Only two out of the three serine-protease inhibitor genes (SPI 2.1, 2.2 and 2.3) expressed in rat liver are tightly controlled by somatotropin acting mainly at the transcriptional level, thus making this gene system particularly suitable to study its molecular mechanism of action. In these studies, we analyzed SPI promoter activities in cultured hepatocytes transfected by electroporation or in cellfree extracts. The proximal SP12.1 promotcr region contains two somatotropin-responsive sites which are functional in intact cells. The more distal clement that maps at positions -175 to ~ 114, and is analogous to the one originally described by Yoon et al. (1990) [Yoon, J. B., Berry, S. A., Seelig, S. & Towle, H. C. (1990) J. Bid. Chem. 265,, behaves as a weak enhancer whose activity is strongly potentiated by proximal 5' downstream sequences that contains potential CCAAT-enhancer binding protein (C/EBP) sites. An additional proximal hormone-sensitive site is located in the close vicinity of the transcription-start site between positions -41 and + 8, and also requires the first C / EBP-binding eleincnt to be active. The distal element appears to contribute more importantly (60%) than the proximal one (40%) to the overall somatotropin stimulation of chimeric gene expression. Nonetheless, both displayed similar dose-dependence, with half-maximal and maximal effects occurring at 0.5-1 nM and 5 -10 nM, respectively. The somatotropin refractoriness of the SPl 2.3 gene appears to be due to the presence of distal (-2300 to -200) inhibitory element(s) in the promoter. Glucocorticoids exert both positive and negative effects on SPI promoter activity. Their stimulatory action appears to involve sequences located between positions -114 and -82, together with a more distal half glucocorticoid-responsive clement, whereas their inhibitory effect is more likely mediated by sequences locatcd between positions -41 and + 8.In vitro transcription assays, performed with promoter-deletion mutants and competitor oligonucleotides, revealed the presence of a major functional C/EBP site located immediately upstream from the transcription-start point. Unfortunately, the regulatory features of SPI gene expression observed in intact cells were completely obliterated by breaking down the cell structure, and could not therefore be studied using cell-free systems.
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