In vivo perimenstrual activation of progelatinase B (proMMP-9) in the human endometrium and its dependence on stromelysin 1 (MMP-3) ex vivo.

Most matrix metalloproteinases (MMPs) are secreted as inactive proenzymes. Their expression is well documented in several human tissues, but their activators in vivo are still unknown. To address this question, the activation of progelatinase B (proMMP-9) in the human endometrium was selected as a model system. ProMMP-9 was detected by gelatin zymography in homogenates of fresh endometrial tissue sampled during all phases of the menstrual cycle, whereas its active form was observed only during the late secretory and menstrual phases. Furthermore, proMMP-9 was expressed and activated in endometrial explants sampled outside the perimenstrual phase and cultured in the absence of both progesterone and oestradiol, mimicking the menstrual condition in vivo. Analysis of such tissue cultures by gelatin zymography and Western blotting showed that activation of proMMP-9 depended on a secreted factor and was selectively inhibited by either a synthetic inhibitor of stromelysin 1 (MMP-3) or a monoclonal antibody that specifically blocks MMP-3, thus providing strong evidence for the activation of proMMP-9 in vivo by MMP-3. The activation of proMMP-3 was itself inhibited by a broad-range MMP inhibitor in most cultures, but seemed to involve multiple pathways, implying both serine proteinases and metalloproteinases, which could operate in parallel or sequentially.

Involvement of fibronectin type II repeats in the efficient inhibition of gelatinases A and B by long-chain unsaturated fatty acids.

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.

Circulating sex hormones and endometrial stromelysin-1 (matrix metalloproteinase-3) at the start of bleeding episodes in levonorgestrel-implant users.

Unpredictable endometrial bleeding is the major side-effect of levonorgestrel-releasing s.c. implants (Norplant), otherwise a method of choice for long-term contraception. The mechanisms responsible for bleeding are still unknown and no reliable treatment is available. Several matrix metalloproteinases (MMP) are expressed and activated in human endometrium only at menstruation and specific synthetic inhibitors of MMP fully prevent the tissue breakdown that occurs in menstrual-like endometrial explants. To investigate whether MMP are inappropriately expressed and activated in Norplant-treated endometria during bleeding episodes, volunteers were recruited to provide blood and endometrial biopsies at the start of bleeding episodes and during non-bleeding intervals. Whereas serum concentrations of levonorgestrel and sex hormones showed no change at bleeding, except for a slight decrease of oestradiol concentration, the expression and activation of stromelysin-1 released by explants cultured for 1 day were consistently increased at the start of bleeding episodes. Furthermore, stromelysin-1 was immunolocalized in stromal cells within breakdown areas of several bleeding endometria, but not in non-bleeding endometria. These observations suggest that the expression and activation of stromelysin-1 participate in the initiation of bleeding episodes upon Norplant contraception. New strategies in the prevention and treatment of abnormal bleeding based on MMP control should be envisaged.

The matrix metalloproteinase-1 (MMP-1) expression in the human endometrium is inversely regulated by interleukin-1 alpha and sex steroids.

OBJECTIVE: To investigate the regulation of perimenstrual MMP-1 expression in human endometrium. DESIGN: In vitro study utilizing epithelial-stromal co-cultures. SETTING: Cell Biology Unit, International Institute of Cellular and Molecular Pathology, and Departments of Pathology and Gynecology, Saint-Luc University Clinics, Louvain University Medical School, Brussels, Belgium. METHODS: Contact-dependent and contact-independent co-cultures were established and resulting MMP-1 gene and protein expression was analyzed by RNase protection assays and soluble-collagen assays. RESULTS: MMP-1 expression in endometrial fibroblasts is markedly stimulated by epithelial cell-conditioned medium. This stimulation can be prevented by antibodies directed against interleukin 1 alpha (IL-1 alpha). Ovarian steroids inhibit MMP-1 production by IL-1 alpha-stimulated fibroblasts in vitro. CONCLUSION: Taken together, our results suggest that epithelium-derived IL-1 alpha is the most important paracrine induced of MMP-1 in endometrial fibroblasts. However, IL-1 alpha-stimulated MMP-1 production in the human endometrium is effectively blocked by ovarian steroids. We believe that this mechanism responsible for the MMP-1 repression that occurs when systemic sex steroid concentrations are high and the MMP-1 production and activity during the perimenstrual phase when estrogen and progesterone concentrations are low.

Temporal and spatial association of matrix metalloproteinases with focal endometrial breakdown and bleeding upon progestin-only contraception.

The pathogenesis of irregular endometrial bleeding, the main reason for stopping contraception with progestins only, is unknown. Based on the recent reappraisal of the mechanisms of menstrual bleeding, we hypothesized that matrix metalloproteinases initiate this disorder. Volunteers upon Norplant treatment provided endometrial biopsies at the start of a bleeding episode and during nonbleeding intervals. Focal stromal breakdown, collagen fiber lysis, and collagenase-1 messenger ribonucleic acid were evidenced in most bleeding endometria, but never in the nonbleeding ones. In the breaking down areas, immunolabeling for gelatinase A was strongly increased, and that of progesterone and estrogen receptors was decreased. Explants from bleeding endometria produced high collagenase and gelatinase activities, whereas release from nonbleeding endometria was negligible. Bleeding endometria released more latent and active forms of collagenase-1 and active gelatinases A and B, but less tissue inhibitor of metalloproteinases-1, than nonbleeding endometria. Collagenase-1 release closely correlated with that of interleukin-1alpha. In contrast, N:-acetyl-beta-hexosaminidase and tissue inhibitor of metalloproteinases-2 were similarly released in both groups. Thus, endometrial bleeding occurs together with focal stromal breakdown, collagen lysis, expression and activation of several matrix metalloproteinases, and decreased production of tissue inhibitor of metalloproteinases-1. These results may lead to new pharmacological treatment of this common medical problem.

Structure of recombinant mouse collagenase-3 (MMP-13).

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1' group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.

Interleukin-1 receptor-ligand interactions modulate interstitial collagenase-1 production by human endometrial fibroblasts.

The expression of interstitial collagenase-1 in the cycling human endometrium is restricted to the perimenstrual phase and is a key event for matrix degradation that initiates menstruation. In the absence of ovarian steroids, collagenase production by endometrial fibroblasts is induced by epithelial cell-derived interleukin-1alpha. Media conditioned by endometrial epithelial cells were found to contain interleukin-1alpha but not interleukin-1beta, and their capacity to induce collagenase production by endometrial fibroblasts correlated with interleukin-1alpha concentration in a saturable manner. Collagenase induction by recombinant interleukin-1alpha was severely inhibited by interleukin-1 receptor antagonist alone and abolished by its combination with soluble interleukin-1 type-II receptor. By contrast, the association of the receptor antagonist with soluble type-I receptor was less effective than each factor alone. Induction of collagenase by epithelial cell-conditioned media was severely inhibited by neutralizing interleukin-1alpha antibodies, whereas the combination of receptor antagonist with soluble type-II receptor proved less effective. We conclude that the collagenase response of endometrial fibroblasts to epithelial cell-derived interleukin-1alpha is effectively blocked in vitro by soluble members of the interleukin-1 family and can thus be modulated in vivo by these or other local factors.

Reelin, the extracellular matrix protein deficient in reeler mutant mice, is processed by a metalloproteinase.

Reelin is the extracellular protein defective in reeler mice. It is believed that reelin acts via the extracellular matrix to influence the development of nearby neurons, but the mechanism remains thus far unknown. In the present work, we present in vivo and in vitro evidence that reelin is cleaved. This processing did not occur in Relnrl-Orl mutant mice in which reelin is not secreted and was prevented in explant cultures by brefeldin treatment, suggesting that it takes place extracellularly or in a postendoplasmic reticulum compartment. Reelin cleavage was inhibited by zinc chelators known to inhibit metalloproteinases but was unaffected by inhibitors of serine, cysteine, or aspartate proteinases. Furthermore, reelin cleavage was insensitive to inhibitors of matrixins, neprilysin, meprin, and peptidyl dipeptidase A, suggesting that the processing enzyme belongs to a different enzyme family. This enzyme and the physiological meaning of reelin processing remain to be characterized further.

Local cytokines induce differential expression of matrix metalloproteinases but not their tissue inhibitors in human endometrial fibroblasts.

The endometrium is the only human tissue to undergo cyclic breakdown and regeneration. This physiological alternation renders it an advantageous system for studying tissue remodelling. Our previous observations indicate that menstrual endometrial breakdown is initiated by matrix metalloproteinases (MMPs), which are controlled overall by ovarian steroids but are also locally regulated by cytokines. We have therefore compared the effect of several endometrial cytokines on the gene expression of eight MMPs and their tissue inhibitors (TIMP)-1, -2 and -3, in primary cultures of human endometrial fibroblasts. Three categories of gene expression were identified: (a) MMP-13, -15 and -16 mRNAs were not detected despite stimulation by various cytokines; (b) MMP-2 and -14 as well as TIMP-1, -2 and -3 mRNAs were constitutively expressed but not markedly affected by the six cytokines tested; (c) mRNAs for MMP-1, -9 and -11 were selectively induced by specific cytokines: insulin-like growth factor-II, epidermal growth factor (EGF), platelet derived growth factor (PDGF)-BB and interleukin (IL)-6 stimulated MMP-11 expression; MMP-1 was induced by EGF, PDGF-BB, tumour necrosis factor (TNF)alpha and IL-1alpha, which also exerted additive effects. In contrast with MMP-1 and MMP-11 gene expression, which was sustained for 48 h, MMP-9 mRNA was quickly induced by TNFalpha, but disappeared within 12 h despite continuing stimulation. These results show that several cytokines are able to induce the selective expression of MMPs in cultured human endometrial fibroblasts and are thus good candidates for involvement in local triggering of menstrual tissue breakdown.

Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation.

The molecular mechanisms predisposing to atherosclerotic aneurysm formation remain undefined. Nevertheless, rupture of aortic aneurysms is a major cause of death in Western societies, with few available treatments and poor long-term prognosis. Indirect evidence suggests that matrix metalloproteinases (MMPs) and plasminogen activators (PAs) are involved in its pathogenesis. MMPs are secreted as inactive zymogens (pro-MMPs), requiring activation in the extracellular compartment. Plasmin, generated from the zymogen plasminogen by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA; refs 14,15), has been proposed as a possible activator in vitro, but evidence for such a role in vivo is lacking. Analysis of atherosclerotic aorta in mice with a deficiency of apoliprotein E (Apoe-/-; ref. 18), singly or combined with a deficiency of t-PA (Apoe-/-:Plat-/-) or of u-PA (Apoe-/-:Plau-/-; ref. 19), indicated that deficiency of u-PA protected against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. This genetic evidence suggests that plasmin is a pathophysiologically significant activator of pro-MMPs in vivo and may have implications for the design of therapeutic strategies to prevent aortic-wall destruction by controlling Plau gene function.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1alpha and its dual block by ovarian steroids.

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1alpha (IL-1alpha) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1alpha and repress MMP-1 production by IL-1alpha-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1alpha and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1alpha is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1alpha, thereby exerting an effective control via a "double-block" mechanism.

Expression of interstitial collagenase (matrix metalloproteinase-1) is related to the activity of human endometriotic lesions.

OBJECTIVE: To determine whether interstitial collagenase (matrix metalloproteinase-1), known to play a pivotal role in the initiation of menstruation, contributes to the pathogenesis of endometriosis. DESIGN: Serial sections of peritoneal red and black endometriotic lesions, ovarian endometriotic cysts, and rectovaginal adenomyotic nodules were analyzed by in situ hybridization for the expression of matrix metalloproteinase-1 by silver staining for the integrity of the fibrillar extracellular matrix and by immunolabeling for the abundance of sex steroid receptors. SETTING: Academic hospital and research laboratory. PATIENT(S): Premenopausal women undergoing laparoscopy for endometriosis. INTERVENTION(S): Biopsy of endometriotic lesions, combined with endometrium whenever possible. MAIN OUTCOME MEASURE(S): Expression of matrix metalloproteinase-1 messenger RNA (mRNA). RESULT(S): Matrix metalloproteinase-1 mRNA was expressed focally in red peritoneal and ovarian endometriosis irrespective of the phase of the menstrual cycle but was not detectable in black peritoneal and rectovaginal lesions. Foci of matrix metalloproteinase-1 expression closely correlated with matrix breakdown and with the absence of P receptors in adjacent epithelial cells. CONCLUSION(S): Correlation of matrix metalloproteinase-1 expression with activity of endometriotic tissue suggests its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions.

The recombinant catalytic domain of mouse collagenase-3 depolymerizes type I collagen by cleaving its aminotelopeptides.

The sequence coding for the catalytic domain of mouse collagenase-3 (MMP-13) was amplified by polymerase chain reaction and expressed in Escherichia coli. The recombinant catalytic domain (CCD), mainly recovered as inclusion bodies, was renatured and purified to homogeneity by preparative SDS-PAGE. The purified CCD degraded gelatin, casein and a synthetic peptide. CCD was not able to cleave the triple-helical domain of type I collagen but conserved the specific property of full-length collagenase-3 to cleave the N-telopeptides. These results show that residues involved in the recognition and cleavage of the aminotelopeptides of type I collagen are located in the catalytic domain of mouse collagenase-3 and that the C-terminal domain is not required for this activity.

Different collagenase gene products have different roles in degradation of type I collagen.

Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in alpha1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (beta components converted to alpha chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.

Regulation and restricted expression of interstitial collagenase suggest a pivotal role in the initiation of menstruation.

Collagenases are the only mammalian enzymes able to cleave, at neutral pH, the triple helical domain of fibrillar collagens, major constituents of the extracellular matrix of the endometrium. Interstitial collagenase is expressed, secreted and activated in human endometrium only just before and during menstruation. The expression of interstitial collagenase is restricted to the areas of the functional layer of the endometrium which are breaking down and to fragments which have been shed. In endometrial explants, combined sex steroids tightly control the expression, secretion and activation of interstitial collagenase, as well as the preservation of the extracellular matrix. These observations imply a pivotal role for this proteinase in the initiation of menstruation.

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.

Focal cellular origin and regulation of interstitial collagenase (matrix metalloproteinase-1) are related to menstrual breakdown in the human endometrium.

Recent studies suggest that interstitial collagenase (MMP-1) is an essential enzyme in the early events leading to menstruation. This study analyses its cellular origin, regulation and relation to extracellular matrix breakdown in the human endometrium, both in cultured and non-cultured samples. The source of MMP-1 was identified by in situ hybridization and by immunohistochemistry on serial sections. This was compared with the immunolocalization of other MMPs, steroid receptors, macrophages, and laminin. In non-cultured endometrium, MMP-1 was only expressed during the perimenstrual period. It was either restricted to superficial foci of stromal cells or extended towards the entire functional layer. MMP-1 expression remarkably correlated with matrix breakdown, as assessed by silver staining, and was prominent at the periphery of shedding fragments and along some arterioles. In cultured non-menstrual explants, MMP-1 expression was induced within two days after deprivation of sex steroids. Both in cultured and non-cultured samples, progesterone receptors were not detectable in epithelial cells at foci of MMP-1 expression. The same stromal cells could synthesize MMP-1, MMP-2 (gelatinase A) and MMP-3 (stromelysin-1), as well as laminin, and did not correspond to macrophages. In conclusion, MMP-1 is focally expressed in stromal cells of the functional layer of the endometrium, when and where steroid receptors disappear, and especially where tissue breakdown is prominent. These observations point to an essential role for MMP-1 in the early stages of menstruation.

Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis.

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.

The expression of interstitial collagenase in human endometrium is controlled by progesterone and by oestradiol and is related to menstruation.

Human endometrial tissue, sampled at different periods of the reproductive cycle, expressed interstitial collagenase mRNA, protein and activity only just before and during the menstrual period. This clear-cut correlation and the inhibition of collagenase expression by progesterone and oestradiol in tissue culture point to a pivotal role of this proteinase in the mechanism of menstrual tissue breakdown and bleeding.

Characterization of the promoter of the gene encoding human tissue inhibitor of metalloproteinases-2 (TIMP-2).

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that control the proteolytic activity of matrix metalloproteinases (MMPs). We report here the cloning and characterization of a 2.5-kb genomic fragment of the human timp-2 gene that includes 519 bp of the 5' flanking region, the first coding exon (432-bp) and part of the first intron. The 5' flanking region has several features of housekeeping genes. It has a high G-C content and is included in a typical CpG island. It also contains a TATA-like element (AATAAAA) located 23 to 37-bp upstream from a cluster of transcription start points (tsp), several Sp1 and one AP-2 motifs, and an AP-1 consensus sequence located at position -590 to -583 from the start codon. When inserted upstream from a promoterless luciferase-encoding gene, a 715-bp fragment of this 5'-flanking sequence behaved as a promoter in transiently transfected NIH3T3 and Rat-1 fibroblasts. The effect of deletions of the promoter suggested the presence of a negative control element located between positions -661 and -575. This element includes the AP-1 consensus sequence. However, treatment with phorbol did not change activity in transfected cells and did not change the timp-2 mRNA content of human HT1080 fibrosarcoma cells. A comparison with the promoter of murine timp-1 revealed several differences consistent with the fact that timp-1 and timp-2 are differentially regulated.