Inhibition of collagenase-3 (MMP-13) expression in transformed human keratinocytes by interferon-γ is associated with activation of extracellular signal-regulated kinase-1,2 and STAT1

Abstract

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate speci®city and restricted expression. MMP-13 is speci®cally expressed by transformed human keratinocytes in squamous cell carcinomas in vivo and its expression correlates with their invasion capacity. Here, we show, that interferon-g (IFN-g) markedly inhibits expression of MMP-13 by human cutaneous SCC cells (UT-SCC-7) and by ras-transformed human epidermal keratinocytes (A-5 cells) at the transcriptional level. In addition, IFN-g inhibits collagenase-1 (MMP-1) expression in these cells. IFN-g abolished the enhancement of MMP-13 and MMP-1 expression by transforming growth factor-b (TGF-b) and tumor necrosis factor-a (TNF-a), and inhibited invasion of A-5 cells through type I collagen. IFN-g also rapidly and transiently activates extracellular signal-regulated kinase 1,2 (ERK1,2) and blocking ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by speci®c MEK1,2 inhibitor PD98059 partially (by 50%) prevents Ser-727 phosphorylation of STAT1 and suppression of MMP-13 expression by IFN-g. Furthermore, Ser-727 phosphorylation of STAT1 by ERK1,2, or independently of ERK1,2 activation is associated with marked reduction in MMP-13 expression. These observations identify a novel role for IFN-g as a potent inhibitor of collagenolytic activity and invasion of transformed squamous epithelial cells, and show that inhibition of MMP-13 expression by IFN-g involves activation of ERK1,2 and STAT1. Oncogene (2000) 19, 248 ± 257.

Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo

“…Together, these data show that suppression of MMP-13 expression by antisense ribozyme results in inhibition of SCC cell proliferation and in apoptosis, suggesting a role for MMP-13 in SCC cell proliferation and survival. These findings are in accordance with our previous observations showing that, in SCC cells treated with interferon-g or infected with p53 adenovirus, downregulation of MMP-13 expression precedes the onset of cell death (Ala-aho et al, 2000(Ala-aho et al, , 2002a. Whether the promoting effect of MMP-13 on cell growth and survival involves proteolytic processing of growth-promoting factors or cleavage of proapoptotic proteins is not known at present.…”

“…The average relative number of proliferating cells was compared to the average tumor sizes in each group Anti-MMP-13 ribozyme inhibits tumor growth R Ala-aho et al suppressor markedly reduces their invasion capacity (Ala-aho et al, 2000(Ala-aho et al, , 2002a. In addition, expression of MMP-13 by human fibrosarcoma (HT-1080) cell line increases its invasion capacity through type I collagen and Matrigel (Ala-aho et al, 2002b).…”

“…The production of MMP-13 and MMP-1 was determined by Western blot analysis (Ala-aho et al, 2000) using monoclonal antibody (181-15A12) against human MMP-13 (Calbiochem, San Diego, CA) (1 : 100) and rabbit polyclonal antibody against human MMP-1 (kindly provided by Dr H BirkedalHansen, NIDR, Bethesda, MD, USA) (1 : 5000), followed by detection of specifically bound primary antibodies with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL; Amersham).…”

“…The gels were washed for 30 min in 50 mM Tris, 0.02% NaN 3 and 2.5% Triton X-100, pH 7.5, and for 30 min in the same buffer supplemented with 5 mM CaCl 2 and 1 mM ZnCl 2 . The gels were then incubated in 50 mM Tris, 0.02% NaN 3 , 5 mM CaCl 2 and 1 mM ZnCl 2 for 24 h at 371C, fixed in 50% methanol/7% acetic acid, stained with 0.2% Coomassie Blue G250, and photographed (Ala-aho et al, 2000).…”

Serpin Peptidase Inhibitor Clade A Member 1 (SerpinA1) Is a Novel Biomarker for Progression of Cutaneous Squamous Cell Carcinoma

“…The functions of the inhibitors PD98059 and SB203580 were verified by Western blot analysis of the cell lysates with antibodies specific for phospho-ERK1/2 and phospho-CREB (both from Cell Signaling Technology Inc., Beverly, MA), respectively. 25,27 …”