Regulation of Expression of the Rat Serine Protease Inhibitor 2.3 Gene by Glucocorticoids and Interleukin‐6

Abstract

The rat serine protease inhibitor 2.3 gene (spi 2.3) is almost completely silent in normal animals and is transiently expressed during acute inflammation. It encodes a potential anti-elastase which is likely to play a major physiological role for the host defense. Two well-known inflammatory mediators, glucocorticoids and interleukin-6 (IL-6) activate the spi 2.3 promoter and increase steady-state levels of mRNA in cultured hepatocytes. GC activation is mediated by a single glucocorticoid-response element which seems to act autonomously. A unique array of four functional IL-6-response sites was identified in the spi 2.3 promoter. Three of them (C-IT-1V) bear structural identity to the CCAAT/enhancer-binding-protein-binding site consensus sequence, whereas the forth closely resembles the consensus KB nuclear factor recognition motif. The C-IV element, which is the most active, contains the motif 5'-CTGGGA and binds the IL-6-inducible acute-phase response factor present in liver nuclear extracts from inflamed rats. Both basal and IL-6-dependent activities of each individual cytokine-response element tcsted separately are strongly down regulated by a recently identified regulatory sequence [Le Cam, A. & Legraverend, C. (1995) Eur: J. Biochem. 231, 620-6271, located in the 3' untranslated region of the spi 2.3 gene. However, this repressor element does not significantly affect overall IL-6-dependent spi 2.3 promoter activity. This suggests that, in the context of the active gene in vivo, all four IL-6-response sites, which are largely redundant, cooperate to overcome the strong repressive effect of the 3' untranslated region silencer and are needed to bring about a maximal TL-6 response.These data reveal a novel type of regulation of an acute-phase gene involving different classes of IL-6-response elements controlled by a repressor and acting in conjunction with a glucocorticoid-response element.

Regulation of expression of the rat SOCS‐3 gene in hepatocytes by growth hormone, interleukin‐6 and glucocorticoids

“…To this end, we inserted single or multiple wild‐type or mutated copies of the G‐rich or the A/T‐rich element in front of a minimal tk promoter linked to the cat gene. As we previouly observed [22], the shortened version of the tk promoter lacking the CCAAT binding site had very little basal activity in hepatocytes and did not respond to hormones including to IL‐6 (Fig. 7a).…”

Characterization of three transcriptional repressor sites within the 3′ untranslated region of the rat serine protease inhibitor 2.3 gene

“…The most abundant complex contained and involves both positive promoter cis-acting elements [15] and mostly C/EBPA since it was largely supershifted or its formation a negative element located in the 3′ UTR [14]. In this study, we inhibited by anti-CEBPA.…”

“…Its inhibitory effect could however be overcome by the specific of the spi 2.3 3′ UTR located between nucleotides 1712 coordinate action of interleukin-6 and glucocorticoids [15]. and 2160 in the cDNA [16], placed upstream of the maximally Thus, the action of this 3′ UTR silencer most likely accounts for active spi 2.3 promoter region (nucleotides Ϫ200 to ϩ8) [15]. the very low level of expression of the spi 2.3 gene in normal Deleted versions of the three PstI fragments contained in the rats and its reversal by inflammatory cytokines explains at least specific 3′ UTR spi 2.3 region were generated using the polyin part, the gene activation observed during acute inflammation merase chain reaction method.…”

“…Its inhibitory effect could however be overcome by the specific of the spi 2.3 3′ UTR located between nucleotides 1712 coordinate action of interleukin-6 and glucocorticoids [15]. and 2160 in the cDNA [16], placed upstream of the maximally Thus, the action of this 3′ UTR silencer most likely accounts for active spi 2.3 promoter region (nucleotides Ϫ200 to ϩ8) [15].…”

“…The 348-bp spi 2.3 3′ UTR fragment derived from box: 5′-tcgaGAGAATTCAGGAG; an oligonucleotide harboring the entire cDNA [16] was subcloned into pUC18. The recombia C/EBP-binding site (bold characters) derived from the rat nant plasmid was linearized with EcoRI or HindIII, allowing hemopexin gene promoter [17], 5′-gtgTCCAGTGATGTAATend-labelling of the 3′ UTR fragment with [A-32 P]dATP and CAGGC ; a mutated version of the glucocorticoid-response eledCTP and the Klenow fragment of DNA polymerase I at either ment (GRE) of the spi 2.3 promoter [15], 5′-tcgaAGAGCACAstrand. Labelled fragments were then excised with EcoRI or GATTTCCCATG.…”