DOI: 10.1111/exd.12556 www.wileyonlinelibrary.com/journal/EXD

Original Article

The MEK/ERK signalling cascade is required for sonic hedgehog signalling pathway-mediated enhancement of proliferation and inhibition of apoptosis in normal keratinocytes Haiyan Liu1,2*, Qiang Jian1*, Ke Xue1*, Cuiling Ma1, Fang Xie3, Rui Wang3, Wenjun Liao1, Yufeng Liu1, Sumin Chi4 and Chengxin Li1,3 1 Department of Dermatology, Xijing Hospital, the Fourth Military Medical University, Xi’an, China; 2Department of Dermatology, Lanzhou General Hospital, Lanzhou Military District, Lanzhou, China; 3Department of Dermatology, Chinese People’s Liberation Army General Hospital, Beijing, China; 4Department of Physiology, the Fourth Military Medical University, Xi’an, China Correspondence: Chengxin Li, Department of Dermatology, Chinese People’s Liberation Army General Hospital, Beijing 100853, China, Tel.: +86-10-66939315, Fax: +86-10-68277283, Department of Dermatology, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032, China; e-mail: [email protected] and Sumin Chi, Department of Physiology, the Fourth Military Medical University, Xi’an 710032, China, Tel.: +86-29-84779060, Fax: +86-29-84774519, e-mail: [email protected] *These authors contributed equally to this work.

Abstract: Keratinocytes (KCs) play a critical role in maintaining the cutaneous structure and are involved in various physiological and pathologic processes of the skin. Many inflammatory skin diseases and skin cancers result from excessive proliferation and insufficient apoptosis of KCs. Recent data suggested that the sonic hedgehog (Shh) signalling pathway plays an essential role in the proliferation and apoptosis of normal KCs. However, the mechanism remains poorly defined. Here, we provide evidence that Shh signalling induces proliferation and inhibits apoptosis in normal KCs via cyclin D1 and Bcl2 in an extracellular signalregulatedkinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent manner. In addition, the effect is independent of phosphoinositide-3 kinase (PI3K)/AKT or Janus kinase/signal transducer and activator of transcription (JAK/STAT) 1/3

pathways. Furthermore, we observed that epidermal growth factor receptor (EGFR) signalling modulates the activity of Shh signalling pathway; besides, Shh and EGFR signalling act additively to induce the ERK activation and the increases in cyclin D1 and Bcl2 thereby affecting proliferation and apoptosis in KCs in vitro. The present study suggests that the MEK/ERK1/2 activation is part of the mechanism of Shh signal-mediated proliferation and apoptosis in normal KCs. Our results may help to elucidate the regulatory mechanisms of the Shh pathway in normal KCs and the pathogenesis of related skin disorders.

Introduction

remains poorly understood (5). Dentice et al. (5) suggested a molecular mechanism whereby Shh-induced type 3 deiodinase can block thyroid hormone action, hence enhancing proliferation of keratinocytes. The Shh signalling pathway controls cellular apoptosis in many kinds of tumor cells, and mouse models with activated Shh signalling have shown that Bcl2 is upregulated in normal keratinocytes, affecting their apoptosis (6,7); however, the mechanism of Shh signalling in normal KC apoptosis is not fully understood. Many studies have provided evidence of an association between Shh signalling and the extracellular signal-regulated kinase (MEK)/ extracellular signal-regulated kinase (ERK) signalling cascade (8). Aberrant activation of Shh pathway combined with activating mutations of K-RAS stimulates the Raf/MEK/ERK cascade (9–11). Furthermore, the Shh pathway mediates invasion and metastasis in hepatocellular carcinoma via the ERK pathway (12). These data suggested that the Shh signalling pathway is closely associated with the MEK/ERK cascade, which is the downstream effector of many important signalling pathways, such as the epidermal growth factor receptor (EGFR) signalling pathway, and is the core of the signalling networks involved in cell proliferation and apoptosis (13). Therefore, the MEK/ERK cascade may be a critical mechanism of

The regulatory mechanisms of keratinocyte (KC) proliferation and apoptosis are a major focus of dermatological research. Under physiological conditions, KC proliferation and apoptosis are kept in balance. However, in the pathologic state, excessive proliferation and insufficient apoptosis lead to a variety of inflammatory skin diseases (such as psoriasis, lichen planus, keratosis seborrheica, etc.) and malignant skin cancers [e.g. Bowen’s disease and squamous cell carcinoma (SCC)]. Many signal transduction pathways and associated molecules have been shown to modulate KC proliferation and apoptosis, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), p38, p53, the phosphoinositide-3 kinase (PI3K)/AKT pathway, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and nuclear factor-kappaB (NF-jB) (1,2). Accumulating data suggest that the sonic hedgehog (Shh) signalling pathway, a classical signalling pathway involved in embryonic development, controls cellular growth, proliferation, differentiation and apoptosis (3). Inhibition of Shh signalling suppresses the growth of tumor cells (4). Recent data suggested that the Shh signalling pathway plays an essential role in the proliferation and apoptosis of normal KCs; however, the mechanism

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Key words: apoptosis – keratinocyte – MEK/ERK – proliferation – sonic hedgehog signalling pathway

Accepted for publication 21 September 2014

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 896–901

The MEK/ERK signalling cascade

Shh-mediated proliferation and apoptosis in normal KCs. Our study aimed to clarify whether the Shh signalling pathway controls the proliferation and apoptosis of normal KC through the MEK/ ERK pathway or other signalling pathways. In this study, we investigated the effects of Shh signalling on the activation of MEK/ERK, (PI3K)/AKT and JAK/STAT 1/3 pathways. Furthermore, we investigated the interactions among the Shh pathway, MEK/ERK and EGFR pathways in the proliferation and apoptosis of normal keratinocytes in vitro, thereby uncovering a potential mechanism by which the Shh pathway contributes to skin disorders and tumor development in response to deregulated Shh signalling.

Methods Cell culture Normal human primary epidermal KCs (NHEKs) were obtained from skin biopsies of healthy volunteers and cultured in serumfree KC growth medium (Gibco-Invitrogen, Carlsbad, CA, USA). Human HaCaT keratinocytes and human A431 cutaneous squamous cell carcinoma (SCC) cells (Japanese RIKEN Bio Resource Center Cell Bank) were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (Gibco-Invitrogen) supplemented with 10% foetal bovine serum (Gibco-Invitrogen) in a humidified atmosphere containing 5% CO2 at 37°C. A431 cells were used as the positive control because the Shh pathway has been shown to be activated in A431 cells and involved in the pathogenesis of SCC (14).

Reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR A Total RNA Extraction Kit (Takara, Shiga, Japan) was used to isolate total cellular RNA, following the manufacturer’s instructions, and the PrimeScriptTM RT reagent Kit (Takara) was used to synthesize cDNA, according to the manufacturer’s protocol. Synthesized cDNA was used as the template for RT-PCR and quantitative PCR. The cDNA and primers (Table S1) were subjected to PCR for 30 amplification cycles at an annealing temperature of 58°C, followed by an extension step at 72°C. The amplification products were separated by electrophoresis on 1% agarose gels, and ethidium bromide staining visualized the separated products. Real-time PCR was conducted with SYBR Premix Ex TaqTM II (Takara) on a Chromo4 continuous fluorescence detector with a PTC-200 DNA Engine Cycler (Bio-Rad, Hercules, CA, USA). The reaction components were 1 ll of forward and reverse primers and b-actin as an internal control. The cycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. All reactions were run in triplicate for at least three independent experiments. Relative quantification was performed according to the DDCT method, and results were expressed in the linear form using the formula 2-DDCT.

Cell growth assay Normal human primary epidermal KCs and HaCaT cells were cultured in 96-well plates overnight and then starved for 12 h, followed by the treatments with recombinant human Shh (rhShh) (Peprotech, Rocky Hill, USA) (1, 2, 4, or 8 lg/ml), cyclopamine (Cell Signaling Technology, Danvers, MA, USA) (1, 2, 5, or 10 lM), U0126 (Sigma, St. Louis, MO, USA) (2.5, 5, 10, or 20 lM) or gefitinib (Sigma) (0.25, 0.5, 1, or 2 lM), respectively, for 24 h; the control group was treated with medium only. The

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 896–901

most optimal concentrations of the above treatments were selected and used to treat the normal epidermal cells for varying times (0, 6, 12, 24 or 48 h). A Cell Counting Kit-8 (CCK-8) (Sigma) assay evaluated the cell viability, according to the manufacturer’s instructions and as previously described (15).

Apoptosis and cell cycle analysis HaCaT cells and NHEKs were plated in six-well plates at a concentration of 2 9 105 cells/well overnight and then starved for 12 h. The cells were then stimulated with rhShh (4 lg/ml), cyclopamine (10 lM), U0126 (10 lM), gefitinib (0.5 lM) or medium, respectively, for 24 h or 48 h. The cells were then harvested, washed and either doubly labelled with Annexin V-FITC/propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA) or labelled with PI only to determine the apoptosis rate or for cell cycle distribution analysis, respectively. Flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) was used to detect the label and analyse the labelled cells, as described previously (16).

Western blotting analysis The treated NHEKs, HaCat or A431 cells were harvested, and proteins from cell lysates were denatured in sodium dodecyl sulphate (SDS). Extracted total protein (50 lg) was applied to separate lanes of 10% SDS-PAGE gels. After electrophoresis, the proteins were transferred to a PVDF membrane (Invitrogen, Carlsbad, CA, USA) and non-specific binding was blocked for 2 h by incubation in blocking buffer. The membranes were incubated with specific antibodies (Abs) against b-actin, Gli-1, Gli-2, Ptch1 (Abcam, Cambridge, UK), SMO, Hhip, Shh (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1, Bcl2 (Cell Signaling Technology), STAT1, STAT3, AKT, ERK1/2, phospho-STAT1, phospho-STAT3, phosphor-AKT or phospho-ERK1/2 (Cell Signaling Technology), respsectively, overnight. Blots were washed with 1 9 TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary Abs (Dako Cytomation, Glostrup, Denmark) for 1 h. Chemiluminescence was used to visualize the bound Abs. Quantity One software (Bio-Rad) was used to quantify the protein band intensities.

Statistical analyses All results were confirmed in at least three independent experiments. SPSS 13.0 software was used to analyse the data. One-way analysis of variance (ANOVA) and Student’s t tests determined statistically significant differences, and linear correlation analysis was used for tendency variation. Differences were considered statistically significant at P-values 0.05).

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increased in response to rhShh (4 lg/ml) and reduced in response to cyclopamine (10 lM) in HaCaT cells at mRNA (Fig. 1g) and protein (Fig. 1e,f) levels (P < 0.05). Taken together, the results showed that the Shh pathway regulated the proliferation and apoptosis of normal KCs, and that the molecular mechanism involved Shh-induced promotion of cyclin D1 and Bcl2.

The Shh signalling pathway specifically activates MEK/ERK signalling, but does not affect the PI3K/AKT or JAK/STAT pathways in KCs in vitro

Figure 1. Shh pathway-mediated cyclin D1 and Bcl2 alterations are involved in the proliferation and apoptosis of KCs in vitro. RT-PCR (a) and Western blotting (b) were used to detect the expression of Ptch-1, Gli1, Shh, Smo and Hhip in NHEKs and HaCaT cells (n = 3); A431 cells were used as a positive control. Changes in cell cycle distributions (c) and apoptosis rates (d) in NHEKs and HaCaT cells after the treatment with 4 lg/ml rhShh or 10 lM cyclopamine for 24 h, as compared with the control (n = 3, *P < 0.05). Real-time PCR (g) and Western blotting analysis (e) of the levels of Gli-1, Gli-2, Ptch-1, cyclin D1 and Bcl2 in HaCaT cells after incubation with rhShh (4 lg/ml), cyclopamine (10 lM) or medium as a control, respectively, for 24 h (n = 3, *P < 0.05). (f) Quantitative analysis of (e) (n = 3, *P < 0.05).

(P < 0.01, Figure S1). Flow cytometry showed that treatment with rhShh (4 lg/ml) significantly promoted proliferation and suppressed cell apoptosis compared with control cells, while treatment with cyclopamine (10 lM) inhibited proliferation and induced cell apoptosis of NHEKs and HaCaT cells (P < 0.05, Fig. 1c,d). Real-time PCR and Western blotting analysis showed that the expression of Gli-1, Gli-2, Ptch-1, cyclin D1 and Bcl2 were

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We observed the influence of Shh signalling on the MEK/ERK, PI3K/AKT and JAK/STAT1/3 pathways in NHEKs and HaCaT cells. The total protein levels of ERK1/2 did not significantly change in response to rhShh (4 lg/ml) or cyclopamine (10 lM) in NHEKs (Fig. 2a,b) and HaCaT cells (Fig. 2c,d). However, the amount of activated (phosphorylated) ERK1/2 (p-ERK1/2) increased in response to Shh and decreased in response to cyclopamine treatment in NHEKs (Fig. 2a,b) and HaCaT cells (Fig. 2c, d), compared with the control. These data suggested that the Shh pathway acted upstream of the MEK/ERK cascade. Notably, the levels of total and phosphorylated AKT, STAT1 and STAT3 did not change in response to treatment with rhShh or cyclopamine (Fig. 2a–d) compared to the control cells, suggesting that Shh signalling’s effects on proliferation and apoptosis in normal KCs are independent of the PI3K/AKT or JAK/STAT1/3 pathways.

MEK/ERK activation is required for Shh signalling-induced proliferation and apoptosis inhibition The MEK1/2-specific inhibitor, U0126, was used to block ERK1/2 activation. CCK-8 assays showed that U0126 suppressed the viability of HaCaT cells in a concentration- and time-dependent manner (P < 0.05, Figure S2a,b). Flow cytometry revealed that U0126

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 896–901

The MEK/ERK signalling cascade

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Notably, Gli1 and Gli2, known transcription factors of Shh pathway in epidermal cells, were obviously reduced at mRNA/protein levels when HaCaT cells were exposed to U0126 (P < 0.05, Fig. 3c,e). In addition, the expression of Ptch-1, the receptor and target gene of Shh signalling pathway, was also strongly inhibited by U0126.

EGFR signalling modulates Shh pathway activity in KCs in vitro

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Figure 3. MEK/ERK activation is required for Shh signalling-induced proliferation and apoptosis inhibition. Flow cytometry was used to analyse the cell cycle distribution (a) and apoptosis rates (b) in HaCaT cells (n = 3, **P < 0.01 and ## P > 0.05). Real-time PCR (e) and Western blotting analysis (c) of the levels of Gli1, Gli-2, Ptch-1, p-ERK1/2, cyclin D1 and Bcl2. HaCaT cells cultured with U0126 (10 lM), rhShh (4 lg/ml), or U0126 + rhShh (10 lM U0126 followed by addition of 4 lg/ml rhShh 1 h later) or medium as the control, respectively, for 6 h (n = 3, *P < 0.05 and ##P > 0.05). (d) Quantitative analysis of (c) (*P < 0.05 and ## P > 0.05).

significantly inhibited HaCaT cell proliferation (P < 0.01, Fig. 3a) and augmented cell apoptosis (P < 0.01, Fig. 3b). Interestingly, there was no significant difference in KC proliferation (P > 0.05, Fig. 3a) or apoptosis (P > 0.05, Fig. 3b) between U0126 + rhShhtreated cells and the cells treated with U0126 alone. The data showed that MEK/ERK played a key role in Shh signallingmediated proliferation and apoptosis inhibition. Real-time PCR and Western blotting analysis showed that the expression of p-ERK1/2, cyclin D1 and Bcl2 decreased in response to U0126 and increased in response to rhShh compared with control cells (P < 0.05, Fig. 3c,d). Interestingly, no significant changes in the mRNA and protein expressions of p-ERK1/2, cyclin D1 and Bcl2 were observed between U0126 + rhShh-treated cells and those treated with U0126 alone (P > 0.05, Fig. 3c,d). The results showed that U0126 blocked the rhShh-induced cyclin D1 and Bcl2, suggesting that cyclin D1 and Bcl2 is critical for Shh signal to modulate the proliferation and apoptosis in KCs.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 896–901

Gefitinib, a specific inhibitor of EGFR, was used to treat HaCaT cells. CCK-8 assays showed that gefitinib reduced the viability of HaCaT cells in a concentration- and time-dependent manner (P < 0.05, Figure S3). Flow cytometry analysis revealed that gefitinib (0.5 lM) decreased the proportion of HaCaT cells in S+G2/M phase (P < 0.05, Fig. 4a) and induced apoptosis in HaCaT cells, compared to the control cells (P < 0.05, Fig. 4b). Interestingly, gefitinib partially reduced the effect of rhShh (4 lg/ ml) on proliferation and apoptosis of HaCaT cells (P < 0.05, Fig. 4a,b), suggesting that this antagonist of EGFR suppressed the Shh pathway-induced proliferation and apoptosis inhibition in KCs. Real-time PCR and Western blotting analysis revealed that the expressions of Gli-1, Gli-2, Ptch-1, p-ERK1/2, cyclinD1 and Bcl2 were significantly reduced or enhanced by gefitinib (0.5 lM; P < 0.05, Fig. 4c,e) or rhEGF (10 ng/ml; P < 0.05, Fig. 4d–f), respectively, compared with the control cells, suggesting that the EFGR signalling modulated the Shh pathway activity in KCs in vitro by affecting the stability of Gli-1 and Gli-2 as well as the level of Ptch-1, the receptor and target gene of Shh pathway. Notably, the transcript/protein levels of p-ERK1/2, cyclinD1 and Bcl2 were lower in gefitinib+rhShh-incubated cells (P < 0.05, Fig. 4c,e) and higher in rhEGF+rhShh-incubated cells (P < 0.05, Fig. 4d,f) than in the rhShh alone-treated cells, suggesting that the upregulation and downregulation of EFGR pathway activity attenuated and augmented, respectively, rhShh-induced ERK phosphorylation and the increases of cyclinD1 and Bcl2, respectively, hence modulating the proliferation and apoptosis in KCs.

Shh and EGFR signalling act additively to induce the ERK activation and the increases in cyclin D1 and Bcl2 in KCs in vitro The combined effects of agonists of the Shh and EGFR pathways, rhShh and rhEGF, were observed on the proliferation and apoptosis of HaCaT cells. Flow cytometry evaluation showed that the proportion of HaCaT cells in the S+G2/M phase significantly increased after treatment with rhShh (4 lg/ml) or rhEGF (10 ng/ ml) for 24 h, respectively, compared with the control cells (P < 0.05, Fig. 4a). However, the proportion of HaCaT cells in the S+G2/M phase was not further augmented by combined treatment with rhShh+rhEGF compared with cells treated with rhShh or rhEGF alone (P > 0.05, Fig. 4a). But as shown by flow cytometry analysis (Fig. 4b), the combined agonists (rhShh+rhEGF) cooperated to further reduce the apoptosis rate compared with cells treated by rhShh or rhEGF alone (P < 0.05, Fig. 4b). The results indicated that Shh acts additively with EGFR signalling to inhibit apoptosis in KCs; however, no combined effects were observed for proliferation of KCs. Real-time PCR analysis showed further increases in p-ERK1/2, cyclinD1 and Bcl2 in rhShh+rhEGF-treated cells compared with cells treated by rhShh or rhEGF alone (P < 0.05, Fig. 4d), suggesting

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Figure 4. Shh acts additively with epidermal growth factor receptor (EGFR) signalling to induce ERK activation and is stimulated by EGFR signalling in KCs in vitro. Flow cytometry in HaCaT cells analysed changes in cell cycle distribution (a) and apoptosis rates (b) after treatment with rhShh (4 lg/ml), gefitinib (0.5 lM), rhEGF (10 ng/ml), gefitinib+rhShh or rhShh+rhEGF for 24 h, respectively, compared with the control (n = 3, *P < 0.05, **P < 0.01 and ##P > 0.05). Real-time PCR (c) and Western blotting analysis (e) of the levels of Gli-1, Gli-2, Ptch-1, p-ERK1/2, cyclin D1 and Bcl2 in HaCaT cells in response to gefitinib (0.5 lM), rhShh (4 lg/ml), gefitinib+rhShh (gefitinib followed by the addition of rhShh 1 h later) or medium as a control, respectively, for 6 h (n = 3, *P < 0.05). Real-time PCR (d) and Western blotting analysis (f) of the levels of Gli-1, Gli-2, Ptch-1, p-ERK1/2, cyclin D1 and Bcl2 in HaCaT cells treated with rhShh (4 lg/ml) alone, rhEGF (10 ng/ml) alone, rhShh+rhEGF (rhEGF followed by the addition of rhShh 1 h later) or medium as a control, respectively, for 6 h (n = 3, *P < 0.05). (g) Quantitative analysis of (e) (*P < 0.05 and ##P > 0.05). (h) Quantitative analysis of (e) (*P < 0.05 and ## P > 0.05).

some degree of additive effects between Shh and EGFR signalling in the activation of ERK1/2 and the induction of cyclinD1 and Bcl2. The same trend was observed in the Western blotting analysis (Fig. 4f,h).

Discussion The Shh signalling pathway, which plays a critical role in early vertebrate development and in tumorigenesis, activates and

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controls proliferation and apoptosis in normal KCs; however, the molecular mechanism is largely undefined. In this report, we present evidence that MEK/ERK1/2 activation is a required cascade for Shh signalling, which induces the expression of cyclin D1 and Bcl2 in normal KCs. In addition, Shh signalling’s effects on KC proliferation and apoptosis are independent of the PI3K/AKT or JAK/STAT1/3 signalling pathways. Moreover, Shh pathway is modulated by EGFR signalling. In addition, Shh and EGFR signalling has additive effects on the activation of ERK and the induction of cyclinD1 and Bcl2 in KCs in vitro. Our results uncovered a potential mechanism by which the Shh pathway may contribute to skin diseases development associated with abnormal KC proliferation and apoptosis. Schnidar et al. (20) previously showed that EGFR signalling acts synergistically with SHH/GLI in oncogenic transformation via EGFR-mediated activation of ERK; moreover, inhibition of ERK with U0126 affected Gli protein stability, and thus, ERK was identified as the upstream activator of the SHH/GLI pathway in KC oncogenic transformation. However, here, we proved that the MEK/ERK is a required downstream cascade for Shh pathwaymediated modulation of KC proliferation and apoptosis; moreover, U0126 was found to reduce the levels of Gli1, Gli2 and Ptch1 in return, which agrees previous reports (8,20,21). Extracellular signal-regulated kinase activation has been shown to lead to cyclin D1 transcriptional induction and protein stabilization (13,22). Furthermore, the ERK activation is required for initiation of cell proliferation and its long-term control. Additionally, sonic hedgehog regulates cell growth and proliferation by inducing the expression of cyclin D and cyclin E, and cyclin D1 has been validated as target of Shh signalling (23). Here, we provide evidence that MEK/ERK activation is required for Shh signalling-induction of cyclin D1 and proliferation of KCs. Raj et al. (24) proposed that Bcl2 family members are involved in apoptosis, including Bcl2, which is involved in apoptosis resistance in KCs. Moreover, the phosphorylation of Bcl2 family proteins is controlled by survival signalling pathways that are predominantly regulated by the MAPK and PI3K/AKT pathways (25). Bigelow et al. showed that activation of the Shh pathway upregulates the transcription of Bcl2, which results in enhanced normal KC anti-apoptotic signalling (26). Recently Russell et al. (22) claimed that ERK is involved in resistance to apoptosis in KCs. However, the potential role of MEK/ERK has not been addressed. Here, we showed that in KCs, Shh signalling induces the increase of Bcl2 in a MEK/ERK-dependent manner. Our results agree with the previous study of Kasper et al. (21) who proposed the EGFR-induced Shh activation in KCs. Besides, Schnidar et al. (20) claimed the synergy of Shh/EGF signalling in the oncogenic transformation in KCs, which is based on EGFRmediated ERK activation; however, in our study, the cooperation of Shh/EGF signalling appears to be based on the combined activation of ERK by Shh and EGF signals. Interestingly, Shh acts additively with EGFR signalling to stimulate ERK phosphorylation, to promote the expression of cyclin D1 and Bcl2 and to inhibit apoptosis; however, the two pathways showed no combined effect on the proliferation of KCs. Our results are consistent with the previous findings of Chang et al. (27) and Squires et al. (28). They claimed in the previous researches that excessive activation of the ERK pathway and

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 896–901

The MEK/ERK signalling cascade

accumulation of cyclin D1 can lead to a cell cycle arrest in the G1 phase, which suppresses cell proliferation. Nevertheless, the combined inhibition of Shh-EGFR signalling is a promising target for developing novel therapeutic strategies based on the cooperation of the two pathways (29) for skin disorders resulting from abnormal KC proliferation and apoptosis. In summary, this study provided evidence that Shh signalling controls the proliferation and apoptosis in normal KCs in a MEK/ ERK-dependent manner. Furthermore, Shh acts additively with EGFR signalling to induce ERK activation and is stimulated by EGFR signalling in KCs in vitro. Our results form a basis for further studies to elucidate the regulatory mechanism of the Shh pathway in normal KCs and the pathogenesis of the keratinocyteassociated skin diseases.

References

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Acknowledgements We thank Dr. Jingwu Xie (Department of Pediatrics, Wells Center for Pediatric Research, Division of Hematology and Oncology and IU Simon Cancer Center) for the comments, Ligang Xiong for excellent technical assistance and selfless help, all the members of the Department of Dermatology of Xijing Hospital lab for the discussion and supports to the study. Haiyan Liu, Qiang Jian and Ke Xue designed the study, performed most part of the experiments and wrote the paper. All authors read and approved the final manuscript. This work was supported by grants from the National Natural Science Foundation (81272984 and 30872264) and the ‘New Century Excellent Talent’ project from the Chinese Ministry of Education (NCET-05-0914).

Conflict of interest The authors have declared no conflicting interests.

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27 Chang F, McCubrey J A. Oncogene 2001: 20: 4354–4364. 28 Squires M S, Nixon P M, Cook S J. Biochem J 2002: 366: 673–680. 29 Eberl M, Klingler S, Mangelberger D et al. EMBO Mol Med 2012: 4: 218–233.

Supporting Information Additional supporting data may be found in the supplementary information of this article. Figure S1. RhShh and cyclopamine promoted or reduced HaCaT cells proliferation in a concentrationdependent manner, respectively. Figure S2. U0126 inhibited the proliferation of HaCaT cells in a concentration- and time-dependent manner. Figure S3. Gefitinib reduced the viability of HaCaT cells in a concentration- and time-dependent manner. Figure S4. Proposed model for the interactions of the Shh pathway with MEK/ERK, the EGFR signalling pathway, and the PI3K/AKT and JAK/STAT1/3 pathways. Table S1. Forward and reverse primers for RT-PCR and real-time PCR.

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ERK signalling cascade is required for sonic hedgehog signalling pathway-mediated enhancement of proliferation and inhibition of apoptosis in normal keratinocytes.

Keratinocytes (KCs) play a critical role in maintaining the cutaneous structure and are involved in various physiological and pathologic processes of ...
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