NR4A1 (Nur77) Mediates Thyrotropin-Releasing Hormone-Induced Stimulation of Transcription of the Thyrotropin β Gene: Analysis of TRH Knockout Mice

et al. (2012) NR4A1 (Nur77) Mediates Thyrotropin-Releasing Hormone-Induced Stimulation of Transcription of the Thyrotropin b Gene: Analysis of TRH Knockout Mice. PLoS ONE 7(7): e40437. doi:10.1371/journal.pone.0040437 NR4A1 (Nur77) Mediates Thyrotropin-Releasing Hormone-Induced Stimulation of Transcription of the Thyrotropin b Gene: Analysis of TRH Knockout Mice Yasuyo Nakajima. 0 Masanobu Yamada 0 Ryo Taguchi 0 Nobuyuki Shibusawa 0 Atsushi Ozawa 0 Takuya Tomaru 0 Koshi Hashimoto 0 Tsugumichi Saito 0 Takafumi Tsuchiya 0 Shuichi Okada 0 Tetsurou Satoh 0 Marian Ludgate, Cardiff University, United Kingdom 0 Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine , Maebashi , Japan Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHb gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHb gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRHdeficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHb gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHb gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHb gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRHinduced TSHb promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHb gene, and 2) TRH mediated induction of the TSHb gene, at least in part by sequential stimulation of the NR4A1-TSHb genes through a PKC and ERK1/2 pathway. - Funding: This study was supported, in part, by grant from Health and Labor Sciences Research Grants, Research on Measures for Intractable Disease Hyptahalamo-Pituitary Dysfunction Research Group (MM), and Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (MY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Thyrotropin-releasing hormone (TRH) was originally isolated as the first hypothalamic hormone [1,2] and a major stimulator of the secretion of thyrotropin (TSH) from the anterior pituitary gland [3]. Subsequently, TRH was also found to promote production of TSH in part by stimulating transcription of the TSHb and a genes. TRH binds to its receptor in the anterior pituitary and activates phospholipase C, leading to calcium mobilization and protein kinase C activation [48] and also stimulation of the MAPK pathway [9,10]. The actions of these intracellular signaling pathways ultimately lead to an increase in transcription of the TSHb and a genes [11,12]. However, precisely how TRH mediates transcription of the TSHb gene in vivo still remains unclear. A pituitary-specific transcription factor, Pit1, was first postulated as a candidate protein that influences TRH-induced stimulation of the TSHb gene. Pit1 which contains two transactivation domains termed the POU-specific domain and POU homeo domain is expressed in somatotrophs, lactotrophs and thyrotrophs, and is critical for the development of pituitary thyrotrophs [13]. In fact, a patient with a mutation of the Pit1 gene exhibited TSH, PRL- and GH- deficiency [14,15]. Pit1 has also been reported to be important for regulation of the TSHb gene by TRH [13]. TRHdependent phosphorylation of Pit1 has been suggested to increase Pit1-binding to low-affinity TSHb promoter-binding sites, and overexpression of a mutant Pit1 containing the DNA-binding domain but lacking the major transactivation domain substantially blocked the TRH-induction of the TSHb promoter activity in GH3 cells [16]. Therefore, TRH may exert its function by changing the state of the Pit1 protein. The second candidate for a protein involved with TRH-induced stimulation of the TSHb gene is GATA2 [17]. GATA2 belongs to a subtype of transcription factors, the GATA family, that binds through its Zn finger domain with the GATA-responsive element (GATA-RE), which has high homology among all GATA family members [18]. GATA2 is expressed in thyrotrophs and gonadotrophs in the pituitary [19]. It has been reported that TRH enhanced GATA2- dependent activation of the TSHb promoter and that this stimulation was abolished by an amino-acid substitution of the GATA2-Zn finger domain or a mutation of the GATA-responsive element of the TSHb gene. In addition, an recent EMSA study by Oba et al revealed that TRH increased the DNA-binding capacity of GATA2 on the gene [20]. We generated TRH-deficient mice using homologous recombination in embryonic stem cells [21]. These mice show characteristic phenotypes, including tertiary hypothyroidism and mild hyperglycemia. The basal serum TSH level was unexpectedly elevated, and the result of the TRH test suggested that the secreted TSH had reduced biological activity. An ontogeny based analysis of these mice demonstrated that there was no requirement for TRH in the development of embryonic thyrotrophs in the pituitary, but TRH was required for the maintenance of the normal function of pituitary thyrotrophs [22]. NR4A1 (also known as Nur77, NGFI-B or TR3) belongs to a superfamily of orphan nuclear receptors and was originally isolated as an immediate-early response gene induced by a nerve growth factor in a pheochromocytoma cell line, PC12 [23]. NR4A1 is also regulated by many physiological stimuli including growth factors, inflammatory signals and hormones, and implicated in a wide range of important biological processes including apoptosis, brain development, glucose metabolism, and vascular remodeling [2427]. Expression of NR4A1 has also been identified in several endocrine organs including the anterior pituitary, ovary, (...truncated)