The expression of matrix metalloproteinases and their inhibitors by pig synovial cells and their regulation by combinations of cytokines and growth factors

• 1.1. Pig synovial fibroblasts in culture were studied to determine if they were an easily reproducible model system for studying the actions of cytokines and growth factors on human synovial cells. The biochemical analyses were conducted by activity assays, enzymography and Northern blot.
• 2.2. Human recombinant interleukin-1α, basic fibroblast growth factor and transforming growth factor-β₁ were studied in combinations because of their known involvement in controlling tissue remodelling.
• 3.3. The response of pig fibroblasts to these agents, in terms of the expression of matrix metalloproteinases (collagenase, gelatinase and stromelysin) and their inhibitors (TIMPs), show that they behave similarly enough to human cells for use when supplies of human primary cells are unavailable.

     

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.     

The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes

Interleukin-1 (IL-1) plays key roles in altering cartilage matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). In the present study, we examined the effect of IL-1beta on cell proliferation, alkaline phosphatase (ALPase) activity, and the expression of MMPs, and TIMPs in chondrocytes derived from normal human femoral cartilage. The cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 0, 1, 10, or 100 U/ml of IL-1beta for up to 28 days. The level of expression of MMPs and TIMPs was estimated by determining mRNA levels using real-time PCR and by determining protein levels using an enzyme-linked immunosorbent assay. Cell proliferation decreased in the presence of IL-1beta after day 21 of culture. ALPase activity decreased significantly in the presence of IL-1beta after day 10 of culture. The expression of MMP-1, -2, and -3 increased markedly in the presence of IL-1beta after day 21 of culture. MMP-13 expression increased markedly in the presence of IL-1beta on day 1 of culture, but decreased markedly after day 7. The expression of TIMP-1 increased significantly after day 14 of culture. The expression of TIMP-2 decreased significantly on day 1, but increased significantly from day 3 to day 14 of culture. These results suggest that IL-1beta may stimulate cartilage matrix turnover by increasing mainly MMP-13 production by the cells.     

Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells

An imbalance in the matrix metalloproteinase (MMP) : tissue inhibitor of MMP (TIMP) ratio may be associated with tissue injury. Here, we studied the regulation of TIMP and MMP gene expression in primary glial cultures to ascertain the factors involved in the regulation of these genes in conditions of inflammatory neuropathology. Astrocytes were found to basally express TIMP-1 and TIMP-3 mRNA while microglia expressed only TIMP-2 mRNA. TIMP-4 mRNA was not detectable in either cell type. Treatment with interferon-alpha (IFN-alpha), IFN-gamma, interleukin-3 (IL-3), IL-6 or tumor necrosis factor-alpha (TNF-alpha) did not alter expression of the TIMP genes. However, in astrocytes, but not in microglia, serum, IL-1beta or lipopolysaccharide (LPS) evoked a dose- and time-dependent increase in TIMP-1 mRNA and a coincident down-regulation of the TIMP-3 gene. Astrocytes were found to express mRNA constitutively for MMPs -3, -11 and -14. In contrast, microglia expressed only MMP-12 mRNA under basal conditions. IL-1beta enhanced MMP-3 mRNA levels while LPS increased the MMP-3, -9, -12, -13 and -14 mRNAs. Our findings reveal that regulatory control of TIMP and MMP gene expression by glial cells is agonist- and cell-type specific, and suggest that innate immune signals govern the temporal and spatial expression patterns of TIMP and MMP genes in neuroinflammatory conditions of the CNS.     

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     

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.     

Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3-E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP-2, -3, -9, -11, -13, and -14 or TIMP-1, -2, and -3 was detected in MC3T3-E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25-dihydroxyvitamin D(3), prostaglandin E(2), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) were added to the cell cultures, MMP-13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP-3 and -9 or TIMP-1 and -3 were found to increase mainly by the cytokines IL-1beta and TNF-alpha. BB94, a nonselective MMP inhibitor, neutralized the (45)Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP-13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP-3 and -9 or TIMP-1 and -3 might modulate matrix degradation by local cytokines only.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.     

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7⁺ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.     

Structural basis of the matrix metalloproteinases and their physiological inhibitors,the tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of multidomain zinc endopeptidases with a metzincin-like catalytic domain, which are involved in extracellular matrix degradation but also in a number of other important biological processes. Under healthy conditions, their proteolytic activity is precisely regulated by their main endogenous protein inhibitors, the tissue inhibitors of metalloproteinases. Disruption of this balance results in pathophysiological processes such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and for rational drug design. Since the first appearance of atomic MMP structures in 1994, a large amount of structural information has become available on the catalytic domains of MMPs and their substrate specificity, interaction with synthetic inhibitors and the TIMPs, the domain organization, and on complex formation with other proteins. This review will outline our current structural knowledge of the MMPs and the TIMPs.     

Modulation of the expression of matrix metalloproteinase and tissue inhibitors of metalloproteinases by TGF-beta1 and IGF-1 in primary human articular and bovine nasal chondrocytes stimulated with TNF-alpha.

Tumour necrosis factor-alpha (TNF-alpha) was able to promote collagen breakdown from bovine cartilage in explant culture. This release was dependent upon matrix metalloproteinases (MMPs) and could be prevented by transforming growth factor-beta1 (TGF-beta1) or insulin-like growth factor-1. Both growth factors reduced the expression and secretion of collagenase enzymes, and TGF-beta1 induced tissue inhibitor of metalloproteinase production. This study shows for the first time that these anabolic growth factors can protect cartilage against TNF-alpha-induced destruction.     

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.     

Differential expression and regulation of cyclooxygenase isozymes in thymic stromal cells

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-gamma up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-gamma down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8- thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.     

Role of interleukin 1 in the regulation of cyclooxygenase gene expression in rat endometrial stromal cells.

Interleukin 1 alpha (IL-1 alpha) stimulates prostaglandin production and cyclooxygenase activity in endometrial stromal cells isolated from the uteri of ovariectomized rats that have been sensitized for the decidual cell reaction. The aim of the present study was to examine the effect of IL-1 alpha on the amount of cyclooxygenase mRNA and protein in these cells. Treatment with IL-1 alpha (20 ng ml-1) for 24 h significantly increased steady-state concentrations of cyclooxygenase 2 (COX-2) mRNA and protein in the cells, as determined by northern and western blot analyses, respectively. Cyclooxygenase 1 (COX-1) mRNA and protein were not detected. Dexamethasone (5 mumol l-1) prevented the IL-1 alpha-induced increase in COX-2 steady-state mRNA. Immunocytochemical staining of COX-2 in the treated cells indicated that IL-1 alpha increased staining, while dexamethasone inhibited this increase. Furthermore, the changes in staining were generalized and not confined to a small subpopulation of cells. These data demonstrate that IL-1 alpha increases steady-state concentrations of COX-2 mRNA and protein in endometrial stromal cells isolated from the uteri of rats that have been sensitized for decidualization.