Expression of matrix metalloproteinases 2 and 9 in the mouse uterus during implantation and oil-induced decidualization

During implantation, matrix metalloproteinases are believed to play roles in the tissue remodelling that accompanies decidualization in the endometrium and in embryo invasion. The objective of this study was to characterize further the expression of matrix metalloproteinases 2 and 9 in the mouse uterus during early pregnancy and oil-induced decidualization. mRNA encoding matrix metalloproteinase 2 was detected in pregnant uteri and uteri undergoing oil-induced decidualization by northern blot analyses. The steady-state concentrations of mRNA encoding matrix metalloproteinase 2 did not change significantly in implantation compared with inter-implantation areas on days 5-8 of pregnancy but were significantly lower in stimulated compared with non-stimulated uterine horns during artificially induced decidualization. mRNA encoding matrix metalloproteinase 9 was also detected in uteri undergoing oil-induced decidualization but not in pregnant uteri. Its concentration was significantly greater in uterine horns undergoing oil-induced decidualization compared with control horns. Immunoreactive matrix metalloproteinases 2 and 9 were detected in the uterus during early pregnancy and oil-induced decidualization by immunohistochemistry, localized to the endometrial stroma, but the staining progressively became weaker and was absent in areas that had undergone decidualization. By day 8 of pregnancy and 72 h after the induction of decidualization, matrix metalloproteinase 2 and 9 proteins remained mainly in the region of non-decidualized stromal cells adjacent to the myometrium. In implantation segments, they were also localized to the region of the trophoblast giant cells. The second objective of the present study was to determine whether endometrial stromal cells isolated from uteri sensitized for decidualization express matrix metalloproteinases 2 and 9. Northern blot analyses and gelatin zymography showed that these cultured cells expressed matrix metalloproteinase 2 and 9, and that transforming growth factor beta1 significantly increased matrix metalloproteinase 9 expression. The results of the present study further characterize matrix metalloproteinases 2 and 9 expression in the uterus during implantation and artificially induced decidualization.     

基质金属蛋白质酶12在子宫内膜癌中的表达及其意义

目的通过研究基质金属蛋白质酶12（MMP-12）在子宫内膜癌中的表达,探讨其与子宫内膜癌浸润转移的关系。方法应用链霉菌抗生物素蛋白——过氧化物酶免疫组织化学方法（S—P法）和蛋白质印迹（Western—blot）对42例子宫内膜腺癌、12例子宫内膜不典型增生及12例正常子宫内膜组织中的MMP-12的表达进行检测,并分析其与子宫内膜癌临床病理特征之间的关系。结果MMP-12蛋白主要表达于子宫内膜癌细胞胞浆中,在问质中有少量表达。MMP-12在子宫内膜癌、子宫内膜不典型增生的阳性表达率与正常子宫内膜相比差异有显著性（P〈0．05）,但子宫内膜癌与子宫内膜不典型增生中的MMP-12阳性表达率之问差异无显著性（P〉0．05）。MMP-12在子宫内膜癌细胞中病理分级为G3、G2强阳性率分别为80．00％及57．14％,均高于G1者的（17．39％）（P〉0．05）;不同肌层浸润深度（〈1／2、≥1／2）者的强阳性率分别为42．86％及53．85％,均高于无肌层浸润者的0％（P〈0．05）;有淋巴结转移者高于无淋巴结者（P〈0．05）;手术病理分期Ⅰ、Ⅱ、Ⅲ期者的强阳性率分别为24．14％、57．14％及83．34％,随着手术病理分期的升高,MMP-12表达强阳性率之间差异有显著性（P〈0．05）。结论MMP-12在子宫内膜癌中的表达随着病理分级的升高、肌层浸润程度的加深以及手术病理分期的升高而增强。     

Relationship between uterine expression of matrix metalloproteinases and their inhibitors and endometrial receptivity

In order to investigate the relationship between the endometrial receptivity and matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1,-3 (TIMP-1,-3) in the endometrium, we used early pregnant mice as the animal model and studied the expression of MMP-2, TIMP-1,-3 in the endometrium in relation to the number of implantation sites after RU486 treatment. The results indicated that RU486 could significantly inhibit embryo implantation and change the expression of MMP-2 and TIMP-1,-3 in a dose-dependent pattern. When the mice were treated with 12 mg/kg RU486, there were a few embryos implanted as compared with the control. The expression of matrix metalloproteinase MMP-2 was low during the period of "implantation window", while the tissue inhibitor of metalloproteinase in the endometrial cells was high, suggesting that the activity of the proteolytic enzyme was strictly controlled by its inhibitors. After RU486 treatment, the generation of TIMP-1,3 was decreased while the MMP-2 was significantly increased, indicating that the normal balance between the activators and their inhibitors in the tissue was broken and the extracellular matrix was excessively degraded, subsequently the embryo implantation was inhibited. Therefore, it is suggested that the anti-implantation effect of RU486 may be mediated by MMPs and their inhibitors TIMPs.     

Relationship between uterine expression of matrix metalloproteinases and their inhibitors and endometrial receptivity

In order to investigate the relationship between the endometrial receptivity and matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 ,-3 (TIMP-1,-3) in the en-dometrium, we used early pregnant mice as the animal model and studied the expression of MMP-2, TIMP-1 ,-3 in the endometrium in relation to the number of implantation sites after RU486 treatment. The results indicated that RU486 could significantly inhibit embryo implantation and change the expression of MMP-2 and TIMP-1,-3 in a dose-dependent pattern. When the mice were treated with 12 mg/kg RU486, there were a few embryos implanted as compared with the control. The expression of matrix metalloproteinase MMP-2 was low during the period of "implantation window", while the tissue inhibitor of metalloproteinase in the endometrial cells was high, suggesting that the activity of the proteolytic enzyme was strictly controlled by its inhibitors. After RU486 treatment, the generation of TIMP-1,3 was decreased while the MMP-2 wa     

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.     

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the hippocampus of lithium-pilocarpine-induced acute epileptic rats

Molecular Biology Reports - Epilepsy is characterised by abnormal neuronal discharges, including aberrant expression of extracellular matrix (ECM) components and synaptic plasticity stabilisation....     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

Synthesis and in vitro evaluation of targeted tetracycline derivatives: effects on inhibition of matrix metalloproteinases

Among other non-antibiotic properties, tetracyclines inhibit matrix metalloproteinases and are currently under study for the treatment of osteoarthritis. Quaternary ammonium conjugates of tetracyclines were synthesized by direct alkylation of the amine function at the 4-position with methyl iodide. When tested in vitro, they inhibited cytokine-induced MMP expression to a lesser extent than parent tetracyclines. This was compensated by an improved inhibition of MMP catalytic activity. Since inhibition of collagen degradation was maintained these derivatives could be potent drug candidates for cartilage-targeted chondroprotective treatment.     

Inhibitory Effect of Aspergillus fumigatus Extract on matrix metalloproteinases Expression

BACKGROUND: Matrix metalloproteinases (MMPs) play a role in several physiologic and pathologic events. There are some evidence indicating the involvement of MMPs in the pathophysiology of fungal infections. METHODS: Here we study somatic extract of Aspergillus fumigatus. The influence of Aspergillus vs. two other fungal extracts on MMPs production by Fibrosarcoma cell line was investigated using an in vitro cytotoxicity assay, sodium dodecyl sulfate-polyacrylamide and gelatin zymography. RESULTS: Comparative dose-dependent inhibitory effects on MMPs were seen by A. fumigatus extract and compared with some steroidal and non-steroidal drugs. Cytotoxicity analysis of our extract revealed much lower cell death than other examined agents. CONCLUSIONS: Since inhibition of MMPs activity has been employed in modality therapy in such diseases as cancer, this extract might be promising in the preparation of anti-MMP therapeutic derivatives.     

In vitro decidualization of rat endometrial stromal cells

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes. This research was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS; no. 18580282, to N. Yamauchi).     

Effect of gamma-synuclein overexpression on matrix metalloproteinases in retinoblastoma Y79 cells

gamma-Synuclein is a small cytoplasmic protein implicated in neurodegenerative diseases and cancer. However, the mechanism of its involvement in diseases is not clear. We studied the role of gamma-synuclein in the regulation of matrix metalloproteinases in retinoblastoma cell culture. Matrix metalloproteinases play important roles in the remodeling of extracellular matrix implicated in tumor progression and in the neurodegenerative diseases. Western blot and zymography data demonstrated a moderate elevation of matrix metalloproteinases-2 and significant upregulation of matrix metalloproteinases-9 in stable cell lines overexpressing gamma-synuclein. No effect of gamma-synuclein overexpression on matrix metalloproteinases-1 level or activity was found. Chloramphenicol-acetyltransferase assay demonstrated that overexpression of gamma-synuclein increases the efficiency of the matrix metalloproteinases-9 promoter. This increment of promoter activity may be mediated by the AP-1 binding site(s), since point mutations in one of these sites (Pr18 or Pr19) and elimination of the distal AP-1 site (Pr14) reduced the increment of promoter activity.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

Effect of glycosaminoglycans on matrix metalloproteinases in type II collagen-induced experimental arthritis

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are involved in tissue remodeling by mediating degradation of extracellular matrix components in both physiology and pathology. As MMPs appear to play a key role in the degradation of cartilage matrix in the progression of arthritic disease, MMPs are considered as potential therapeutic targets. The effect of chondroitin sulfate A (CSA) on MMPs in type II collagen-induced experimental arthritis was studied. The anti-arthritic effect of CSA was evidenced by a decrease in marker activities like lysosomal beta-hexosaminidase and beta-glucuronidase. Arthritic animals showed significantly higher activity of MMP2 and MMP9 and increased levels of other MMPs, including MMP3 and MT-1 MMP in cartilage and serum. Treatment with CSA significantly decreased the activity of MMPs, particularly MMP9 in serum and synovial effusate and cartilage. The effect of CSA was further studied by fragmenting CSA into low-molecular-weight oligosaccharides. The oligosaccharide-treated animals showed considerably lower MMP activity (particularly MMP9) compared with arthritic controls. The CSA (and the oligosaccharides derived from it) not only reduced the activity of MMPs but also decreased the protein level expression of MMPs, indicating that the production of MMPs is affected. These results indicate that the antiarthritic effect of CSA involves down-regulation of MMPs, which are critically involved in the progression of arthritic disease.     

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Matrix metalloproteinases and endometrial remodelling.

Menstruation, angiogenesis, embryo implantation and placentation are natural processes involving degradation of the extracellular matrix within the endometrium. A number of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) have now been identified in association with these processes. In particular, mRNA for proMMP-1 and proMMP-3 and for TIMP-1 and TIMP-2 are elevated in human endometrial tissue at menstruation compared with other times of the female reproductive cycle. ProMMPs -1, -2, -3 and -9 are released from cultured human endometrial stromal cells, production of all but proMMP-2 being regulated by cytokines known to be expressed in human endometrial epithelium. At least two gelatinases are released by ovine trophoblast in culture and can be flushed from the uterine lumen at the time of implantation. Thus, matrix metalloproteinases clearly have roles in normal endometrial functions.     

In vitro decidualization of human endometrial stromal cells.

Stromal cells isolated from proliferative human endometrium undergo morphologic and biochemical changes when exposed to a mixture of ovarian hormones, acquiring characteristics of decidual cells. In addition to the previously reported progestin-induced secretion of prolactin (PRL) by explants of human proliferative endometrium, and of PRL and laminin by stromal cells in culture, "in vitro" induction of several other decidual cell products was demonstrated in the present study, using cultures of stromal cells isolated from proliferative endometrium. Incubation of stromal cells with a mixture of estradiol, medroxyprogesterone acetate and relaxin, at a concentration reported to yield maximal stimulation of PRL production, resulted in changes from elongated to rounder cells, approx. 90% of which showed immunostaining for PRL under these conditions. Immunocytochemical procedures were carried out on cytospins of decidual cells isolated from decidual tissue adherent to fetal membranes collected at delivery (positive controls), and on stromal cells cultured in Lab-Tek chamber-slides, in the absence (negative controls) or in the presence of added hormones. Antibodies to 24K (a heat-shock protein also named HRP27), desmin (present in intermediate filaments), p29 (a protein associated with the estrogen receptor), and PP12 (an insulin growth factor-1 binding protein), did not react with stromal cells isolated from proliferative endometrium but showed immunostaining of the rounder cells obtained after hormonal treatment when tested with the peroxidase-labeled second antibody complex. In another series of similar experiments, in which the same decidualization end-points were employed, changes in 24K, desmin and PP12 expression were obtained by adding to the insulin-containing medium PRL instead of the hormonal mixture, a finding suggesting sequential steps during the decidualization process.     

Dehydroepiandrosterone inhibits glucose flux through the pentose phosphate pathway in human and mouse endometrial stromal cells, preventing decidualization and implantation

Endometrial stromal cells (ESC) must undergo a hormone-driven differentiation to form decidual cells as a requirement of proper embryo implantation. Recent studies from our laboratory have demonstrated that decidualizing cells require glucose transporter 1 expression and an increase in glucose use to complete this step. The present study focuses on the glucose-dependent molecular and metabolic pathways, which are required by ESC for decidualization. Inhibition of glycolysis had no effect on decidualization. However, blockade of the pentose phosphate pathway (PPP) with pharmacologic inhibitors 6-aminonicotinamide or dehydroepiandrosterone (DHEA), and short hairpin RNA-mediated knockdown of glucose-6-phosphate dehydrogenase, the rate-limiting step in the PPP, both led to strong decreases in decidual marker expression in vitro and decreased decidualization in vivo. Additionally, the studies demonstrate that inhibition is due, at least in part, to ribose-5-phosphate depletion, because exogenous nucleoside administration restored decidualization in these cells. The finding that PPP inhibition prevents decidualization of ESC is novel and clinically important, because DHEA is an endogenous hormone produced by the adrenal glands and elevated in a high proportion of women who have polycystic ovary syndrome, the most common endocrinopathy in reproductive age women. Together, this data suggest a mechanistic link between increased DHEA levels, use of glucose via the PPP, and pregnancy loss.