Regulation of two rat serine‐protease inhibitor gene promoters by somatotropin and glucocorticoids

Abstract

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.

The role of STAT proteins in growth hormone signaling

“…The expression of liver-speci®c spi 2.1 is tightly controlled by GH at the transcriptional level (LeCam et al, 1987;Paquereau et al, 1992). This GH-regulated gene was originally identi®ed through the screening of rat hepatic cDNA libraries (LeCam et al, 1987;Yoon et al, 1987).…”

Transcriptional Repression, a Novel Function for 3' Untranslated Regions

“…count for the lack of basal expression and the loss of GH responsiveness of this gene in vivo [5]. We have now investigated whether the 3' UTR extension of the spi 2.3 gene could carry such functions.…”

Enhanced proteolytic activity directed against the N-terminal of IGF-I in diabetic rats

“…Of interest in this regard is the observation that Spi 2·1 inhibits this protease activity (Maake et al 1997). This serine protease inhibitor is expressed predominantly in the liver and is growth hormone dependent (Paquereau et al 1992). As discussed above, reduced growth hormone levels are observed in diabetic rats (Tannenbaum 1981).…”

“…Furthermore, it is far from clear whether Spi 2·1 is the major inhibitor of the protease in serum or indeed physiologically important in regulating the proteolytic activity that generates des(1-3)IGF-I. The available polyvalent antisera against Spi 2·1 recognizes multiple proteins in serum which presumably represent other members of the serpin family (Paquereau et al 1992). Thus, it is possible that changes in other inhibitors rather than changes in the enzyme concentration may account for the increased protease activity observed in diabetic rats.…”

“…A rat Spi 2·1 cDNA obtained from Dr LeCam, Montpellier, France (Paquereau et al 1992) was radiolabeled by nick-translation using 32 P-dCTP (New England Nuclear, Lachire, Quebec, Canada). For quantitation of Spi 2·1 mRNA, serial dilutions of RNA (5-20 µg) from individual rat liver samples were immobilized on nitrocellulose paper using a filtration vacuum manifold (Schleicher and Schuell, Keene, NH, USA) and hybridized under stringent conditions.…”