Global, but not chondrocyte-specific, MT1-MMP deficiency in adult mice causes inflammatory arthritis.

Membrane-type I metalloproteinase (MT1-MMP/MMP14) plays a key role in various pathophysiological processes, indicating an unaddressed need for a targeted therapeutic approach. However, mice genetically deficient in Mmp14 show severe defects in development and growth. To investigate the possibility of MT1-MMP inhibition as a safe treatment in adults, we generated global Mmp14 tamoxifen-induced conditional knockout (Mmp14kd) mice and found that MT1-MMP deficiency in adult mice resulted in severe inflammatory arthritis. Mmp14kd mice started to show noticeably swollen joints two weeks after tamoxifen administration, which progressed rapidly. Mmp14kd mice reached a humane endpoint 6 to 8 weeks after tamoxifen administration due to severe arthritis. Plasma TNF-α levels were also significantly increased in Mmp14kd mice. Detailed analysis revealed chondrocyte hypertrophy, synovial fibrosis, and subchondral bone remodeling in the joints of Mmp14kd mice. However, global conditional knockout of MT1-MMP in adult mice did not affect body weight, blood glucose, or plasma cholesterol and triglyceride levels. Furthermore, we observed substantial expression of MT1-MMP in the articular cartilage of patients with osteoarthritis. We then developed chondrocyte-specific Mmp14 tamoxifen-induced conditional knockout (Mmp14chkd) mice. Chondrocyte MT1-MMP deficiency in adult mice also caused apparent chondrocyte hypertrophy. However, Mmp14chkd mice did not exhibit synovial hyperplasia or noticeable arthritis, suggesting that chondrocyte MT1-MMP is not solely responsible for the onset of severe arthritis observed in Mmp14kd mice. Our findings also suggest that highly cell-type specific inhibition of MT1-MMP is required for its potential therapeutic use.

Multienzyme activity profiling for evaluation of cell-to-cell variability of metabolic state.

In solid organs, cells of the same "type" can vary in their molecular phenotype. The basis of this state variation is being revealed by characterizing cell features including the expression pattern of mRNAs and the internal distribution of proteins. Here, the variability of metabolic state between cells is probed by enzyme activity profiling. We study individual cells of types that can be identified during the post-mitotic phase of oogenesis in Xenopus laevis. Whole-cell homogenates of isolated oocytes are used for kinetic analysis of enzymes, with a focus on the initial reaction rate. For each oocyte type studied, the activity signatures of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase 1 (MDH1) vary more between the homogenates of single oocytes than between repeat samplings of control homogenates. Unexpectedly, the activity signatures of GAPDH and MDH1 strongly co-vary between oocytes of each type and change in strength of correlation during oogenesis. Therefore, variability of the kinetic behavior of these housekeeping enzymes between "identical" cells is physiologically programmed. Based on these findings, we propose that single-cell profiling of enzyme kinetics will improve understanding of how metabolic state heterogeneity is related to heterogeneity revealed by omics methods including proteomics, epigenomics, and metabolomics.

Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor.

Low-density lipoprotein receptor (LDLR) mediates clearance of plasma LDL cholesterol, preventing the development of atherosclerosis. We previously demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) cleaves LDLR and exacerbates the development of atherosclerosis. Here, we investigated determinants in LDLR and MT1-MMP that were critical for MT1-MMP-induced LDLR cleavage. We observed that deletion of various functional domains in LDLR or removal of each of the five predicted cleavage sites of MT1-MMP on LDLR did not affect MT1-MMP-induced cleavage of the receptor. Removal of the hemopexin domain or the C-terminal cytoplasmic tail of MT1-MMP also did not impair its ability to cleave LDLR. On the other hand, mutant MT1-MMP, in which the catalytic domain or the MT-loop was deleted, could not cleave LDLR. Further Ala-scanning analysis revealed an important role for Ile at position 167 of the MT-loop in MT1-MMP's action on LDLR. Replacement of Ile167 with Ala, Thr, Glu, or Lys resulted in a marked loss of the ability to cleave LDLR, whereas mutation of Ile167 to a non-polar amino acid residue, including Leu, Val, Met, and Phe, had no effect. Therefore, our studies indicate that MT1-MMP does not require a specific cleavage site on LDLR. In contrast, an amino acid residue with a hydrophobic side chain at position 167 in the MT-loop is critical for MT1-MMP-induced LDLR cleavage.