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Matrix Metalloproteinase-19 Expression in Dermal Wounds and by Fibroblasts in Culture
ORIGINAL ARTICLE See related Commentary on page xix
Matrix Metalloproteinase-19 Expression in Dermal Wounds and by Fibroblasts in Culture Niina Hieta,w Ulla Impola,z Carlos Lo¤pez-Ot|¤ n,y Ulpu Saarialho-Kere,z and Veli-Matti Khriw8
Centre for Biotechnology, University of Turku and Abo Akademi University,Turku, Finland; wDepartment of Dermatology and 8Department of Medical
Biochemistry, University of Turku,Turku, Finland; yDepartamento Bioquimica y BiologIØ a Molecular, Instituto Universitario de Oncologia, Universidad de Oviedo, Oviedo, Spain; zDepartment of Dermatology, University of Helsinki and Biomedicum Helsinki, Helsinki
Here, we have examined the expression of matrix metalloproteinase-19 (MMP-19) in human cutaneous wounds and by human skin ¢broblasts in culture. Expression of MMP-19 was detected by immunohistochemistry in ¢broblasts, capillary endothelial cells, and macrophages in the dermal layer of large granulating wounds, as well as in chronic venous and decubitus ulcers. MMP-19 mRNA expression and pro-MMP-19 production by dermal ¢broblasts in culture was potently enhanced by tumor necrosis factor-a (TNF-a). Induction of MMP-19 expression by TNF-a was prevented partially by blocking the activation of extracellular signal-regulated kinase (ERK)-1/2 by PD98059 and p38 activity by SB203580. Activation of ERK1/2 by adenovirus-mediated delivery of constitutively active MAPK/ERK kinase 1 resulted in the induction of MMP-19 expression. Activation of p38
alone by adenovirally delivered constitutively active MAPK kinase 3b (MKK3b) and MKK6b also enhanced MMP-19 production, and the most potent induction of MMP-19 expression was noted when ERK1/2 was activated in combination with p38. Activation of c-Jun Nterminal kinase (JNK). Abundant pro-MMP-19 production by ¢broblasts was associated with proteolytic processing of secreted pro-MMP-19. These results suggest a role of MMP-19 in cutaneous wound repair and identify three distinct signaling pathways, which coordinately mediate induction of MMP-19 expression in ¢broblasts: mitogen-activated ERK1/2 pathway and stress-activated JNK and p38 pathways, of which control proteolytic activity of dermal ¢broblasts. Key word: mitogen-activated protein kinase. J Invest Dermatol 121:997 ^1004, 2003
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normal tissue architecture, e.g., in rheumatoid arthritis, osteoarthritis, autoimmune blistering skin disorders, cutaneous photoaging, and tumor invasion and metastasis (Johansson et al, 2000a). Human MMP-19 cDNA was initially cloned from liver and mammary gland and was also identi¢ed as an autoantigen in in£amed rheumatoid synovium (Cossins et al, 1996; Pends et al, 1997; Sedlacek et al, 1998). The catalytic domain of MMP-19 hydrolyzes type IV collagen, laminin, nidogen, large tenascin-C, ¢bronectin, type I gelatin, aggrecan, and cartilage oligomeric matrix protein, but not triple-helical type I collagen (Stracke et al, 2000a, b). The activity of MMP-19 catalytic domain is inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2), TIMP-3, and TIMP- 4 and less e⁄ciently by TIMP-1 (Stracke et al, 2000b). Latent MMP-19 is activated by autoproteolysis, and in contrast to several other MMPs, MMP-19 is not known to activate any other latent MMPs (Stracke et al, 2000b). Expression of MMP-19 has been detected in injured and in£amed synovium, especially in endothelial cells of synovial capillaries, suggesting a role for MMP-19 in angiogenesis (Kolb et al, 1999; Konttinen et al, 1999). Expression of MMP-19 is also enhanced in blood mononuclear cells of patients with multiple sclerosis (Ramanathan et al, 2001). In myoepithelial cells and epidermal keratinocytes, the expression of MMP-19 is lost upon malignant transformation (Djonov et al, 2001; Impola et al, 2003). In culture, MMP-19 is expressed by several types of malignant and normal cells, including epidermal keratinocytes and dermal ¢broblasts (Grant et al, 1999; Impola et al, 2003). In this study, we have examined the role and regulation of MMP-19 in dermal wound repair in vivo and by human skin
atrix metalloproteinases (MMP) are a family of structurally related zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the extracellular matrix (ECM) (see Nagase and Woessner, 1999; Johansson et al, 2000a). At present, 24 human members of MMP gene family have been identi¢ed, and they are often classi¢ed into subgroups of collagenases, gelatinases, stromelysins, membrane-type MMP, and other MMPs based on their structure and substrate speci¢city (Nagase and Woessner, 1999). There is considerable amount of evidence that MMPs play an important role in connective tissue remodeling in physiologic situations including developmental tissue morphogenesis, tissue repair, and angiogenesis. In addition, MMPs also regulate cellular growth factor response and in£ammatory reaction by cleavage of growth factors, cytokines, chemokines, and their receptors (Sternlicht and Werb 2001). MMPs also play an important role in destruction of
Manuscript received December 11, 2002; revised March 14, 2003; accepted for publication May 6, 2003 Reprint requests to: Veli-Matti Kahari, MD, PhD, Center for Biotechnology, University of Turku, Tykist˛katu 6B, FIN-20520 Turku, Finland. Email: veli-matti.kahari@utu.¢ Abbreviations: DMEM, Dulbecco’s modi¢ed Eagle’s medium; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; MKK, MAPK kinase; MEK, MAPK/ ERK kinase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; TNF-a, tumor necrosis factor-a.
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 997
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¢broblasts in culture. MMP-19 was noted in dermal ¢broblasts, macrophages, and endothelial cells during normal cutaneous wound repair and in chronic cutaneous ulcers. We also show, that tumor necrosis factor-a (TNF-a) potently enhances MMP-19 expression and that this is mediated coordinately via extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results provide evidence for the role of MMP-19 in remodeling of dermal ECM during wound repair. In addition, our results identify two distinct mechanisms of inducing proteolytic capacity of dermal ¢broblasts: mitogen-responsive ERK1/2 pathway and stress-activated p38 MAPK, suggesting that both play a role in controlling the MMP-19 expression and proteolytic phenotype of ¢broblasts, e.g., during wound repair. MATERIALS AND METHODS Materials Human recombinant TNF-a and epidermal growth factor (EGF), MAPK/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 (20 -amino30 -methoxy£avone), and p38 inhibitor SB203580 ([4 -(£uorophenyl)-2-(4 methylsul¢nylphenyl)-5-(4 -pyridyl)1H-imidazole]) were obtained from Calbiochem (San Diego, CA). Human recombinant transforming growth factor-b and platelet-derived growth factor-AA were obtained from Sigma Chemical Co. (St. Louis, MO). Human recombinant interleukin-1b (IL1b) was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Wound tissues Human wound samples comprising the epithelial wound edge and the corresponding part of nonre-epithelialized dermis were collected from patients with chronic venous and decubitus ulcers (n ¼ 9), who underwent excision and grafting procedures at the Helsinki University Central Hospital. In addition, biopsies from well-granulating ulcers (n ¼ 5) (all less than 2 months old) that required skin grafting because of their large size were also examined. The procedures were approved by the local Ethical Committee. Informed consent was obtained from each patient prior to the biopsy of wounds. Immunohistochemistry Immunostainings were performed on formalin-¢xed para⁄n-embedded tissue sections using the peroxidase^ antiperoxidase technique and diaminobenzidine as chromogenic substrate (Saarialho-Kere et al, 1993). Rabbit polyclonal antiserum against human MMP-19 (Research Diagnostics Inc., Flanders, NJ) was diluted 1:80 and reacted overnight at 41C. On serial sections activated ¢broblasts were stained with a monoclonal antibody to the N-terminus of the type I procollagen (PC-I) molecule (1:500; MAB 1912; Chemicon, Temecula, CA) (Vaalamo et al, 1997) and tissue macrophages with CD- 68 (KP-1, 1:300, M814; Dako, Carpinteria, CA). Harris hematoxylin was used as counterstain. Cell cultures Normal human skin ¢broblast cultures were established from punch biopsy obtained from a voluntary healthy male donor (age 23) and maintained in Dulbecco’s modi¢ed Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU per mL penicillin G, and 100 mg per mL of streptomycin. For experiments with growth factors and cytokines, ¢broblasts were maintained in culture medium supplemented with 0.5% FCS for 18 h, growth factors and cytokines were then added, and the incubations were continued for indicated periods of time. In experiments involving MAPK inhibitors, these were added 1 h before growth factors or cytokines. RNA analysis Total cellular RNA was isolated from cells using the single-step method (Chomczynski and Sacchi, 1987). Northern blot hybridizations were performed as described previously (Reunanen et al, 1998) using a 1.5-kb cDNA for MMP-19 (Pendas et al, 1997). The 32 P-cDNA^mRNA hybrids were visualized by autoradiography and quantitated by scanning densitometry, and MMP-19 mRNA levels were corrected for the levels of 18S rRNA in the same samples. Assay of MMP-19 and TIMP-2 production Equal aliquots of the conditioned medium of ¢broblasts were analyzed for the amount of MMP-19 by western blotting, as described previously (Reunanen et al, 1998) using mouse monoclonal antibody against human MMP-19 (1:500) (Pendas et al, 1997) or polyclonal rabbit antiserum against TIMP-2 (1:1000) (Chemicon International Inc., Temecula, CA). For TIMP-2 western blot, samples were reduced with 5% mercaptoethanol before electrophoretic
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fractionation. Speci¢c binding of antibodies was detected with peroxidaseconjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL) (Amersham Corp., UK). The levels of immunoreactive MMP-19 were quantitated by densitometric scanning of the X-ray ¢lms. Assay of MAPK activation The levels of activated ERK1/2, c-Jun Nterminal kinase (JNK), and p38 were determined by western blot analysis using antibodies speci¢c for phosphorylated, activated forms of the corresponding MAPKs (Cell Signaling Technology, Beverly, MA). Fibroblasts were maintained in DMEM with 0.5% FCS for 18 h, incubated with TNF-a for di¡erent periods of time, and then lyzed in 100 mL of Laemmli sample bu¡er. The samples were sonicated, fractionated by 10% SDS^PAGE, and transferred to Hybond ECL membrane (Amersham Corp., UK). Western blotting was performed as described previously (Reunanen et al, 1998), with phospho-speci¢c antibodies in dilution 1:1000. As control, the levels of total p38 were determined in the same samples using a speci¢c antibody (Cell Signaling Technology). Binding of primary antibodies was detected with peroxidase-conjugated secondary antibodies and visualized by ECL. Infection of ¢broblasts with recombinant adenoviruses Recombinant replication-de¢cient adenovirus RAdlacZ (RAd35) (Wilkinson and Akrigg, 1992), which contains the Escherichia coli b-galactosidase (lacZ) gene under the control of CMV IE promoter, and empty adenovirus RAd66 (Wilkinson and Akrigg, 1992) were kindly provided by G.W.G. Wilkinson (University of Cardi¡, Wales, UK). Adenovirus containing the coding regions of mutated, constitutively active human MEK1 (RAdMEK1ca) (Foschi et al, 1997) was kindly provided by M. Foschi (University of Florence, Italy). Adenoviruses for constitutively active MAPK kinase 7 (MKK7; RAdMKK7D) (Wang et al, 1998b), MKK3b (RAdMKK3bE) (Wang et al, 1998a), and MKK6b (RAdMKK6bE) (Wang et al, 1998a) genes driven by CMV IE promoter were provided by J. Han (Scripps Institute, La Jolla, CA). In experiments, 5 105 ¢broblasts in suspension were infected as previously described with recombinant adenoviruses at a multiplicity of infection of 500, which gives 100% transduction e⁄ciency (Reunanen et al, 2000), plated, and incubated for 18 h. Culture medium (DMEM with 1% FCS) was changed, and the cultures incubated for 24 h. Aliquots of conditioned medium were analyzed for the levels of MMP-19 and TIMP-2, as described above. Cell layers were harvested and used for RNA extraction or for determination of MAPK activation.
RESULTS Expression of MMP-19 by ¢broblasts, endothelial cells, and macrophages in cutaneous wounds MMP-19 is a recently discovered MMP that cleaves various ECM components including basement membrane constituents and is expressed by a wide array of malignant and normal cells, including human skin ¢broblasts in culture (Grant et al, 1999). To examine the possible role and regulation of MMP-19 in cutaneous ECM remodeling, we ¢rst performed immunostainings on samples from ¢ve human normally healing cutaneous wounds and nine chronic dermal ulcers. Expression of immunoreactive MMP-19 was detected in ¢broblast-like cells in the wound stroma of well granulating wounds (Fig 1A). MMP-19 expression was also detected in chronic venous and decubitus ulcers (Fig 1B). Staining of parallel sections for type I procollagen (PC-I) revealed, that MMP-19-positive cells embedded in dermal layer were ¢broblasts (Fig 1C,D). Interestingly, ¢broblasts expressing MMP-19 were often found in the vicinity of in£ammatory cells (Fig 1A). MMP-19 expression was also detected in microvascular endothelial cells and mononuclear cells devoid of type I procollagen (Fig 1C,D). Furthermore, MMP-19 expression was noted particularly at the surface of the wound bed, but also deeper in the dermis, in CD- 68-positive mononuclear cells representing macrophages (Fig 1E,F). Induction of ¢broblast MMP-19 expression by TNF-a To examine the possible regulation of ¢broblast MMP-19 expression by in£ammatory cell-derived factors, we treated normal human skin ¢broblasts in culture with di¡erent in£ammatory
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Figure 1. MMP-19 is expressed by ¢broblasts, endothelial cells, and macrophages in cutaneous wounds. Human skin samples were obtained from chronic venous and decubitus ulcers (n ¼ 9) and from clinically well granulating ulcers (less than 2 months old) (n ¼ 5). Immunostainings were performed on formalin-¢xed para⁄n-embedded tissue sections using the peroxidase^antiperoxidase technique and diaminobenzidine as chromogenic substrate. MMP19 expression was detected with rabbit polyclonal antiserum against human MMP-19. Activated ¢broblasts were stained with a monoclonal antibody to the N-terminus of the type I procollagen (PC-I) molecule and macrophages with a monoclonal antibody against CD- 68. Harris hematoxylin was used as counterstain. (A) MMP-19-positive ¢broblasts in the wound stroma of a well-granulating ulcer containing in£ammatory cells. (B) A chronic venous ulcer with ¢broblasts stained for MMP-19 protein. (C) Staining for MMP-19 in another chronic venous ulcer with positive ¢broblasts and endothelial cells. (D) Staining for PC-I in a serial section. Arrows, corresponding MMP-19- and PC-I-positive ¢broblasts; arrowhead, a MMP-19-positive and PC-I-negative mononuclear cell. (E) A 1-month-old well granulating ulcer stained for MMP-19. (F) Staining for CD- 68 in a serial section to (E). Arrows, corresponding MMP-19- and CD- 68 -positive macrophages; asterisks, corresponding regions. Bars, 24 mm.
cell-derived growth factors, which play a role in cutaneous wound repair. Treatment of cells with TNF-a and IL-1b for 24 h enhanced the expression of MMP-19 mRNA (by 1.9- and 2.0fold, respectively), as detected by northern blot hybridizations (Fig 2A). MMP-19 expression by dermal ¢broblasts was also enhanced by transforming growth factor-b, epidermal growth factor, and platelet-derived growth factor-AA, but slightly less potently (1.7- to 1.8-fold) than with TNF-a and IL-1b (Fig 2A). Because the most potent induction of MMP-19 expression in dermal ¢broblasts was obtained with TNF-a, we examined the molecular mechanism of this response in detail. As shown in Fig 2B, the production of pro-MMP-19 by dermal ¢broblasts was potently enhanced by a 24 -h incubation with TNF-a, whereas the production of TIMP-2 was not altered by TNF-a treatment. Induction of ¢broblast MMP-19 expression by TNF-a is mediated by ERK1/2 and p38 MAPK Our recent observations indicate, that activation of ¢broblast collagenase-1 (MMP-1) expression by TNF-a is mediated by p38 MAPK (Reunanen et al, 2002). In this study, we elucidated the speci¢c
roles of these MAPK cascades in the regulation of the expression of MMP-19 by human skin ¢broblasts. Initially, the cells were treated with TNF-a (20 ng/mL) for di¡erent periods of time and the activation of ERK1/2, JNK, and p38 was determined by western blot analysis using antibodies against activated forms of these MAPKs. As shown in Fig 3A, exposure of ¢broblasts to TNF-a resulted in rapid and transient activation of ERK1/2 and JNK, detected at 15 and 30 min of incubation. Interestingly, p38 MAPK was also rapidly (15 min) activated by TNF-a, and the activation was still detectable after 6 h (Fig 3A). The levels of total p38 were not altered by TNF-a in the same samples. As noted above, treatment of dermal ¢broblasts with TNF-a resulted in potent enhancement of MMP-19 mRNA abundance and this e¡ect was in part (by 43%) prevented by MEK1/2 inhibitor PD98059 (20 ng/mL) (Fig 3B,C). In addition, TNF-aelicited induction of MMP-19 mRNA levels was in part (by 35%) prevented by p38 inhibitor SB203580 (10 mM) (Fig 3B,C). These results provide evidence that both the ERK1/2 pathway and the p38 MAPK cascade mediate enhancement of MMP-19 expression by TNF-a. Nevertheless, ¢broblasts treated with
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Figure 2. Fibroblast MMP-19 expression is induced by TNF-a. (A) Human skin ¢broblasts in culture were treated for 24 h with IL-1b (10 U/mL), TNF-a (20 ng/mL), transforming growth factor-b1 (TGF-b1) (5 ng/mL), epidermal growth factor (EGF) (25 ng/mL), and platelet-derived growth factor-AA (PDGF-AA) (20 ng/mL). Aliquots (15 mg) of total RNA were analyzed for levels of MMP-19 mRNA by northern blot hybridizations. 18S rRNA was visualized by ethidium bromide staining. (B) Human skin ¢broblasts were treated with TNF-a (20 ng/mL) and incubated for 24 h. The levels of proMMP-19 and TIMP-2 in the conditioned medium of the cells were determined by western blot analysis.
TNF-a expressed MMP-19 mRNA when both ERK1/2 and p38 pathways were blocked by PD98059 and SB203580, respectively (Fig 3B,C), indicating involvement of other TNF-a-activated signaling pathways, e.g., JNK, which could not be examined in this context by a chemical inhibitor. Activation of ERK1/2 and p38 results in induction of MMP19 expression by dermal ¢broblasts To directly examine the role of ERK1/2, JNK, and p38 MAPK cascades in the regulation of the expression of the endogenous MMP-19 gene, we utilized adenovirus-mediated gene delivery of constitutively active MEK1, MKK7, MKK3b, and MKK6b to ¢broblasts. Our previous observations show that transduction of ¢broblasts with recombinant adenovirus RAdMEK1ca harboring constitutively active mutant of MEK1 resulted in marked activation of ERK1/ 2, but not p38 or JNK (Ravanti et al, 1999; Reunanen et al, 2000, 2002). Infection of ¢broblasts with adenovirus RAdMKK7D coding for constitutively active MKK7 speci¢cally activates JNK (Reunanen et al, 2002). In addition, infection of cells with adenoviruses for constitutively active MKK3b (RAdMKK3bE) or MKK6b (RAdMKK6bE) alone or in combination results in activation of p38, but not ERK1/2 or JNK (Ravanti et al, 1999; Reunanen et al, 2000, 2002). As shown in Fig 4A,B, activation of ERK1/2 by RAdMEK1ca resulted in induction of MMP-19 mRNA abundance in dermal ¢broblasts and this e¡ect was augmented by simultaneous activation of p38 by adenoviral delivery of constitutively active MKK3b and MKK6b. Interestingly, infection of cells with RAdMKK6bE alone also potently upregulated MMP-19 mRNA, whereas the e¡ect of RAdMKK3bE was less potent (Fig 4A,B). Infection of cells with the control adenovirus RAdlacZ had no e¡ect on MMP-19 mRNA levels in ¢broblasts (Fig 4A,B). Adenoviral delivery of constitutively active MKK7D alone had no e¡ect on MMP-19 mRNA abundance, but it markedly (twofold) augmented the upregulatory e¡ect of constitutively active MEK1 on MMP-19 mRNA levels (Fig 4C).
Figure 3. Induction of ¢broblast MMP-19 expression by TNF-a is mediated by ERK1/2 and p38 MAPK. (A) Human skin ¢broblasts were treated with TNF-a (20 ng/mL) for di¡erent periods of time, as indicated. The levels of activated ERK1/2 (p-ERK1/2), JNK (p-JNK), and p38 (p-p38) were determined by western blot analysis using phosphospeci¢c antibodies for the corresponding MAP kinases. The levels of total p38 were determined in the same samples by western blot analysis using a speci¢c antibody. (B) Human skin ¢broblasts were treated for 24 h with TNF-a (20 ng/mL) alone or in combination with PD98059, a speci¢c inhibitor of ERK1/2 kinases MEK1/2, or with SB203580, a selective inhibitor of p38 MAPK, added 1 h before TNF-a in concentrations (mM) indicated. Aliquots (15 mg) of total RNA were analyzed for levels of MMP-19 by northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. (C) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels. The values are shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments.
Transduction of ¢broblasts with RAdMEK1ca also resulted in marked induction of proMMP-19 production, and this e¡ect was potently inhibited by MEK1/2 inhibitor PD98059 (Fig 4D,E). The stimulatory e¡ect of RAdMEK1ca on proMMP-19 production was augmented by simultaneous expression of constitutively
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active MKK7 and the most abundant production of proMMP-19 was noted when ERK1/2 was activated in combination with p38 by constitutively active MKK3b and MKK6b (Fig 4D,E). Production of pro-MMP-19 was also induced by RAdMKK3bE
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and RAdMKK6bE, and this e¡ect was entirely inhibited by p38 inhibitor SB203580 (Fig 4D,E). Interestingly, in cells infected with RAdMEK1ca in combination with RAdMKK7D, RAdMKK3bE, or RAdMKK6bE, abundant pro-MMP-19
Figure 4. Activation of ERK1/2 and p38 results in induction of MMP-19 expression by ¢broblasts. (A) Human skin ¢broblasts (5 105) were transduced with replication-de¢cient empty control adenovirus (RAd66) or with adenoviruses coding for constitutively active forms of MEK1 (RAdMEK1ca), MKK3b (RAdMKK3bE), or MKK6b (RAdMKK6bE), as indicated at a multiplicity of infection of 500 and incubated for 18 h in DMEM supplemented with 1% FCS. Medium was then changed, the incubations were continued for 24 h, and the cells were harvested. Aliquots (10 mg) of total RNA were analyzed for the levels of MMP-19 mRNA with northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. (B) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels and shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments. (C) Human skin ¢broblasts (5 105) were transduced with replication-de¢cient empty control adenovirus (RAd66) or with adenoviruses RAdMEK1ca and RAdMKK7D coding for constitutively active MKK7, as in (A). Aliquots 12 mg of total RNA were analyzed for the levels of MMP-19 mRNA with northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. Densitometric quantitation of MMP-19 mRNA levels corrected for 18S rRNA levels in the same samples are shown below the panels relative to levels in untreated control cultures (1.0). (D) Human skin ¢broblasts were infected with empty control adenovirus (RAd66) or with adenoviruses RAdMEK1ca and RAdMKK7D and with adenoviruses harboring constitutively active MKK3b (RAdMKK3bE) and MKK6b (RAdMKK6bE), as indicated at a multiplicity of infection of 500, and incubated for 18 h in DMEM supplemented with 1% FCS. MEK1/2 inhibitor PD98059 (40 mM) and p38 inhibitor SB203580 (20 mM) were added to cultures indicated at the time of infection. After 18 h, medium was changed, fresh PD98059 and SB203580 were added, and incubations were continued for 24 h. The levels of MMP-19 and TIMP-2 in the conditioned medium of the cells were determined by western blot analysis. Migration positions of molecular weight markers are shown on the right. (E) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels. The values are shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments.
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production was associated with proteolytic processing of proMMP-19 with an estimated molecular weight of 58 kDa to active MMP-19, noted as the appearance of approximately 10-kDa smaller immunoreactive protein in western blot (Fig 4D). Infection of ¢broblasts with the empty control adenovirus RAd66 had no e¡ect on the production of MMP-19 (Fig 4D,E). In the same cells, the production of TIMP-2 was not induced in parallel with MMP-19 (Fig 4D). DISCUSSION
In this study, we have examined the role and regulation of MMP19, a newly discovered MMP with catalytic activity against several ECM components including constituents of basement membrane, in dermal ¢broblasts in vivo and in culture. Our results show that MMP-19 is expressed by ¢broblasts in the dermal layer of normally healing and chronic cutaneous wounds. The expression of MMP-19 was also detected in capillary endothelial cells in the wounds. Furthermore, expression of MMP-19 was detected in mononuclear in£ammatory cells negative for procollagen I and positive for CD68 and therefore representing macrophages. These results are in accordance with previous observations showing expression of MMP-19 on the surface of blood mononuclear cells (Sedlacek et al, 1998) and myeloid cells (Mauch et al, 2002). Our results also show that the expression of MMP-19 in dermal ¢broblasts is potently enhanced by TNF-a, suggesting a role for MMP-19 in degradation of dermal ECM in in£ammatory conditions. This conclusion is supported by our ¢nding that MMP-19 is produced in wound stroma by ¢broblasts often located in areas in¢ltrated by in£ammatory cells. Further evidence for the role for MMP-19 in wound repair is also provided by our observations that the expression of MMP-19 by ¢broblasts in healthy intact skin is very low (Impola et al, 2003). We also dissected the role of three distinct MAPK signaling pathways, ERK1/2, JNK, and p38, in the regulation of MMP-19 expression in normal human skin ¢broblasts in culture. We show that treatment with TNF-a simultaneously activates ERK1/2, JNK, and p38 in ¢broblasts and that these pathways play a distinct role in the induction of MMP-19 expression. The up-regulation of MMP-19 expression by TNF-a was mediated by coordinate activation of ERK1/2 and p38. Nevertheless, blocking both ERK1/2 and p38 pathways by chemical inhibitors did not block the e¡ect of TNF-a on MMP-19 mRNA levels, indicating that other TNF-a-activated signaling pathways, e.g., JNK or NFkB, may be involved. Interestingly, we have recently noted that TNF-a also enhances expression of MMP-19 by epidermal keratinocytes, suggesting that this proin£ammatory cytokine plays an important role in up-regulation of MMP-19 expression both in the dermal and in the epidermal compartment during wound repair (Impola et al, 2003). We also utilized adenovirus-mediated gene delivery of constitutively active MEK1, MKK7, MKK3b, and MKK6b to examine the e¡ect of the activation of corresponding endogenous MAPKs on the expression of MMP-19 in normal human skin ¢broblasts. Our results showed that activation of ERK1/2 by constitutively active MEK1 resulted in marked induction of MMP-19 expression, the most abundant expression noted when ERK1/2 was activated in combination with JNK or p38. Interestingly, activation of p38 alone by adenovirus-mediated delivery of constitutively active MKK3b and MKK6b also resulted in induction of the expression of MMP-19, whereas speci¢c activation of JNK alone by constitutively active MKK7 was not su⁄cient to induce expression of MMP-19. Activation of ERK1/2 by constitutively active MEK1 and p38 MAPK by MKK3b and MKK6b also resulted in marked induction of pro-MMP-19 production, and the most potent induction was noted when ERK1/2 was activated in combination with JNK and p38. Interestingly, abundant pro-MMP-19 production by ¢broblasts also resulted in proteolytic processing of pro-MMP-19, most likely owing to autoproteolytic activation
(Stracke et al, 2000b). Taken together, these results indicate that both ERK1/2 and p38 signaling pathways play an important role in regulating the production and activation of proMMP-19 by ¢broblasts. MAPK play an important role in regulating cell growth, differentiation, survival, and death (Garrington and Johnson, 1999; Westermarck and Khri, 1999). To date, three mammalian MAPK pathways have been characterized in detail: mitogenactivated ERK1/2 pathway (Raf-MEK1/2-ERK1/2), JNK (MEKK1- 4-MKK4/7-JNK1-3), and p38 (MAPK kinase kinase-MKK3/6-p38a/b) pathways, activated by in£ammatory cytokines and cellular stress (see Garrington and Johnson, 1999; Westermarck and Kahari, 1999). Activation and nuclear translocation of MAPKs results in phosphorylation and activation of their downstream e¡ectors, nuclear protein kinases, e.g., MAPKactivated protein kinases-1, -2, and -3 or transcription factors, e.g., Elk-1, c-Jun, activating transcription factor 2, and cyclic AMP response element-binding protein, which in turn regulate, e.g., expression of the components of AP-1 complex (see Garrington and Johnson, 1999; Westermarck and Kahari, 1999). Recent observations have provided evidence that ERK1/2, JNK, and p38 MAPK regulate the proteolytic capacity of ¢broblasts and squamous carcinoma cells by mediating the activation of MMP-1, stromelysin-1 (MMP-3), gelatinase-B (MMP9), and collagenase-3 (MMP-13) expression (Gum et al, 1997; Ridley et al, 1997; Reunanen et al, 1998, 2002; Simon et al, 1998; Ravanti et al, 1999; Johansson et al, 2000b; Westermarck et al, 2001). Constant activation of ERK1/2 by active mutants of Raf-1 or MEK1 results in transformation of ¢broblasts (Cowley et al, 1994; Janulis et al, 1999; Mansour et al, 1994). Furthermore, activation of ERK1/2 pathway has been documented in renal and breast carcinomas in vivo (Oka et al, 1995; Sivaraman et al, 1997). The consequences of ERK1/2 activation are cell-speci¢c, because activation of ERK1/2 cascade results in growth arrest in small-cell lung carcinoma cells (Ravi et al, 1998) and suppresses MMP-13 expression by SCC cells (Ala-aho et al, 2000). Our recent observations showed that ERK1/2 activity increases the expression of MMP-1 and MMP-3 in ¢broblasts (Reunanen et al, 2002). Our results here show that MMP-19 production by ¢broblasts expressing constitutively active MEK1 is further enhanced, when constant activation of ERK1/2 is superimposed on persistent activation of JNK or p38. This phenomenon may play an important role in wound repair, in which dermal ¢broblasts are exposed to cytokines produced by in£ammatory cells, resulting in coordinate activation of ERK1/2, JNK, and p38. Our recent observations show that activation of p38 MAPK by constitutively active MKK3b and MKK6b has no marked e¡ect on human MMP-1 promoter activity, but that activation of p38a results in marked stabilization of both MMP-1 and MMP-3 mRNAs (Westermarck et al, 2001; Reunanen et al, 2002).We have also recently shown that p38a mediates activation of protein phosphatases 1 and 2 A resulting in inhibition of ERK1, 2 cascade at the level of MEK1,2 resulting in suppression of MMP-1 promoter activity (Westermarck et al, 2001). The results presented here show that activation of p38 also induces expression of MMP-19 by dermal ¢broblasts. It is therefore possible that activation of p38a also enhances the expression of MMP-19 expression via mRNA stability. Controlled turnover of collagenous ECM and basement membranes is an important feature in connective tissue remodeling in physiologic situations, such as tissue repair and angiogenesis, as well as in pathologic conditions, including rheumatoid arthritis, chronic ulcers, and tumor invasion (Johansson et al, 2000a). The ability to degrade type I collagen is essential for migration of epidermal keratinocytes (Pilcher et al, 1997). In addition, cleavage of the surrounding collagenous ECM by ¢broblasts alters their cell^ matrix interactions and facilitates their migration capacity (Messent et al, 1998). It has also been suggested that imbalance in the production of MMP and TIMP-2 may inhibit keratinocyte mi-
VOL. 121, NO. 5 NOVEMBER 2003
gration and lack of TIMP-1 and TIMP-3 expression by proliferating keratinocytes may compromise stability of basement membrane in chronic ulcers (Vaalamo et al, 1999). In this context, it is interesting to note that MMP-19 has been suggested to play a role in migration of myeloid cells, which express MMP-19 on their surface (Mauch et al, 2002). The results of this study do not allow conclusions to be made on the role of MMP-19 expression in the ¢broblast phenotype during wound repair. Nevertheless, our recent observation show that MMP-19 is not expressed by migrating keratinocytes, but in proliferating keratinocytes during cutaneous wound repair, indicating that MMP-19 expression is not necessarily associated with cell migration (Impola et al, 2003). This notion is further supported by our recent observation that MMP-19 is expressed by proliferating keratinocytes in psoriatic epidermis (Suomela et al, 2003). Our results identify ERK1/2 and p38 as two distinct MAPK pathways independently mediating the activation of the expression of the basement membrane degrading MMP-19 in ¢broblasts. Constant activation of ERK1/2 in dermal ¢broblasts also results in marked suppression in their production of type I collagen and collagen ¢bril-associated proteoglycan, decorin (Laine et al, 2000; Reunanen et al, 2000), and enhances the expression of ECM degrading MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) (Reunanen et al, 2002). Together these results provide evidence that ERK1/2 serves as an important molecular switch, the activation of which alters the ECM productive phenotype of normal ¢broblasts to proteolytic phenotype characterized by production of ECM cleaving MMPs and suppression of ECM deposition. Nevertheless, in the presence of the activation of stress-activated JNK and p38 MAPK, ERK1/2 activity is not essential for induction of ¢broblast MMP-19 mRNA and protein by TNF-a. Furthermore, activation of p38 is alone su⁄cient to induce the expression of MMP-19 mRNA and protein. Nevertheless, our results clearly show that coordinate activation of ERK1/2 in combination with JNK or p38 MAPK results in most potent induction of MMP-19 expression and apparently plays a crucial role in stimulation of the proteolytic capacity of normal ¢broblasts in vivo, e.g., during wound repair and tumor invasion. The expert technical assistance of Hanna Haavisto, Marjo Hakkarainen, and Sari Pitkanen is gratefully acknowledged. We also thank Dr P. Fort for plasmids. This study was supported by grants from the Academy of Finland; the Sigrid Juselius Foundation; the Cancer Research Foundation of Finland,Turku University Central Hospital; by research contract with Finnish Life and Pension Insurance Companies; by Turku Graduate School of Biomedical Sciences; and by personal grants (to N.H.) from the Finnish Culture Foundation, the Cancer Foundation, and the Finnish-Norwegian Medical Foundation.
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J Biol Chem 274:801^813, 1999 Johansson N, Ahonen M, Khri VM: Matrix metalloproteinases in tumor invasion. Cell Mol Life Sci 57:5^15, 2000a Johansson N, Ala-aho R, Uitto V, Grenman R, Fusenig NE, Lopez-Otin C, Khri VM: Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J Cell Sci 113:227^235, 2000b Kolb C, Mauch S, Krawinkel U, Sedlacek R: Matrix metalloproteinase-19 in capillary endothelial cells: Expression in acutely, but not in chronically, in£amed synovium. Exp Cell Res 250:122^130, 1999 KonttinenYT, Ainola M,Valleala H, et al: Analysis of 16 di¡erent matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: Di¡erent pro¢les in trauma and rheumatoid arthritis. Ann Rheum Dis 58:691^697, 1999 Laine P, Reunanen N, Ravanti L, Foschi M, Santra M, Iozzo RV, Khri VM: Activation of extracellular signal-regulated protein kinase1,2 results in down-regulation of decorin expression in ¢broblasts. Biochem J 349:19^25, 2000 Mansour SJ, Matten WT, Hermann AS, et al: Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966^970, 1994 Mauch S, Kolb C, Kolb B, Sadowski T, Sedlacek R: Matrix metalloproteinase-19 is expressed in myeloid cells in an adhesion-dependent manner and associates with the cell surface. J Immunol 168:1244^1251, 2002 Messent A, Tuckwell D, Knupr V, Humphries M, Murphy G, Gavrilovic J.: E¡ects of collagenase-cleavage of type I collagen on a2b1 integrin-mediated cell adhesion. J Cell Sci 111:1127^1135, 1998 Nagase H, Woessner JF Jr: Matrix metalloproteinases. J Biol Chem 274:21491^21494, 1999 Oka H, Chatani Y, Hoshino R, et al: Constitutive activation of mitogen-activated protein (MAP) kinases in human renal cell carcinoma. Cancer Res 55:4182^ 4187, 1995 Pendas AM, Knuper V, Puente XS, et al: Identi¢cation and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location, and tissue distribution. J Biol Chem 272:4281^4286, 1997 Pilcher BK, Dumin JA, Sudbeck BD, Krane SM,Welgus HG, Parks WC: The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J Cell Biol 137:1445^1457, 1997 Ramanathan M, Weinstock-Guttman B, Nguyen LT, et al: In vivo gene expression revealed by cDNA arrays: The pattern in relapsing-remitting multiple sclerosis patients compared with normal subjects. J Neuroimmunol 116:213^219, 2001 Ravanti L, Heino JL, Lopez-Otin C, Khri VM: Induction of collagenase-3 (MMP13) expression in human skin ¢broblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase. J Biol Chem 274:2446^ 2455, 1999 Ravi RK, Weber E, McMahon M, et al: Activated Raf-1 causes growth arrest in human small cell lung cancer cells. J Clin Invest 101:153^159, 1998 Reunanen N, Foschi M, Han J, Kahari VM.: Activation of extracellular signal-regulated kinase 1/2 inhibits type I collagen expression by human skin ¢broblasts. J Biol Chem 275:34634^34639, 2000 Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Khri VM: Activation of p38a mitogen-activated protein kinase enhances collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. 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detected as an autoantigen in rheumatoid arthritis. Immunobiology 198:408^423, 1998 Simon C, Goepfert H, Boyd D: Inhibition of the p38 mitogen-activated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion. Cancer Res 58:1135^1139, 1998 Sivaraman V, Wang H, Nuovo G, Malbon C: Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest 99:1478^1483, 1997 Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17:463^516, 2001 Stracke JO, Fosang AJ, Last K, et al: Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP). FEBS Lett 478:52^56, 2000a Stracke JO, Hutton M, Stewart M, et al: Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19: Evidence for a role as a potent basement membrane degrading enzyme. J Biol Chem 275:14809^14816, 2000b Suomela S, Kariniemi AL, Impola U, et al: Matrix metalloproteinase-19 is expressed by keratinocytes in psoriasis. Acta Dermato-Venereol 83:108^114, 2003 Vaalamo M, Leivo T, Saarialho-Kere U: Di¡erential expression of tissue inhibitors of metalloproteinases (TIMP-1-2-3, and - 4) in normal and aberrant wound healing. Hum Pathol 30:795^802, 1999
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Vaalamo M, Mattila L, Johansson N, et al: Distinct populations of stromal cells express collagenase-3 (MMP-13) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds. J Invest Dermatol 109:96^ 101, 1997 Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, Chien KR: Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 273:2161^2168, 1998a Wang Y, Su B, Sah VP, Brown JH, Han J, Chien KR: Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a speci¢c activator for c-June NH2-terminal kinase in ventricular muscle cells. J Biol Chem 273:5423^5426, 1998b Westermarck J, Khri VM: Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13:781^792, 1999 Westermarck J, Li SP, Kallunki T, Han J, Khri VM: p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 MEK2 activity and collagenase-1 (MMP-1) gene expression. Mol Cell Biol 21:2373^2383, 2001 Wilkinson GW, Akrigg A: Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. Nucleic Acids Res 20:2233^2239, 1992
Matrix Metalloproteinase-19 Expression in Dermal Wounds and by Fibroblasts in Culture Niina Hieta,w Ulla Impola,z Carlos Lo¤pez-Ot|¤ n,y Ulpu Saarialho-Kere,z and Veli-Matti Khriw8
Centre for Biotechnology, University of Turku and Abo Akademi University,Turku, Finland; wDepartment of Dermatology and 8Department of Medical
Biochemistry, University of Turku,Turku, Finland; yDepartamento Bioquimica y BiologIØ a Molecular, Instituto Universitario de Oncologia, Universidad de Oviedo, Oviedo, Spain; zDepartment of Dermatology, University of Helsinki and Biomedicum Helsinki, Helsinki
Here, we have examined the expression of matrix metalloproteinase-19 (MMP-19) in human cutaneous wounds and by human skin ¢broblasts in culture. Expression of MMP-19 was detected by immunohistochemistry in ¢broblasts, capillary endothelial cells, and macrophages in the dermal layer of large granulating wounds, as well as in chronic venous and decubitus ulcers. MMP-19 mRNA expression and pro-MMP-19 production by dermal ¢broblasts in culture was potently enhanced by tumor necrosis factor-a (TNF-a). Induction of MMP-19 expression by TNF-a was prevented partially by blocking the activation of extracellular signal-regulated kinase (ERK)-1/2 by PD98059 and p38 activity by SB203580. Activation of ERK1/2 by adenovirus-mediated delivery of constitutively active MAPK/ERK kinase 1 resulted in the induction of MMP-19 expression. Activation of p38
alone by adenovirally delivered constitutively active MAPK kinase 3b (MKK3b) and MKK6b also enhanced MMP-19 production, and the most potent induction of MMP-19 expression was noted when ERK1/2 was activated in combination with p38. Activation of c-Jun Nterminal kinase (JNK). Abundant pro-MMP-19 production by ¢broblasts was associated with proteolytic processing of secreted pro-MMP-19. These results suggest a role of MMP-19 in cutaneous wound repair and identify three distinct signaling pathways, which coordinately mediate induction of MMP-19 expression in ¢broblasts: mitogen-activated ERK1/2 pathway and stress-activated JNK and p38 pathways, of which control proteolytic activity of dermal ¢broblasts. Key word: mitogen-activated protein kinase. J Invest Dermatol 121:997 ^1004, 2003
M
normal tissue architecture, e.g., in rheumatoid arthritis, osteoarthritis, autoimmune blistering skin disorders, cutaneous photoaging, and tumor invasion and metastasis (Johansson et al, 2000a). Human MMP-19 cDNA was initially cloned from liver and mammary gland and was also identi¢ed as an autoantigen in in£amed rheumatoid synovium (Cossins et al, 1996; Pends et al, 1997; Sedlacek et al, 1998). The catalytic domain of MMP-19 hydrolyzes type IV collagen, laminin, nidogen, large tenascin-C, ¢bronectin, type I gelatin, aggrecan, and cartilage oligomeric matrix protein, but not triple-helical type I collagen (Stracke et al, 2000a, b). The activity of MMP-19 catalytic domain is inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2), TIMP-3, and TIMP- 4 and less e⁄ciently by TIMP-1 (Stracke et al, 2000b). Latent MMP-19 is activated by autoproteolysis, and in contrast to several other MMPs, MMP-19 is not known to activate any other latent MMPs (Stracke et al, 2000b). Expression of MMP-19 has been detected in injured and in£amed synovium, especially in endothelial cells of synovial capillaries, suggesting a role for MMP-19 in angiogenesis (Kolb et al, 1999; Konttinen et al, 1999). Expression of MMP-19 is also enhanced in blood mononuclear cells of patients with multiple sclerosis (Ramanathan et al, 2001). In myoepithelial cells and epidermal keratinocytes, the expression of MMP-19 is lost upon malignant transformation (Djonov et al, 2001; Impola et al, 2003). In culture, MMP-19 is expressed by several types of malignant and normal cells, including epidermal keratinocytes and dermal ¢broblasts (Grant et al, 1999; Impola et al, 2003). In this study, we have examined the role and regulation of MMP-19 in dermal wound repair in vivo and by human skin
atrix metalloproteinases (MMP) are a family of structurally related zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the extracellular matrix (ECM) (see Nagase and Woessner, 1999; Johansson et al, 2000a). At present, 24 human members of MMP gene family have been identi¢ed, and they are often classi¢ed into subgroups of collagenases, gelatinases, stromelysins, membrane-type MMP, and other MMPs based on their structure and substrate speci¢city (Nagase and Woessner, 1999). There is considerable amount of evidence that MMPs play an important role in connective tissue remodeling in physiologic situations including developmental tissue morphogenesis, tissue repair, and angiogenesis. In addition, MMPs also regulate cellular growth factor response and in£ammatory reaction by cleavage of growth factors, cytokines, chemokines, and their receptors (Sternlicht and Werb 2001). MMPs also play an important role in destruction of
Manuscript received December 11, 2002; revised March 14, 2003; accepted for publication May 6, 2003 Reprint requests to: Veli-Matti Kahari, MD, PhD, Center for Biotechnology, University of Turku, Tykist˛katu 6B, FIN-20520 Turku, Finland. Email: veli-matti.kahari@utu.¢ Abbreviations: DMEM, Dulbecco’s modi¢ed Eagle’s medium; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase; MAPK, mitogen activated protein kinase; MKK, MAPK kinase; MEK, MAPK/ ERK kinase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; TNF-a, tumor necrosis factor-a.
0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 997
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¢broblasts in culture. MMP-19 was noted in dermal ¢broblasts, macrophages, and endothelial cells during normal cutaneous wound repair and in chronic cutaneous ulcers. We also show, that tumor necrosis factor-a (TNF-a) potently enhances MMP-19 expression and that this is mediated coordinately via extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results provide evidence for the role of MMP-19 in remodeling of dermal ECM during wound repair. In addition, our results identify two distinct mechanisms of inducing proteolytic capacity of dermal ¢broblasts: mitogen-responsive ERK1/2 pathway and stress-activated p38 MAPK, suggesting that both play a role in controlling the MMP-19 expression and proteolytic phenotype of ¢broblasts, e.g., during wound repair. MATERIALS AND METHODS Materials Human recombinant TNF-a and epidermal growth factor (EGF), MAPK/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 (20 -amino30 -methoxy£avone), and p38 inhibitor SB203580 ([4 -(£uorophenyl)-2-(4 methylsul¢nylphenyl)-5-(4 -pyridyl)1H-imidazole]) were obtained from Calbiochem (San Diego, CA). Human recombinant transforming growth factor-b and platelet-derived growth factor-AA were obtained from Sigma Chemical Co. (St. Louis, MO). Human recombinant interleukin-1b (IL1b) was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Wound tissues Human wound samples comprising the epithelial wound edge and the corresponding part of nonre-epithelialized dermis were collected from patients with chronic venous and decubitus ulcers (n ¼ 9), who underwent excision and grafting procedures at the Helsinki University Central Hospital. In addition, biopsies from well-granulating ulcers (n ¼ 5) (all less than 2 months old) that required skin grafting because of their large size were also examined. The procedures were approved by the local Ethical Committee. Informed consent was obtained from each patient prior to the biopsy of wounds. Immunohistochemistry Immunostainings were performed on formalin-¢xed para⁄n-embedded tissue sections using the peroxidase^ antiperoxidase technique and diaminobenzidine as chromogenic substrate (Saarialho-Kere et al, 1993). Rabbit polyclonal antiserum against human MMP-19 (Research Diagnostics Inc., Flanders, NJ) was diluted 1:80 and reacted overnight at 41C. On serial sections activated ¢broblasts were stained with a monoclonal antibody to the N-terminus of the type I procollagen (PC-I) molecule (1:500; MAB 1912; Chemicon, Temecula, CA) (Vaalamo et al, 1997) and tissue macrophages with CD- 68 (KP-1, 1:300, M814; Dako, Carpinteria, CA). Harris hematoxylin was used as counterstain. Cell cultures Normal human skin ¢broblast cultures were established from punch biopsy obtained from a voluntary healthy male donor (age 23) and maintained in Dulbecco’s modi¢ed Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU per mL penicillin G, and 100 mg per mL of streptomycin. For experiments with growth factors and cytokines, ¢broblasts were maintained in culture medium supplemented with 0.5% FCS for 18 h, growth factors and cytokines were then added, and the incubations were continued for indicated periods of time. In experiments involving MAPK inhibitors, these were added 1 h before growth factors or cytokines. RNA analysis Total cellular RNA was isolated from cells using the single-step method (Chomczynski and Sacchi, 1987). Northern blot hybridizations were performed as described previously (Reunanen et al, 1998) using a 1.5-kb cDNA for MMP-19 (Pendas et al, 1997). The 32 P-cDNA^mRNA hybrids were visualized by autoradiography and quantitated by scanning densitometry, and MMP-19 mRNA levels were corrected for the levels of 18S rRNA in the same samples. Assay of MMP-19 and TIMP-2 production Equal aliquots of the conditioned medium of ¢broblasts were analyzed for the amount of MMP-19 by western blotting, as described previously (Reunanen et al, 1998) using mouse monoclonal antibody against human MMP-19 (1:500) (Pendas et al, 1997) or polyclonal rabbit antiserum against TIMP-2 (1:1000) (Chemicon International Inc., Temecula, CA). For TIMP-2 western blot, samples were reduced with 5% mercaptoethanol before electrophoretic
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fractionation. Speci¢c binding of antibodies was detected with peroxidaseconjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL) (Amersham Corp., UK). The levels of immunoreactive MMP-19 were quantitated by densitometric scanning of the X-ray ¢lms. Assay of MAPK activation The levels of activated ERK1/2, c-Jun Nterminal kinase (JNK), and p38 were determined by western blot analysis using antibodies speci¢c for phosphorylated, activated forms of the corresponding MAPKs (Cell Signaling Technology, Beverly, MA). Fibroblasts were maintained in DMEM with 0.5% FCS for 18 h, incubated with TNF-a for di¡erent periods of time, and then lyzed in 100 mL of Laemmli sample bu¡er. The samples were sonicated, fractionated by 10% SDS^PAGE, and transferred to Hybond ECL membrane (Amersham Corp., UK). Western blotting was performed as described previously (Reunanen et al, 1998), with phospho-speci¢c antibodies in dilution 1:1000. As control, the levels of total p38 were determined in the same samples using a speci¢c antibody (Cell Signaling Technology). Binding of primary antibodies was detected with peroxidase-conjugated secondary antibodies and visualized by ECL. Infection of ¢broblasts with recombinant adenoviruses Recombinant replication-de¢cient adenovirus RAdlacZ (RAd35) (Wilkinson and Akrigg, 1992), which contains the Escherichia coli b-galactosidase (lacZ) gene under the control of CMV IE promoter, and empty adenovirus RAd66 (Wilkinson and Akrigg, 1992) were kindly provided by G.W.G. Wilkinson (University of Cardi¡, Wales, UK). Adenovirus containing the coding regions of mutated, constitutively active human MEK1 (RAdMEK1ca) (Foschi et al, 1997) was kindly provided by M. Foschi (University of Florence, Italy). Adenoviruses for constitutively active MAPK kinase 7 (MKK7; RAdMKK7D) (Wang et al, 1998b), MKK3b (RAdMKK3bE) (Wang et al, 1998a), and MKK6b (RAdMKK6bE) (Wang et al, 1998a) genes driven by CMV IE promoter were provided by J. Han (Scripps Institute, La Jolla, CA). In experiments, 5 105 ¢broblasts in suspension were infected as previously described with recombinant adenoviruses at a multiplicity of infection of 500, which gives 100% transduction e⁄ciency (Reunanen et al, 2000), plated, and incubated for 18 h. Culture medium (DMEM with 1% FCS) was changed, and the cultures incubated for 24 h. Aliquots of conditioned medium were analyzed for the levels of MMP-19 and TIMP-2, as described above. Cell layers were harvested and used for RNA extraction or for determination of MAPK activation.
RESULTS Expression of MMP-19 by ¢broblasts, endothelial cells, and macrophages in cutaneous wounds MMP-19 is a recently discovered MMP that cleaves various ECM components including basement membrane constituents and is expressed by a wide array of malignant and normal cells, including human skin ¢broblasts in culture (Grant et al, 1999). To examine the possible role and regulation of MMP-19 in cutaneous ECM remodeling, we ¢rst performed immunostainings on samples from ¢ve human normally healing cutaneous wounds and nine chronic dermal ulcers. Expression of immunoreactive MMP-19 was detected in ¢broblast-like cells in the wound stroma of well granulating wounds (Fig 1A). MMP-19 expression was also detected in chronic venous and decubitus ulcers (Fig 1B). Staining of parallel sections for type I procollagen (PC-I) revealed, that MMP-19-positive cells embedded in dermal layer were ¢broblasts (Fig 1C,D). Interestingly, ¢broblasts expressing MMP-19 were often found in the vicinity of in£ammatory cells (Fig 1A). MMP-19 expression was also detected in microvascular endothelial cells and mononuclear cells devoid of type I procollagen (Fig 1C,D). Furthermore, MMP-19 expression was noted particularly at the surface of the wound bed, but also deeper in the dermis, in CD- 68-positive mononuclear cells representing macrophages (Fig 1E,F). Induction of ¢broblast MMP-19 expression by TNF-a To examine the possible regulation of ¢broblast MMP-19 expression by in£ammatory cell-derived factors, we treated normal human skin ¢broblasts in culture with di¡erent in£ammatory
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Figure 1. MMP-19 is expressed by ¢broblasts, endothelial cells, and macrophages in cutaneous wounds. Human skin samples were obtained from chronic venous and decubitus ulcers (n ¼ 9) and from clinically well granulating ulcers (less than 2 months old) (n ¼ 5). Immunostainings were performed on formalin-¢xed para⁄n-embedded tissue sections using the peroxidase^antiperoxidase technique and diaminobenzidine as chromogenic substrate. MMP19 expression was detected with rabbit polyclonal antiserum against human MMP-19. Activated ¢broblasts were stained with a monoclonal antibody to the N-terminus of the type I procollagen (PC-I) molecule and macrophages with a monoclonal antibody against CD- 68. Harris hematoxylin was used as counterstain. (A) MMP-19-positive ¢broblasts in the wound stroma of a well-granulating ulcer containing in£ammatory cells. (B) A chronic venous ulcer with ¢broblasts stained for MMP-19 protein. (C) Staining for MMP-19 in another chronic venous ulcer with positive ¢broblasts and endothelial cells. (D) Staining for PC-I in a serial section. Arrows, corresponding MMP-19- and PC-I-positive ¢broblasts; arrowhead, a MMP-19-positive and PC-I-negative mononuclear cell. (E) A 1-month-old well granulating ulcer stained for MMP-19. (F) Staining for CD- 68 in a serial section to (E). Arrows, corresponding MMP-19- and CD- 68 -positive macrophages; asterisks, corresponding regions. Bars, 24 mm.
cell-derived growth factors, which play a role in cutaneous wound repair. Treatment of cells with TNF-a and IL-1b for 24 h enhanced the expression of MMP-19 mRNA (by 1.9- and 2.0fold, respectively), as detected by northern blot hybridizations (Fig 2A). MMP-19 expression by dermal ¢broblasts was also enhanced by transforming growth factor-b, epidermal growth factor, and platelet-derived growth factor-AA, but slightly less potently (1.7- to 1.8-fold) than with TNF-a and IL-1b (Fig 2A). Because the most potent induction of MMP-19 expression in dermal ¢broblasts was obtained with TNF-a, we examined the molecular mechanism of this response in detail. As shown in Fig 2B, the production of pro-MMP-19 by dermal ¢broblasts was potently enhanced by a 24 -h incubation with TNF-a, whereas the production of TIMP-2 was not altered by TNF-a treatment. Induction of ¢broblast MMP-19 expression by TNF-a is mediated by ERK1/2 and p38 MAPK Our recent observations indicate, that activation of ¢broblast collagenase-1 (MMP-1) expression by TNF-a is mediated by p38 MAPK (Reunanen et al, 2002). In this study, we elucidated the speci¢c
roles of these MAPK cascades in the regulation of the expression of MMP-19 by human skin ¢broblasts. Initially, the cells were treated with TNF-a (20 ng/mL) for di¡erent periods of time and the activation of ERK1/2, JNK, and p38 was determined by western blot analysis using antibodies against activated forms of these MAPKs. As shown in Fig 3A, exposure of ¢broblasts to TNF-a resulted in rapid and transient activation of ERK1/2 and JNK, detected at 15 and 30 min of incubation. Interestingly, p38 MAPK was also rapidly (15 min) activated by TNF-a, and the activation was still detectable after 6 h (Fig 3A). The levels of total p38 were not altered by TNF-a in the same samples. As noted above, treatment of dermal ¢broblasts with TNF-a resulted in potent enhancement of MMP-19 mRNA abundance and this e¡ect was in part (by 43%) prevented by MEK1/2 inhibitor PD98059 (20 ng/mL) (Fig 3B,C). In addition, TNF-aelicited induction of MMP-19 mRNA levels was in part (by 35%) prevented by p38 inhibitor SB203580 (10 mM) (Fig 3B,C). These results provide evidence that both the ERK1/2 pathway and the p38 MAPK cascade mediate enhancement of MMP-19 expression by TNF-a. Nevertheless, ¢broblasts treated with
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Figure 2. Fibroblast MMP-19 expression is induced by TNF-a. (A) Human skin ¢broblasts in culture were treated for 24 h with IL-1b (10 U/mL), TNF-a (20 ng/mL), transforming growth factor-b1 (TGF-b1) (5 ng/mL), epidermal growth factor (EGF) (25 ng/mL), and platelet-derived growth factor-AA (PDGF-AA) (20 ng/mL). Aliquots (15 mg) of total RNA were analyzed for levels of MMP-19 mRNA by northern blot hybridizations. 18S rRNA was visualized by ethidium bromide staining. (B) Human skin ¢broblasts were treated with TNF-a (20 ng/mL) and incubated for 24 h. The levels of proMMP-19 and TIMP-2 in the conditioned medium of the cells were determined by western blot analysis.
TNF-a expressed MMP-19 mRNA when both ERK1/2 and p38 pathways were blocked by PD98059 and SB203580, respectively (Fig 3B,C), indicating involvement of other TNF-a-activated signaling pathways, e.g., JNK, which could not be examined in this context by a chemical inhibitor. Activation of ERK1/2 and p38 results in induction of MMP19 expression by dermal ¢broblasts To directly examine the role of ERK1/2, JNK, and p38 MAPK cascades in the regulation of the expression of the endogenous MMP-19 gene, we utilized adenovirus-mediated gene delivery of constitutively active MEK1, MKK7, MKK3b, and MKK6b to ¢broblasts. Our previous observations show that transduction of ¢broblasts with recombinant adenovirus RAdMEK1ca harboring constitutively active mutant of MEK1 resulted in marked activation of ERK1/ 2, but not p38 or JNK (Ravanti et al, 1999; Reunanen et al, 2000, 2002). Infection of ¢broblasts with adenovirus RAdMKK7D coding for constitutively active MKK7 speci¢cally activates JNK (Reunanen et al, 2002). In addition, infection of cells with adenoviruses for constitutively active MKK3b (RAdMKK3bE) or MKK6b (RAdMKK6bE) alone or in combination results in activation of p38, but not ERK1/2 or JNK (Ravanti et al, 1999; Reunanen et al, 2000, 2002). As shown in Fig 4A,B, activation of ERK1/2 by RAdMEK1ca resulted in induction of MMP-19 mRNA abundance in dermal ¢broblasts and this e¡ect was augmented by simultaneous activation of p38 by adenoviral delivery of constitutively active MKK3b and MKK6b. Interestingly, infection of cells with RAdMKK6bE alone also potently upregulated MMP-19 mRNA, whereas the e¡ect of RAdMKK3bE was less potent (Fig 4A,B). Infection of cells with the control adenovirus RAdlacZ had no e¡ect on MMP-19 mRNA levels in ¢broblasts (Fig 4A,B). Adenoviral delivery of constitutively active MKK7D alone had no e¡ect on MMP-19 mRNA abundance, but it markedly (twofold) augmented the upregulatory e¡ect of constitutively active MEK1 on MMP-19 mRNA levels (Fig 4C).
Figure 3. Induction of ¢broblast MMP-19 expression by TNF-a is mediated by ERK1/2 and p38 MAPK. (A) Human skin ¢broblasts were treated with TNF-a (20 ng/mL) for di¡erent periods of time, as indicated. The levels of activated ERK1/2 (p-ERK1/2), JNK (p-JNK), and p38 (p-p38) were determined by western blot analysis using phosphospeci¢c antibodies for the corresponding MAP kinases. The levels of total p38 were determined in the same samples by western blot analysis using a speci¢c antibody. (B) Human skin ¢broblasts were treated for 24 h with TNF-a (20 ng/mL) alone or in combination with PD98059, a speci¢c inhibitor of ERK1/2 kinases MEK1/2, or with SB203580, a selective inhibitor of p38 MAPK, added 1 h before TNF-a in concentrations (mM) indicated. Aliquots (15 mg) of total RNA were analyzed for levels of MMP-19 by northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. (C) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels. The values are shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments.
Transduction of ¢broblasts with RAdMEK1ca also resulted in marked induction of proMMP-19 production, and this e¡ect was potently inhibited by MEK1/2 inhibitor PD98059 (Fig 4D,E). The stimulatory e¡ect of RAdMEK1ca on proMMP-19 production was augmented by simultaneous expression of constitutively
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active MKK7 and the most abundant production of proMMP-19 was noted when ERK1/2 was activated in combination with p38 by constitutively active MKK3b and MKK6b (Fig 4D,E). Production of pro-MMP-19 was also induced by RAdMKK3bE
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and RAdMKK6bE, and this e¡ect was entirely inhibited by p38 inhibitor SB203580 (Fig 4D,E). Interestingly, in cells infected with RAdMEK1ca in combination with RAdMKK7D, RAdMKK3bE, or RAdMKK6bE, abundant pro-MMP-19
Figure 4. Activation of ERK1/2 and p38 results in induction of MMP-19 expression by ¢broblasts. (A) Human skin ¢broblasts (5 105) were transduced with replication-de¢cient empty control adenovirus (RAd66) or with adenoviruses coding for constitutively active forms of MEK1 (RAdMEK1ca), MKK3b (RAdMKK3bE), or MKK6b (RAdMKK6bE), as indicated at a multiplicity of infection of 500 and incubated for 18 h in DMEM supplemented with 1% FCS. Medium was then changed, the incubations were continued for 24 h, and the cells were harvested. Aliquots (10 mg) of total RNA were analyzed for the levels of MMP-19 mRNA with northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. (B) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels and shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments. (C) Human skin ¢broblasts (5 105) were transduced with replication-de¢cient empty control adenovirus (RAd66) or with adenoviruses RAdMEK1ca and RAdMKK7D coding for constitutively active MKK7, as in (A). Aliquots 12 mg of total RNA were analyzed for the levels of MMP-19 mRNA with northern blot hybridization. 18S rRNA was visualized by ethidium bromide staining. Densitometric quantitation of MMP-19 mRNA levels corrected for 18S rRNA levels in the same samples are shown below the panels relative to levels in untreated control cultures (1.0). (D) Human skin ¢broblasts were infected with empty control adenovirus (RAd66) or with adenoviruses RAdMEK1ca and RAdMKK7D and with adenoviruses harboring constitutively active MKK3b (RAdMKK3bE) and MKK6b (RAdMKK6bE), as indicated at a multiplicity of infection of 500, and incubated for 18 h in DMEM supplemented with 1% FCS. MEK1/2 inhibitor PD98059 (40 mM) and p38 inhibitor SB203580 (20 mM) were added to cultures indicated at the time of infection. After 18 h, medium was changed, fresh PD98059 and SB203580 were added, and incubations were continued for 24 h. The levels of MMP-19 and TIMP-2 in the conditioned medium of the cells were determined by western blot analysis. Migration positions of molecular weight markers are shown on the right. (E) Densitometric quantitation of MMP-19 mRNA levels in northern blot hybridizations corrected for 18S rRNA levels. The values are shown relative to levels in untreated control cultures (1.0). The values represent means of two independent experiments.
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production was associated with proteolytic processing of proMMP-19 with an estimated molecular weight of 58 kDa to active MMP-19, noted as the appearance of approximately 10-kDa smaller immunoreactive protein in western blot (Fig 4D). Infection of ¢broblasts with the empty control adenovirus RAd66 had no e¡ect on the production of MMP-19 (Fig 4D,E). In the same cells, the production of TIMP-2 was not induced in parallel with MMP-19 (Fig 4D). DISCUSSION
In this study, we have examined the role and regulation of MMP19, a newly discovered MMP with catalytic activity against several ECM components including constituents of basement membrane, in dermal ¢broblasts in vivo and in culture. Our results show that MMP-19 is expressed by ¢broblasts in the dermal layer of normally healing and chronic cutaneous wounds. The expression of MMP-19 was also detected in capillary endothelial cells in the wounds. Furthermore, expression of MMP-19 was detected in mononuclear in£ammatory cells negative for procollagen I and positive for CD68 and therefore representing macrophages. These results are in accordance with previous observations showing expression of MMP-19 on the surface of blood mononuclear cells (Sedlacek et al, 1998) and myeloid cells (Mauch et al, 2002). Our results also show that the expression of MMP-19 in dermal ¢broblasts is potently enhanced by TNF-a, suggesting a role for MMP-19 in degradation of dermal ECM in in£ammatory conditions. This conclusion is supported by our ¢nding that MMP-19 is produced in wound stroma by ¢broblasts often located in areas in¢ltrated by in£ammatory cells. Further evidence for the role for MMP-19 in wound repair is also provided by our observations that the expression of MMP-19 by ¢broblasts in healthy intact skin is very low (Impola et al, 2003). We also dissected the role of three distinct MAPK signaling pathways, ERK1/2, JNK, and p38, in the regulation of MMP-19 expression in normal human skin ¢broblasts in culture. We show that treatment with TNF-a simultaneously activates ERK1/2, JNK, and p38 in ¢broblasts and that these pathways play a distinct role in the induction of MMP-19 expression. The up-regulation of MMP-19 expression by TNF-a was mediated by coordinate activation of ERK1/2 and p38. Nevertheless, blocking both ERK1/2 and p38 pathways by chemical inhibitors did not block the e¡ect of TNF-a on MMP-19 mRNA levels, indicating that other TNF-a-activated signaling pathways, e.g., JNK or NFkB, may be involved. Interestingly, we have recently noted that TNF-a also enhances expression of MMP-19 by epidermal keratinocytes, suggesting that this proin£ammatory cytokine plays an important role in up-regulation of MMP-19 expression both in the dermal and in the epidermal compartment during wound repair (Impola et al, 2003). We also utilized adenovirus-mediated gene delivery of constitutively active MEK1, MKK7, MKK3b, and MKK6b to examine the e¡ect of the activation of corresponding endogenous MAPKs on the expression of MMP-19 in normal human skin ¢broblasts. Our results showed that activation of ERK1/2 by constitutively active MEK1 resulted in marked induction of MMP-19 expression, the most abundant expression noted when ERK1/2 was activated in combination with JNK or p38. Interestingly, activation of p38 alone by adenovirus-mediated delivery of constitutively active MKK3b and MKK6b also resulted in induction of the expression of MMP-19, whereas speci¢c activation of JNK alone by constitutively active MKK7 was not su⁄cient to induce expression of MMP-19. Activation of ERK1/2 by constitutively active MEK1 and p38 MAPK by MKK3b and MKK6b also resulted in marked induction of pro-MMP-19 production, and the most potent induction was noted when ERK1/2 was activated in combination with JNK and p38. Interestingly, abundant pro-MMP-19 production by ¢broblasts also resulted in proteolytic processing of pro-MMP-19, most likely owing to autoproteolytic activation
(Stracke et al, 2000b). Taken together, these results indicate that both ERK1/2 and p38 signaling pathways play an important role in regulating the production and activation of proMMP-19 by ¢broblasts. MAPK play an important role in regulating cell growth, differentiation, survival, and death (Garrington and Johnson, 1999; Westermarck and Khri, 1999). To date, three mammalian MAPK pathways have been characterized in detail: mitogenactivated ERK1/2 pathway (Raf-MEK1/2-ERK1/2), JNK (MEKK1- 4-MKK4/7-JNK1-3), and p38 (MAPK kinase kinase-MKK3/6-p38a/b) pathways, activated by in£ammatory cytokines and cellular stress (see Garrington and Johnson, 1999; Westermarck and Kahari, 1999). Activation and nuclear translocation of MAPKs results in phosphorylation and activation of their downstream e¡ectors, nuclear protein kinases, e.g., MAPKactivated protein kinases-1, -2, and -3 or transcription factors, e.g., Elk-1, c-Jun, activating transcription factor 2, and cyclic AMP response element-binding protein, which in turn regulate, e.g., expression of the components of AP-1 complex (see Garrington and Johnson, 1999; Westermarck and Kahari, 1999). Recent observations have provided evidence that ERK1/2, JNK, and p38 MAPK regulate the proteolytic capacity of ¢broblasts and squamous carcinoma cells by mediating the activation of MMP-1, stromelysin-1 (MMP-3), gelatinase-B (MMP9), and collagenase-3 (MMP-13) expression (Gum et al, 1997; Ridley et al, 1997; Reunanen et al, 1998, 2002; Simon et al, 1998; Ravanti et al, 1999; Johansson et al, 2000b; Westermarck et al, 2001). Constant activation of ERK1/2 by active mutants of Raf-1 or MEK1 results in transformation of ¢broblasts (Cowley et al, 1994; Janulis et al, 1999; Mansour et al, 1994). Furthermore, activation of ERK1/2 pathway has been documented in renal and breast carcinomas in vivo (Oka et al, 1995; Sivaraman et al, 1997). The consequences of ERK1/2 activation are cell-speci¢c, because activation of ERK1/2 cascade results in growth arrest in small-cell lung carcinoma cells (Ravi et al, 1998) and suppresses MMP-13 expression by SCC cells (Ala-aho et al, 2000). Our recent observations showed that ERK1/2 activity increases the expression of MMP-1 and MMP-3 in ¢broblasts (Reunanen et al, 2002). Our results here show that MMP-19 production by ¢broblasts expressing constitutively active MEK1 is further enhanced, when constant activation of ERK1/2 is superimposed on persistent activation of JNK or p38. This phenomenon may play an important role in wound repair, in which dermal ¢broblasts are exposed to cytokines produced by in£ammatory cells, resulting in coordinate activation of ERK1/2, JNK, and p38. Our recent observations show that activation of p38 MAPK by constitutively active MKK3b and MKK6b has no marked e¡ect on human MMP-1 promoter activity, but that activation of p38a results in marked stabilization of both MMP-1 and MMP-3 mRNAs (Westermarck et al, 2001; Reunanen et al, 2002).We have also recently shown that p38a mediates activation of protein phosphatases 1 and 2 A resulting in inhibition of ERK1, 2 cascade at the level of MEK1,2 resulting in suppression of MMP-1 promoter activity (Westermarck et al, 2001). The results presented here show that activation of p38 also induces expression of MMP-19 by dermal ¢broblasts. It is therefore possible that activation of p38a also enhances the expression of MMP-19 expression via mRNA stability. Controlled turnover of collagenous ECM and basement membranes is an important feature in connective tissue remodeling in physiologic situations, such as tissue repair and angiogenesis, as well as in pathologic conditions, including rheumatoid arthritis, chronic ulcers, and tumor invasion (Johansson et al, 2000a). The ability to degrade type I collagen is essential for migration of epidermal keratinocytes (Pilcher et al, 1997). In addition, cleavage of the surrounding collagenous ECM by ¢broblasts alters their cell^ matrix interactions and facilitates their migration capacity (Messent et al, 1998). It has also been suggested that imbalance in the production of MMP and TIMP-2 may inhibit keratinocyte mi-
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gration and lack of TIMP-1 and TIMP-3 expression by proliferating keratinocytes may compromise stability of basement membrane in chronic ulcers (Vaalamo et al, 1999). In this context, it is interesting to note that MMP-19 has been suggested to play a role in migration of myeloid cells, which express MMP-19 on their surface (Mauch et al, 2002). The results of this study do not allow conclusions to be made on the role of MMP-19 expression in the ¢broblast phenotype during wound repair. Nevertheless, our recent observation show that MMP-19 is not expressed by migrating keratinocytes, but in proliferating keratinocytes during cutaneous wound repair, indicating that MMP-19 expression is not necessarily associated with cell migration (Impola et al, 2003). This notion is further supported by our recent observation that MMP-19 is expressed by proliferating keratinocytes in psoriatic epidermis (Suomela et al, 2003). Our results identify ERK1/2 and p38 as two distinct MAPK pathways independently mediating the activation of the expression of the basement membrane degrading MMP-19 in ¢broblasts. Constant activation of ERK1/2 in dermal ¢broblasts also results in marked suppression in their production of type I collagen and collagen ¢bril-associated proteoglycan, decorin (Laine et al, 2000; Reunanen et al, 2000), and enhances the expression of ECM degrading MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) (Reunanen et al, 2002). Together these results provide evidence that ERK1/2 serves as an important molecular switch, the activation of which alters the ECM productive phenotype of normal ¢broblasts to proteolytic phenotype characterized by production of ECM cleaving MMPs and suppression of ECM deposition. Nevertheless, in the presence of the activation of stress-activated JNK and p38 MAPK, ERK1/2 activity is not essential for induction of ¢broblast MMP-19 mRNA and protein by TNF-a. Furthermore, activation of p38 is alone su⁄cient to induce the expression of MMP-19 mRNA and protein. Nevertheless, our results clearly show that coordinate activation of ERK1/2 in combination with JNK or p38 MAPK results in most potent induction of MMP-19 expression and apparently plays a crucial role in stimulation of the proteolytic capacity of normal ¢broblasts in vivo, e.g., during wound repair and tumor invasion. The expert technical assistance of Hanna Haavisto, Marjo Hakkarainen, and Sari Pitkanen is gratefully acknowledged. We also thank Dr P. Fort for plasmids. This study was supported by grants from the Academy of Finland; the Sigrid Juselius Foundation; the Cancer Research Foundation of Finland,Turku University Central Hospital; by research contract with Finnish Life and Pension Insurance Companies; by Turku Graduate School of Biomedical Sciences; and by personal grants (to N.H.) from the Finnish Culture Foundation, the Cancer Foundation, and the Finnish-Norwegian Medical Foundation.
REFERENCES Ala-aho R, Johansson N, GrEnman R, Fusenig NE, Lopez-Otin C, Kahari VM: Inhibition of collagenase-3 (MMP-13) expression in transformed human keratinocytes by interferon-g is associated with activation of extracellular signalregulated kinase-1,2 and STAT1. Oncogene 19:248^257, 2000 Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate^phenol^chloroform extraction. Anal Biochem 162:156^159, 1987 Cossins J, Dudgeon TJ, Catlin G, Gearing AJ, Clements JM: Identi¢cation of MMP18, a putative novel human matrix metalloproteinase. Biochem Biophys Res Commun 228:494^498, 1996 Cowley S, Paterson H, Kemp P, Marshall CJ: Activation of MAP kinase kinase is necessary and su⁄cient for PC12 di¡erentiation and for transformation of NIH 3T3 cells. Cell 77:841^852, 1994 Djonov V, Hogger K, Sedlacek R, Laissue J, Draeger A: MMP-19. cellular localization of a novel metalloproteinase within normal breast tissue and mammary gland tumours. J Pathol 195:135^137, 2001 Foschi M, Chari S, Dunn MJ, Sorokin A: Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3 -kinase. EMBO J 16:6439^6451, 1997
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