Southwestern histochemical localization of regulatory element binding proteins for rat anterior pituitary hormones.

对于一个基因的激活或抑制，在基因的调控元件（RE）和调控元件的结合蛋白之间形成稳定的分子复合物通常是一个先决条件（REBP）。Pit-1/GHF-1与生长激素（GH） （GH-1）的RE形成复合物，激活大鼠GH基因，并与催乳素（PRL） （P1）的RE结合，激活PRL基因。同时，生长激素基因启动子近端抑制因子结合蛋白（PREB）与生长激素基因启动子近端抑制因子（PRE）结合抑制生长激素基因。在体外GH3细胞和大鼠垂体前叶切片中，采用新引入的西南组织化学方法（SWH）定位Pit-1/GHF-1和PREB。免疫组化（IHC）检测Pit-1/GHF-1蛋白、GH和PRL。部分组织切片采用SWH和IHC双染色。REBP的定位使用了大鼠GH-1（-89和-70）和大鼠PRE（-169和-152）对应的寡脱氧核苷酸（oligo- dna）。SWH结果显示，Pit-1/GHF-1主要存在于GH细胞和PRL细胞的细胞核中，以及除促性腺细胞外的其他细胞的细胞核中，而PREB的核定位主要存在于PRL细胞和除GH细胞和促性腺细胞外的其他细胞中。SWH表达Pit-1/GHF-1阳性的细胞核在IHC中也表达Pit-1/GHF-1蛋白。在体外培养的GH3细胞中，结果与垂体前叶切片相似。我们的研究结果支持GH基因激活需要GH-1和Pit-1/GHF-1之间复合物的形成，PRL基因激活需要P1和Pit-1/GHF-1之间复合物的形成。在PRL产生细胞中，PRE和PREB复合物的形成抑制了GH基因。在生长激素产生细胞中抑制PRL基因的机制仍有待研究。

For the activation or suppression of a gene, the formation of stable molecule complexes between regulatory elements (RE) of the gene and binding proteins for the regulatory elements is often a prerequisite (REBP). Pit-1/GHF-1 form complex with RE of growth hormone (GH) (GH-1) and activates rat GH gene and it also binds to RE of prolactin (PRL) (P1) and activates PRL gene. At the same time, the binding of GH gene promoter proximal repressor element binding protein (PREB) to GH gene promoter proximal repressor element (PRE) suppress GH gene. In GH3 cells in vitro and in sections of rat anterior pituitary, Pit-1/GHF-1 and PREB were localized by utilizing a newly introduced Southwestern histochemical method (SWH). Pit-1/GHF-1 protein, GH and PRL were localized by immunohistochemistry (IHC). Some tissue sections were used for the double staining by SWH and IHC.For the localization of REBP,oligodeoxynucleotides (oligo-DNAs) correspond to rat GH-1 (-89 and -70) and rat PRE (-169 and -152) were used. SWH revealed that Pit-1/GHF-1 was mainly found in nuclei of GH or PRL cells as well as in nuclei of some other cells except gonadotrophs, whereas nuclear localization of the PREB was mainly in PRL cells and some other cells except GH cells and gonadotrophs in the rat pituitary tissue sections. Cell nuclei positive for Pit-1/GHF-1 by SWH were also positive for Pit-1/GHF-1 proteins by IHC.In GH3 cells in vitro, the results similar to that seen in sections of anterior pituitary were found.Our findings support a notion that the formation of complex between GH-1 and Pit-1/GHF-1 is needed for the activation of GH gene and that the formation of complex between P1 and Pit-1/GHF-1 is needed for the activation of PRL gene. If appears that the formation of complex between PRE and PREB suppressed GH gene in PRL producing cells. A mechanism that suppresses PRL gene in GH producing cells remains to be investigated.