There is a common polymorphism in the promoter sequence of the human stromelysin-1 gene, with one allele having a run of six adenosines (6A) and the other five adenosines (5A). We have previously reported, in a 3-year follow-up study of patients with coronary atherosclerosis, that those patients who are homozygous for the 6A allele show a more rapid progression of the disease. In this study, we have investigated whether the 5A/6A promoter polymorphism plays a role in the regulation of stromelysin-1 gene expression. In transient transfection experiments, a stromelysin-1 promoter construct with 6A at the polymorphic site was found to express less of the chloramphenicol acetyltransferase reporter gene than a construct containing 5A. Electrophoretic mobility shift assay and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding of one of the nucleoprotein factors was more readily detectable with an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells. Thus, the common 5A/6A polymorphism of the human stromelysin-1 promoter appears to play an important role in regulating stromelysin-1 gene expression and may be involved in the progression of coronary heart disease.Stromelysin-1 is a key member of the matrix metalloproteinase (MMP) 1 family, with a broad substrate specificity. It can degrade types II, IV, and IX collagen, proteoglycans, laminin, fibronectin, gelatins, and elastin (1-3). In addition, stromelysin-1 can also activate other MMPs such as collagenase, matrilysin, and gelatinase B, rendering stromelysin-1 crucial in connective tissue remodeling (4 -6). Expression of stromelysin-1 is primarily regulated at the level of transcription, where the promoter of the gene responds to various stimuli, including growth factors, cytokines, tumor promoters, and oncogene products (7-10). The regulatory effects of such stimuli are mediated through a number of cis-elements located in the stromelysin-1 promoter. For instance, the activator protein-1 binding site at positions Ϫ63 to Ϫ70 is necessary for the basal expression of the gene and is also involved in interleukin-1 induction (11-13). A promoter element located between Ϫ1218 and Ϫ1202 is responsible for the induction of stromelysin-1 expression by platelet-derived growth factor B/B (14, 15), whereas three sequences that share strong homology with the glucocorticoid-responsive consensus element are likely to be involved in the dexamethasone suppression (16).Over the last few years, MMPs have been implicated in the connective tissue remodeling during atherogenesis (17)(18)(19)(20)(21)(22). By in situ mRNA hybridization, we originally demonstrated the presence of stromelysin-1 in coronary atherosclerotic plaques (18). E...

In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complex in vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a. a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1-1097 and 1678-2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

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The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.

Of the five classes of intermediate filaments found in vertebrate tissues, the cytokeratins are considered unique to epithelial tissues, while vimentin is expressed by endothelial and mesenchymal cells. In neither case is the precise function of the filament system known. Epithelial cells in culture often express vimentin as well as cytokeratins, but co-expression in vivo, as reported for pleomorphic adenomas of the parotid gland and metastatic carcinoma cells in ascites or pleural fluid, is still controversial. Here we report the co-expression of cytokeratins and vimentin in situ, in the parietal endoderm of the mouse embryo 8.5-13.5 days old. This population of individual, motile cells seems to be derived from a conventional epithelium by migration and differentiation. Our results support the idea that vimentin expression is specifically related to reduced cell-to-cell contact, and to the independent existence of a cell following detachment from an epithelial sheet.