Resistance of collagenase-2 (matrix metalloproteinase-8)-deficient mice to TNF-induced lethal hepatitis

Acute fulminant liver failure is a serious worldwide health problem. Despite maximal supportive intensive care treatment, the disease offers a poor prognosis, with mortality rates of >80%. We have previously shown that a broad-spectrum inhibitor of matrix metalloproteinases (MMPs) confers complete protection in a mouse model of TNF-induced lethal hepatitis, thereby suggesting the possibility of protecting cancer patients against the deleterious side effects of TNF therapy. In our search for the individual matrix metalloproteinases involved, we found that the recently generated MMP-8-deficient mice are significantly protected against TNF-induced acute hepatitis. In contrast to their wild-type counterparts, MMP-8-null mice display very little hepatocyte necrosis and apoptosis, resulting in a much better survival outcome. We found that these animals clearly display impaired leukocyte influx into the liver and no release of the neutrophil-specific, LPS-induced CXC chemokine. Our findings provide evidence that MMP-8 plays an essential role in acute liver failure and might be a promising new target for the treatment for this illness.     

Smad3 mediates transforming growth factor-beta-induced collagenase-3 (matrix metalloproteinase-13) expression in human gingival fibroblasts. Evidence for cross-talk between Smad3 and p38 signaling pathways

Transforming growth factor-beta (TGF-beta) is a potent inducer of collagenase-3 (MMP-13) gene expression in human gingival fibroblasts, and this requires activation of the p38 mitogen-activated protein kinase pathway. Here, we have constructed recombinant adenoviruses harboring genes for hemagglutinin-tagged Smad2, Smad3, and Smad4 and used these in dissecting the role of Smads, the signaling mediators of TGF-beta, in regulation of endogenous MMP-13 gene expression in human gingival fibroblasts. Adenoviral expression of Smad3, but not Smad2, augmented the TGF-beta-elicited induction of MMP-13 expression. In addition, adenoviral gene delivery of dominant negative Smad3 blocked the TGF-beta-induced MMP-13 expression in gingival fibroblasts. Co-expression of Smad3 with constitutively active MKK3b and MKK6b, the upstream activators of p38, resulted in nuclear translocation of Smad3 in the absence of TGF-beta and in induction of MMP-13 expression. The induction of MMP-13 expression by Smad3 and constitutively active mutants of MKK3b or MKK6b was blocked by specific p38 inhibitor SB203580 and by the dominant negative form of p38alpha. These results show that TGF-beta-induced expression of human MMP-13 gene in gingival fibroblasts is dependent on the activation of two distinct signaling pathways (i.e. Smad3 and p38alpha). In addition, these findings provide evidence for a novel type of cross-talk between Smad and p38 mitogen-activated protein kinase signaling cascades, which involves activation of Smad3 by p38alpha.     

Molecular modeling of non-covalent binding of homochiral (3S,3'S)-astaxanthin to matrix metalloproteinase-13 (MMP-13)

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of various important chronic illnesses, including cancer, arthritis, and cardiovascular disease (CVD). In particular, MMP-13 is currently being probed as a potential key target in CVD and malignant disease due to its documented effects on extracellular matrix (ECM) remodeling, important in the pathophysiology of these diseases. Within the family of related mammalian MMP enzymes, MMP-13 possesses a large hydrophobic binding pocket relative to that of other MMPs. Homochiral astaxanthin (3S,3'S-AST; 3S,3'S-dihydroxy-beta,beta-carotene-4,4'-dione), an important antioxidant and anti-inflammatory xanthophyll carotenoid, is an active metabolite of several novel soft drugs in clinical development; it is also extensively used and tested as a human nutraceutical. In the current study, the prediction of the geometry and energetics of its binding to human MMP-13 was conducted with molecular modeling. The method used was found to predict the energy of binding of known ligands of MMP-13 with great precision. Blind docking using the whole protein target was then used in order to identify the possible binding site(s) of AST. AST was predicted to bind at several sites in close proximity to the active center. Subsequent analyses focused on the binding site at the atomic (i.e., amino acid sequence) level suggested that AST can bind to MMP-13 with high affinity and favorable energetics. Therefore, the modeling study predicts potential direct enzyme-inhibitory activity of AST against MMP-13, a behavior that may be exploited in mammalian systems in which pathological upregulation of MMP activity is paramount.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

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.     

Interferonregulatoryfactor-8(IRF-8) regulates the expression of matrix metalloproteinase-13 (MMP-13) in chondrocytes

Low levels of inflammation-induced expression of matrix metalloproteinase (MMP) play a crucial role in articular cartilage matrix destruction in osteoarthritis (OA) patients. Interferon regulatory factor-8 (IRF-8), an important member in the IRF family, plays a key role in regulating the inflammation-related signaling pathway. The aim of this study is to investigate the physiological roles of IRF-8 in the pathological progression of OA. We found that IRF-8 was expressed in human primary chondrocytes. Interestingly, the expression of IRF-8 was upregulated in OA chondrocytes. In addition, IRF-8 was increased in response to interleukin-1β (IL-1β) treatment, mediated by the Janus kinase 2 (JAK2) pathway. Overexpression of IRF-8 in human chondrocytes by transduction with lentiviral-IRF-8 exacerbated IL-1β-induced expression of matrix metalloproteinase-13 (MMP-13) in human chondrocytes. In contrast, knockdown of IRF-8 inhibited IL-1β-induced expression of MMP-13. Importantly, IRF-8 could bind to the promoter of MMP-13 and stimulate its activity. Additionally, overexpression of IRF-8 exacerbated IL-1β-induced degradation of type II collagen. However, silencing IRF-8 abrogated the degradation of type II collagen. Taken together, our findings identified a novel function of IRF-8 in regulating articular cartilage matrix destruction by promoting the expression of MMP-13.     

Structure-based design of potent and selective inhibitors of collagenase-3 (MMP-13)

Computer aided drug design led to a new class of spiro-barbiturates (e.g., 4a, MMP-13 K(i)=4.7 nM) that are potent inhibitors of MMP-13.     

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.     

Involvement of matrix metalloproteinase-9 in platelet-activating factor-induced angiogenesis

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-kappaB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-kappaB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-kappaB inhibitors. Transfected NF-kappaB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-kappaB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-kappaB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-kappaB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.     

Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.     

Inhibition of gelatinase B (matrix metalloproteinase-9) by dihydrolipoic acid

Alpha-lipoic acid (LA) is a disulphide-containing fatty acid that is absorbed from the diet and transported to tissues. Once it has been taken up by mammalian cells, LA is reduced to dihydrolipoic acid (DHLA), a vicinal dithiol, and rapidly effluxed into the extracellular milieu. We hypothesized that DHLA may be an effective inhibitor of human gelatinase B (GelB). Purified human GelB was incubated with 0 to 200 micromol/L DHLA, and residual enzyme activity was measured by HPLC using a fluorogenic substrate (matrix metalloproteinase substrate III). DHLA inhibited GelB in a dose-dependent fashion with an IC50 of 20 micromol/L. Oxidation of DHLA resulted in a loss of DHLA's capacity to inhibit GelB. The DHLA-mediated inhibition of GelB was independent of the zinc concentration in the reaction buffer. DHLA had no inhibitory effect on gelatinase A. Zymographs of activated neutrophil lysates demonstrated that higher concentrations of DHLA also prevent the activation of GelB proenzyme. Bronchoalveolar lavage fluid from mice fed a diet enriched with LA showed significantly increased GelB inhibitory capacity (p = 0.0002 vs. regular diet). We conclude that DHLA can modulate neutrophil-derived GelB activity through direct inhibition of enzyme activity and by preventing the activation of GelB proenzyme.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.