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

Discoidin domain receptor 2 mediates collagen-induced activation of membrane-type 1 matrix metalloproteinase in human fibroblasts

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity, including fibroblasts and invasive cancer cells. However, pathways of MT1-MMP up-regulation are not clearly understood. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it up-regulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation and its physiological relevance are not known. In this study, we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not the β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited the collagen-induced MT1-MMP-dependent activation of pro-MMP-2 and up-regulation of MT1-MMP at the gene and protein levels. Interestingly, DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited the MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell-surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signaling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without the non-helical telopeptide region compared with intact collagen fibrils. Furthermore, DDR2-dependent MT1-MMP activation by cartilage was found to be more efficient when the tissue was partially damaged. These data suggest that DDR2 is a microenvironment sensor that regulates fibroblast migration in a collagen-rich environment

Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity▿

The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 μM is shown to bind with about 50 μM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a β-structure (39.8% ± 2.2%, based on 5 samples) with negligible contribution of α-helix structure. When bound to Chlide, the β-structure content in the complex is reduced to 21.6% ± 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 μM, and 21.6 μM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity

The Dimer Interface of the Membrane Type 1 Matrix Metalloproteinase Hemopexin Domain: CRYSTAL STRUCTURE AND BIOLOGICAL FUNCTIONS*

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion