MT3-MMP in Matrix-Dependent Melanoma Invasion Figure 3. MT3-MMP silencing inhibits fibrin invasion and enhances collagen invasion of WM852 cells. (A) MT1-MMP and MT3-MMP mRNA expression in WM852 cells transfected with control siRNA (Ctrl) or siRNAs targeting MT1-MMP (siMT1) or MT3-MMP (siMT3-1 and siMT3-2) were quantified by qPCR (n = 3). (B and C) The protein levels of MT1-MMP (B) and MT3-MMP (C) were assessed by immunoblotting after transient transfection of the siRNAs or MT3-MMP cDNA (MT3-MMP) as indicated. MT1-MMP was detected as a 60 kDa band and MT3-MMP as 65/60 kDa bands that were downregulated by the corresponding siRNAs. An additional MT3-MMP fragment of 37 kDa in size was detected in the cells after transient transfection with MT3-MMP cDNA. Ponceau-staining served as a loading control. Asterix marks a non-specific band. (D) Cell surface MT3-MMP was detected by surface biotinylation of cells transfected with control siRNA or the most efficient MT3-MMP siRNA (siMT3-1), followed by immunoprecipitation. Asterix marks a non-specific band. (E) Light micrographs of fibrin and collagen cross-sections visualize the invasion of WM852 cells transfected with the indicated siRNAs. The cells were plated atop 3D fibrin and type I collagen 1 d after siRNA transfections and allowed to invade for 5 d. Quantitative results are expressed as the number of invasive foci per microscopic field (n = 3). doi:10.1371/journal.pone.0028325.g003 MT3-MMP facilitates Bowes melanoma cell invasion into fibrin but restricts collagen invasion Given the increased collagen-invasive activity of WM852 cells after MT3-MMP silencing, we next sought to define the effects of stable MT3-MMP expression on the invasion of Bowes melanoma cells, which normally display almost undetectable levels of the transcript. Stable transfectants increased MT3-MMP mRNA expression ,9-fold (a level ,50% lower than endogenous expression in WM852 cells) while stimulating fibrin invasion 2.560.3 fold (n = 3; p = 0.02; Fig. 4A and B). Importantly, increasing MT3-MMP expression markedly reduced collagen invasion by ,80% (0.1760.07, n = 3; p = 0.021; Fig. 4A), thus shifting the invasive phenotype of Bowes cells towards that displayed by WM852 cells. In these stable Bowes cells, MT3-MMP was detected predom- inantly as a ,37 kDa cleaved fragment by immunoblotting (Fig. 4C), and GM6001 enhanced the levels of 60–65 kDa proteins that most likely correspond to the full-length and activated or differentially glycosylated forms of MT3-MMP protein (Fig. 4C; [34]). After inhibition of lysosomal degradation by bafilomycin A, mostly the cleaved 37-35 kDa fragments of MT3-MMP were increased (Fig. 4C). Nevertheless, the major MT3-MMP form exposed to biotinylation on the surface of Bowes cells was the full-length/activated MT3-MMP, whereas only a faint band corresponding to the 37 kDa fragment was detectable by surface biotinylation (Fig. 4D). Notably, the cleavage to 37- 35 kDa fragments was inhibited in tandem with dramatically increased full-length/activated MT3-MMP by simultaneous treatment with GM6001 and bafilomycin A (Fig. 4C). Since both GM6001 and the inactivating E247A mutation (MT3E/A, Fig. S2) that stabilized the 60–65 kDa MT3-MMP also enhanced the levels of MT3-MMP on the cell surface (Fig. 4D and E), these results suggest that constitutive autocatalytic processing of the cell surface MT3-MMP followed by internalization and lysosomal degradation of the membrane-bound fragments resulted in unexpectedly rapid MT3-MMP turn-over. MT3-MMP also slightly reduced the levels of endogenous MT1-MMP protein in cells cultured on fibrin by ,26% (0.7460.2), which was reversed by the MMP inhibitor GM6001 (1.1460.2; Fig. 3C). Likewise, MT3-MMP enhanced the cleavage of MT1-MMP to inactive 37–43 kDa fragments, which were observed after bafilomycin treatment (1.760.2 -fold increased processed/total ratio relative to mock; Fig. 4C). Moreover, MT3- MMP, but not MT3E/A, induced the processing of overexpressed MT1-MMP mainly to the ,37 kDa fragment after transient co- transfection (Fig. 4F). This raised the possibility of in trans processing of the MT-MMPs by a mechanism that would resemble the autocatalytic cleavages in MT1-MMP homo-dimers or oligomers [35,36]. Since such processing would require close physical proximity or interactions, co-immunoprecipitation anal- yses were carried out. Consistently with an MT1-MMP cleavage by MT3-MMP, MT1-MMP was co-immunoprecipitated in the same complexes with MT3-MMP and MT3E/A (Fig. 4F). However, MT1-MMP expression was less affected by MT3- MMP in cells cultured on collagen (,20% reduction of 60 kDa MT1-MMP; 1.460.2 -fold increased processed/total ratio), suggesting that the regulation of MT1-MMP protein levels may not be the only mechanism how MT3-MMP alters collagen invasion. MT3-MMP reduces cell surface MT1-MMP and functions as a matrix composition-dependent effector of cell invasion To define the interrelated functions of MT1-MMP and MT3- MMP in cell invasion, COS-1 cells, which are largely devoid of endogenous MMPs, were engineered to express MT1-MMP, PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28325