ERK1/ATF-2 signaling axis contributes to interleukin-1β-induced MMP-3 expression in dermal fibroblasts

Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling by degrading the extracellular matrix (ECM) components. This mechanism is implicated in a variety of physiological and pathological cellular processes including wound healing. One of the key proteins involved in this process is the proinflammatory cytokine interleukin-1β (IL-1β, which induces the expression of MMP-3 mRNA and the secretion of MMP-3 protein by dermal fibroblasts. In this study, we first investigated the contribution of activating transcription factor 2 (ATF-2) to IL-1β-induced MMP-3 expression in dermal fibroblasts. Our results showed that in cells transfected with ATF-2 siRNA or treated with the ATF-2 inhibitor SBI-0087702, IL-1β-induced MMP-3 mRNA expression was reduced. We also demonstrated that IL-1β stimulates the phosphorylation of ATF-2. These observations suggest that ATF-2 plays an important role in IL-1β-induced MMP-3 expression. Next, we investigated the role of MAPK signaling in ATF-2 activation. In cells treated with the extracellular signal-regulated kinase (ERK) inhibitor FR180240, as well as in cells transfected with ERK1 and ERK2 siRNAs, IL-1β-induced MMP-3 mRNA expression was reduced. In addition, we showed that IL-1β induced the phosphorylation of ERK1/2. These observations suggest that ERK1 and ERK2 are involved in IL-1β-induced MMP-3 expression. However, ERK1 and ERK2 do seem to play different roles. While the ERK inhibitor FR180204 inhibited IL-1β-induced ATF-2 phosphorylation, only in cells transfected with ERK1 siRNA, but not ERK2 siRNA, IL-1β-induced ATF-2 phosphorylation was reduced. These findings suggest that the ERK1/ATF-2 signaling axis contributes to IL-1β-induced MMP-3 expression in dermal fibroblasts.


Cell culture
This study was approved by Nihon University Animal Care and Use Committee (AP13B051). All the experiments were performed in accordance with the relevant guidelines and regulations. Dog dorsal skin samples (n = 3, healthy 3-year-old beagle dogs, male) were collected after local anesthesia with 1% lidocaine and 10 μg/mL adrenaline. To minimize potential pain and infection, remifentanil hydrochloride (3 to 5 μg/kg/min; Janssen Pharmaceutical K.K, Tokyo, Japan) and cefazolin (22 mg/kg; Nichi-Iko Pharmaceutical Co., Ltd, Toyama, Japan) were administered intravenously before the time of awakening after anesthesia. Dermal fibroblasts were isolated by explant culture as reported previously [37]. Briefly, canine dermis from the dorsal skin was collected and cut into 3-mm 2 sections. Each explant was placed into 90-mm Petri dish. The attached explants were maintained in α-MEM supplemented with 10% FBS, inside a static-culture incubator at 5% CO 2 and 37˚C. The medium was changed once a week and dermal fibroblasts were obtained as outgrowth cells. The cells were harvested using 0.25% trypsin-EDTA once they reached 90-95% confluence. The collected cells were suspended using CELLBANKER 1 supplemented with medium at a density of 2×10 6 cells/500 μL, and 500 μL of the cell suspension was placed into a sterilized serum tube. The tubes were then placed into a freezing vessel and cryopreserved at -80˚C. Before experiments, serum tubes were removed from the freezing vessel and immersed into a water bath at 37˚C. The thawedout cell suspension was transferred into a centrifuge tube containing α-MEM supplemented with 10% FBS, and then centrifuged at 300 g for 3 min. After removal of the supernatant, the pellet was suspended in α-MEM 10% FBS and transferred into a 75-cm 2 culture flask. Static cultures were then maintained under the same conditions as before the cryopreservation. Cells were harvested using 0.25% trypsin-EDTA once they reached approximately 90% confluency. Then, the detached cells were seeded at a density of 1×10 6 cells/75-cm 2 culture flask. The fourth-passaged canine dermal fibroblasts were used for all following experiments. Cells from different animals were used in different experiments. Cells were characterized by detecting the mRNA expression of chemotropic factors such as: Netrin-1, Netrin-3, Ephrin-A3, Ephrin-A4, and Semaphorin-4D as reported previously [37]. The mRNA expression of chemotropic factors in dermal fibroblasts was lower compared to mesenchymal stem cells, confirming that the cells are dermal fibroblasts.

Real-time RT-PCR
Total RNA was extracted from canine dermal fibroblasts with TRIzol reagent. The first-strand cDNA synthesis was carried out with 500 ng of total RNA using PrimeScript RT Master Mix. Real-time RT-PCR was performed with 2 μL of the first-strand cDNA in 25 μL (total reaction volume) with SYBR Premix Ex Taq II. Primers were designed for canine MMP-3, while the house keeping protein TATA box binding protein (TBP), was used as a control. Table 1 shows the sequences of primers used for real-time RT-PCR. Real-time RT-PCR of no-template controls was performed with 2 μL of RNase-and DNA-free water. In addition, real-time PCR of no-reverse transcription control was performed with 2 μL of each RNA sample. All PCR reactions were conducted using Thermal Cycler Dice Real Time System II with the following protocol: 1 cycle of denaturation at 95˚C for 30 s, 40 cycles of denaturation at 95˚C for 5 s and annealing/extension at 60˚C for 30 s. The results were analyzed by the second derivative maximum method and the comparative cycle threshold (ΔΔCt) method using the real-time RT-PCR analysis software. The amplification of TBP from the same amount of cDNA was used as an endogenous control, while cDNA amplification from canine dermal fibroblasts at time 0 was used as a calibration standard.

Western blotting
The cells were lysed with a lysis buffer containing 20 mM HEPES, 1 mM PMSF, 10 mM sodium fluoride, and a complete mini EDTA-free protease inhibitor cocktail at pH 7.4. Protein concentrations were adjusted using the Bradford method [38]. Extracted proteins were boiled at 95˚C for 5 min in SDS buffer. Samples were loaded into separate lanes of 12% Mini-PRO-TEAN TGX gel and separated by electrophoresis. Separated proteins were transferred to PVDF membranes, treated with Block Ace for 50 min at room temperature, and incubated with primary antibodies [p-ERK1/2 (1:1000), t-ERK1/2 (1:1000), p-ATF-2 (1:1000), t-ATF-2 (1:1000)] for 120 min at room temperature. After washing, the membranes were incubated with an HRP-conjugated anti-rabbit or a mouse IgG antibody (1:10000) for 90 min at room temperature. Immunoreactivity was detected using ECL Western Blotting Analysis System. Chemiluminescent signals of the membranes were measured using ImageQuant LAS 4000 mini.

MMP-3 activity assay
Canine dermal fibroblasts were seeded at a density of 3.0×10 5 cells per well in 6-well culture plates. The cells were treated with IL-1β after starvation for 24 h, and culture supernatants were collected. To activate pro-MMP, samples were incubated with 4-aminophenylmercuric acetate at 37˚C for 24 h. MMP-3 activity in the culture supernatant were measured using the MMP-3 activity kit according to the manufacturer's instructions. The activity of MMP-3 was detected by a fluorescence microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific K.K., Kanagawa, Japan) at excitation/emission wavelengths of 360 nm/460 nm.

siRNA transfection
Dermal fibroblasts seeded at a density of 1×10 5 cells/35-mm dish or 5 × 10 5 cells/90-mm dish, were transfected using Opti-MEM containing 5 μl/mL Lipofectamine 2000 and 50 nM ATF-2 or scrambled siRNA for 6 h [37]. The sequences of the siRNA are indicated in the Supplementary Table 2. The efficiency of siRNA was confirmed by western blotting.

Wound healing assay
Culture-Insert 2 Well (ibidi GmbH, Am Klopferspitz, Germany) was placed on the 35-mm dish. 70 μl of cell suspension (1×10 6 /ml) were applied into each well of the Culture-Insert 2 Well. After the incubation for 24 h, cell culture inserts were carefully removed followed by stimulation of IL-1β. The phase-contrast images were analyzed using the MRI Wound Healing Tool for ImageJ [39].

Statistical analysis
The data from these experiments are presented as the mean ± standard error of measurement. Statistical analysis was performed using StatMate IV. The data from the time course study were analyzed using two-way analysis of variance, and the data from other experiments were analyzed using one-way analysis of variance. Tukey's test was used as post-hoc analysis. P-values less than 0.05 were considered statistically significant.

IL-1β induces MMP-3 expression and secretion
We first examined the effect of the inflammatory cytokine IL-1β on MMP-3 protein secretion in dermal fibroblasts. Cells treated with IL-1β (100 pM) for 24 h showed a time-dependent increase in MMP-3 activity in the culture medium ( Fig 1A). The cells treated with various concentration of IL-1β (0-100 pM) for 24 h showed a dose-dependent increase in MMP-3 activity in the culture medium ( Fig 1B). Then, we examined the mRNA expression of MMP-3 using real-time RT-PCR. Incubation with 100 pM IL-1β enhanced MMP-3 mRNA expression in a time-dependent manner ( Fig 1C) with the maximum effect observed at 6 h, followed by a decrease in the MMP-3 mRNA expression. The cells treated with various concentration of IL-1β (0-100 pM) for 6 h showed a dose-dependent induction of the MMP-3 mRNA expression ( Fig 1D), which was similar to the effect of IL-1β on MMP-3 secretion. These observations suggest that IL-1β stimulated MMP-3 secretion by upregulating MMP-3 expression in canine dermal fibroblasts. We checked whether MMP-3 implicated in the cellular biological function, such as cellular migration. When the cells were treated with IL-1β (100 pM), cell migration occurred in a time-dependent manner. The MMP-3 inhibitor UK356618 (2 μM, 2 h) significantly inhibited Table 2. Sequences for siRNA transfection.

ATF-2 contributes to IL-1β-induced MMP-3 expression
The expression of MMPs has been considered to be primarily regulated at the transcriptional level, because most members of the MMP family share common cis-elements in their promoter sequences [23][24][25]. The transcription factor ATF-2 is known to be involved in the regulation of numerous proteins and plays a role in the IL-1β-induced increase in their expression [40,41]. We sought to establish if this previously observed modulatory effect of ATF-2 occurs in dermal fibroblasts. Fibroblasts pretreated with the ATF-2 inhibitor, SBI-0087702 (10 μM) for 24 h failed to show IL-1β-induced increase in MMP-3 mRNA expression (Fig 3A), suggesting that ATF-2 facilitates this phenomenon. Then, we examined ATF-2 phosphorylation in cells treated with IL-1β and found it was increased with the peak levels occurring 15 min post-treatment, whereas no change in total-ATF-2 (t-ATF-2) expression was observed. After reaching the peak, the ATF-2 phosphorylation steadily declined to the basal level within 30 min (Fig 3B). These observations indicate that ATF-2 was activated by IL-1β stimulation and is likely to contribute to the effect of IL-1β on MMP-3 expression in dermal fibroblasts. We confirmed that the ATF2 inhibitor SBI-0087702 reduced IL-1β-induced ATF2 phosphorylation (Fig 3D and 3E). Next we performed ATF-2 knockdown experiment using siRNA transfection. The protein expression of ATF-2 was significantly reduced in the cells transfected with ATF-2 siRNA, but not in scramble siRNA-transfected cells (Fig 4A and 4B). In the ATF-2 siRNA-transfected cells, IL-1β-induced MMP-3 mRNA expression was attenuated (Fig 4C). We confirmed that IL-1β-induced cellular migration was attenuated in the cells transfected with ATF-2 siRNA (Fig 4D and 4E). These observations suggest that ATF-2 contributes to IL-1β-induced MMP-3 expression.

ERK signaling pathway is involved in IL-1β-mediated MMP-3 expression
IL-1β has been demonstrated to induce MMP-3 expression via the mitogen-activated protein (MAPK) signaling pathways [42][43][44], and ATF-2 has also been shown to be regulated via MAPK signaling pathways in response to inflammatory cytokines [40,41,45,46]. Then, as the following study, we examined the relationship between ATF-2 and MAPK signaling pathways. Regarding MAPK signaling on IL-1β-induced MMP-3 expression, we investigated the effects of inhibitors of ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK, which are the enzymes representing the three main MAPK pathway [47,48]. In the cells treated with the ERK inhibitor FR180204 (25 μM) for 1 h, IL-1β failed to induce MMP-3 mRNA expression, whereas SB203963 (20 μM) and SP600125 (10 μM), inhibitors of p38 MAPK and JNK, respectively, showed less effects on IL-1β-induced MMP-3 mRNA expression (Fig 5A). Since activated MAPKs subsequently phosphorylate and activate downstream targets such as transcription factors and regulators of cell functions, we next examined whether IL-1β activated ERK by detecting the phosphorylation status using immunoblotting with an anti-phospho-ERK1/2 (p-ERK1/ 2) antibody. The cells stimulated with 100 pM IL-1β showed an increase in p-ERK1/2 but not total-ERK1/2 (t-ERK1/2) expression (Fig 5B and 5C). The maximum phosphorylation levels were observed 5 min after stimulation and then they gradually decreased.
To confirm the contribution of ERK1/2 in IL-1β-induced MMP-3 mRNA expression, we performed ERK1/2 knockdown experiment using siRNA transfection. The protein expression of ERK1 or ERK2 was significantly reduced by the transfection with respective siRNAs, but not with scramble siRNA as a control (Fig 6A-6C). IL-1β-induced MMP-3 mRNA expression ERK1/ATF-2 signaling axis and interleukin-1β-induced MMP-3 expression was attenuated in the ERK1 and ERK2 siRNA-transfected cells compared with the scramble siRNA-transfected cells (Fig 6D). Subsequently, we performed the experiments with ERK1 and ERK2 siRNAs together. In the ERK1 and 2 double-knockdown cells, IL-1β-induced MMP-3 mRNA expression was also attenuated (Fig 6D). We observed that IL-1β-induced cellular migration was attenuated in the cells transfected with siRNA for ERK1 or ERK2. In the ERK1 and 2 double-knockdown cells, IL-1β-induced cellular migration was also attenuated. However, there is no significant difference between the cells transfected with siRNA for ERK1, ERK2 and ERK1/  (Fig 7A and 7B). These observations strongly suggest that the ERK1/2 activation contributes to the upregulation of MMP-3 mRNA expression induced by IL-1β in dermal fibroblasts.

ATF-2 contributes to IL-1β-induced MMP-3 expression as a downstream target of ERK1
In the following study, we examined whether ERK signaling regulated the activation of ATF-2 in IL-1β-treated dermal fibroblasts. We first confirmed that the ERK inhibitor FR180204  (Fig 8A). In the cells treated with the ERK inhibitor, the phosphorylation of ATF-2 induced by IL-1β was attenuated (Fig 8B). To examine the contribution of ERK subtypes to ATF-2 phosphorylation, we performed ERK1/2 knockdown experiment. IL-1β-induced ATF-2 phosphorylation was clearly inhibited ERK1/ATF-2 signaling axis and interleukin-1β-induced MMP-3 expression by ERK1 siRNA transfection, but not by scramble siRNA transfection (Fig 8B and 8D). We also observed that ERK2 siRNA had no effect on IL-1β-induced ATF-2 phosphorylation. Therefore, we examined the effect of ERK1 and ERK2 double knockdown on IL-1β-induced ATF-2 phosphorylation. In the cells transfected with ERK1 and ERK2 siRNAs, IL-1β-induced ATF-2 phosphorylation was reduced compared with a control (Fig 8C and 8D). Taken together, it is most likely that ERK1 signaling is dominantly involved in the activation of ATF-2 in IL-1β-induced MMP-3 expression.

Discussion
Several observations suggest that MMP-3 has an important role in wound healing [49]. It was shown that the expression of MMP-3 in wound effluent of patients with impaired wound healing was lower than in those with normal wound healing [50]. Additionally, In MMP-3 null mice, delayed wound healing has been demonstrated. Finally, in MMP-3-deficient fibroblasts, the ability of fibroblasts to contract collagen gels was reduced. Moreover, MMP-3 has been also demonstrated to contribute to type I collagenolysis [17]. In this study, we observed that IL-1β failed to induce cellular migration in the presence of UK356618, a specific inhibitor for protease activity of MMP-3, suggesting that the protease activity of MMP-3 plays a crucial role in dermal fibroblasts. Therefore, MMP-3 is considered to have a key role in wound contraction and tissue remodeling during wound repair [51,52].
In this study, we demonstrated that the ATF-2 inhibitor SBI-0087702 inhibited IL-1βinduced MMP-3 mRNA expression and that IL-1β failed to induce MMP-3 mRNA expression in ATF-2 knockdown cells. Taken together, these observations strongly suggest that ATF-2 is involved in IL-1β-stimulated MMP-3 expression in dermal fibroblasts. In unstimulated cells, ATF-2 is maintained in a transcriptionally inactive form by an intramolecular inhibitory interaction. Namely, the DNA binding domain is folded and binds to the amino-terminal transactivation domain [53]. In response to stimuli, N-terminal phosphorylation has been demonstrated to release the intramolecular inhibition and lead to the activation of ATF-2 [54][55][56]. Activated ATF-2 regulates gene expression through homo-or hetero-dimerization with other AP-1 family members, such as the CREB, Fos, Maf, or Jun family transcription factors [57,58]. Additionally, the phosphorylation of ATF-2 has also been demonstrated to control its degradation [59]. It is possible that such mechanisms can influence MMP-3 expression induced by IL-1β, because IL-1β induces ATF-2 phosphorylation in dermal fibroblasts.
MAPK pathways have been reported to phosphorylate ATF-2. In response to stimuli including inflammatory cytokines, JNK and p38 MAPK, phosphorylate ATF-2 at amino acids Thr69 and Thr71 [45,55,60]. ERK1/2 was also reported to phosphorylate ATF-2 at Thr71 in the ultraviolet response [61]. In cells stimulated with growth factors such as insulin and epidermal growth factor, the cooperative interaction between ERK and p38 pathways has been suggested as a two-step mechanism, in which the Ras-Raf-MEK-ERK pathway triggers ATF-2 Thr71 phosphorylation, and the Ras-Ral-Src-p38 pathway subsequently stimulates Thr69 phosphorylation [62]. In our study, the ERK inhibitor significantly inhibited IL-1β-induced MMP-3 mRNA expression, but not p38 and JNK inhibitors, and attenuated IL-1β-induced ATF-2 phosphorylation. Therefore, it is likely that ATF-2 Thr71 phosphorylation is involved in IL-1β-induced MMP-3 expression in dermal fibroblasts.
Multiple MAPK signaling pathways are activated by several stimuli including cytokines such as IL-1β [48]. In the previous study, it has been reported that three major MAPK signaling pathways, ERK, JNK, and p38 MAPK, are activated in IL-1β-treated periadipocytes [63]. In cardiac fibroblasts, IL-1β-induced p38 MAPK activation was reported to contribute to MMP3 expression [64]. In human dermal fibroblasts, ultraviolet B induces the activation of JNK and p38 MAPK [65]. We previously reported that IL-1β activated ERK, JNK and p38 MAPK and subsequently induced COX-2 expression in feline synovial fibroblasts [66]. On the other hand, in canine dermal fibroblasts, we observed that IL-1β induced COX-2 expression via the activation of ERK signaling [37]. These observations strongly suggest that the response of MAPKs is highly dependent on the cellular context. ERK1 and ERK2, ERK isoforms, possess 83% amino acid identity and are co-expressed in most tissues [67,68]. These two isoforms are generally co-activated in cells stimulated with multiple extracellular stimuli [69][70][71]. Recently, the functional differences between the two isoforms were demonstrated using antisense techniques or siRNA transfection [66,68,[72][73][74][75][76]. Here, we performed ERK-knockdown experiments by treatment with ERK isoform-specific siRNA. Both in ERK1 and ERK2 knockdown cells, IL-1β-induced MMP-3 mRNA expression was attenuated. However, IL-1β-induced ATF-2 phosphorylation was attenuated in ERK1 knockdown cells, but not in ERK2-knockdown cells. These results suggest that ERK1 contributes to MMP-3 expression via transcriptional activation of ATF-2 in dermal fibroblasts. On the other hand, transcriptional regulation of MMP-3 via ERK2 pathway remained unclear, which appears to cause the partially inhibitory effect of ATF-2 inhibitor on IL-1β-induced MMP-3 mRNA expression.
In cells treated with an ATF-2 inhibitor and transfected with ATF-2 siRNA, IL-1β-induced MMP-3 mRNA expression was partially inhibited. These results suggest that other transcription factors may probably contribute to IL-1β-induced MMP-3 mRNA expression as well as ATF-2. Genes of MMP family including MMP-3 contain several cis-elements in their promoter sequences, which allow a tight control of cell-specific MMP gene expression by a diverse set of trans-activators, such as polyomavirus enhancer-A binding protein-3 (PEA3) and NF-κB, as well as ATF-2 [24,77]. In this study, in cells treated with an ERK1/2 inhibitor and transfected with ERK1 or 2 siRNA, the effect of IL-1β on MMP-3 mRNA expression was inhibited. However, the IL-1β-induced ATF2 phosphorylation was attenuated in the cells transfected ERK1 siRNA, but not ERK2 siRNA. Therefore, ERK2 pathway appears to be necessary for activating other transcription factors in fully MMP-3 expression induced by IL-1β. In dermal fibroblasts, ERK1 contributes to IL-1β evoked the activation of ERK1/ATF-2 signaling axis, which contributes to cellular migration via MMP-3 expression. ERK2 pathway appears to be necessary for activating other transcription factors in fully MMP-3 expression induced by IL-1β.

Conclusions
In conclusion, the findings of this study are significantly helping the understanding of the ERK1 MAPK signaling axis, and the role of transcription factor ATF-2, in respect to IL-1βinduced MMP-3 expression in dermal fibroblasts (Fig 9). Such mechanism regulating MMP-3 expression, appears to be important for the occurrence of wound healing in the skin.