title: The thyrotropin-releasing hormone gene 1998: cloning, characterization, and transcriptional regulation in the central nervous system, heart, and testis.
abstract: Mechanisms of triiodothyronine (T3) negative regulation of the human thyrotropin-releasing hormone (TRH) gene were investigated with a chimeric construct of the 5' flanking region fused to a luciferase reporter gene, transfected into human neuroblastoma cells (HTB-11). Maximum negative regulation was achieved with constructs containing bases -242 to +54. Four sequences in this region exhibited homology with half sites of thyroid hormone response elements (TRE) (AGGTCA). The most important site was a sequence with an overlapping TRE/CRE, involving bases -53 to -60 (TGACCTCA). Potential combinatorial interactions of thyroid hormone receptors and CREB at this site were explored. Modest promoter stimulation was achieved with dibutyryl cyclic adenosine monophosphate (cAMP) (10(-3) M) plus IBMX (0.5 mM). Stimulation was greatly enhanced (+820%) by cotransfection of a constitutively activated protein kinase A (pPKA) construct. Cotransfection with pCREB increased stimulation further to 1350% above control. Stimulation of pPKA and pCREB interfered with stimulation by unliganded TRbeta1, and co-transfected pPKA and pCREB blocked T3 negative inhibition by TRbeta1-T3 complexes. When this site was mutated by polymerase chain reaction (PCR) mutagenesis, the mutant construct failed to respond to unliganded TRbeta1, and stimulation by pPKA and/or pCREB was inhibited markedly, from 12.5- to 2.1-fold, p < 0.001. Moreover, TRbeta1-T3 complexes failed to show any inhibition of the mutated promoter. These results suggest that negative regulation is achieved by inhibition of CREB stimulation of the TRH promoter at this overlapping TRE/CRE site. The two cosuppressors, NCoR and SMRT, were able to augment stimulation of the TRH promoter by unliganded TRbeta1 and enhance the magnitude of T3 inhibition. The potential role of the TRH gene and the pathophysiology of thyroid hormone resistance was investigated with three mutant TRbeta1 constructs. Thyroid hormone resistance was found to be expressed at the level of TRH gene regulation, due to lowered inhibition by mutant TRbeta1-T3 complexes and by their dominant negative effects on wild-type TRbeta1-T3 inhibition. TRH gene expression has been identified in the heart. Cardiac TRH mRNA was not regulated by T3, in contrast to HTB-11 cells, but cardiac TRH mRNA density could be augmented by glucocorticoids and by testosterone. TRH receptors were identified using Scatchard blots that showed a kilodalton of 1.4 nM and a bmax of 10 pmol/mg protein. TRH-R mRNA was identified also by reverse transcription polymerase chain reaction (RT-PCR). Enhanced ventricular contractility by TRH was demonstrated in both an open-chested dog preparation and in ex vivo ventricular myocytes, using video edge cinematography. Under controlled conditions, myocyte shortening was 13.3%, and TRH (10(-6) M) caused muscle shortening to increase 140%, (p < 0.005). TRH gene expression was demonstrated exclusively in Leydig cells of the testis. High affinity binding sites were identified in testicular membranes with a kilodalton of 1.6 x 10(-6) M. TRH was able to inhibit LH and HCG-activated testosterone secretion significantly. Thus, one paracrine role of TRH in the testis may be to serve as inhibitory modulator of gonadotropin-stimulated testosterone secretion.
authors.affiliation: Division of Endocrinology, University of Maryland Medical System, Baltimore 21201-1595.
authors.forename: J F
authors.initials: JF
authors.lastname: Wilber
authors.forename: A H
authors.initials: AH
authors.lastname: Xu
mesh_terms: D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism; D013737:Testis / Q000378:metabolism*; D018382:Thyroid Hormone Resistance Syndrome / Q000235:genetics; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D013973:Thyrotropin-Releasing Hormone