Gonadotropin surge-induced differential upregulation of collagenase-1 (MMP-1) and collagenase-3 (MMP-13) mRNA and protein in bovine preovulatory follicles

The ovulatory process is characterized by focalized extracellular matrix degradation at the apex of preovulatory follicles. Many studies have implicated the matrix metalloproteinases (MMPs) as potential mediators of follicle rupture. Objectives of this study were to determine localization and effect of the gonadotropin surge on temporal expression of MMP-1 and MMP-13 in bovine preovulatory follicles. Samples were collected at 0, 6, 12, 18, 24, and 48 h (corpora lutea) after GnRH injection (n = 5-6 per time point) and amounts of MMP-1 and MMP-13 mRNA and protein determined using dot blot or semiquantitative RT-PCR and Western blot analyses. Samples were also collected at 0 and 20 h after GnRH injection for immunohistochemical localization of MMP-1 and MMP-13. Results indicate that follicular expression of MMP-1 and MMP-13 increased following the gonadotropin surge. Abundance of MMP-1 mRNA increased at 6, 12, and 48 h post-GnRH injection. Immunoreactive MMP-1 was localized to granulosal and thecal layers of preovulatory follicles. Amounts of MMP-1 protein increased in both the apex and the base of preovulatory follicles. Abundance of MMP-13 mRNA increased at 6, 24, and 48 h post GnRH injection. Amounts of MMP-13 protein also increased in the follicular apex and base. Immunoreactive MMP-13 was localized to granulosal and thecal layers of preovulatory follicles. Results indicate MMP-1 and MMP-13 are increased in bovine preovulatory follicles following the gonadotropin surge but do not support a requirement for differential up-regulation of MMP-1 and MMP-13 (follicular apex vs. base) for the preovulatory collagenolysis required for follicle rupture.     

Potent and selective 2-naphthylsulfonamide substituted hydroxamic acid inhibitors of matrix metalloproteinase-13

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.     

Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.     

High-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor

The high-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a sulfonamide derivative of a hydroxamic acid compound (WAY-151693) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated for residues 7-164 by means of hybrid distance geometry-simulated annealing using a total of 3280 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures is 0.43(+/-0.05) A for the backbone atoms, 0.80(+/-0.09) A for all atoms, and 0.47(+/-0.04) A for all atoms excluding disordered side-chains. The overall structure of MMP-13 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and anti-parallel arrangement and three alpha-helices where its overall fold is consistent with previously solved MMP structures. A comparison of the NMR structure of MMP-13 with the published 1.6 A resolution X-ray structure indicates that the major differences between the structures is associated with loop dynamics and crystal-packing interactions. The side-chains of some active-site residues for the NMR and X-ray structures of MMP-13 adopt distinct conformations. This is attributed to the presence of unique inhibitors in the two structures that encounter distinct interactions with MMP-13. The major structural difference observed between the MMP-13 and MMP-1 NMR structures is the relative size and shape of the S1' pocket where this pocket is significantly longer for MMP-13, nearly reaching the surface of the protein. Additionally, MMP-1 and MMP-13 exhibit different dynamic properties for the active-site loop and the structural Zn-binding region. The inhibitor WAY-151693 is well defined in the MMP-13 active-site based on a total of 52 distance restraints. The binding motif of WAY-151693 in the MMP-13 complex is consistent with our previously reported MMP-1:CGS-27023A NMR structure and is similar to the MMP-13: RS-130830 X-ray structure.     

MMP-13 is induced during chondrocyte hypertrophy

During development, mRNA for matrix metalloproteinase-13 (MMP-13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP-13 expression and the transition to hypertrophy. When hypertrophy was induced by serum-free culture with ascorbate and bone morphogenetic protein-2 (BMP-2), MMP-13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP-13, confirming that MMP-13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP-13 reacted with the 59-kDa protein, confirming that it is MMP-13. Although mRNA for MMP-13 peaked at days 4-5 of culture, only low levels of MMP-13 activity were present, and the activity increased gradually in parallel with later increases in MMP-2. These results suggest that MMP-13 is activated by MMP-2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification.     

Inhibition of matrilysin expression by antisense or RNA interference decreases lysophosphatidic acid-induced epithelial ovarian cancer invasion

Our previous reports show that matrilysin [matrix metalloproteinase (MMP)-7] is overexpressed in epithelial ovarian cancer (EOC) and recombinant MMP-7 promotes EOC invasion in vitro. In the present study, we further evaluated the correlation of MMP-7 expression to EOC invasiveness and examined its role in lysophosphatidic acid (LPA)-induced invasion. By sense and antisense gene transfection in vitro, we show that overexpression of MMP-7 in all MMP-7 stably transfected DOV13 clones significantly enhanced their invasiveness, although MMP-7 antisense transfection caused a 91% decrease of MMP-7 expression (P < 0.01) and 87% decrease of invasion (P < 0.05) in geneticin (G418)-selected DOV13 clone P47-M7As-3 compared with vector-transfected control. As assessed by MMP-7 ELISA, LPA treatment at 10 to 80 micromol/L significantly stimulated the secretion of total MMP-7 in DOV13 conditioned medium (P < 0.01). In addition, LPA apparently induced the activation of MMP-7 in DOV13 cells as detected by gelatin zymography. In the antisense MMP-7-transfected DOV13 clone (P47-M7As-3), LPA-increased invasion was significantly decreased compared with vector control. Moreover, knocking down of MMP-7 by small interfering RNA also suppressed LPA-induced invasion in two EOC cell lines (DOV13 and R182). Altogether, our results show that MMP-7 expression is correlated with EOC invasiveness and LPA-induced MMP-7 secretion/activation may represent a new mechanism that facilitates ovarian cancer invasion besides the well-known induction of MT1-MMP-mediated proMMP-2 activation by LPA.     

MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1

Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.     

Involvement of H-Ras and reactive oxygen species in proinflammatory cytokine-induced matrix metalloproteinase-13 expression in human articular chondrocytes

Proinflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) enhance degradation of cartilage-specific, type II collagen by matrix metalloproteinase-13 (MMP-13). We investigated the previously unknown role of H-Ras and reactive oxygen species (ROS) in the cytokine induction of MMP-13 gene expression in human articular chondrocytes by using pharmacological inhibitors, RNA interference (RNAi) and antioxidants. Manumycin A, an inhibitor of H-Ras farnesylation by farnesyltransferase, suppressed IL-1β- and TNF-α-induced MMP-13 mRNA and protein expression. Small interfering RNA (siRNA)-mediated H-Ras silencing down-regulated MMP-13 mRNA and protein induction by IL-1β and TNF-α. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) inhibitor, diphenyleneiodonium (DPI) suppressed cytokine-induced MMP-13 expression and superoxide production. Apocynin, another NOX inhibitor, also diminished MMP-13 induction. Deoxyglucose an antimetabolite of glucose metabolism reduced MMP-13 increase. Role of NOX-mediated ROS production was reaffirmed by the observation that the antioxidants, trolox, nordihydroguaiaretic acid (NDGA), quercetin and resveratrol downregulated cytokine-induced MMP-13 mRNA and protein expression. These results provide strong pharmacological and genetic evidence for the implication of H-Ras and NADPH oxidase-generated superoxide production in MMP-13 gene regulation by IL-1β and TNF-α. These proteins could be potentially targeted for therapeutic inhibition of MMP-13-driven cartilage erosion by using H-Ras and NOX inhibitors and antioxidants.     

YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site

Matrix metalloproteinases (MMPs) are key enzymes that implement degradation of the extracellular matrix during cellular invasion in development, tissue remodeling, and pathogenic disease states. MMP-13 has pivotal roles in the pathogenesis of invasive cancers and arthritis. Here we report the identification of Y-box binding protein-1 (YB-1) as a new repressor of MMP-13 transactivation. YB-1 binds in vitro in DNA affinity chromatography to the activator protein-1 (AP-1) DNA sequence within the MMP-13 promoter. Chromatin immunoprecipitation assays reveal that YB-1 binds in living cells to the MMP-13 gene promoter to a region of the MMP-13 promoter containing the AP-1 site. YB-1 represses tumor promoter-induced MMP-13 promoter transactivation at the AP-1 site. This is the first report demonstrating YB-1 binding in vitro and in living cells to a mammalian AP-1 target gene, and the first report of YB-1 regulation of the MMP-13 promoter.     

Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.