Sotelo-Rivera Israim, Cote-Vélez Antonieta, Uribe Rosa-María, Charli Jean-Louis, Joseph-Bravo Patricia
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico.
Endocrine. 2017 Mar;55(3):861-871. doi: 10.1007/s12020-016-1223-z. Epub 2017 Jan 6.
Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference.
Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry.
Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone.
Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.
皮质酮可防止寒冷诱导的大鼠促甲状腺激素释放激素（Trh）表达的刺激，以及二丁酰环磷酸腺苷（dB - cAMP）对下丘脑培养物中Trh转录的刺激作用。我们探寻了这种干扰的机制。
对Wistar大鼠室旁核（PVN）、17日龄大鼠下丘脑细胞培养物或神经母细胞瘤SH - SY5Y细胞进行磷酸化环磷酸腺苷反应元件结合蛋白（pCREB）的免疫组织化学分析。培养物分别与dB - cAMP、地塞米松及两种药物联合孵育1小时；其核提取物用于染色质免疫沉淀；胞质或核提取物用于蛋白激酶A（PKAc）催化亚基和糖皮质激素受体（GR）的共免疫沉淀分析；通过免疫细胞化学分析它们的亚细胞分布。
冷暴露增加了大鼠PVN中TRH神经元的pCREB水平，预先注射皮质酮可减弱该效应。地塞米松干扰了福斯高林引起的核pCREB增加及其与Trh启动子的结合；抗组蛋白脱乙酰酶 - 3抗体沉淀了用三碘甲状腺原氨酸处理而非dB - cAMP +地塞米松处理的下丘脑细胞核提取物中的染色质，排除了染色质浓缩作为相关机制。胞质或核提取物的共免疫沉淀分析显示活化的GR和PKAc之间存在蛋白质 - 蛋白质相互作用。下丘脑或SH - SY5Y细胞的免疫细胞化学分析显示，与单独使用福斯高林或地塞米松相比，用福斯高林 +地塞米松孵育的细胞中PKAc和GR的核转位减少。
糖皮质激素和cAMP通过活化的糖皮质激素受体与蛋白激酶A催化亚基的相互作用对Trh转录发挥相互抑制作用，减少它们的核转位，限制环磷酸腺苷反应元件结合蛋白的磷酸化及其与Trh启动子的结合。
