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.     

Collagenase-3 (MMP-13) expression in cutaneous malignant melanoma

BACKGROUND: Matrix metalloproteases (MMPs), enzymes with the ability to degrade the extracellular matrix, play an important role in tissue invasion by cutaneous malignant melanoma (CMM). One specific MMP, collagenase-3 (MMP-13), is thought to have a key function in the activation of MMP. AIMS: To evaluate the expression of MMP-13 in CMM and assess its possible relationship to clinical and pathological parameters. METHODS: MMP-13 expression was analyzed in 51 paraffin-embedded tumor samples from patients with invasive CMM, ten samples from in situ melanomas, and in eight samples from benign lesions (three dermal melanocytic nevi, three compound melanocytic nevi and two atypical melanocytic nevi) using immunohistochemical techniques. The median follow-up period in patients with invasive CMM was 50 months. RESULTS: Benign lesions were consistently negative for MMP-13, whereas three of the ten in situ melanomas (30%) and 23 of the 51 invasive CMMs (45%) showed positive immunostaining for MMP-13. The percentage of MMP-13-positive tumors correlated significantly and positively with the mitotic index (p=0.002) in invasive CMM. However, our results did not show any significant association between tumoral MMP-13 expression and relapse-free survival in patients with invasive CMM. CONCLUSIONS: MMP-13 appears to be a factor associated with tumor aggressiveness in CMM. It seems to eliminate an important barrier not only against tumoral invasion but also against proliferation.     

Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.     

Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.     

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.     

Identification of novel, exosite-binding matrix metalloproteinase-13 inhibitor scaffolds

Matrix metalloproteinase-13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). Compounds that inhibit the metalloproteinase at the Zn binding site typically lack specificity among MMP family members. Analogs of the low-micromolar lead MMP-13 inhibitor 4, discovered through high-throughput screening, were synthesized to investigate structure-activity relationships in this inhibitor series. Systematic modifications of 4 led to the discovery of MMP-13 inhibitors 20 and 24 which are more selective than 4 against other MMPs. Compound 20 is also approximately fivefold more potent as an MMP-13 inhibitor than the original HTS-derived lead compound 4.     

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.     

ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3

Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.     

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.     

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

A systematic computational analysis of human matrix metalloproteinase 13 (MMP-13) crystal structures and structure-based identification of prospective drug candidates as MMP-13 inhibitors repurposable for osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis with no available disease-modifying treatments, and is a major cause of disability. Matrix metalloproteinase 13 (MMP-13) is vital for OA progression and thus, inhibition of MMP-13 is an effective strategy to treat OA. Since the past few decades, drug repurposing has gained substantial popularity worldwide as a time- and cost-effective approach to find new indications for the existing drugs. Therefore, more than 40 X-ray co-crystal structures of the human MMP-13 with bound inhibitors are investigated to gain the structural insights such as conserved direct interactions with binding site residues, namely Ala-238, Thr-245 and Thr-247. Afterwards, enrichment study using active and decoy set of ligands revealed three MMP-13 structures (PDB-IDs: 1XUC, 3WV1 and 5BPA) with optimal enrichment performance. Docking-based screening of existing drugs against the three crystal structures followed by binding free-energy calculation suggested drugs namely eltrombopag, cilostazol and domperidone as potential MMP-13 inhibitors that need further experimental validation. These insights may serve as a potential starting point of further experimental validation and structure-based drug design/repurposing of MMP-13 inhibitors for the treatment of OA. Abbreviations 2D two-dimensional

3D three-dimensional

FDA Food and Drug Administration

MM-GBSA Molecular Mechanics Generalized Born Surface Area

MMPs matrix metalloproteinases

MMP-13 matrix metalloproteinase 13

NMR nuclear magnetic resonance

OA osteoarthritis

PDB Protein Data Bank

PDB-ID Protein Data Bank ID

PLIP protein–ligand interaction profiler

ROC receiver operating characteristic,

RMSD root mean square deviation

Communicated by Ramaswamy H. Sarma     

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.     

Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.     

Development of a receptor-based 3D-QSAR study for the analysis of MMP2, MMP3, and MMP9 inhibitors

The ability of Gold software to predict the binding disposition of matrix metalloproteinase (MMP) inhibitors was evaluated using MMP3 and MMP8. The best procedure was subsequently employed to dock into MMP2, MMP3 and MMP9 nearly 70 compounds that were tested for their inhibitory activity against the three MMP subtypes. The best binding poses were used as an alignment tool for the development of 3D-QSAR studies. Evaluation of the three resulting 3D-QSAR models allowed us to indicate the ligand properties and residues important for activity and selectivity. MMP2 is an important anticancer drug target, while MMP3 and MMP9 are considered to be anti-targets for tumor pathologies. As such, our results could predict the binding affinities of new MMP2 inhibitors, providing additional information regarding the selectivity against MMP3 and MMP9. Furthermore, this strategy may be used also for the investigation of other MMPs.