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[Cancer Research 60, 7156-7162, December 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Activation of Fibroblast Collagenase-1 Expression by Tumor Cells of Squamous Cell Carcinomas Is Mediated by p38 Mitogen-activated Protein Kinase and c-Jun NH2-terminal Kinase-21

Jukka Westermarck2, Songping Li, Panu Jaakkola, Tuula Kallunki, Reidar Grénman and Veli-Matti Kähäri

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University [J. W., S. L., P. J., V-M. K.], MediCity Research Laboratory [J. W.] and Department of Medical Biochemistry [V-M. K.], University of Turku, and Department of Dermatology [V-M. K.] and Otorhinolaryngology-Head and Neck Surgery [R. G.], Turku University Central Hospital, FIN-20520 Turku, Finland, and Apoptosis Laboratory, Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark [T. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Collagenase-1 [matrix metalloproteinase (MMP)-1] is expressed by stromal fibroblasts of various invasive malignant tumors. Here, we have examined the molecular mechanisms of tumor-induced expression of MMP-1 by stromal fibroblasts. Treatment of fibroblasts with conditioned media of tumor cells derived from squamous cell carcinomas (SCCs) of the oral cavity and larynx resulted in activation of fibroblast MMP-1 expression at the transcriptional level. The induction of MMP-1 expression correlates with activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase and phosphorylation of c-Jun and activating transcription factor-2 (ATF-2) and is dependent on the activity of p38 mitogen-activated protein kinase. Furthermore, using fibroblasts derived from JNK2-/- mice, we show that JNK2 is required for induction of fibroblast collagenase-3 expression in response to conditioned SCC tumor cell medium. Together, these results provide evidence that stress-activated p38 and JNK pathways play a crucial role in paracrine regulation of collagenolytic capacity of stromal fibroblasts in SCCs and suggest JNK2 as a novel target for inhibition of MMP-1 expression and tumor invasion.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor invasion is a multistage process in which cellular motility is associated with controlled proteolysis and that involves interactions between tumor cells and the ECM.3 During this process, malignant cells detach from the primary tumor, migrate, and invade through structural barriers, e.g., basement membranes and stromal ECM (1 , 2) . This requires proteolytic degradation of the ECM, and, accordingly, enhanced expression of distinct classes of proteinases by tumor and stromal cells has been documented in malignant tumors with distinct histogenetic background. Recent studies provide evidence that ECM degradation by MMPs plays an important role in tumor growth, invasion, and metastasis, as well as in tumor-induced angiogenesis (see Refs. 1, 2, 3 ). The human MMP gene family consists of at least 20 structurally related zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the ECM. According to their substrate specificity and structure, MMPs can be divided to subgroups of collagenases, stromelysins, gelatinases, membrane-type MMPs, and other MMPs (1 , 2) .

Collagenase-1 (MMP-1) is one of the few proteinases capable of degrading fibrillar collagens, and it is expressed by several types of normal and malignant cells (1 , 2) . In addition, enhanced expression of MMP-1 has been shown to correlate with poor prognosis of several types of malignant tumors (4, 5, 6) . The expression of MMP-1 in invasive neoplastic tumors, e.g., SCCs, of the head and neck and vulva is detected primarily in the stromal compartment (7 , 8) , suggesting, that the expression of MMP-1 in peritumoral fibroblasts is induced in paracrine manner by tumor cells and tumor-infiltrating inflammatory cells. A single AP-1 element located at -65 to -72 in the promoter region of human MMP-1 gene plays a critical role in the activation of MMP-1 gene transcription in response to a variety of extracellular signals (9) . In addition, other regulatory cis-elements, including the ETS element adjacent to AP-1 binding site, play a role in the regulation of AP-1-dependent MMP-1 gene transcription (10 , 11) .

MAPK signaling modules mediate the induction of the expression and the activity of AP-1 and ETS transcription factors in response to extracellular stimuli (12 , 13) . At present, three distinct MAPK pathways are known in detail: (a) ERK1/2; (b) JNK/SAPK; and (c) p38 MAPK. The ERK1/2 pathway (Raf{Rightarrow}MEK1/2{Rightarrow}ERK1/2) is activated by mitogenic growth factors via Ras and by phorbol esters via protein kinase C. In contrast, the stress-activated MAPK pathways JNK/SAPK (MEK kinase 1–3{Rightarrow}MAPK kinase 4 and 7{Rightarrow}JNK/SAPK) and p38 (MAPK kinase kinase{Rightarrow}MAPK kinase 3 and 6{Rightarrow}p38) are activated by cellular stress, e.g., UV light and osmotic and oxidative stress, and by inflammatory cytokines. The ERK1/2 pathway has been shown to mediate activation of minimal MMP-1 promoter by serum, phorbol ester, insulin, and oncostatin M (14, 15, 16) . Activity of p38 MAPK is required for IL-1-elicited induction of MMP-1 expression in fibroblasts and endothelial cells (17) , whereas enhancement of fibroblast MMP-1 expression by lipid second messenger ceramide and tumor promoter okadaic acid involves coordinate activation of the ERK1/2, JNK/SAPK, and p38 MAPK pathways (18 , 19) .

In this study, we have examined the role of MAPK pathways in regulation of fibroblast MMP-1 expression using an experimental approach that would mimic the environment of peritumoral fibroblasts in the invasive SCCs of the head and neck. We show that SCC tumor cell-derived soluble factors activate fibroblast MMP-1 expression at the transcriptional level and that this is mediated by p38 MAPK. In addition, tumor cell-elicited induction of collagenase-3 expression in murine fibroblasts is dependent on activation of JNK2. These results show for the first time that stress-activated MAPK pathways play an important role in tumor cell-induced activation of collagenolytic phenotype of fibroblasts and identify the JNK2 pathway as a novel therapeutic target for inhibition of SCC invasion.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies.
Recombinant TNF-{alpha}, IL-1ß, TGF-{alpha}, GM-CSF, HB-EGF, EGF, and TGF-ß were obtained from Sigma. Blocking antibodies against TNF-{alpha} and IL-1ß were obtained from Genzyme. MEK1/2 inhibitor PD98059, p38 inhibitor SB203580, and PI3k inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). Phosphospecific ERK1/2, JNK, p38, c-Jun, ATF-2, and CREB antibodies and antibody against total p38 were from New England Biolabs (Beverly, MA). batimastat (BB-94) was provided by British Biotech.

Cell Cultures.
Tumor cell lines were established from primary SCCs of the floor of the mouth (UT-SCC-2), supraglottic larynx (UT-SCC-8), tongue (UT-SCC-14), and gingiva (UT-SCC-18); from recurrent SCC of glottic larynx (UT-SCC-6A); and from the metastasis of the same tumor (UT-SCC-6B) at the time of operation in Turku University Central Hospital. Stromal fibroblasts were established from the same SCCs from which tumor cell lines UT-SCC-2 and UT-SCC-8 were derived. Normal human skin fibroblast cultures were established from a volunteer healthy male donor (age, 24 years). Human neonatal foreskin fibroblasts were obtained from American Type Culture Collection. Establishment and characterization of embryonal fibroblast cultures from JNK2-/- mice have been described previously (20) . All cell cultures were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 IU/ml penicillin G, and 100 µg/ml streptomycin.

Tumor Cell Media.
SCC tumor cells in subcultures 6–12 were incubated in culture medium with 1% FCS for 48 h, and conditioned media were collected, assayed for total protein concentration (21) , and stored at -20°C. Confluent fibroblast cultures were maintained in DMEM supplemented with 1% FCS for 18 h before replacing the medium with DMEM containing different concentrations of the conditioned TM and 1% FCS. In experiments involving signaling pathway inhibitors, these were added 1 h before replacing the medium with TM containing an equal concentration of the inhibitor. In experiments with batimastat (BB-94), this was added to the culture medium of tumor cells in the beginning of the 48-h incubation or simultaneously when tumor media collected without BB-94 were added to fibroblast cultures.

RNA Analysis.
Total cellular RNA was isolated from cells using the RNeasy kit (Qiagen,Valencia, CA). Aliquots of total RNA (5–15 µg) were analyzed by Northern blot hybridization, as described previously (19) . The probes used were 2.0-kb human collagenase-1 (MMP-1) cDNA (22) , a 2.0-kb mouse collagenase-3 (MMP-13) cDNA (23) , and 1.3-kb cDNA for rat GAPDH (24) . The cDNA-mRNA hybrids were visualized by autoradiography, and the levels of MMP-1 mRNA were quantitated by scanning densitometry and corrected for the levels of GAPDH mRNA or abundance of 28S rRNA in the same samples.

Transient Transfections.
Human neonatal skin fibroblast cultures were transiently transfected with 0.5 µg of human MMP-1 promoter/CAT 5'-deletion constructs [2278CLCAT, -95CLCAT, -72CLCAT, and -55CLCAT (Ref. 25 ; kindly provided by Dr. William C. Parks; Washington University, St. Louis, MO)]. Cells were transfected with FuGene6 cationic lipid transfection reagent following the manufacturer’s instructions (Roche, Mannheim, Germany), and medium was replaced 8 h after transfection with medium containing 50% of conditioned medium of SCC tumor cell line UT-SCC-2, and the incubations were continued for 36 h. CAT activity was measured as an index of promoter activity (11) .

DNase I Footprinting.
Nuclear protein extract was prepared as described previously (11) . For DNaseI footprinting, plasmid -178CLCAT (25) was linearized with NarI, end-labeled with [{alpha}-32P]dCTP using Klenow DNA polymerase (Promega), and digested with XhoI to release human MMP-1 promoter fragment extending from -60 to -178, which was purified by PAGE. Labeled DNA fragment was incubated for 10 min at room temperature with 30 µg of nuclear extract and 2 µg of poly(dI-dC) (Boehringer Mannheim) in a reaction buffer [10 mM Tris-HCl (pH 8), 5 mM MgCl2, 1 mM CaCl2, 2 mM DTT, 50 mg/ml BSA, and 100 mM KCl)], 0.5 unit of DNaseI (Boehringer Mannheim) was added, and the reaction was stopped after 2 min. The digestions were fractionated on an 8% polyacrylamide sequencing gel in parallel with a chemical G+A sequencing ladder (26) .

Determination of MAPK and Transcription Factor Activation.
The activation of ERK1/2, JNK, p38, c-Jun, ATF-2, and CREB was determined by Western blot analysis using antibodies specific for phosphorylated forms of these proteins. Confluent fibroblast cultures were treated with tumor cell media for different periods of time, washed, and lysed in 100 µl of Laemmli sample buffer. Thereafter, proteins were sonicated and separated in 10% SDS-PAGE gel and transferred to nitrocellulose membrane (Amersham). Western blot analysis with phosphospecific antibodies was performed as described previously (18 , 19) , using enhanced chemiluminescence detection system (Amersham).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Fibroblast MMP-1 Expression by SCC Tumor Cell Media.
To study the regulation of fibroblast MMP-1 gene expression by SCC tumor cells, we collected conditioned media (TM) of six low-passage tumor cell lines established from SCCs of the oral cavity and larynx and used them to treat SCC stromal fibroblasts in culture. Initially, fibroblasts were incubated with medium containing 30% or 50% of SCC TM, and their MMP-1 mRNA expression was studied by Northern blot hybridizations. As shown in Fig. 1ACitation , conditioned media of tumor cell lines UT-SCC-2, UT-SCC-14, and UT-SCC-18 (called +TM below) potently enhanced MMP-1 mRNA expression, whereas treatment with conditioned media of tumor cell lines UT-SCC-6A, UT-SCC-6B, and UT-SCC-8 (called -TM below) did not markedly alter MMP-1 mRNA levels in fibroblasts. Nearly maximal activation of fibroblast MMP-1 mRNA expression was obtained with 30% of +TM, and this concentration was therefore used in additional experiments. Similar results were also obtained with another SCC tumor fibroblast line and normal human skin fibroblasts incubated with same media (data not shown).



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  		Fig. 1. Induction of stromal fibroblast MMP-1 expression by SCC tumor cells. A, human head and neck SCC stromal fibroblasts were treated for 24 h with medium containing 30% or 50% of conditioned medium of six different human SCC tumor cell lines, as indicated. B, SCC stromal fibroblasts were treated for 24 h with different ratios of conditioned media of SCC tumor cell lines, as indicated. A and B, total RNA was isolated, and aliquots (12 µg) were analyzed for expression of MMP-1 and GAPDH mRNAs by Northern blot hybridizations.

 
Next, stromal fibroblasts were treated simultaneously with +TM and -TM in different ratios to rule out the possibility that inability of -TM to activate fibroblast MMP-1 expression was due to the presence of inhibitory factor(s) that could block activation of MMP-1 expression by stimulatory factor(s) in the same media. Interestingly, treatment with a high concentration (70% or 90%) of -TM (of UT-SCC-6A) also enhanced fibroblast MMP-1 expression, and a mixture of +TM (10%) and -TM (90%) activated MMP-1 expression 3.3-fold more potently than 10% +TM (of UT-SCC-2) alone (Fig. 1B)Citation . Furthermore, total protein concentration of tumor media did not correlate with the capacity to activate MMP-1 expression (data not shown), suggesting that the difference between -TM and +TM is based on the difference in the concentration of stimulatory factor(s) in the media.

Next, we compared the level of induction of fibroblast MMP-1 mRNA expression by 30% +TM to that obtained by treatment with TNF-{alpha}, IL-1ß, TGF-{alpha}, EGF, HB-EGF, and GM-CSF. As shown in Fig. 2ACitation , IL-1ß and TNF-{alpha} stimulate MMP-1 mRNA expression nearly as potently as +TM, whereas EGF, HB-EGF, and TGF-{alpha} were clearly less potent in enhancing MMP-1 mRNA abundance in fibroblasts, and GM-CSF had no marked effect. To study, whether the inducing factor in the +TM would be TNF-{alpha} or IL-1ß, we treated tumor fibroblasts with +TM preincubated with specific blocking antibody against these cytokines. As shown in Fig. 2BCitation , induction of fibroblast MMP-1 mRNA levels by TNF-{alpha} and IL-1ß was entirely inhibited by the corresponding blocking antibody, whereas neither antibody altered the induction of MMP-1 mRNA expression by 10% or 5% +TM.



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  		Fig. 2. Characterization of the conditioned SCC TM. A, SCC stromal fibroblasts were incubated for 24 h with 30% of UT-SCC-2 medium (+TM), or with IL-1ß (10 units/ml), GM-CSF (100 µM), TNF-{alpha} (20 ng/ml), EGF (10 ng/ml), HB-EGF (25 ng/ml), and TGF-{alpha} (10 ng/ml), as indicated. Total RNA was isolated, and expression of MMP-1 and GAPDH mRNAs was determined by Northern blot hybridizations, quantitated by densitometry, and corrected for the levels of GAPDH mRNA. Data shown represent the effect of the individual factor shown relative to the effect of tumor medium (+TM) treatment in the same experiment. B, SCC tumor fibroblasts were incubated for 24 h with medium containing the indicated proportion of conditioned medium of tumor cell line UT-SCC-2 (+TM), IL-1ß (10 units/ml), or TNF-{alpha} (20 ng/ml), preincubated for 2 h with specific blocking antibody against IL-1ß or TNF-{alpha}. The abundance of MMP-1 and GAPDH mRNAs was determined by Northern blot hybridizations. C, SCC tumor cell lines UT-SCC-2 and UT-SCC-14 were cultured for 48 h in medium containing 1% FCS with or without metalloproteinase inhibitor BB-94 (6 µM), and SCC stromal fibroblasts were treated with medium containing 30% of this medium (Lanes 1–5). In parallel, tumor fibroblasts were treated with conditioned media of cells incubated without BB-94, which was added to fibroblast cultures simultaneously with tumor cell media (Lanes 6 and 7). The expression of MMP-1 and GAPDH mRNAs was examined by Northern blot hybridizations.

 
To examine whether the MMP-1-inducing capacity of +TM is dependent on proteolytic shedding or cleavage of tumor cell-derived factors, we incubated SCC tumor cells in the presence of batimastat (BB-94), a specific inhibitor of metalloproteinase activity (27) . Treatment of fibroblasts with +TM collected after a 48-h incubation of SCC tumor cells in the presence of BB-94 resulted in an equally potent activation of MMP-1 mRNA expression as seen with conditioned media of tumor cells incubated without BB-94 (Fig. 2Citation C, Lanes 1–5). Furthermore, the addition of BB-94 to fibroblasts simultaneously with +TM had no effect on the induction of fibroblast MMP-1 expression by +TM (Fig. 2Citation C, Lanes 6 and 7).

Activation of MMP-1 Gene Transcription by SCC Tumor Cell Media.
We and others have shown that the MMP-1 gene promoter segment at -95 to -65, which contains the adjacent AP-1 and ETS binding sites, plays an important role in the activation of MMP-1 gene transcription in fibroblasts (10 , 11) . To examine the regulation of MMP-1 gene promoter activity by SCC TM, we transiently transfected neonatal human foreskin fibroblasts with 5' deletion constructs of the human MMP-1 promoter linked to CAT reporter gene and measured CAT activity as an indicator of promoter activity after a 36-h treatment of fibroblasts with +TM. As shown in Fig. 3ACitation , the activity of -55CLCAT, which contains the basal promoter region of the MMP-1 gene, was only slightly (1.3-fold) enhanced by treatment of fibroblasts with +TM, whereas the activity of -72CLCAT, which contains the AP-1 element, was stimulated 4.4-fold. Interestingly, the presence of the ETS element in -95CLCAT or other upstream elements in -2278CLCAT did not increase MMP-1 promoter activation by +TM treatment (Fig. 3A)Citation , providing evidence that the AP-1 site plays a major role in stimulation of MMP-1 promoter activity in response to SCC TM.



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  		Fig. 3. Activation of MMP-1 promoter by SCC TM. A, human neonatal foreskin fibroblasts were transiently transfected with distinct human MMP-1 promoter/CAT constructs and treated for 36 h with 50% of conditioned medium of SCC tumor cell line UT-SCC-2 (+TM), followed by an assay of CAT activity as an indicator of promoter activity. Induction of promoter activity compared with untreated cells (1.00) represents the mean of five independent experiments. B, DNase I footprinting was performed with end-labeled MMP-1 promoter fragment (-60 to -178) alone (NAKED) or together with nuclear extracts from untreated control cells (CTL) or from cells treated with 50% of conditioned medium of UT-SCC-2 tumor cells (+TM). The samples were fractionated on an 8% polyacrylamide sequencing gel in parallel with the A+G sequencing ladder of the same promoter fragment. The location of the AP-1 and ETS binding sites is indicated, and the sequence of the elements in antisense orientation is shown. A representative experiment of four independent experiments with identical results is shown.

 
To further study the role of AP-1 and ETS cis-elements in tumor cell-elicited activation of the MMP-1 promoter, we performed in vitro DNase footprinting using a MMP-1 promoter fragment extending from -60 to -115 as a probe. Interestingly, the AP-1 element at -72 to -65 was clearly protected with nuclear proteins from untreated control fibroblasts, and no alteration in protection was observed after treatment with +TM for 6, 8, and 12 h (Fig. 3BCitation ; data not shown), indicating that the activation of MMP-1 promoter by +TM is not associated with increased occupancy of the AP-1 binding site. In accordance with the data above, the ETS element at position -89 to -83 was not protected by nuclear proteins from control or +TM-treated fibroblasts (Fig. 3B)Citation . These results indicate that the activation of MMP-1 gene expression by +TM occurs at the promoter level and suggest that it is mediated through activation of prebound AP-1 transcription factors but not through the ETS element.

Regulation of AP-1 Expression and Activity by Tumor Cell Media.
To study the regulation of AP-1 expression by tumor media, we treated stromal fibroblasts with both +TM and -TM for different periods of time and determined MMP-1 and AP-1 mRNA abundance by Northern blot hybridizations. Interestingly, treatment with both +TM and -TM induced expression of junB, c-fos (Fig. 4, A and B)Citation , and fra-1 (data not shown) mRNA equally potently, whereas induction of c-jun mRNA by +TM declined markedly later on when compared with induction by -TM (Fig. 4, A and B)Citation . Neither ets-1 nor ets-2 mRNA was induced by tumor cell media (data not shown).



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  		Fig. 4. Regulation of AP-1 expression and phosphorylation by SCC TM. A, tumor fibroblasts were incubated with 30% of +TM or -TM for different time periods, as indicated. Total RNA was isolated, and the levels of MMP-1, c-jun, junB, c-fos, and GAPDH mRNA were determined by Northern blot hybridizations. B, quantitation of AP-1 mRNA data representing the mean of two experiments in which conditioned tumor cell media of cell lines UT-SCC-2 and UT-SCC-6A or UT-SCC-14 and UT-SCC-8 were used in pairs. C, tumor fibroblasts were incubated with 30% +TM or -TM for the indicated time period. Thereafter, cells were lysed to sample buffer, and equal aliquots were subjected to Western blot analysis using antibodies specific for phosphorylated forms of c-Jun (p-Jun) and ATF-2 (p-ATF2; top) or CREB (p-CREB; bottom). As loading controls, the levels of total p38 were determined from the same filters. A representative experiment of three independent experiments with identical results is shown.

 
Activation of c-jun transcription is mediated by JNK and p38 MAPK, which phosphorylate ATF-2 and c-Jun bound to the AP-1 site in the promoter of c-jun (12) . To study the activation of c-Jun and ATF-2 by SCC tumor cell media, we treated tumor fibroblasts with +TM and -TM and examined phosphorylation of c-Jun and ATF-2 by Western blot analysis using phosphospecific antibodies. As shown in Fig. 4CCitation , treatment of fibroblasts with +TM induced phosphorylation of c-Jun from 30–120 min, whereas no phosphorylated form of c-Jun was observed in response to -TM treatment. Moreover, ATF-2 phosphorylation was clearly increased at 15 and 30 min of incubation with +TM, as compared with -TM. In contrast, treatment with -TM induced phosphorylation of transcription factor CREB slightly more potently (Fig. 4C)Citation . Taken together, these results show that activation of signaling pathways that induce c-Jun and ATF-2 phosphorylation specifically correlates with induction of MMP-1 expression by SCC tumor cell-derived factors and suggest that MMP-1 gene expression is regulated by mechanisms involving posttranscriptional regulation of AP-1 activity.

Induction of MMP-1 Expression by SCC TM Is Mediated by p38.
To study the role of distinct MAPK pathways in the induction of fibroblast MMP-1 expression by tumor cell media, we first examined the activation of ERK1/2, JNK, and p38 by Western blot analysis using phosphospecific antibodies. Interestingly, ERK1/2 was equally activated by both +TM and -TM in a time range from 15 min to 1 h (Fig. 5, A and B)Citation . In contrast, JNK and p38 were clearly more potently activated by +TM (Fig. 5, A and B)Citation , suggesting a role for JNK and p38 MAPK in the activation of fibroblast MMP-1 gene expression by SCC tumor cells.



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  		Fig. 5. Activation of fibroblast JNK and p38 by SCC TM. Tumor fibroblasts were treated with 30% of conditioned medium of UT-SCC-2 or UT-SCC-14 cells (+TM) or with conditioned medium of UT-SCC-8 or UT-SCC-6A cells (-TM) for the indicated periods of time. Thereafter, cells were lysed to sample buffer, and activation of ERK1/2, JNK, and p38 MAPK was determined by Western blot analysis using phosphospecific antibodies for the corresponding MAPKs. Analysis of total p38 was performed as a loading control. Cell lysate of HaCaT keratinocytes treated with EGF (20 ng/ml) for 20 min were used as a positive control B, quantitative data from four independent experiments. The levels of activated ERK1/2 (p-ERK), JNK (p-JNK), and p38 MAPK (p-p38), as well as total ERK1/2, JNK, and p38, are shown relative to the levels at time point 0 h (1.0).

 
To directly examine the role of distinct MAPK pathways in the tumor medium-elicited activation of MMP-1 expression, we blocked ERK1/2 and p38 MAPK pathways by chemical inhibitors previously shown to block MMP-1 gene activation by diverse stimuli (17, 18, 19) . As seen in Fig. 6ACitation , activation of fibroblast MMP-1 mRNA abundance by +TM was potently inhibited by SB203580, a specific inhibitor of p38 activity. In contrast, treatment of fibroblasts with PD98059, an inhibitor of ERK1/2 activators MEK1/2, had no marked effect on the induction of MMP-1 mRNA levels by +TM (Fig. 6, A and B)Citation , although it potently abrogated induction of MMP-1 mRNA abundance by C2-ceramide (Fig. 6B)Citation . In parallel, blocking the activity of PI3k by specific inhibitor LY294002 had no marked effect on the activation of fibroblast MMP-1 expression by +TM (Fig. 6A)Citation .



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  		Fig. 6. TM-elicited induction of fibroblast MMP-1 expression is mediated by p38 MAPK. A, tumor fibroblasts were incubated for 24 h with 30% of conditioned medium of SCC tumor cell line UT-SCC-2 (+TM) without or with specific MEK1/2 inhibitor PD98059 (PD; 10 µM), specific p38 inhibitor SB203580 (SB; 10 µM), or specific PI3k inhibitor LY294002 (LY; 10 µM), all of which were added 1 h before +TM. B, tumor fibroblasts were incubated for 24 h with 30% of +TM as described in A or with C2-ceramide (Cer; 50 µM) without or with MEK1/2 inhibitor PD98059 (PD) added at the concentrations indicated 1 h before +TM. A and B, total RNA was analyzed for expression of MMP-1 and GAPDH mRNAs by Northern blot hybridizations. The levels of MMP-1 mRNA were quantitated by densitometry and corrected for GAPDH mRNA levels and are shown below the panels relative to the levels in control cells (1).

 
Induction of Mouse Fibroblast Collagenase-3 Expression by SCC TM Is Mediated by JNK2.
To study the role of the JNK pathway in the TM-elicited induction of fibroblast MMP-1 expression, we treated embryonal fibroblasts derived from a JNK2 knockout mouse (JNK2-/-) and from a corresponding wild-type mouse (JNK2+/+) with +TM (of UT-SCC-2 and UT-SCC-14) and determined the expression of murine collagenase-3 (MMP-13), the only murine fibroblast collagenase. Interestingly, +TM treatment clearly induced the expression of mouse collagenase-3 mRNA in JNK2+/+ fibroblasts, but not in JNK2-/- fibroblasts (Fig. 7A)Citation . Similar results were obtained with two embryonal fibroblast cell lines from two distinct strains of JNK2+/+ and JNK2-/- mice. To confirm that the lack of mouse collagenase-3 mRNA induction by +TM in JNK2-/- cells is not due to general suppression of gene regulation, we treated JNK2-/- and JNK2+/+ mouse fibroblasts with okadaic acid, which is known to activate MMP-1 gene expression through MAPK signaling (19) , and with TGF-ß, a down-regulator of fibroblast MMP-1 expression (28) . As shown in Fig. 7BCitation , treatment with okadaic acid up-regulated mouse MMP-13 mRNA levels, and treatment with TGF-ß down-regulated mouse MMP-13 mRNA levels in both JNK2-/- and JNK2+/+ mouse fibroblasts. The specific role of JNK signaling in tumor medium-induced mouse fibroblast MMP-13 expression was further confirmed by showing that ERK1/2 was similarly activated in both JNK2+/+ and JNK2-/- cell lines (Fig. 7C)Citation . This is in accordance with the data above showing that activation of the ERK1/2 pathway is not required for induction of MMP-1 gene expression by SCC tumor cell media. In addition, the expression of JNK1 is similar in JNK2-/- and JNK2+/+ cell lines, confirming that lack of the JNK2 gene in JNK2-/- fibroblast cells has no effect on the expression of JNK1 (Fig. 7C)Citation .



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  		Fig. 7. TM-elicited induction of fibroblast MMP-1 expression is mediated by JNK2. A, embryonal fibroblasts from JNK2+/+ and JNK2-/- mouse were treated for 24 h with 50% of SCC tumor cell media, as indicated. B, JNK2+/+ and JNK2-/- fibroblasts were treated for 24 h with okadaic acid (OA; 10 ng/ml) or TGF-ß (TGF; 5 ng/ml). A and B, aliquots (15 µg) of total RNA were analyzed for expression of murine collagenase-3 (mMMP-13) mRNA by Northern blot hybridizations. 28S rRNA was visualized by ethidium bromide staining as a loading control. Representative blots of two experiments, with fibroblasts derived from two distinct mouse strains, are shown. C, JNK2+/+ and JNK2-/- mouse embryonal fibroblasts were treated with 50% of conditioned medium of UT-SCC-2 cells (+TM) for the time periods indicated. Thereafter, cells were lysed to sample buffer, and activation of ERK1/2 (p-ERK) was determined by Western blot analysis using phosphospecific antibody. The filter was stripped and subjected to analysis of total JNK1 and JNK2 using specific antibodies. Cell lysates of HaCaT keratinocytes treated with EGF (20 ng/ml) for 20 min were used as a positive control for ERK1/2 activation.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor growth and invasion is a complex process that involves interactions between tumor cells and fibroblasts of the surrounding stromal tissue. Degradation of stromal ECM by MMPs apparently plays an important role in growth and invasion of malignant tumors, and in general, MMPs are produced by tumor cells, peritumoral stromal fibroblasts, and tumor-infiltrating inflammatory cells (1, 2, 3) . Malignant tumor cells also induce expression of distinct MMPs in peritumoral fibroblasts. For example, breast carcinoma cell-derived IL-1 potently enhances expression of MMP-13 in stromal fibroblasts (29) . Interestingly, recent findings also suggest that stromal fibroblasts play a role in regulation of the malignant epithelial cell phenotype and that this may be achieved by fibroblast-derived MMPs (30, 31, 32) . Previous studies using recombinant growth factors and cytokines and purified tumor promoters have been successful in elucidating signaling mechanisms involved in the regulation of MMP gene expression, but this experimental approach does not take into account the in vivo condition, in which tumor cells and stromal fibroblasts are exposed to a variety of tumor cell-derived factors. In the present study, we show that a subgroup of low-passage primary tumor cell lines established from human head and neck SCCs secretes factor(s) that induce MMP-1 expression in normal and tumor fibroblasts. Although we were not able to identify the collagenolytic phenotype-inducing factor in TM, we show that it is neither TNF-{alpha} nor IL-1ß and that the effect is not dependent on MMP activity and requires activity of p38 MAPK and JNK2.

Increased AP-1 activity has been shown to transform benign cells and enhance tumor cell invasion and metastasis (33) . Although enhanced expression of AP-1 genes in malignant tumors in vivo has been reported, there is no consistent pattern that would serve as a marker for increased invasion or malignancy (34, 35, 36) . Interestingly, even reduced expression of c-jun, junB, and c-fos genes was observed in human lung carcinomas, as compared with normal tissue (37) . In the present study, prolonged activation of fibroblast c-jun expression by SCC tumor cell media is associated with stimulation of MMP-1 gene expression, whereas equal activation of c-fos, junB, and fra-1 mRNA expression by -TM and +TM suggests that these AP-1 genes do not play a role in the induction of fibroblast MMP-1 gene expression. Our results also show that the increased phosphorylation of c-Jun in cells treated with +TM was more pronounced than the difference seen in the regulation of c-jun expression, further suggesting that phosphorylation of AP-1 components plays a key role in the regulation of MMP-1 transcription. Furthermore, induction of c-jun expression by -TM treatment in the absence of c-Jun phosphorylation suggests that activation of c-jun expression may be mediated, in part, by c-Jun phosphorylation-independent mechanisms such as the ERK5{Rightarrow}MEF2 pathway (38) .

We have recently shown that the activation of fibroblast MMP-1 expression by ceramide and okadaic acid is mediated by coordinate activation of ERK1/2, JNK/SAPK, and p38 MAPK pathways (18 , 19) . Furthermore, MEK1 activity is required for increased expression of MMP-1 in Ras-transformed human fibroblasts.4 Moreover, it has been shown that constitutive activation of ERK1/2 results in transformation of fibroblasts (39) and that the ERK1/2 pathway is activated in malignant tumors in vivo (40 , 41) . Interestingly, the results of the present study clearly show that ERK1/2 activation is not required for the induction of fibroblast MMP-1 expression by +TM and that, in the absence of JNK2, activation of the ERK1/2 pathway is not sufficient for induction of murine fibroblast MMP-13 expression. Our results also show that ERK1/2 activation and induction of junB and c-fos mRNA expression are similar with both -TM and +TM, which is in accordance with a recent study showing that the expression of c-fos and junB is regulated primarily by the ERK1/2 pathway (42) . Taken together, these results clearly show that, in contrast to other stimuli (18 , 19) , ERK1/2 activation does not play a role in the paracrine regulation of fibroblast MMP-1 expression by SCC tumor cells. Our results also show that activity of p38 MAPK is critical in induction of fibroblast MMP-1 expression by SCC cell media. These results are in accordance with our previous observations showing the importance of the p38 MAPK pathway in induction of MMP-1 expression in fibroblasts and in SCC cells (18 , 19 , 43) .

Our observations, which suggest that induction of MMP-1 promoter activity in response to +TM treatment takes place without alterations in the occupancy of MMP-1 promoter AP-1 binding element, are in accordance with previous results showing that inhibition of phorbol ester-elicited MMP-1 promoter activation by dexamethasone does not alter AP-1 site occupation (44) . Interestingly, it was recently reported that dexamethasone inhibits JNK activity and phosphorylation of c-Jun (45) , emphasizing the role of JNK in the activation of prebound AP-1 complexes by phosphorylation, as has been shown previously in the activation of c-jun promoter (46) . Because JNK2 displays a higher affinity for c-Jun than other JNK isoforms (47) , we assessed the role of JNK signaling in tumor medium-elicited enhancement of collagenolytic phenotype of fibroblasts using embryonal fibroblasts from JNK2 knockout mice. No murine homologue for human MMP-1 has been identified; therefore, we determined the expression of murine collagenase-3 (MMP-13), the only murine fibroblast collagenase in these cells. Murine MMP-13 promoter contains certain conserved regulatory elements, including AP-1 and ETS-binding elements, similar to those in the human MMP-1 promoter, but in general, the two promoters do not show remarkable homology (23) . However, the expression pattern of murine MMP-13 and human MMP-1 is similar in cutaneous wound repair in vivo because both are expressed by migrating keratinocytes and dermal fibroblasts (48) , providing evidence for similar regulatory mechanisms for these MMPs, at least in fibroblasts and keratinocytes. Our results show that JNK2 is required for +TM-elicited induction of mouse MMP-13 expression, providing for the first time direct genetic evidence for the role of JNK signaling in the regulation of fibroblast MMP expression. In contrast, lack of JNK2 did not markedly alter the induction of mouse MMP-13 expression by okadaic acid or down-regulation of MMP-13 expression by TGF-ß. This suggests that specific inhibition of JNK2 activity could be used to inhibit induction of MMP-1 expression by tumor cell-derived factors without interfering with regulation of physiological collagen turnover regulated by inflammatory cytokines and growth factors such as TGF-ß. Interestingly, a recent study showed that inhibition of JNK2 expression by antisense oligonucleotides blocked EGF-induced transformation of human lung carcinoma cells (49) , providing further evidence that specific inhibition of signaling via JNK2 may serve as a novel approach to inhibit tumor growth and invasion.

In conclusion, the results above show that low-passage tumor cells from SCCs of the head and neck secrete soluble factor(s), which dose-dependently activate MMP-1 expression in stromal fibroblasts. Furthermore, this effect is not due to the presence of TNF-{alpha} or IL-1ß and is not dependent on MMP activity. Our results show that activation of fibroblast collagenolytic phenotype by SCC tumor cells is mediated by p38 MAPK and JNK2, which may serve as novel targets for therapy aimed at inhibiting malignant tumor invasion.


   ACKNOWLEDGMENTS
 
The expert technical assistance of Hanna Haavisto, Tarja Heikkilä, and Marita Potila is gratefully acknowledged. We also thank Dr. S. E. Bauer, P. Angel, J. Minna, and P. Fort for plasmids.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Academy of Finland (Projects 30985 and 45996), the Sigrid Jusélius Foundation, the Cancer Research Foundation of Finland, and Turku University Central Hospital (Project 13336) and by a research contract with Finnish Life and Pension Insurance Companies. Back

2 To whom requests for reprints should be addressed, at Centre for Biotechnology, University of Turku, Tykistökatu 6B, FIN-20520 Turku, Finland. Fax: 358-2-3338000; E-mail: jukwes{at}utu.fi Back

3 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/ERK kinase; SAPK, stress-activated protein kinase; TNF-{alpha}, tumor necrosis factor {alpha}; IL, interleukin; TGF, transforming growth factor; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF; GM-CSF, granulocyte macrophage colony-stimulating factor; TM, tumor cell medium; SCC, squamous cell carcinoma; ATF-2, activating transcription factor-2; AP-1, activator protein 1; PI3k, phosphatidylinositol 3'-kinase; CREB, cAMP-responsive element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase. Back

4 J. Westermarck, S-P. Li, T. Kallunki, J. Han, and V-M. Kähäri. p38 MAPK dependent activation of protein phosphatase-1 and 2A inhibits MEK1,2 activity and collagenase-1 (MMP-1) gene expression, submitted for publication. Back

Received 5/31/00. Accepted 10/18/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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