LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages.

The metalloproteinase ADAM17 plays a pivotal role in initiating inflammation by releasing TNF from its precursor. Prolonged TNF release causes many chronic inflammatory diseases, indicating that tight regulation of ADAM17 activity is essential for resolution of inflammation. In this study, we report that the endogenous ADAM17 inhibitor TIMP-3 inhibits ADAM17 activity only when it is bound to the cell surface and that cell surface levels of TIMP-3 in endotoxin-activated human macrophages are dynamically controlled by the endocytic receptor LRP1. Pharmacological blockade of LRP1 inhibited endocytic clearance of TIMP-3, leading to an increase in cell surface levels of the inhibitor that blocked TNF release. Following LPS stimulation, TIMP-3 levels on the surface of macrophages increased 4-fold within 4 h and continued to accumulate at 6 h, before a return to baseline levels at 8 h. This dynamic regulation of cell surface TIMP-3 levels was independent of changes in TIMP-3 mRNA levels, but correlated with shedding of LRP1. These results shed light on the basic mechanisms that maintain a regulated inflammatory response and ensure its timely resolution.

YAP is essential for tissue tension to ensure vertebrate 3D body shape.

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

Distinct biological events generated by ECM proteolysis by two homologous collagenases.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.

Engineered Tissue Inhibitor of Metalloproteinases-3 Variants Resistant to Endocytosis Have Prolonged Chondroprotective Activity.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity.

The MET/Vascular Endothelial Growth Factor Receptor (VEGFR)-targeted Tyrosine Kinase Inhibitor Also Attenuates FMS-dependent Osteoclast Differentiation and Bone Destruction Induced by Prostate Cancer.

The tyrosine kinase inhibitor TAS-115 that blocks VEGF receptor and hepatocyte growth factor receptor MET signaling exhibits antitumor properties in xenografts of human gastric carcinoma. In this study, we have evaluated the efficacy of TAS-115 in preventing prostate cancer metastasis to the bone and bone destruction using the PC3 cell line. When PC3 cells were injected into proximal tibiae in nude mouse, severe trabecular and cortical bone destruction and subsequent tumor growths were detected. Oral administration of TAS-115 almost completely inhibited both PC3-induced bone loss and PC3 cell proliferation by suppressing osteoclastic bone resorption. In an ex vivo bone organ culture, PC3 cells induced osteoclastic bone resorption when co-cultured with calvarial bone, but TAS-115 effectively suppressed the PC3-induced bone destruction. We found that macrophage colony-stimulating factor-dependent macrophage differentiation and subsequent receptor activator of NF-κB ligand-induced osteoclast formation were largely suppressed by adding TAS-115. The phosphorylation of the macrophage colony-stimulating factor receptor FMS and osteoclast related kinases such as ERK and Akt were also suppressed by the presence of TAS-115. Gene expression profiling showed that FMS expression was only seen in macrophage and in the osteoclast cell lineage. Our study indicates that tyrosine kinase signaling in host pre-osteoclasts/osteoclasts is critical for bone destruction induced by tumor cells and that targeting of MET/VEGF receptor/FMS activity makes it a promising therapeutic candidate for the treatment of prostate cancer patients with bone metastasis.

Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors.

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis.

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1(-/-)) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4(-/-) mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.

Anti-arthritic actions of ß-cryptoxanthin against the degradation of articular cartilage in vivo and in vitro.

An inverse correlation between the morbidity of rheumatoid arthritis and daily intake of ß-cryptoxanthin has been epidemiologically shown. In this study, we investigated the effects of ß-cryptoxanthin on the metabolism of cartilage extracellular matrix in vivo and in vitro. Oral administration of ß-cryptoxanthin (0.1-1 mg/kg) to antigen-induced arthritic rats suppressed the loss of glycosaminoglycans in articular cartilage, which is accompanied by the interference of aggrecanase-mediated degradation of aggrecan. Inhibition of the interleukin 1α (IL-1α)-induced aggrecan degradation by ß-cryptoxanthin was also observed with porcine articular cartilage explants in culture. ß-Cryptoxanthin (1-10 µM) dose-dependently down-regulated the IL-1α-induced gene expression of aggrecanase 1 (ADAMTS-4) and aggrecanase 2 (ADAMTS-5) in cultured human chondrocytes. Moreover, ß-cryptoxanthin was found to augment the gene expression of aggrecan core protein in chondrocytes. These results provide novel evidence that ß-cryptoxanthin exerts anti-arthritic actions and suggest that ß-cryptoxanthin may be useful in blocking the progression of rheumatoid arthritis and osteoarthritis.

BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice.

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERß. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male.

Abrogation of prostaglandin E-EP4 signaling in osteoblasts prevents the bone destruction induced by human prostate cancer metastases.

The metastasis of tumors to bone is known to be promoted by prostaglandin E2 (PGE2) produced by the tumor host stromal tissue. Although bone metastases frequently occur in prostate cancer patients, the significance of PGE2 in stromal responses to the tumor is not known. In this study, we report that PGE2 and its receptor EP4 play a pivotal role in bone destruction and metastasis in an experimental metastasis model of prostate cancer in nude mice. Using human prostate cancer PC-3 cells that are stably transfected with luciferase, we showed that the development of bone metastasis was accompanied by increased osteoclastic bone resorption in the bone metastasis microenvironment, and could be abrogated by an EP4 receptor antagonist. The growth of PC-3 cells in vitro was not influenced by PGE2 or by the EP4 receptor. However, cell-cell interactions between fixed PC-3 cells and host osteoblasts induced PGE2 production and RANKL expression in the osteoblasts. Addition of an EP4 antagonist suppressed both PGE2 and RANKL expression induced by the PC3-osteoblast interaction, which would have consequent effects on osteoclast activation and osteolysis. These results indicate that the blockage of PGE2-EP4 signaling prevents the bone destruction required for prostate cancer metastases, and that this is, in part due to the abrogation of bone cell responses. The study provides further evidence that an EP4 antagonist is a candidate for the treatment of prostate cancer in the blockade of bone metastasis.

A personal journey with matrix metalloproteinases.

I was given the honor of delivering the 2015 Lifetime Membership Award lecture at the International Proteolysis Society's annual meeting held in Penang, Malaysia in October 2015. It gave me an opportunity to look back on how I started my research on matrix metalloproteinases (MMPs) and how I continued to work on these proteinases for the next 42 years. This is a series of sketches from the personal journey that I took with MMPs, starting from the purification of metalloproteinases, cloning, structural studies, then to a more recent encounter, endocytic regulation of matrix-degrading metalloproteinases.

Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice.

UNLABELLED: Due to its ability to inhibit prometastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients, suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse. In mice, high systemic TIMP-1 levels increased the liver susceptibility towards metastasis by triggering the formation of a premetastatic niche. This promoted hepatic metastasis independent of origin or intrinsic metastatic potential of tumor cells. High systemic TIMP-1 led to increased hepatic SDF-1 levels, which in turn promoted recruitment of neutrophils to the liver. Both inhibition of SDF-1-mediated neutrophil recruitment and systemic depletion of neutrophils reduced TIMP-1-induced increased liver susceptibility towards metastasis. This indicates a crucial functional role of neutrophils in the TIMP-1-induced premetastatic niche. CONCLUSION: Our results identify TIMP-1 as an essential promoter of hepatic premetastatic niche formation.

Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2).

Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes.

Low density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytic clearance of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4): functional differences of non-catalytic domains of ADAMTS-4 and ADAMTS-5 in LRP1 binding.

Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

Metalloproteinase-dependent and -independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

DOTAM derivatives as active cartilage-targeting drug carriers for the treatment of osteoarthritis.

Targeted drug-delivery methods are crucial for effective treatment of degenerative joint diseases such as osteoarthritis (OA). Toward this goal, we developed a small multivalent structure as a model drug for the attenuation of cartilage degradation. The DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide)-based model structure is equipped with the cathepsin D protease inhibitor pepstatin A, a fluorophore, and peptide moieties targeting collagen II. In vivo injection of these soluble probes into the knee joints of mice resulted in 7-day-long local retention, while the drug carrier equipped with a scrambled peptide sequence was washed away within 6-8 h. The model drug conjugate successfully reduced the cathepsin D protease activity as measured by release of GAG peptide. Therefore, these conjugates represent a promising first drug conjugate for the targeted treatment of degenerative joint diseases.

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.

Generation of a mouse line harboring a Bi-transgene expressing luciferase and tamoxifen-activatable creER(T2) recombinase in cartilage.

We have used an aggrecan gene enhancer to generate a transgenic murine line (Acan-CreER-Ires-Luc) expressing firefly luciferase and tamoxifen activatable Cre recombinase (Cre-ER(T2) ). The expression and efficiency of the inducible Cre recombinase activity were tested in double transgenic mice created by crossing the Acan-CreER-Ires-Luc line with a Rosa26-lacZ reporter mouse. The expression pattern of the transgene of our line was restricted to cartilage from embryonic to adult stages. ß-galactosidase staining was observed in growth plate, articular cartilage, as well as fibrocartilage of meniscus, trachea, and intervertebral discs. Similar staining was observed in a previously described Agc1 (tm(IRES-creERT2)) murine line. The presence of luciferase in our transgene allows the visualization of the transgene expression in live animals. Weekly measurements from 2 to 8 weeks of age showed a reduction in luminescence in knee joints between 2 and 4 weeks of age, but stabilization thereafter. Following the surgical induction of osteoarthritis at 12 weeks of age, the level of luminescence remained the same in the knee joints for 8 weeks. This Acan-CreER-Ires-Luc murine line allows indirect monitoring of the transcriptional activity of the Acan gene via expression of luciferase, while the inducible Cre recombinase activity facilitates studies involving gain or loss of gene expression in cartilage.