Peinado J R, Cruz-García D, Vázquez-Martínez R, Anouar Y, Tonon M C, Vaudry H, Gracia-Navarro F, Castaño J P, Malagón M M
Department of Cell Biology, Physiology and Immunology, University of Cordoba, 14014 Cordoba, Spain.
Gen Comp Endocrinol. 2006 Jun;147(2):222-30. doi: 10.1016/j.ygcen.2006.01.004. Epub 2006 Feb 14.
The frog intermediate lobe comprises two functionally distinct cell subtypes, referred to as secretory and storage melanotropes, which differ in their ultrastructure, secretory, and synthetic rates, and display dissimilar responses to hypothalamic regulatory factors. All these differences make melanotrope subtypes an excellent model to analyze the expression and regulation of genes involved in the control and maintenance of the secretory state of endocrine cells. However, quantification of the expression levels of genes involved in the secretory process requires the characterization of a gene whose expression remains constant irrespective of the secretory state of the cells. In this study, we have cloned the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from frog pituitary and have evaluated its suitability as internal standard in gene expression studies in melanotropes. A semiquantitative RT-PCR system developed to this end revealed that secretory melanotropes and storage melanotropes possess similar expression levels of GAPDH, whereas, as expected, secretory melanotropes showed higher levels of POMC transcripts than storage cells. Furthermore, we found that the expression of the convertase PC1, an intracellular protease involved in POMC processing, parallels that of POMC, thus suggesting that the higher secretory rate of the POMC-derived peptide alpha-MSH exhibited by secretory melanotropes is supported by their higher PC1 expression levels. In addition, we have shown that both POMC and PC1 mRNAs are up-regulated by the hypothalamic factor TRH in melanotrope cell cultures. In contrast, the inhibitory factor NPY reduced the expression level of the convertase but did not modify that of POMC. Taken together, these results demonstrate that PC1 expression is regulated in melanotropes by both stimulatory (TRH) and inhibitory (NPY) hypothalamic signals, in a manner which essentially parallels that observed for the precursor POMC.
青蛙垂体中间叶包含两种功能不同的细胞亚型，即分泌型和储存型促黑素细胞，它们在超微结构、分泌和合成速率方面存在差异，并且对下丘脑调节因子表现出不同的反应。所有这些差异使得促黑素细胞亚型成为分析参与内分泌细胞分泌状态控制和维持的基因表达及调控的理想模型。然而，对参与分泌过程的基因表达水平进行定量需要鉴定一个其表达不受细胞分泌状态影响而保持恒定的基因。在本研究中，我们从青蛙垂体中克隆了甘油醛-3-磷酸脱氢酶（GAPDH）基因，并评估了其作为促黑素细胞基因表达研究内参的适用性。为此开发的半定量RT-PCR系统显示，分泌型促黑素细胞和储存型促黑素细胞的GAPDH表达水平相似，而正如预期的那样，分泌型促黑素细胞的POMC转录本水平高于储存型细胞。此外，我们发现参与POMC加工的细胞内蛋白酶转化酶PC1的表达与POMC的表达平行，这表明分泌型促黑素细胞中POMC衍生肽α-MSH的较高分泌速率是由其较高的PC1表达水平所支持的。此外，我们还表明，在促黑素细胞培养物中，下丘脑因子TRH可上调POMC和PC1的mRNA水平。相反，抑制因子NPY降低了转化酶的表达水平，但未改变POMC的表达水平。综上所述，这些结果表明，在促黑素细胞中，PC1的表达受下丘脑刺激信号（TRH）和抑制信号（NPY）的调控，其方式与前体POMC基本相似。
