Differential Stromal Regulation of MMP-1 Expression in Benign and Malignant Keratinocytes  Kristiina Airola, Norbert E. Fusenig  Journal of Investigative Dermatology  Volume 116, Issue 1, Pages 85-92 (January 2001) DOI: 10.1046/j.1523-1747.2001.00223.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Regulation of MMP-1 and MMP-13 expression by collagen and cocultured fibroblasts in transformed keratinocytes. (a) Northern blot analysis of MMP-1 (transcript size 2.1 kb) and MMP-13 (2.0 and 2.5 kb) as well as the reference gene GAPDH (1.3 kb) expression by HaCaT, A-5, and A-5RT3 cells cultured on tissue culture plastic, on type I collagen gel, and under organotypic coculture conditions (collagen concentration always 3.2 mg per ml). (b) The effect of type I collagen on the expression of MMP-1 and MMP-13 by malignant SCC clones. RT-PCR was performed with 1 μg RNA of A-5RT1, II-4, SCL-I, and SCL-II cells isolated from cultures grown on plastic or on type I collagen gels. PCR amplification with primers for MMP-1 and β-actin was carried out semiquantitatively in one reaction tube and resulted in a 409 bp MMP-1 fragment and a 244 bp β-actin fragment. The 462 bp MMP-13 fragment was amplified separately. Journal of Investigative Dermatology 2001 116, 85-92DOI: (10.1046/j.1523-1747.2001.00223.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Matrix-dependent expression of MMP-1 in A-5RT3 cells. A-5RT3 cells were cultivated on tissue culture plastic, type I collagen gels, gelatin, and Matrigel. The expression of MMP-1 was analyzed by RT-PCR (a) and by western blot analysis (b). For western blot hybridization, equal aliquots of culture medium were fractioned in a 10% SDS-PAGE gel, and a monoclonal MMP-1 antibody was used to detect 52 and 54 kDa proteins corresponding to latent nonglycosylated and glycosylated forms of pro-MMP-1. Journal of Investigative Dermatology 2001 116, 85-92DOI: (10.1046/j.1523-1747.2001.00223.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Induction of MMP-1 expression in both epithelial and fibroblastic compartments of A-5RT3 organotypic cocultures. (a) Northern blot analysis of MMP-1 expression by epithelial cells and fibroblasts in organotypic cocultures. HaCaT, A-5, and A-5RT3 cells were cultivated in a coculture system on collagen gels populated with fibroblasts. At 14 d, epithelium was mechanically separated from the fibroblast-containing collagen gel and the RNA was isolated from both compartments. Fibroblast monocultures within a collagen gel were used as a control for the effect of three-dimensional collagen on collagenase expression by fibroblasts. (b) Western blot analysis of MMP-1 protein secretion. Equal aliquots of culture medium representing total MMP-1 production by the cocultures at time periods of 2–4 d and 12–14 d have been analyzed. Journal of Investigative Dermatology 2001 116, 85-92DOI: (10.1046/j.1523-1747.2001.00223.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 MMP-1 analysis in the organotypic cocultures. At 14 d, one coculture with HaCaT (a), A-5 (b), and A-5RT3 (c) cells was cut into two halves, to obtain one formalin-fixed specimen for histologic analysis (a-c) and one freshly frozen tissue for in situ hybridization (d-h). Histology of the A-5RT3 coculture shows a collagen gel significantly reduced in thickness (c), compared with that with HaCaT and A-5 cells. (d) In situ hybridization of an A-5RT3 coculture with an MMP-1 antisense probe. Dark-field exposure shows the expression of MMP-1 mRNA in both epithelium (arrows) and fibroblasts (arrowheads). (e) Bright-field exposure of (d) with higher magnification showing the cellular localization of the signal, and (f) A-5RT3 coculture hybridized with MMP-1 sense probe for the purpose of negative control. Scale bars: (a-c, e) 25 μm; (d, f) 50 μm. Journal of Investigative Dermatology 2001 116, 85-92DOI: (10.1046/j.1523-1747.2001.00223.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 MMP-1 mRNA is expressed by A-5RT3 cells in vivo. (a) Schematic picture of the transplantation model. Transplants were prepared by seeding 2 × 105 cells on a type I collagen gel and were transplanted the following day onto the dorsal muscle fascia of nude mice (Swiss/c nu/nu backcrosses). (b) In situ hybridization of a 4 d transplant of A-5RT3 tumor cells and (c) corresponding dark-field exposure with lower magnification showing expression of MMP-1 mRNA in the A-5RT3 cells residing on top of a collagen gel (arrows). (d), (e) At 15 d, invasion of the collagen gel by tumor cells and activation of the mouse stroma is observed. (d) Dark-field exposure of MMP-1 expressing A-5RT3 cells (arrows), and (e) the corresponding bright-field exposure. (f) – (h) At 4 wk tumor cells are in contact with the stroma. MMP-1 mRNA is expressed at the tumor-stroma border (arrows) in dark-field (f) and bright-field (g) exposures. Comparable to the induction of MMP-1 expression by fibroblasts in the A-5RT3 cocultures, mouse collagenase (MMP-13) expression is induced in fibroblastic cells (arrowheads) at the stroma bordering the tumor, as detected with an antisense mouse MMP-13 probe (h). (i), (j) An A-5 transplant at 6 wk. In contrast to the A-5RT3 cells, no MMP-1 mRNA is detected in the benign epithelium formed on the mouse granulation tissue: (i) bright-field and (j) dark-field exposure. Scale bars: (b, d-j) 25 μm; (c) 50 μm. Journal of Investigative Dermatology 2001 116, 85-92DOI: (10.1046/j.1523-1747.2001.00223.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions