Coexpression of Integrin αvβ3 and Matrix Metalloproteinase-2 (MMP-2) Coincides with MMP-2 Activation: Correlation with Melanoma Progression  Uta B. Hofmann, Johan R. Westphal, Erwin T. Waas, Jürgen C. Becker, Dirk J. Ruiter, Goos N.P. van Muijen  Journal of Investigative Dermatology  Volume 115, Issue 4, Pages 625-632 (October 2000) DOI: 10.1046/j.1523-1747.2000.00114.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of membrane-bound and soluble MMP-2 in human melanoma cell lines. (A) Zymography of cell lysates prepared from cultured untransfected (MV3, BLM) or mock-transfected (MV3-neo, BLM-neo), or β3-transfected (MV3-β3, BLM-β3) cells. Functionally active MMP-2 is present exclusively in the αvβ3 transfectants. SFCM was analyzed by zymography (B) and western blotting (C). Secretion of active MMP-2 is restricted to the β3-negative cell line as determined by zymography. Purified MMP-2 protein was used as controls (right lane). Position and molecular size of markers are indicated. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of MT1-MMP and TIMP-2 in human melanoma cell lines. Western blot analysis for MT1-MMP of SFCM (A) and cell lysates (B) prepared from cultured untransfected (MV3) or mock-transfected (MV3-neo), or β3-transfected (MV3-β3) cells. (C) Western blot analysis of soluble TIMP-2 in SFCM of cell lines. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of MMP-2 mRNA in xenografts derived from parental, neo- and β3-transfected MV3 and BLM cells. Reverse transcription–PCR analysis was performed on RNA extracted from frozen xenograft sections. An incubation in which template cDNA was omitted was used as a negative control; RNA from the MV3 cell line was used as a positive control. Reverse transcription–PCR of phorphobilinogen-deaminase mRNA was used as a control (lower panel). Reverse transcription–PCR was performed in duplicate. Length of PCR products are indicated. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of MMP-2 in xenografts derived from parental, neo- and β3-transfected MV3 and BLM cells. Zymography was performed on xenograft tissue extracts (20 μg). (A) Increased levels of activated MMP-2 were present in xenografts of the β3-transfected cell lines. (B) Expression of different processing forms of MMP-2 as a percentage of total MMP-2 protein. Intermediate (64 kDa) and active (62 kDa) MMP-2 are clearly increased in xenografts derived from the β3-transfected cell lines. Purified MMP-2 protein was used as control in zymograghy (right lane). Position and molecular size of marker is indicated. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunostaining of MMP-2 and αvβ3 in human melanoma lesions. No staining of tumor cells in MIS for MMP-2 (A) and αvβ3 (B); arrows indicate isolated MMP-2-positive dendritic cells in the epidermis and αvβ3-positive blood vessels. Strong staining of MMP-2 (C) and αvβ3 (D) in tumor cells in an aPM. Double labeling for MMP-2 (blue) and αvβ3 (red). Note heterogeneous expression of MMP-2 (blue), αvβ3 (red) and MMP-2/αvβ3 (purple) in an aPM (E) and a MM (F); high percentage of tumor cells show coexpression of MMP-2 and αvβ3 (purple, arrows), in these sections no counterstaining was used. Colocalization of MMP-2 and αvβ3 in an aPM (G–I) and MM (J–L) with double immunofluorescent labeling and confocal laser microscopy analysis. Green indicates αvβ3 expression, and red indicates MMP-2 expression. Yellow indicates colocalization. Scale bar: (A, B) 90 μm; (C, D) 60 μm; (E, F) 30 μm; (G–L) 10 μm. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 MMP-2 and αvβ3 expression in melanoma lesions as a percentage of immunoreactive melanocytic cells. All melanoma in situ lesions are negative for MMP-2 and αvβ3. Both, the percentage of MMP-2- or αvβ3-positive tumor cells and the percentage of double-stained tumor cells were increased in aPM and MM. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Lysates prepared from tissue extracts of 11 different MM were analyzed by zymography. Purified MMP-2 protein was used as control in zymograghy (right lane). Position and molecular size of marker is indicated. Journal of Investigative Dermatology 2000 115, 625-632DOI: (10.1046/j.1523-1747.2000.00114.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions