Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium.

The human endometrium is a dynamic tissue, which undergoes extensive tissue remodelling during the menstrual cycle. Due to their involvement in such processes, several well-characterized matrix metalloproteinases (MMP) have previously been studied in the endometrium. MMP-26 is a newly described matrilysin. We studied MMP-26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Tissue distribution and cycle variation was examined using in-situ hybridization, Northern blot analyis and real time PCR. The probes for Northern blot analysis and real time PCR recognized non-overlapping sequences. MMP-26 was localized exclusively in epithelial cells of both glands and the luminal surface. Expression increased during the proliferative phase to a maximum at mid-cycle, then decreased to non-detectable levels in the late secretory and menstrual phases. Expression of MMP-26 mRNA in endometrial tissue explants in vitro required stimulation with both estradiol and progesterone. The tissue content of c-jun mRNA was assayed, since c-jun, as part of the enhancer complex AP-1, may be involved in regulation of MMP-26 gene transcription. The pattern of c-jun expression over the menstrual cycle was similar to that of MMP-26. Epithelial expression in the peri- and post-ovulatory stages of the menstrual cycle suggests the involvement of MMP-26 in reproductive processes.

Allele-specific regulation of matrix metalloproteinase-7 promoter activity is associated with coronary artery luminal dimensions among hypercholesterolemic patients.

An enhanced expression of matrix metalloproteinase (MMP)-7 has previously been demonstrated in atherosclerotic and aneurysmal tissue. Because perturbed regulation of MMP-7 may influence the development of these diseases, we searched the MMP-7 promoter for functional polymorphisms. An A to G substitution at position -181 (-181 A/G) and a C to T substitution at position -153 (-153 C/T) with frequencies of 0.50 and 0.10, respectively, were identified. Allele-specific associations were studied in 350 patients undergoing percutaneous transluminal coronary angioplasty. Hypercholesterolemic patients carrying the -181G allele or the -153T allele had smaller reference luminal diameters before percutaneous transluminal coronary angioplasty. Reverse transcription-polymerase chain reaction demonstrated that expression of MMP-7 was confined to differentiated U937 cells. Northern blot analysis could not detect an effect of native or oxidatively modified low density lipoprotein on MMP-7 expression. Thus, the limitation of allele-specific effects on vessel wall remodeling to hypercholesterolemic patients may be secondary to lipid-mediated accumulation of MMP-7-expressing monocyte-derived macrophages within the vessel wall. Both polymorphisms influenced the binding of nuclear proteins. Furthermore, in transient transfection studies, the combination of the 2 rare alleles conferred an increased promoter activity. In conclusion, the present study identified and characterized 2 common polymorphisms in the promoter region of the MMP-7 gene that are functional in vitro and seem to influence coronary arterial dimensions in hypercholesterolemic patients with manifest coronary artery disease.

Polymorphisms of the human matrix gla protein (MGP) gene, vascular calcification, and myocardial infarction.

The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5'-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A-7 and Ala 83 alleles more frequent in patients with MI than in controls (P<0.003). In the AXA Study among subjects with femoral atherosclerosis, the same alleles were more common in the presence than the absence of plaque calcification (P<0.025). The other MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions -814, -138, and -7 relative to the start of transcription. When compared with the -138 T allele, the minor -138 C: allele consistently conferred a reduced promoter activity of -20% (P<0.0001) in rat vascular smooth muscle cells and of -50% (P<0.004) in a human fibroblast cell line, whereas the other polymorphisms, including -7, displayed no evidence of in vitro functionality. We conclude that the A-7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Genetic diversity in the matrix metalloproteinase family. Effects on function and disease progression.

Atherosclerosis is an example of a complex trait, where the course of the disease is influenced by a combination of common variation in a constellation of genes and the effect of a wide range of environmental variables. Thus, the underlying disease mechanisms will be modulated by genetic diversity and the effect this diversity has on an individual's response to environmental challenges such as smoking, diet, and exercise. Unlike the consequences of mutations in severe single-gene disorders on protein function, the impact of individual common, functionally important sequence changes in genes contributing to multifactorial diseases is likely to be very small. The challenge is to dissect the contribution that each of these genes makes to the disease process. We have tackled this by identifying common genetic variants, studying their effects on function, and applying them to the analysis of association in appropriately structured and suitably powered studies. Even with our incomplete understanding of the disease, the list of potential candidate genes we could study is vast; but, we do know from pathological studies that a wide spectrum of structural architecture exists in atherosclerotic plaques, suggesting that remodeling of vascular connective tissue is fundamentally important. Matrix remodeling is controlled by a complex network of cell and matrix interactions, the net outcome of which is the product of a balance between synthetic and degradative processes. Our work has focused on the family of enzymes and inhibitors most directly associated with matrix turnover--the matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs, tissue inhibitors of MPs). We specifically searched for functionally relevant genetic variants that might modulate the delicate control of matrix turnover. Using these molecular genetic strategies to investigate the impact of natural genetic variation on vascular matrix remodeling has begun to shed new light on the importance of these genes in atherogenesis.

Human stromelysin gene promoter activity is modulated by transcription factor ZBP-89.

Matrix metalloproteinase expression is under strict regulation in physiological conditions. Disruption of the regulatory mechanisms can lead to tissue destruction and is associated with tumour invasion and metastasis. Using the one-hybrid assay technique with a cis-element in the promoter region of the stromelysin (matrix metalloproteinase-3) gene, a cDNA encoding a transcription factor termed ZBP-89 was obtained. The interaction between ZBP-89 and the stromelysin promoter element was confirmed by electrophoretic mobility shift assays with a recombinant ZBP-89. Reporter gene expression under the control of the stromelysin promoter in transiently transfected cells was significantly increased when the cells were cotransfected with a ZBP-89 expression construct. These results indicate that ZBP-89 interacts with the stromelysin promoter and upregulates its activity. As ZBP-89 expression is known to be increased in gastric carcinoma cells, induction of stromelysin expression may be a significant factor in tumour metastasis.

A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2).

whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

The Streptomyces coelicolor sporulation-specific sigma WhiG form of RNA polymerase transcribes a gene encoding a ProX-like protein that is dispensable for sporulation.

In the non-motile mycelial organism Streptomyces coelicolor A3(2), the sporulation gene whiG encodes a protein that closely resembles RNA polymerase sigma factors such as sigma D of Bacillus subtilis, which mainly control motility and chemotaxis genes. Here, we show that the whiG gene product, purified from an Escherichia coli strain carrying an expression construct, could activate E. coli core RNA polymerase in vitro to transcribe a sigma D-dependent motility-related promoter from B. subtilis. Such RNA polymerase holoenzyme preparations could also transcribe from an S. coelicolor promoter, PTH4, previously shown to require an intact whiG gene for in-vivo transcription. The in-vivo dependence on whiG was therefore shown to be direct. Unusually, the initiation of PTH4 transcription in vitro depended on the provision of appropriate dinucleotides. The whiG-dependent PTH4 transcription unit consisted of a single gene, orfTH4. Sequence comparisons suggested that the gene product was a member of a small group of proteins that include the B. subtilis and E. coli ProX proteins. Though none of these proteins shared more than about 30% of extended primary sequence identity, they had similar size and hydropathy profiles, and could be aligned end to end to reveal a mosaic of similarities. The ProX proteins of B. subtilis and E. coli are implicated in glycine betaine transport in response to hyperosmotic stress. However, disruption of orfTH4 did not cause any obvious phenotypic changes in growth or development on media of varying osmotic strengths.

Preelectrophoresis of agarose plugs containing bacterial chromosomal DNA prepared for analysis by pulsed field gel electrophoresis can improve the clarity of restriction patterns.

Pulsed field gel electrophoresis has indicated that chromosomal DNA isolated from stationary phase Pseudomonas fluorescens, Pseudomonas putida, and Escherichia coli cells immobilized in agarose can be fragmented during its release. For P. fluorescens it was demonstrated that the entire chromosome is affected and that there are no specifically fragile sites. The extent of the damage increased both during storage of the DNA and also when magnesium ions were provided, suggesting that nucleases may be involved. Chromosomal DNA isolated from exponentially growing cultures was subjected to significantly less damage during release and storage. Removal of damaged DNA by electrophoresis prior to digestion was shown to result in clearer restriction patterns of chromosomal DNA.

The effect of bacterial products on human fibroblast and keratinocyte detachment and viability.

An in vitro model has been developed to study the effect of soluble bacterial products on the viability and detachment of skin cell types utilized cultured grafts. Microbial products prepared from clinical isolates of bacterial species which most commonly colonize burn lesions showed marked variation in their ability to detach and kill both keratinocytes and fibroblasts. All three isolates of Acinetobacter spp. tested were effective in causing detachment and death of keratinocytes and fibroblasts, whereas Escherichia coli, Proteus mirabilis and Enterobacter spp. tested had little, or no, effect on detachment or viability for either skin cell type. Four Staphylococcus aureus isolates elicited variable strain-dependent results with regard to detachment and viability. One isolate possessed activity specific for keratinocyte detachment and death. These results indicate the possible undesirable effects such bacterial species may have on graft success in colonized burn wounds.