Dalla Valle Luisa, Toffolo Vania, Vianello Silvia, Belvedere Paola, Colombo Lorenzo
Comparative Endocrinology Laboratory, Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
J Steroid Biochem Mol Biol. 2004 Jan;88(1):79-89. doi: 10.1016/j.jsbmb.2003.11.001.
The expression of cytochrome P450scc, encoded by the CYP11A gene, was investigated in the rat kidney from birth to adulthood. In the male and female rat kidneys, the corresponding mRNA was detected by semi-quantitative reverse transcriptase and polymerase chain reaction (RT-PCR) analysis with specific primers, resulting in higher levels of expression during the first 15 days from birth. RT-PCR and sequence analysis showed that the P450scc mRNA coding region was the same for both kidney and testis, whereas 5'-RACE analysis (rapid amplification of cDNA ends) demonstrated that the renal transcription utilizes a distal transcription start site (TSS) located 76 b upstream of that used in ovarian and testicular P450scc mRNA expression, which is placed 43 b upstream of the first ATG. The 5'-UTR sequence of renal P450scc cDNA exactly matched the contiguous upstream untranslated region of the gene, suggesting that alternative splicing was not involved in the synthesis of this transcript. Northern hybridization detected a specific transcript only in the newborn male, but not in adult rat kidney, confirming the higher levels of expression in the first days of the rat's life. Positive immunodetections of cytochrome P450scc were found in renal cortical distal tubules and the results were confirmed by Western blotting analysis. As demonstrated by semi-quantitative RT-PCR, the male kidney also expresses the messengers corresponding to the steroidogenic acute regulatory (StAR) and steroidogenic factor 1 (SF-1) proteins, which are normally required for steroidogenesis in steroidogenic tissues, such as gonads and adrenal cortex. These studies suggest that the rat kidney has the capability for local steroid hormone production, although the physiological significance of the pregnenolone eventually produced remains to be established.
研究了CYP11A基因编码的细胞色素P450scc在大鼠从出生到成年期肾脏中的表达情况。在雄性和雌性大鼠肾脏中，使用特异性引物通过半定量逆转录酶和聚合酶链反应（RT-PCR）分析检测到相应的mRNA，结果显示出生后的前15天表达水平较高。RT-PCR和序列分析表明，肾脏和睾丸中P450scc mRNA的编码区相同，而5'-RACE分析（cDNA末端快速扩增）表明，肾脏转录利用的是一个位于卵巢和睾丸P450scc mRNA表达所用转录起始位点（TSS）上游76 bp处的远端转录起始位点，该位点位于第一个ATG上游43 bp处。肾脏P450scc cDNA的5'-UTR序列与该基因相邻的上游非翻译区完全匹配，提示该转录本的合成不涉及可变剪接。Northern杂交仅在新生雄性大鼠中检测到特异性转录本，而在成年大鼠肾脏中未检测到，证实了大鼠出生后最初几天的表达水平较高。在肾皮质远端小管中发现了细胞色素P450scc的阳性免疫检测结果，并通过蛋白质免疫印迹分析得到了证实。如半定量RT-PCR所示，雄性大鼠肾脏还表达与类固醇生成急性调节蛋白（StAR）和类固醇生成因子1（SF-1）相对应的信使，这些蛋白通常是性腺和肾上腺皮质等类固醇生成组织中类固醇生成所必需的。这些研究表明，大鼠肾脏具有局部产生类固醇激素的能力，尽管最终产生的孕烯醇酮的生理意义仍有待确定。
