Rat hepatocytes have the potential to secrete three similar acidic glycoproteins, serine protease inhibitors 1, 2 and 3 (SPI-1, SPI-2, SPI-3), recognized by the same antibodies. They were synthesized as precursors of comparable sizes (45 kDa), which were post-translationally modified by N-glycosylation at three (SPI-3) or four (SPI-1 and SPI-2) sites. This appeared to account for the size difference of mature proteins. The mRNA sequences, derived from cDNA clones, displayed a high degree of similarity (70-90%), except the sequence of the antiproteasereactive centers which were completely divergent. SPI-1 and SPI-2 mRNAs were of similar sizes (1.8 kb), and were smaller than that of SPI-3 (2.2 kb); the difference corresponded to a longer, 3'-end untranslated sequence. Production of SPI-1 and SPI-2, which was constitutive in the normal animal, could be abolished by hypophysectomy and was strongly decreased during acute inflammation. In contrast, production of SPI-3, which was barely detectable in normal rats, was transiently induced during inflammation.The family of serine proteinase inhibitors, referred to as serpins (for a review, see [l]), appears to display, from a functional point of view, a great deal of diversity. While many members of this continuously growing family (e. g. cc,-antitrypsin, ccl-antichymotrypsin, antithrombin 111, serine proteinase inhibitors, a,-antiplasmin, plasminogen activator inhibitor 1 and horse leukocyte elastase inhibitor) have the wellestablished property of inhibiting serine proteases by forming irreversible complexes in a 1 : 1 molar ratio with proteinases [2], several others are not proteinase inhibitors. This subclass includes precursors of biologically active peptides such as angiotensinogen [3,4], carrier proteins like thyroxine-binding protein [5] and corticosteroid-binding globulin 161, and proteins like ovalbumin [7] and the barley protein Z [S], which do not possess any well-defined function. Another interesting feature of the serpins is the fact that a variety of factors have been shown to control the expression of their genes. Thus, genes encoding ovalbumin and related proteins are regulated by steroid hormones [9], those encoding antiproteases that belong to the group of acute-phase proteins [lo, 111 are under the control of glucocorticoids and inflammatory cytokines (for a review, see [12]) and the gene encoding the plasminogen activator inhibitor type 1 is regulated by various effectors including hormones and growth factors (for a review, see 1131). Serine proteinase inhibitors (SPI), secreted by rat liver, represent a remarkable example of the aforementioned feature. Rat hepatocytes have been shown to secrete proteins Correspondence to A.
A 92-kDa polypeptide present in rabbit and dog cardiac muscle was purified to homogeneity and some of its properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar, if not identical to macrophage gelsolin; it cross-reacts immunologically with anti-rabbit macrophage gelsolin antibody, has a Ca2+-sensitive shortening effect on the actin filaments as judged by the high shear viscometry and sedimentation experiments, and has a similar amino acid composition. In addition, immunoblot and SDS polyacrylamide gel analysis of cardiac muscle extracts obtained at high and low ionic strength showed that this protein is tightly bound to myofibrils, both in the absence and presence of Ca2+, in ventricular as well as in atrial muscle cells. Indirect immunofluorescence microscopy revealed a striated gelsolin staining pattern analogous to that previously observed for the skeletal muscle gelsolin, suggesting that in the muscle cell this protein is sharing the same localisation as actin. Because of its severing and nucleating properties the gelsolin may play a major role in the organization, assembly and turnover of the thin filaments within the muscle cells.
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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