Molecular characterization of three rat liver serine‐protease inhibitors affected by inflammation and hypophysectomy

Abstract

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.

Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes

“…It has been shown that inflammation causes a decrease in the Spi 2 expression. 26 AdCTGF and AdTGF␤1 up-regulate Spi 2 RNA in mouse liver compared with the adenoviral control AdE1. Spi 2 is constitutively expressed in liver tissue of normal rats and significantly decreased during inflammation.…”

“…Spi 2 is constitutively expressed in liver tissue of normal rats and significantly decreased during inflammation. 26 The up-regulation of protease inhibitors is a common feature of overexpression of TGF␤1 and CTGF and may reflect the role that these genes play in fibrosis. 6,18 It has been shown that synthetic serine protease inhibitors, in combination with a metalloprotease 1 inhibitor, prevent the invasion and metastasis of tumor cells 27 by inhibiting the degradation of the extra-cellular matrix.…”

Transcriptional Repression, a Novel Function for 3' Untranslated Regions

“…The procedure for run-on analysis, which includes incubation of the nuclei with [a-"PIUTP, purification of total cellular RNA and hybridization to templates immobilized on a nylon membrane (Hybond NC, Amersham) has been previously described [16]. The following plasmids were used as hybridization templates: pUC19 bearing 0.52 kb of the spi 2.1 cDNA 5' region [2]; pUC19 bearing the 0.35-kb 3' UTR specific extension of the spi 2.3 gene [2]; pBR322 harboring 0.65 kb of the rat a,-macroglobulin cDNA [17]; pBR322 bearing 1.6 kb of the mouse p-actin cDNA [18]; pBR322 containing 1.2 kb of the rat albumin cDNA [19]; Bluescript SK+ bearing 0.7 kb of the rat a,-acid glycoprotein cDNA [20]; pUC 19 bearing 1.25 kb of the rat insulin receptor tyrosine kinase inhibitor cDNA @p63) [21]. The amount of labeled transcripts newly synthetized by control and inflamed rat liver nuclei used for hybridization to templates corresponded to 20 X 1 O6 trichloroacetic-acid-precipitable counts.…”

“…Nuclei isolated from the liver [15] of normal or turpentine inflamed rats [2] were used to evaluate the in vivo rates of transcription initiation. The procedure for run-on analysis, which includes incubation of the nuclei with [a-"PIUTP, purification of total cellular RNA and hybridization to templates immobilized on a nylon membrane (Hybond NC, Amersham) has been previously described [16].…”

“…Site-specific mutations were introduced using asymmetric PCR [13]. The 348-bp extension specific of the spi 2.3 3' UTR region located at nucleotides 1712-2060 of the cDNA [2] or the three restriction fragments of 64, 125 and 159 bp generated by its cleavage with Pstl, or two elements of the coding spi 2.3 region (A, nucleotides 390-824; B, nucleotides 824-1020) were subcloned at various positions into CAT vectors. A pEMBL construct bearing the aldolase B promoter (nucleotides -190 to f14) [14] was kindly provided by M. Raymondjean (ICGM, Paris, France).…”