Identification of an enhancer involved in tissue-specific regulation of the rat fibronectin gene.

Fibronectin (FN) is a widely distributed extracellular matrix protein that is essential for cell adhesion in a variety of biological processes such as wound healing, tissue development and remodeling and oncogenic transformation. Appropriate FN levels are obtained by induction or repression of the FN gene in response to specific factors or circumstances in vivo. In order to identify regulatory regions involved in tissue-specific expression of FN, we have examined the transcriptional activity of overlapping fragments, within 4 kb upstream of the rat FN gene, following transfection into different cell types. Two regions conferred increases in transcription. The region between -1.08 and -2.6 displayed tissue-specificity and was active in fibroblasts but not hepatoma cells. The second region, between -3.2 and -3.9, was active in both cell types. Further characterization of the -1.08 to -2.6 segment demonstrated that it acts as an enhancer. Exonuclease III deletions of the 3' and 5' ends of the enhancer localized essential sequences between -1.5 and -1.7 and indicate that this fragment acts in concert with other sites between -1.08 and -2.6 to provide maximum enhancer activity. Gel mobility shift assays demonstrated fibroblast-specific binding of nuclear protein(s) to a 65 bp fragment within the essential region and DNase I footprinting localized this binding to a 27 bp sequence. Deletion of the sequence abolished the activity of the 1.5 kb enhancer. These studies show that a novel DNA sequence at -1688 is involved in regulating transcription of the FN gene in fibroblasts.

cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium.

A cDNA encoding a receptor antagonist of interleukin 1 (IL-1ra), secreted from human monocytes, has recently been isolated and sequenced [Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. H. & Thompson, R. C. (1990) Nature (London) 343, 341-346]. We have identified another version of this IL-1ra, which is predominantly expressed in epithelial cells. This IL-1ra lacks a leader sequence and, thus, is probably intracellular. Both proteins are derived from the same gene through use of an alternative transcriptional start site and internal splice-acceptor site. Expression of intracellular IL-1ra cDNA in COS cells demonstrated that the intracellular product specifically inhibited exogenous interleukin 1-dependent responses. Keratinocytes were shown to contain significant amounts of nonsecreted IL-1ra protein. Constitutive expression of the intracellular IL-1ra may be an intracellular defensive mechanism in exposed epithelial cells and/or may serve to regulate autocrine interleukin 1-mediated pathways of differentiation.

Identification of three related human GRO genes encoding cytokine functions.

The product of the human GRO gene is a cytokine with inflammatory and growth-regulatory properties; GRO is also called MGSA for melanoma growth-stimulatory activity. We have identified two additional genes, GRO beta and GRO gamma, that share 90% and 86% identity at the deduced amino acid level with the original GRO alpha isolate. One amino acid substitution of proline in GRO alpha by leucine in GRO beta and GRO gamma leads to a large predicted change in protein conformation. Significant differences also exist in the 3' untranslated region, including different numbers of ATTTA repeats associated with mRNA instability. A 122-base-pair region in the 3' region is conserved among the three GRO genes, and a part of it is also conserved in the Chinese hamster genome, suggesting a role in regulation. DNA hybridization with oligonucleotide probes and partial sequence analysis of the genomic clones confirm that the three forms are derived from related but different genes. Only one chromosomal locus has been identified, at 4q21, by using a GRO alpha cDNA clone that hybridized to all three genes. Expression studies reveal tissue-specific regulation as well as regulation by specific inducing agents, including interleukin 1, tumor necrosis factor, phorbol 12-myristate 13-acetate, and lipopolysaccharide.

Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair.

Adherence of monocytes to endothelial cells or extracellular matrices is likely to play a critical role in triggering monocyte activation in extravascular sites of infection, chronic inflammatory disorders, tissue damage and neoplastic growth. We have constructed a cDNA library from monocytes adhered for 30 min on plastic and have screened it by differential hybridization for mRNA rapidly induced by adherence. Two of the cDNA isolated have been identified as IL-1 beta and superoxide dismutase. Sequence data from three other adherence specific clones demonstrates the presence of ATTTA mRNA instability sequences in their 3' untranslated regions signifying inflammation-associated genes. The deduced amino acid sequences indicate the presence of open reading frames with sequence homologies to a family of host defense cytokines, one of them being identified as IL-8. Of the 14 clones initially identified, 4 have been analyzed for induction of mRNA expression. Although 3 of the 4 clones were equally induced by PMA and LPS under nonadherent conditions, all 4 cDNA showed distinct patterns of induction with adherence to extracellular matrix components or endothelial cells. The deduced amino acid sequence homologies indicate that we have isolated cDNA that code for three unique gene products. These cDNA belong to a gene family of early host defense cytokines involved in inflammation and cell growth, but which are differentially regulated by adherence to different surfaces.