MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration

Matrix metalloproteinase-3 (MMP-3) expression is promoted after pulpotomy, and application of MMP-3 to dental pulp after pulpotomy accelerates angiogenesis and hard tissue formation. However, the mechanism by which MMP-3 promotes dental pulp wound healing is still unclear. Connective tissue growth factor/CCN family 2 (CTGF/CCN2), a protein belonging to the CCN family, is considered to participate in wound healing, angiogenesis, and cell migration. In this study, we examined the involvement of CTGF/CCN2 in MMP-3-induced cell migration in human dental pulp (fibroblast-like) cells. In human dental pulp cells, MMP-3 promoted cell migration, but this effect was clearly blocked in the presence of anti-CTGF/CCN2 antibody. MMP-3 provoked mRNA and protein expression and secretion of CTGF/CCN2 in a concentration- and time-dependent manner. The MMP-3 inhibitor NNGH failed to suppress MMP-3-induced CTGF/CCN2 protein expression. The potent dynamin inhibitor dynasore clearly inhibited MMP-3-induced CTGF/CCN2 expression. These results strongly suggest that MMP-3 induces CTGF/CCN2 production independently of the protease activity of MMP-3 and dependently on dynamin-related endocytosis, which is involved in cell migration in human dental pulp cells.     

Unexpected Transcellular Protein Crossover Occurs During Canonical DNA Transfection

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP‐LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30–50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection. J. Cell. Biochem. 115: 2047–2054, 2014. © 2014 Wiley Periodicals, Inc.