From the INSERM U532, Institut de Recherche sur la Peau, Université Paris VII, Hôpital Saint-Louis, Pavillon Bazin, 75010 Paris, France
Received for publication, February 24, 2003 , and in revised form, April 16, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extracellular stimuli elicit specific intracellular signals via activation of a family of so-called mitogen-activated protein (MAP) kinases, consisting of extracellular signal-regulated kinases, p38 MAP kinases, and the JNKs (46). These MAP kinases phosphorylate transcription factors within the cell nucleus, thereby activating a number of cellular functions, including proliferation, apoptosis, differentiation, and regulation of gene expression. It has been reported that the extracellular signal-regulated kinases and p38 MAP kinases are activated in fibroblasts during collagen matrix contraction under isometric tension (7) and that they cooperate in contraction-stimulated activation of the immediate early gene c-fos, although they are not required for lattice contraction per se (8). In mechanically unloaded collagen lattices, on the other hand, extracellular signal-regulated kinase signaling is disrupted, a mechanism that may be responsible for the entry of fibroblast into a quiescent state after several days under these conditions (9).
The JNK group of MAP kinases, also known as stress-activated kinases, are activated upon exposure of cells to cytokines, growth factors, and environmental stresses such as UV irradiation or heat shock (10). Three distinct genes, jnk1, jnk2, and jnk3, have been identified to encode JNKs. The former two genes are ubiquitously expressed, whereas jnk3 is selectively expressed in the heart, testis, and brain. Dual Thr and Tyr phosphorylation of JNK by the MAP kinase kinases (MKKs), MKK4 and MKK7, results in JNK activation and nuclear translocation. In the nucleus, JNKs phosphorylate transcription factors such as c-Jun (11), a process that leads to maximal transcriptional activity of the latter (12). Thus far, little is known about the role of JNK in the context of fibroblast ability to remodel collagen matrices, except that it was shown recently that JNK regulates the phenotypic modulation of lung fibroblasts into myofibroblasts induced by TGF- (13), interleukin 4, and interleukin 13 (14).
Recent data from our laboratory have indicated that basal JNK activity in fibroblasts maintains a limited yet significant pool of phosphorylated c-Jun protein (15). In this report, using pharmacologic and genetic approaches aimed at interfering with basal JNK activity, we demonstrate a critical role for the latter in allowing fibroblast motility, whereas it inhibits the ability of fibroblasts to contract free-floating collagen matrices and does not modify the expression of fibrillar collagen genes or their modulation by TGF-. Details are provided herein.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Collagen Matrix ContractionFibroblasts were harvested from monolayer culture with 0.25% trypsin and 1 mM EDTA, and then trypsin was neutralized with 10% fetal calf serum-containing medium. Collagen lattices in 60-mm dishes were prepared with 7 ml of a mixture containing 106 fibroblasts and 1 mg/ml native type I collagen (BiocoatTM; BD Biosciences) in medium supplemented with 10% fetal calf serum. When needed, TGF- was added to the mixture before polymerization of the collagen matrix. Polymerization of collagen matrices required
60 min at 37 °C. To initiate lattice contraction, freshly polymerized matrices were released from the underlying culture dish with a few gentle taps on the dish.
Wound Closure and TranswellTM Motility AssaysFor wound closure assays, confluent cell monolayers were wounded by manually scraping the cells with a pipette tip. After wounding, wound size was verified to ensure that all wounds were the same width (see corresponding figures). The cell culture medium was then replaced with fresh medium, and wound closure was monitored by microscopy at various times. TranswellTM migration assays were performed utilizing 8-µm pore, 6.5-mm polycarbonate TranswellTM filters (Falcon, Franklin Lakes, NJ). In some experiments, a type I collagen solution (10 µg in 100 µl) was allowed to polymerize in the upper well for 1 h at 37 °C. Single cell suspensions were seeded onto the upper surface of the filters in medium containing 10% fetal calf serum (without or with prior collagen coating) and allowed to migrate through the membrane. After a 16-h incubation period, cells on the upper surface of the filter were wiped off with a cotton swab, and the cells that had migrated to the underside of the filter were fixed, stained with DiffQuickTM (Dade Behring, Düdingen, Switzerland), and counted by bright-field microscopy at x200 in six random fields.
Northern BlottingTotal RNA was obtained using an RNeasy kit (Qiagen GmbH, Hilden Germany) and analyzed by Northern hybridization (20 µg/lane) with 32P-labeled cDNA probes for COL1A1 (20), COL1A2 (21), COL3A1 (22), and GAPDH (23). Hybridization signal was revealed with a PhosphorImager (Storm 840; Amersham Biosciences).
Western BlottingWhole cell extracts were prepared in 10 mM Tris, pH 7.4, 1% SDS, and 1 mM sodium vanadate; treated with Benzon nuclease (Sigma) for 5 min at room temperature; and denatured by heating at 95 °C for 3 min. Protein concentration in each lysate was assayed with a one-step colorimetric method (Bio-Rad protein reagent; Bio-Rad), and 25 µg of protein was resolved by SDS-PAGE. After electrophoresis, proteins were transferred to Hybond ECL nitrocellulose filters (Amersham Biosciences). Filters were placed in blocking solution (1x Tris-buffered saline and 5% nonfat milk) for 1 h and immunoblotted with either goat anti-type I collagen (Southern Biotech, Birmingham, AL), rabbit anti-phospho-c-Jun (Upstate Biotechnologies, Lake Placid, NY), anti-JNK1 (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-phospho-JNK1 (Cell Signaling Technology Inc., Beverly, MA) at a 1:1000 dilution in 1x Tris-buffered saline, 0.1% Tween 20, and 5% nonfat milk for 1 h. A mouse anti--actin (Sigma) antibody at a 1:10,000 dilution in 5% nonfat milk was used as a control. For detection of phosphorylated proteins, nonfat milk was replaced by bovine serum albumin. After incubation, filters were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h. Filters were then washed, developed according to chemiluminescence protocols (ECL; Amersham Biosciences), and revealed with a PhosphorImager (Storm 840; Amersham Biosciences).
Transient Cell Transfections and ElectroporationTo determine basal JNK activity, we used a reporter system derived from the mammalian one-hybrid system, consisting of a reporter plasmid, Gal4-lux, and a trans-activator plasmid encoding a chimeric trans-activator protein (Gal4BD-c-Jun) consisting of the DNA binding domain of Gal4 (Gal4BD) and the transactivation domain of c-Jun that requires phosphorylation by JNK to fully transactivate Gal4-lux (Stratagene, La Jolla, CA). Transfections were performed using the calcium phosphate/DNA co-precipitation procedure with a commercial assay kit (Promega, Madison, WI). pRSV--galactosidase was co-transfected in every experiment to monitor transfection efficiencies. Luciferase activity was determined with a commercial kit (Promega). For high transfection efficiency of a dominant-negative (D/N) MKK4 expression vector (a kind gift from Dr. A. Atfi; INSERM U482, Paris, France) (24), human dermal fibroblasts were electroporated with a NucleofectorTM (Amaxa GmbH, Koeln, Germany) according to the manufacturer's protocol. Transfection efficiency was estimated to be around 80% (data not shown) by fluorescence-activated cell-sorting analysis of a co-transfected green fluorescent protein expression vector.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Once the JNK status was clearly defined in our experiments, we examined the role of its basal activity on fibroblast motility. Initially, we compared the motility of wt, jnk/, and junAA immortalized mouse embryo fibroblasts in a wound closure assay. As shown in Fig. 2, wt fibroblasts migrated into the wound area and completely closed the wound within 14 h (top left panels). On the other hand, in jnk/ fibroblast cultures, the wounds remained open (Fig. 2, middle left panels). Variations in cell proliferation did not account for the differences between wt and jnk/ fibroblasts because wt fibroblasts closed the wound equally rapidly in the absence or presence of mitomycin C (4 µg/ml), a compound that blocks cell division (data not shown).
|
Because c-Jun is a key substrate for JNK, junAA fibroblasts, in which c-Jun Ser63 and Ser73 have been mutated into alanines so that c-Jun can no longer be phosphorylated by JNK, were used to determine whether c-Jun phosphorylation by JNK was important for the effect of basal JNK activity on fibroblast motility. As shown in Fig. 2 (bottom panels), junAA fibroblasts were identified as poorly motile in the same wound closure assay described above, undistinguishable from jnk/ fibroblasts (Fig. 2, middle panels). These data indicate that the basal state of c-Jun phosphorylation by JNK is critical for cell migration.
Next, because TGF- is a known activator of fibroblast motility (25), we tried to determine whether TGF-
would be able to accelerate fibroblast migration in the absence of basal JNK activity. When either jnk/ or junAA fibroblasts were treated with TGF-
(Fig. 2, right panels), no significant difference in migration within the wound could be observed as compared with untreated cultures, indicating that TGF-
cannot overcome the impairment in motility induced by the loss of basal JNK activity or by the absence of basal levels of phospho-c-Jun.
The striking difference in cell motility between wt and jnk/ immortalized fibroblasts raised the question of whether such a phenomenon may be specific for either the knockout or the immortalized phenotype. This led us to determine whether pharmacologic inhibition of JNK activity in either wt immortalized fibroblasts or human dermal fibroblasts would impair their motility in the same wound closure assay. For this purpose, we used SP600125, a specific JNK inhibitor (18, 19), at a concentration of 20 µM, which is sufficient to inhibit basal JNK activity, as measured by either Western blotting for phospho-JNK or in a modified mammalian two-hybrid system specific for phospho-c-Jun-driven transactivation (see Fig. 1). Under these conditions, wt fibroblasts treated with SP600125 were unable to close a mechanically generated wound in a confluent cell layer (Fig. 3A, bottom panels), whereas cells treated with the solvent alone did (top panels). Moreover, consistent with the results obtained using jnk/ fibroblasts, TGF- was unable to reverse the inhibitory effect of SP600125 in wt fibroblasts. Similar results were obtained with normal human skin fibroblasts (Fig. 3B): Me2SO-treated dermal fibroblasts rapidly migrated into the wound, and the latter was completely closed within 20 h (top panels). On the other hand, SP600125-treated cultures were blocked in their ability to close the wound, even when treated with TGF-
(bottom panels).
|
Together, these experiments identify basal JNK activity as a critical component of the cell machinery allowing fibroblast migration. Interestingly, these results prolong our recent identification of a role for basal JNK activity in maintaining a pool of transcriptionally active, phosphorylated c-Jun protein in fibroblasts, leading to detectable phospho-c-Jun-dependent gene transactivation under unstimulated conditions (15).
We next attempted to determine whether the JNK pathway is involved during collagen gel contraction by fibroblasts, a phenomenon that involves both extracellular matrix remodeling and cell motility (reviewed in Ref. 26). To this end, we compared the behavior of wt, jnk/, and junAA fibroblasts placed in free-floating, mechanically unloaded, collagen gels. The kinetics of collagen gel contraction was then recorded over a 7-day period. As shown in Fig. 4, jnk/ and junAA fibroblasts were significantly more potent that their wt counterparts in contracting collagen gels. Maximal lattice contraction by jnk/ and junAA fibroblasts was submaximal 2 days after plating, leading to a 70% reduction of the original lattice size, whereas gel contraction by wt fibroblasts never exceeded 30%, even over a 15-day period (data not shown). No significant changes in cell proliferation within the collagen gels were detected with either cell type over the time course of the experiments, as measured by counting cells after bacterial collagenase digestion of the lattices (data not shown). When added immediately after mixing the cell suspension in the collagen solution, TGF-
significantly accelerated gel contraction by wt, jnk/, and junAA fibroblasts. A representative experiment is shown in Fig. 4A. Under such experimental conditions, lattice contraction reached the same extent with either wt fibroblasts or jnk/ and junAA fibroblasts, although the maximum was attained slightly later in the case of wt fibroblasts (Fig. 4B). Together, these results demonstrate that basal JNK activity reduces the capacity of fibroblasts to contract collagen gels but does not alter the potentiation of this phenomenon by TGF-
.
|
It is noteworthy that we recently identified JunB, another JNK target, as a potential substitute for c-Jun to mediate antagonistic effects of TNF- against TGF-
-dependent gene transcription in junAA fibroblasts (15). Specifically, we determined that TNF-
efficiently blocks TGF-
signaling in junAA fibroblasts, whereas it does not do so in jnk/ fibroblasts. A dominant-negative mutant form of MKK4 that prevents JNK activation efficiently blocked the effect of TNF-
against TGF-
in junAA cells, indicating that this inhibitory mechanism is dependent on JNK function and utilizes a substrate other than c-Jun. By means of antisense approaches, we determined that JunB substitutes for JunAA to mediate the inhibitory activity of TNF-
against Smad signaling in a JNK-dependent manner. Contrasting with the latter results, in the present investigation, which was carried out in the absence of cytokine stimulation (i.e. when only basal JNK activity may be involved), junAA and jnk/ fibroblasts both exhibit impaired motility (Fig. 2) and increased contractile activity (Fig. 4), as compared with their wt counterparts. Such identical behavior suggests that no other JNK substrate is able to replace the JunAA mutant in the context of either cell motility or capacity of fibroblasts to contract collagen gels. One plausible explanation would be that basal JunB expression (i.e. not induced by cytokines) in fibroblasts is very low and is not sufficient to drive detectable JNK-dependent transcriptional effects, whereas c-Jun is expressed at levels sufficient to transmit basal JNK activity. Another possibility may derive from the fact that JunB is a weaker transcriptional activator than c-Jun (2729). In the context of TNF-
-induced JunB neosynthesis, the latter relays, at least in part, some of the JNK signals when c-Jun expression or activation by JNK is blocked (15).
Mechanistically, the small G protein Rac has been implicated in both cell motility and floating matrix contraction (30). Furthermore, a parallel decrease in both fibroblast motility and fibroblast capacity to contract free-floating gels has been correlated to the aging process (31). Specifically, poorly contractile fibroblasts derived from older tissue donors display lower motility than fibroblasts from younger donors, the latter of which also exhibit a better capacity to contract free-floating collagen gels. It appears that the link between collagen gel contraction and cell motility is largely circumstantial, and no definitive mechanistic proof has been established thus far to link these two phenomena. Because the results presented above go against such scheme, we tried to establish another assay capable of addressing the issue of whether cell contact to collagen may modify fibroblast motility. For this purpose, we utilized another motility assay based on the TranswellTM system, which is able to measure the cell migratory potential through nylon membrane pores in the absence or presence of a collagen coating (see "Materials and Methods"). As shown in Fig. 5, jnk/ fibroblasts exhibited a much lower capacity to migrate through the TranswellTM membrane than their wt counterparts, and this capacity was not influenced, either positively or negatively, by the collagen coating. These data, in full compliance with those obtained using the in vitro wound closure assay presented in Figs. 2 and 3, allow us to conclude that basal JNK activity is an important parameter allowing fibroblast motility.
|
We next examined whether basal JNK activity may influence basal fibrillar collagen gene expression and its up-regulation by TGF-.
As a first attempt to address this hypothesis, confluent wt and jnk/ fibroblast cultures were incubated for 24 h in the absence or presence of TGF-, after which expression of type I and type III collagen genes was determined by Northern analysis of total RNA. As shown in Fig. 6A, remarkably similar basal steady-state mRNA levels for COL1A1, COL1A2, and COL3A1 were detected in wt and jnk/ fibroblasts. A 24-h incubation with TGF-
resulted in marked up-regulation of the expression of each of these genes, which was identical in its magnitude in both cell types. Type I collagen production, estimated by Western blotting (Fig. 6B), showed identical basal levels in both wt and jnk/ fibroblasts, as well as similar induction in response to TGF-
.
|
Secondly, human dermal fibroblasts were transfected with either empty or D/N MKK4 expression vectors by means of a NucleofectorTM (see "Materials and Methods"), after which collagen gene expression was measured by Northern hybridization. Results presented in Fig. 6C demonstrate that D/N MKK4 expression does not modify either basal (lane 3 versus lane 1)or TGF--induced (lane 4 versus lane 2) collagen mRNA steady-state levels. Together with the results obtained using jnk/ fibroblasts, these data demonstrate that basal JNK activity in fibroblasts is not a determinant for either basal fibrillar collagen gene expression or the extent of its activation by TGF-
, contrasting sharply with the critical role played by JNK activation in the ability of inflammatory cytokines to antagonize the up-regulation of fibrillar collagen genes by TGF-
(15).
ConclusionsCell migration has been considered a key event responsible for collagen gel contraction by fibroblasts under mechanically unloaded conditions. The results presented in this report suggest that a correlation between the two phenomena is only coincidental because cellular signaling driven by basal JNK activity differentially modulates these two cellular functions. Specifically, we provide definitive evidence for a critical role for basal JNK activity in allowing fibroblasts to migrate, whereas, at the same time, it severely alters their capacity to contract collagen gels and does not affect fibrillar collagen gene expression.
Our data identify the basal activity of the JNK pathway as highly selective in controlling several fibroblast functions essential for tissue repair, namely, cell migration, matrix contraction, and collagen biosynthesis. In vivo relevance to wound healing requires further investigations.
![]() |
FOOTNOTES |
---|
Recipient of a postdoctoral fellowship from the Association pour la Recherche contre le Cancer (France).
Recipient of a postdoctoral fellowship from Association des Sclérodermiques de France and Laboratories Actelion (Basel, Switzerland), awarded by Groupe Français de Recherche sur la Sclérodermie (France).
¶ To whom correspondence should be addressed: INSERM U532, Institut de Recherche sur la Peau, Pavillon Bazin, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75475 Paris cedex 10, France. Tel.: 33-1-53-72-20-69; Fax: 33-1-53-72-20-51; E-mail: mauviel{at}chu-stlouis.fr.
1 The abbreviations used are: TGF, transforming growth factor; JNK, c-Jun NH2-terminal kinase; wt, wild-type; MAP, mitogen-activated protein; MKK, MAP kinase kinase; D/N, dominant-negative.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|