Membrane Type 1 Matrix Metalloproteinase Regulates Cellular Invasiveness and Survival in Cutaneous Epidermal Cells Usha Nagavarapu, Kenneth Relloma,

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Membrane Type 1 Matrix Metalloproteinase Regulates Cellular Invasiveness and Survival in Cutaneous Epidermal Cells Usha Nagavarapu, Kenneth Relloma, G. Scott Herron Journal of Investigative Dermatology Volume 118, Issue 4, Pages 573-581 (April 2002) DOI: 10.1046/j.1523-1747.2002.01713.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 In situ hybridization of keratinocyte MT1-MMP transcripts. Cells transduced with MT1-as were grown on chamber slides and probed with FITC-labeled MT1-MMP anti-sense and sense RNA probe. The levels of MT1-MMP transcripts show a decrease in MT1-as transduced cells after 48 h of infection as compared with control and mock-transduced cells when probed with the anti-sense probe. The sense probe gave only a background signal in control and mock-transduced cells (data not shown), whereas a strong signal for MT1-as transcript was seen relative to controls and was further decreased at 72 h of transduction (data not shown). Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunofluorescence of MT1-MMP protein expression. Cells transduced with MT1-as and MT1-s constructs were grown on chamber slides, and fixed with 3 M glycine, 0.9% NaCl, pH 3.0 for 3 min and then blocked with 0.5% bovine serum albumin in phosphate-buffered saline for 30 min. Slides were incubated with MT1-MMP monoclonal antibody (Ab-4, Calbiochem) at 20 µg per ml. MT1-as-transduced cells show a significant decrease in cell associated MT1-MMP expression as compared with control, and MT1-s- and mock-transduced cells after 48 h of infection. Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Western blots of cellular MT1-MMP. Total cell lysates were loaded into each lane and probed with MT1-MMP rabbit polyclonal antibody. Lane 1: control (nontransduced cells). Lane 2: cells transduced with sense construct (MT1-s). Lane 3: cells transduced with anti-sense construct (MT1-as). Lane 4: cells transduced with mock construct. β-actin was used as an internal control. MT1-as-transduced cells (lane 3, anti-sense) show a significant decrease in cell-associated MT1-MMP expression as compared with controls (lane 1), MT1-s-transduced cells (lane 2), and mock-transduced cells (lane 4) after 48 h of infection. Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Zymographic analysis of pro-MMP-2 activation in conditioned media. (A) NHK cells were transduced with MT1-s, mock, and MT1-as constructs. Conditioned media was collected after 48 h and 72 h after transduction and analyzed by gelatin zymography. Quantitative results of zymograms were obtained by densitometry of the gels. Results represent the mean ± SD of two separate zymograms. Relative changes in pro-MMP-2 activation were calculated based on densitometry. An increase in activation was observed in MT1-s-transduced cells, whereas MT1-as-transduced cells showed a decrease in pro-MMP-2 activation as compared with control and mock-transduced cells. *p < 0.05 compared with mock-transduced cells. No significant difference was observed in the active MMP-2 band in control cells (nontransduced cells and mock-transduced cells). The intensity of the active MMP-2 band in control cells was approximately 3.5-fold higher than the MT1-as lane in NHK cells at 48 h after transduction. Furthermore, analysis of the conditioned media from NIK and SCC-25 cells also showed that MMP-2 activity was inhibited in MT1-as expressing cells (see B and C). Conversely, overexpression of MT1-MMP via LzrsMT1-MMP sense induces pro-MMP-2 activation. The MMP-2 activity in MT1-s was approximately 6–7-fold more than MT1-as in NIK cells at 48 h and 72 h after transduction. (B) NIK were transduced with MT1-s, mock, and MT1-as constructs. Conditioned media was collected after 48 h and 72 h after transduction and analyzed by gelatin zymography. Quantitative results of zymograms were obtained by densitometry of the gels. Results represent the mean ± SD of two separate zymograms. Relative changes in pro-MMP-2 activation were calculated based on densitometry. *p < 0.05 compared with mock-transduced cells. (C) SCC-25 cells were transduced with MT1-s, mock, and MT1-as constructs and conditioned media analyzed as described for NHK and NIK. *p < 0.05 compared with mock-transduced cells. Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Boyden chamber analysis of cellular invasion through collagen and fibronectin. NHK (A), NIK (B), and SCC-25 (C) cells were transduced with MT1-s and MT1-as constructs and 48 h and 72 h after transduction cells were analyzed for migration. Cells were also mock transduced with a mock vector. Results represent the mean ± SD of four separate experiments. Each bar represents the mean ± SD for cell migration of five wells. An increase in migration was observed in MT1-s-transduced cells, whereas MT1-as-transduced cells showed a decrease in migration as compared with control and mock-transduced cells on collagen and fibronectin in all three cell types. *p < 0.05 compared with mock-transduced cells. Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 DNA nuclear fragmentation and cell survival analysis. Basal level of apoptotic nuclear fragmentation in NHK (A-a), NIK (B-a), and SCC-25 (C-a) showed cells transduced with MT1-as had significantly higher internucleosomal DNA-histone fragment signals as compared with MT1-s, mock, and control cells. A decrease in the number of viable cells measured by the NHK (A-b), NIK (B-b), and SCC-25 (C-b) cells transduced by MT1-as as measured by a cell proliferation assay. Results represent the mean ± SD of four separate experiments. *p < 0.05 compared with mock-transduced cells. Journal of Investigative Dermatology 2002 118, 573-581DOI: (10.1046/j.1523-1747.2002.01713.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions