Inflammatory cytokines tumor necrosis factor-␣ and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C 2 -and C 6 -ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C 2 -ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C 2 -ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C 2 -ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/ JNK activator SEK1, or SAPK␤. In addition, ceramidedependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.

Here, we have examined the role of distinct MAPK pathways in the regulation of collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by human skin fibroblasts. Tumor necrosis factor-␣ rapidly and transiently activated ERK1/2 and JNK in fibroblasts, whereas the activation of p38 MAPK was more persistent. Inhibition of p38 activity by SB203580 markedly (by 80 -90%) inhibited induction of MMP-1 and MMP-3 expression by tumor necrosis factor-␣, whereas blocking the activation of ERK1/2 by PD98059 had no effect. Activation of endogenous ERK1/2 by adenovirusmediated transfer of constitutively active MEK1 resulted in potent induction of MMP-1 and MMP-3 expression. Activation of endogenous or adenovirally expressed p38␣ by adenovirally delivered constitutively active MKK3b and MKK6b also enhanced MMP-1 and MMP-3 expression and augmented the up-regulatory effect of ERK1/2 activation on the expression of these MMPs. Activation of ERK1/2 resulted in induction of c-jun, junB, and c-fos expression, whereas activation of p38 alone had no effect. In contrast, activation of p38␣ resulted in marked stabilization of MMP-1 and MMP-3 mRNAs. These results identify two distinct and complementary signaling mechanisms mediating induction of MMP-1 and MMP-3 expression in dermal fibroblasts: AP-1-dependent transcriptional activation via the ERK1/2 pathway and AP-1-independent enhancement via p38␣ MAPK by mRNA stabilization. It is conceivable that both modes of action play an important role in controlling the proteolytic phenotype of fibroblasts, e.g. in wound repair and tumor invasion.

Treatment with the lipid second messenger, ceramide, activates extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase, and p38 in human skin fibroblasts and induces their collagenase-1 expression (Reunanen, N., Westermarck, J., Hä kkinen, L., Holmströ m, T. H., Elo, I., Eriksson, J. E., and Kä hä ri, V.-M. (1998) J. Biol. Chem. 273, 5137-5145). Here we show that C 2 -ceramide inhibits expression of type I and III collagen mRNAs in dermal fibroblasts, suppresses pro␣2(I) collagen promoter activity, and reduces stability of type I collagen mRNAs. The down-regulatory effect of C 2 -ceramide on type I collagen mRNA levels was abrogated by protein kinase C inhibitors H7, staurosporine, and Ro-31-8220 and potently inhibited by a combination of MEK1,2 inhibitor PD98059 and p38 inhibitor SB203580. Activation of ERK1/2 by adenovirus-mediated expression of constitutively active MEK1 resulted in marked down-regulation of type I collagen mRNA levels and production in fibroblasts, whereas activation of p38 by constitutively active MAPK kinase-3b and MAPK kinase-6b slightly up-regulated type I collagen expression. These results identify the ERK1/2 signaling cascade as a potent negative regulatory pathway with respect to type I collagen expression in fibroblasts, suggesting that it mediates inhibition of collagen production in response to mitogenic stimulation and transformation. Fibrillar type I collagen is an abundant component of the extracellular matrix (ECM)1 of various human connective tissues. Type I collagen is a heterotrimeric molecule consisting of two ␣1 chains and one ␣2 chain. The expression of pro␣1(I) and pro␣2(I) collagen genes is coordinately regulated during tissue development, growth, and repair resulting in their synthesis in a 2:1 ratio (1). The expression of type I collagen in fibroblasts is stimulated by transforming growth factor-␤ (TGF-␤) (2), interleukin-4 (3), and connective tissue growth factor (4) and inhibited by epidermal growth factor (5), tumor necrosis factor-␣ (TNF-␣) (2), interferon-␥ (2), glucocorticoids (6), and tumor promoters (7,8) and by contact with three-dimensional collagen (9). Excessive deposition of type I collagen is observed e.g. in fibrosis of skin, lungs, and liver (see Ref. 1). TNF-␣ is a proinflammatory cytokine that inhibits the formation of ECM by suppressing the expression of type I collagen and by inducing the production of matrix metalloproteinases (MMPs) by fibroblasts (2, 10). Binding of TNF-␣ to its 55-kDa cell surface receptor activates neutral sphingomyelinase, which hydrolyzes cell membrane sphingomyelin to the lipid second messenger, ceramide. We have recently shown that synthetic cell-permeable C 2 -ceramide enhances fibroblast collagenase-1 (MMP-1) expression via coordinate activation of the following three distinct mitogen activated protein kinase (MAPK) pathways: extracellular signal-regulated kinase-1/2 (ERK1/2), cJun N-terminal kinase (JNK), and p38 (11).Here we show that activation of ceramide signaling pathway potently inhibits the ex...

Expression of collagenase‐3 [matrix metalloproteinase‐13 (MMP‐13)] has been previously demonstrated in squamous‐cell carcinomas of both the head and neck and the vulva, cutaneous basal‐cell carcinomas, chondrosarcomas and melanomas. Using in situ hybridization, MMP‐13 mRNA expression was detected in 13 of 23 (52%) urinary bladder transitional‐cell carcinomas (TCCs). Expression was restricted to cells in the invading edges of tumors. No expression of MMP‐13 mRNA could be detected in normal urothelium. As detected by immunohistochemistry, MMP‐13 protein showed an expression pattern similar to that of MMP‐13 mRNA. Expression of MMP‐13 mRNA and protein was also detected in 2 bladder carcinoma cell lines (RT4 and T24). In these cell lines, TNF‐α potently induced MMP‐13 mRNA expression. Retinoids and a selective p38 inhibitor, SB203580, potently inhibited MMP‐13 mRNA expression. Our results demonstrate MMP‐13 expression in human urinary bladder carcinoma cells in vivo and in vitro and suggest that MMP‐13 may serve as a marker for transformation and invasion in urinary bladder TCCs. Int. J. Cancer 88:417–423, 2000. © 2000 Wiley‐Liss, Inc.