TRE. TRH mRNA expression of CA77 cells was increased about 2-2.5 fold 30 min after TPA stimulation. When PC12 cells were stimulated by TPA after transfection of the plasmids containing serially deleted 5'upstream of the rat TRH gene ligated to luciferase gene, the transcription of luciferase gene was increased more than 3.2 fold with the plasmid pTRH(-600/84)Luc and pTRH(-113/84)Luc. However, the transcriptional activation was remarkably decreased less than 1.6 fold with pTRH(-77/84)Luc, pTRH(-47/84)Luc, and pTRH(6/84)Luc. The plasmid containing the sequence of -108/-79 did not show any significant activation in both of basal and TPA-stimulated transcription, whereas the plasmid containing the sequence of -70/-41 showed a slight but significant transcriptional activation by TPA. The plasmid containing the sequence of -114/-47 showed remarkable increase in basal transcription and TPA induced transcription of luciferase gene. Gel shift assay revealed that the oligonucleotides spanning -108/-79 and -70/-41 bound to c-Jun, whereas the oligonucleotides spanning -40/1, 1/30, 31/60, 61/84 did not bind. The oligonucleotide of -70/-41 bound to c-Jun with higher affinity compared to that of -108/-79. The one base pair mutant of -70/-41(deletion of C from the middle of TGACCTCA) bound to c-Jun with higher affinity, whereas the one base pair replaced mutant(C to G) bound with lower affinity compared to the wild type oligonucleotide. These results suggest that the rat TRH gene expression is stimulated by TPA to a smaller degree compared to that of other genes, and the two elements act cooperatively as TRE. The downstream TRE variant is mainly responsible for TPA response and c-Jun binding, and the upstream variant play a permissive role for transcriptional activation. The addition of one nucleotide C in the downstream element may be responsible for the relatively lower response of the rat TRH gene to TPA.

Characterization of Cyclic AMP Response Element (CRE) in the Promoter of the Rat Thyrotropin Releasing Hormone (TRH) Gene.: Woon Won Jung, In Myung Yang, Kwang Sik Seo, Seok Won Jung; J Korean Endocr Soc. 1994;9(3):190-199. Published online November 6, 2019

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Abstract PDF: We investigated whether the two variant elements of CRE(TGcCGTCA[5'CRE], TGACcTCA[3'CRE]) in the 5'flanking region of the rat TRH gene, which are different from the CRE consensus sequence(5'-TGACGTCA-3') by one base pair, are responsive to cAMP, and whether the one base pair difference is responsible for the degree of cAMP responsiveness of the gene. When CA 77 cells were stimulated with forskolin and isobutylmethylxanthine for 4 hours, the level of TRH mRNA was increased by only two fold. The transient gene expression study using serial 5'deletion of the TRH gene in PC12 cells showed that the region between-113 and-77, which includes 5'CRE, was crucial for the cAMP resonsiveness. When the plasmid, which contains the 30 bp oligonucleotide including either 5'CRE or 3'CRE ligated to the enhancerless RSV promoter, was transfected into PC12 cells, it did not significantly affect not only the basal transcription but cAMP responsiveness. The 65 bp oligonucleotide including both 5'CRE and 3'CRE, however, increased both of the basal transcription and cAMP-stimulated transcription by 2-3 fold. When the sequence of 5'CRE was converted to that of the CRE consensus by replacing one base pair, the cAMP responsiveness was increased by two fold although the basal transcription was not increased. The one base pair mutant of 3'CRE increased both of the basal and cAMP-stimulated transcription by 3-4 fold. These results suggest that there are the two variant CREs in rat TRH gene, which are relatively weak CRE compared to the CREs of other neuropeptide genes and cooperative for the activation of both the basal and cAMP-stimulated transcription. The one base pair difference of the variant CREs from the CRE consensus sequence is responsible for the weak responsiveness to cAMP.

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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Abstract PDF: BACKGROUND
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.

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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Abstract PDF: BACKGROUND
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.

Analysis of Glucocorticoid Response Element and TPA Response Element of Rat Thyrotropin-Releasing Hormine Gene by Site-Directed Mutagenesis.: Sung Woon Kim, Jin Woo Kim, Young Seol Kim, Young Kil Choi, In Myoung Yang, Jung Taek Woo, Woon Won Chung; J Korean Endocr Soc. 1999;14(2):278-292. Published online January 1, 2001

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Abstract PDF: BACKGROUND
We previously demonstrated that a GRE/TRE composite sequence, which is located between 200 bp and 220 bp relative to the transcriptional start site of rat TRH gene, is responsible for the dexamethasone (DEX)- and TPA-induced transcriptional activation, and the transcriptional activation by DEX is mediated by interaction between glucocorticoid receptor (GR) and a TRE-binding transcriptional factor such as c-Jun. However, a non-specific binding with the transciption factors can not be excluded as the mutants used in the previous report could not inhibit the binding of GR and c-Jun completely, and it remains unclear which one of the two TRE-like sequences is critical for the interaction of the two transcription factors. METHODS: Luciferase expressing plasmids that contain a part of rat TRH promoter including the composite GRE sequence or its mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX or/and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX and TPA increased the transcriptional activity of the wild type composite sequence by 3 folds and 4 folds, respectively, and the combined stimulation increased the activity by 10 folds. The mutants of which all 6 nucleotides of the GRE half site were replaced and removed almost did not bind to GR and eould not enhance the transcriptional activity at all in response to DEX. The GRE-deleted mutant bound to c-Jun with a remarkably lower affinity and showed a lower response to TPA, whereas the GRE-replaced mutant bound to c-Jun with a similar affinity and showed a similar response to TPA compared to those of the wild type. In response to the combined simulation with DEX and TPA, the mutants showed 30-40% of the trancriptional activity of the wild type. Basal transcriptional activity of all the TRE mutants was significantly lower than that of the wild type. While they almost could not bind to c-Jun, their binding affinity to GR was comparable to that of the wild type. Whereas the DEX- and TPA-induced transcriptional activity of 5 TRE mutant was 10% and 15% of that of the wild type, it responded to those agents in a similar pattern as the wild type. The 3 TRE mutant and the mutant of both TRE sites did not respond to DEX and TPA. The GRE-deleted mutant hardly formed the DNA-protein complex as did the wild type, while the GRE -replaced mutant could form the complex in a less amount with nuclear extract of HeLa celL CONCLUSION: These results suggest that GRE/TRE composite sequence of rat TRH gene specifically binds to GR and c-Jun, providing a site for interaction between the two transcription factors, and that both TRE sites play an important role in basal transcription, and that the 3 TRE site is more critical in the interaction between GRE and TRE for DEX-induced transcriptional activation. (J Kor Endocrinol 14:278-292, 1999)

The Characterization of Glucocoritcoid Response Element(GRE) on the Promoter of Thyrotropin-Releasing Hormone(TRH) Gene.: Sung Woon Kim, Jin Woo Kim, Young Seol Kim, Young Kil Choi, In Myoung Yang, Jung Taek Woo, Woon Won Chung; J Korean Endocr Soc. 1999;14(2):265-277. Published online January 1, 2001

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Abstract PDF: BACKGROUND
We previously demonstrated that the promoter of rat TRH gene has GRE half site (TGTTCT) between -210 bp and -205 bp flanking with similar sequences of TPA response element (TRE), TAGTCA, at a distance of several base pairs from the GRE half site. It promps us to hypothesize that this composite GRE/TRE sequence can provide a site for interaction between glucocorticoid receptor (GR) and c-Jun. Thus, we investigated whether the composite sequence mediates transcriptional regulation induced by dexamethasone (DEX) and 12-O-tetradecanoyl phobol-13-acetate (TPA), and whether it binds GR and c-Jun. METHODS: A luciferase expressing plasmids that contain a part of rat TRH promoter including the composite sequence or their mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX increased the transcriptional activity of the plasmid containing the wild type GRE by 2.5 folds, and TPA increased the transcriptional activity by 4 folds. The simultaneous stimulation with DEX and TPA synergistically increased the transcriptional activity by 10 folds. Two mutants whose GRE half sits were altered showed no responses to DEX, and suppressed the TPA-induced or both agents-induced transcriptional activity by 50%. Two mutants whose TRE-like sites were altered suppressed the DEX-induced transcriptional activity by 20%, TPA-induced trarptional activity by 25%, and both agents-induced transcriptional activity by 50%. Gel retardation assay showed that the composite sequence fonned a complex with GR and its mutants bound to GR with remarkably less affinity. c-Jun also bound to the composite sequence to form two cornplexes with less affinity compared to the AP-1 consensus sequence. The mutants of the TRE-like sequence bound to c-Jun with a significantly lower affinity compared to that of the wild type. Simulateous binding of the composite sequence with GR and c-Jun did not form any larger complex. The complex of GR and the composite sequence was much smaller than that formed by c-Jun, suggesting that GR binds to the composite sequence as a monomer. CONCLUSION: These results suggest that the composite sequence of GRE half site and TRE-like site on the promoter of rat TRH gene provides binding sites for GR and c-Jun, which mediate the interaction between two signal transduction pathways. (J Kor Soc Endocrinol 14:265-277, 1999)

Effect of Ga2 gene mutation on the Expression of Thyrotropin-Releasing Hormone ( TRH ) Receptor Gene in GH3 Cells.: Seung Joon Park, In Myung Yang, Jeong Hwa Ryu, Joo Ho Chung, Jee Chang Jung, Kye Chang Ko, Young Seol Kim, Young Kil Choi; J Korean Endocr Soc. 1997;12(3):357-363. Published online January 1, 2001

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Expression of TRH Receptor Gene in GH-Secreting Piruitary Adenomas.: In Myung Yang, Seung Joon Park, Jeong Wha Ryu, Joo Ho Chung, Mee Sook Ryu, Jeong Taek Woo, Sung Woon Kim, Jin Woo Kim, Young Seol Kim, Young Kil Choi; J Korean Endocr Soc. 1997;12(3):349-356. Published online January 1, 2001

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Abstract PDF: Background
To test the hypothesis that Galphas gene mutation may suppress the expression of TRH-R gene, we investigated whether hTRH-R gene expression is lower in human GH-secreting pituitary adenomas with Galphas mutation than in tumors without the mutation. Method: TRH-induced paradoxical response of GH was observed in 8 acromegalic patients. The mutation of gene was identified by direct sequencing of the genomic DNA prepared from GH-secreting pituitary adenomas. The expression of hTRHT mRNA was quantitated by RT-PCR. Results: The transcript of hTRH-R gene was detected in 6 of 8(75%) tumors. Three of these(50%) showed the paradoxical GH response to TRH and the other three patients did not show the response. The relative expression of hTRH-R mRNA in the tumors from patients with the paradoxical response of GH to TRH did not differ from that in the tumors from patients without the paradoxical response. Direct PCR sequencing of Galphas disclosed a mutant allele and a normal allele only at codon 201 in 4 of 8 tumors. The paradoxical response to TRH was observed in 2 of 4 patients without the mutation, and 2 of 4 patients with the mutation. The hTRH-R gene expression of pituitary adenomas did not differ between the tumors without the mutation and those with mutation. Conclusion: This study suggests that the expression of TRH-R gene is not likely to be a main determinant for the paradoxical response of GH to TRH, and that Galphas mutation does not seem to suppress the gene expression of TRH-R in GH secreting adenoma.

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