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¹ The First Department of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
² Department of Hematology, Fourth Hospital of Hebei Medical University, Shijiazhuang, P.R. China
³ Cancer Chemotherapy Center, University of Occupational and Environmental Health, Kitakyushu, Japan
⁴ Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Sciences, Tokyo, Japan

	Abstract

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Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 mast cells expressing constitutively activated c-kit catalytic domain mutations. HMC-1 showed constitutive activation of JNK/c-Jun, and the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G₁ phase and apoptosis accompanied by cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis. Following SP600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Thus, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with mutations in the catalytic domain of c-kit.

	Introduction

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c-Jun N-terminal kinase (JNK), known as a family of stress-activated protein kinases (SAPK), plays a pivotal role in the cellular response to extracellular stimuli including UV, ionizing radiation, heat shock and inflammatory cytokines. JNK phosphorylates the critical serine residues, Ser63 and Ser73 in activation domain of c-Jun/activator protein 1 (AP-1) transcription factor. Activation of JNK has been implicated in both cell survival and cell death (Davis 2000; Kyriakis & Avruch 2001). The JNK-dependent pathway is required for TNF - and UV-induced apoptosis (Tournier et al. 2000; Deng et al. 2003). In addition, the JNK pathway has also been implicated in the transformation of pre-B cell by Bcr-Abl and in the transformation of fibroblasts by the Met oncogene (Dickens et al. 1997; Rodrigues et al. 1997), suggesting that the JNK pathway can play a significant role in cellular transformation and tumor cell growth. The JNK protein kinases are coded by three genes: jnk1, jnk2, and jnk3. The jnk1 and jnk2 genes are ubiquitously expressed, while the jnk3 gene is expressed only in the brain, heart, and testis (Davis 2000).

JNK proteins phosphorylate serine 63 and 73 of c-Jun and increase the ability of c-Jun to activate transcription (Smeal et al. 1991). JNK1 was first identified as a serine/threonine kinase that phosphorylated the AP1 family member c-Jun in response to UV (Derijard et al. 1994). Activated c-Jun/AP1 complex by JNK regulates cell proliferation by promoting cell-cycle progression, which links to p53 and cyclin D1 (Schreiber et al. 1999; Bakiri et al. 2000). Thus, signals relayed by JNK through c-Jun regulate a range of cellular process including cell proliferation, tumorigenesis, apoptosis and embryonic development (Dunn et al. 2002; Wiltshire et al. 2002). JNK is known to be activated by a number of growth factors including stem cell factor (SCF), a ligand of c-kit (Foltz & Schrader 1997), that promote proliferation and survival of hematopoietic cells.

c-kit, a member of receptor tyrosine kinase family is expressed on a variety of cell types, including mast cells, hematopoietic stem cells, melanocytes, and germ cells (Yarden et al. 1987; Ashman 1999). SCF, known as mast cell growth factor or steel factor, is the ligand of c-kit. Signaling mediated by SCF and c-kit is essential for regulation of the proliferation, differentiation, survival, chemotaxis and functional activation of mast cells (Galli et al. 1994; Nilsson & Metcalfe 1996). Activated c-kit stimulates proliferation of mast cells and prevents their apoptosis in vitro and in vivo (Mekori et al. 1993; Iemura et al. 1994). Gain-of-function mutations in c-kit are associated with factor-independent proliferation of mast cells, causing mastocytosis (Nagata et al. 1995; Longley et al. 1996). The mutations result in autophosphorylation of c-kit, subsequent activation of downstream signaling molecules including the serine/threonine kinases, JNK1 and JNK2, phosphatidylinositol 3 (PI3) kinase, and signal transducer and activator of transcription 3 (STAT3) (Chian et al. 2001; Ning et al. 2001). Mastocytosis with the c-kit mutations is resistant to tyrosine kinase inhibitor imatinib mesylate (STI571) (Frost et al. 2002; Ma et al. 2002; Zermati et al. 2003). Therefore, agents that specifically block the critical downstream signaling pathway of c-kit receptor may have clinical benefit in treatment of diseases associated with the c-kit mutations. It has been demonstrated that the JNK pathway plays a critical role in mediating c-kit-induced cell proliferation in bone marrow-derived mast cells (BMMC). Moreover, JNK may have both mitogenic and pro-apoptotic roles in BMMC (Timokhina et al. 1998). JNK1 and JNK2 were constitutively activated by D816V c-kit mutant; however, the function of JNK has not been completely understood.

Human HMC-1 mast cells have been established from a patient with mast cell leukemia with two point mutations in the intracellular juxtamemberane domain (Val560Gly) and in the catalytic domain (Asp816Val) of the c-kit receptor (Furitsu et al. 1993). We report here that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK (Bennett et al. 2001), inhibits proliferation of HMC-1 mast cells and induces apoptosis of HMC-1 cells in a caspase-3-dependent manner. Our results demonstrate the importance of constitutive activated JNK/c-Jun in proliferation and survival of HMC-1 cells.

	Results

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Spontaneous activation of JNK/c-Jun in HMC-1 cells

PI3K and MAP kinase have been implicated in SCF-mediated survival and proliferation in mast cells. We therefore determined the activation status of Akt and MAP kinases in HMC-1 cells that have ability in SCF-independent proliferation. As shown in Fig. 1, neither phospho-Akt nor phospho-ERK was detected in the absence of SCF. Treatment of HMC-1 cells with SCF significantly induced phosphorylation of Akt and ERK. In contrast, although SCF treatment slightly enhanced phosphorylation of JNK, constitutive phosphorylation of JNK, p38 and c-Jun was observed in untreated HMC-1 cells.

View larger version (25K): [in this window] [in a new window]   Figure 1  JNK/c-Jun is constitutively phosphorylated in HMC-1 cells. HMC-1 cells were treated with SCF (100 ng/mL) for 15 min or left untreated. Cell lysates were analyzed by Western blotting using antibodies to phospho-Akt (Ser473), phospho-ERK1/2, phospho-JNK, phospho-c-Jun and phospho-p38 and then stripped and reprobed for actin. Data from three different experiments.

As shown in Fig. 2A, a dose-dependent reduction in phosphorylation of c-Jun was observed in HMC-1 cells treated with JNK inhibitor SP600125. In contrast, no significant change in c-Jun and JNK protein expression levels was observed. Moreover, it is interesting to note that SP600125 partially inhibited phosphorylation of JNK. A previous study has also reported inhibition of phospho-JNK by SP600125, suggesting inhibition of JNK autophosphorylating actitivty (Bennett et al. 2001). In order to evaluate the specificity of JNK inhibitor SP600125, HMC-1 cells were pretreated with SP600125 for 1 h before SCF stimulation and phosphorylation status of Akt, ERK and p38 in HMC-1 cells was assessed (Fig. 2B). As a result, SP600125 had no effect on phosphorylation of Akt, ERK and p38.

View larger version (43K): [in this window] [in a new window]   Figure 2  SP600125 inhibits constitutive phosphorylation of c-Jun in HMC-1 cells. (A) Cells were treated with various concentrations of SP600125 for 1 h, and then cell lysates were analyzed by Western blotting as described in Experimental procedures. (B) Cells were pretreated with SP600125 1 h before SCF (100 ng/mL) stimulation for 15 min.

HMC-1 cells were treated with increasing concentrations of JNK inhibitor SP600125 (1–20 µM) for 24 and 48 h in order to evaluate a role of JNK activation in cell proliferation. Cell proliferation was assessed using the TetraColor One assay. As show in Fig. 3A, SP600125 inhibited cell proliferation in a dose-dependent manner. In contrast, PD98059 showed no significant effect on cell proliferation even at a concentration of 20 µM (Fig. 3B), showing the importance of JNK/c-Jun in HMC-1 cell proliferation. Moreover, 20 µM of PD98059 almost completely blocked SCF-induced activation of ERK in HMC-1 cells (Fig. 3C).

View larger version (22K): [in this window] [in a new window]   Figure 3  Growth inhibition of HMC-1 mast cells by SP600125 JNK inhibitor. (A) Cells were treated with various concentrations of SP600125 for 24 h (left) or 48 h (right), and proliferation activities of cells were assessed by the TetraColor One assay kit. (B) Cells were incubated with either SP600125 (filled diamond) or PD98059 ERK inhibitor (filled triangle) at 5–20 µM for 48 h. Proliferation activities of cells were assessed by TetraColor One. The optical density (OD) was measured by ELISA plate reader at 450 nm. Data are expressed as mean ± standard deviation (SD) of five experiments. (C) Cells were pretreated with 20 µM PD98059 for 30 min, and then stimulated with SCF (100 ng/mL) for 15 min and lyzed. The blot was probed with anti-phospho-ERK1/2 antibody. This blot was subsequently stripped and reprobed with anti-ERK1/2 antibody.

We next investigated the effect of JNK inhibitor SP600125 on apoptosis (Fig. 4). To analyze the early phase apoptosis of cell population and to obtain quantitative data, annexin V/PI staining followed by FCM analysis were performed. We measured the percentage of early apoptotic cells after treatment with SP600125. As shown in Fig. 4A, treatment with various concentrations (1–10 µM) of SP600125 for 24 h resulted in increase in apoptotic cells. In the absence of SP600125, 3.8% of cells showed early apoptosis. The percentage of early apoptotic cells was increased to 15.2% in the presence of 5 µM of SP600125. Early apoptosis was further accompanied by the time exposed to SP600125 (Fig. 4B). These data clearly showed that SP600125 induces early apoptosis.

View larger version (49K): [in this window] [in a new window]   Figure 4  SP600125 induces early apoptosis of HMC-1 mast cells in a dose- and a time-dependent manner. (A) Cells (5 x 105 cells/mL) were incubated with 0.1% DMSO (control) or 1–10 µM of SP600125 for 24 h. (B) Cells were treated with 5 µM SP600125 for 12 or 24 h. Apoptotic cells were detected by double staining with PI and Annexin V, and analyzed by FACScan. Data represent early apoptotic cells of five different experiments.

There are several pathways involved in apoptosis, including caspase-dependent and caspase-independent mechanisms. Caspases-3 is a central effector in executing the apoptotic process of caspase-dependent manner. To determine whether caspase-3 is involved in apoptosis induced by SP600125 in HMC-1 cells, we examined activation of caspase-3 by Western blotting using a mAb that specifically recognizes the activated form of caspase-3. Cells were stimulated with SP600125 for the indicated time and at the indicated concentration. As shown in Fig. 5A, active fragments (17, 19 kDa) of caspase-3 cleaved from 35-kDa pro-caspase-3 were generated following treatment with 5 µM of SP600125 in Western blot analysis using a caspase-3-specific Ab. In addition, cleavage of a specific substrate of caspase-3, PARP was also observed in the presence of 5 µM SP600125 (Fig. 5B).

View larger version (66K): [in this window] [in a new window]   Figure 5  SP600125 induces cleavage of caspase-3 and PARP. HMC-1 cells were treated with 5 µM of SP600125. At different time points, cells were harvested and lyzed in 250 µL of lysis buffer. Cell extracts were subjected to Western blotting for caspase-3 and PARP. (A) Caspase-3 (35 kDa) was cleaved (19 and 17 kDa). (B) PARP was cleaved by SP600125. Data from three different experiments.

View larger version (10K): [in this window] [in a new window]   Figure 6  Caspase-3 inhibitor, but not caspase-9 inhibitor blocks SP600125-induced early apoptosis of HMC-1 cells. Cells were treated with 100 µM of Z-DEVD-FMK caspase-3 inhibitor or Z-LEHD-FMK caspase-9 inhibitor for 2 h prior to treatment with 5 µM SP600125 for 24 h 0.6% DMSO was used as a control. Apoptotic cells were detected by double stained with PI and Annexin V, and analyzed by FACScan. Data represent the mean of percentage of Annexin V positive staining cells of four different experiments.

It is known that c-Jun is required for progression through the G₁ phase of the cell cycle (Wisdom et al. 1999); next we determined whether SP600125 would induce cell cycle arrest by inhibition of c-Jun activity. Cells were untreated or treated with 5 µM SP600125 for 8 h, and cell cycle distribution was determined by PI staining. As shown in Fig. 7, a significant G₁ arrest was observed in HMC-1 cells treated with SP600125. In addition, there was an increase in sub-G₁ phase cells, which was consistent with our annexin V/PI staining data, showing increased apoptotic cells after treatment with SP600125. These results demonstrated that SP600125 induces cell cycle arrest as well as apoptosis, and both events in combination are likely to contribute to the proliferation inhibition of HMC-1 cells by SP600125.

View larger version (28K): [in this window] [in a new window]   Figure 7  Suppressive effect of SP600125 on cell cycle progression of HMC-1 cells. Cells were untreated or treated with 5 µM SP600125 for 8 h as indicated and cellular DNA content was determined by PI staining and flow cytometry. Cell cycle parameters were determined using ModFit software. Data from four independent experiments.

View larger version (81K): [in this window] [in a new window]   Figure 8  Inhibition of cyclin D3 protein expression by SP600125 in HMC-1 cells. Cells were incubated with various concentrations of SP600125. After 24 h, cells were harvested and lyzed in 100 µL lysis buffer. Western blotting was carried out as described in Experimental procedures. Results shown are representative of those of three different experiments.

	Discussion

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SP600125 is a reversible ATP competitive inhibitor with more than 20-fold selectivity over other kinases including ERK, p38 MAPKs, MKKs and PKCs. SP600125 dose-dependently inhibits the phosphorylation of c-Jun, a component of the transcription factor AP-1, which is phosphorylated at Ser 63/73 by JNK (Bennett et al. 2001). c-Jun is a specific and important functional endpoint of JNK signaling. We demonstrated that c-Jun is constitutively activated in HMC-1 cells, which is inhibited by SP600125. In addition, we provide evidence that HMC-1 cell proliferation was suppressed in a dose- and a time-dependent manner when cells were treated with SP600125. Thus, spontaneous activation of the JNK/c-Jun pathway is essential for proliferation in mast cells.

Apoptosis, executed by caspase, is critical for maintaining tissue homeostasis, and impaired apoptosis is now recognized to be a key step in tumorigenesis. There are several pathways involved in apoptosis, including caspase-dependent and caspase-independent mechanisms (Lockshin & Zakeri 2002). In the present study, HMC-1 cells underwent apoptosis following treatment with JNK inhibitor SP600125, showing that JNK/c-Jun activity is required for cell survival. In addition, SP600125-induced apoptosis was dependent upon caspase-3, but not caspase-9. Spontaneous protein expression of c-myc was also observed in HMC-1 cells. c-myc is required for efficient response to a variety of apoptosis stimuli, including transcription and translation inhibitors, glucose deprival, and DNA damage (Prendergast 1999). It has been also proposed that c-myc acts to sensitize cells to a variety of apoptotic triggers (Capeans et al. 1998).

In the present study, SP600125-induced G₁ arrest of HMC-1 human mast cells was observed, and cyclin D3 expression was reduced with SP600125 treatment in HMC-1 cells. Entry into the S phase is regulated by cell cycle regulatory molecules including cyclins, cyclin-dependent kinases CDKs and pRb. Association of cyclin D with CDKs induces phosphorylation of pRb (Bartek et al. 1996; Ezhevsky et al. 1997; Sherr 2000). The expression of cyclin D3 has been reported to be accelerated in BMMC incubated with SCF (Itakura et al. 2001). In this regard, Tanaka et al. (2005) demonstrated dependency of cell cycle progression on cyclin D3 in mast cells.

HMC-1 cell growth was almost completely blocked in presence of 5 µM SP600125. In this regard, Chian et al. (2001) have reported that the 85-kDa regulatory subunit of PI3K is constitutively associated with D816V c-Kit. Interestingly, they further demonstrated that although PI3K generally contributes to the activation of Akt and JNKs, Jnk 1 and Jnk 2 were activated but Akt was not in the D816V c-Kit mutant. A recent study has also reported that c-kit mutation did not lead to constitutive phosphorylation of Akt or ERK in HMC-1 cells (Sundstrom et al. 2003). Consistent with these studies, we observed no constitutive activation of Akt and ERK in HMC-1 cells in the absence of SCF. Moreover, PI3K and Src kinase signaling pathways have been shown to converge to activate Rac 1 and JNK, which play a critical role in SCF-induced proliferation of BMMC (Timokhina et al. 1998). These arguments support the importance of JNK/c-Jun in HMC-1 cell proliferation and survival.

Inhibitors specific for signal transduction cascades can be used in the treatment of several malignancies. SP600125 selectively inhibits JNK activity and blocks c-Jun phosphorylation in a competitive manner (Bennett et al. 2001). Mast cell diseases associated with c-kit mutant (most commonly Asp 816 Val) are resistant to imatinib mesylate STI571, because this mutant interferes with binding of STI571 to the enzymatic site of the c-kit (Akin & Metcalfe 2004). Thus, blocking the JNK/c-Jun pathway is a more selective approach for suppression of pathological processes induced by mastocytosis-associated c-kit mutation.

	Experimental procedures

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Cell cultures

Human mast cell line HMC-1 was cultured in Iscove's modified Dulbecco's medium (IMDM; Gibco BRL, 12440-053, Grand Island, NY, USA) with 10% fetal bovine serum in a humidified 5% CO₂ incubator at 37 °C.

Reagents

JNK inhibitor SP600125 (Seikagaku; Tokyo, Japan), ERK inhibitor PD98059 (Funakoshi; Tokyo, Japan) and SCF (PeproTech EC; London, UK) were used in the present study. Z-DEVD-FMK (caspase-3 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) were purchased from R & D Systems (Minneapolis, MN, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) Ab and HRP-conjugad anti-mouse IgG Ab were purchased from Amersham Biosciences (Fairfield, CT, USA). Abs against JNK, phospho-JNK (Thr183/Try185), c-Jun, phospho-c-Jun (Ser63/73), ERK1/2, phospho-ERK1/2 (Thr202/Try204), phospho-Akt (Ser473), phospho-p38 MAKP (Thr180/Try182), caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, PARP, cleaved PARP (Asp214), c-myc, p53, cyclin D3 for Western blot were purchased from Cell Signaling Technology (Beverly, MA, USA).

Protein extraction and Western blotting

Cells were pelleted by centrifugation and lyzed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 2 mM sodium orthovanadate, 5 mM sodium pyrophosphate) with protease inhibitor mixture (complete mini) and 1% Triton X-100. Same amount of 2xSDS (sodium dodecyl sulfate) buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue) with 2-mercaptoethanol was added to each sample, then boiled for 5 min. Equal amount of samples were run on Tris-Glycine polyacrylamide gel and proteins were transferred on to nitrocellulose membrane. The membranes were blocked in PBS (phosphate-buffered saline) with 0.1% Tween20 (PBS-T) and 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary Ab (1 : 500 dilution). The membranes were washed several times with PBS-T, and then incubated for 1 h with appropriate HRP-conjugated secondary Ab (against rabbit or mouse). After washing several times with PBS-T, proteins on the membrane were visualized by ECL chemiluminescence system (Amersham Pharmacia Biotech; Piscataway, NJ, USA) as directed in manufacturer's instruction.

Proliferation assay

The proliferation assay was performed as previously described (Tanaka et al. 2002). Briefly, HMC-1 cells (1 x 10⁴) were incubated with various concentration of SP600125 on 96-well flat-bottomed microfilter plates in IMDM and 10% FCS for 24–48 h at 37 °C. After cells were stained with TetraColor One kit including tetrazolium and electron carrier mixture (Seikagaku) for 1 h at 37 °C, the optical density value of each well was measured by a enzyme-linked immunosorbent assay plate reader at 450 nm.

Apoptosis assay

Apoptosis was evaluated by flow cytometry utilizing cellular annexin V binding (Becton Dickinson; Franklin Lakes, NJ, USA; Annexin V: FITC Apoptosis Detection KitI). Briefly, cells (5 x 10⁵ per well) were incubated with various concentrations of SP600125. Apoptotic cells were measured by using an Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions, and sorted using a FACScan flow cytometer (Becton Dickinson). All PI-positive cells were considered dead. PI-negative and annexin V-positive cells were considered early apoptotic cells and the double negative remaining cells were considered viable.

Cell cycle analysis

HMC-1 cells were cultured in IMDM containing 10% FCS and treated with SP600125 5 µM. After 8 h, cells were washed with ice-cold PBS, and fixed in 70% ethanol at least 2 h at 4 °C. After treatment of cells with 10 µg/mL RNase A (Wako, Osaka, Japan) for 20 min at 37 °C, the cells were stained with 50 µg/mL of propidium iodide (PI) (Sigma Aldrich, Chicago, IL, USA) for 3 min at room temperature and analyzed by flow cytometry. The data were analyzed using the ModFit software (Verity Software House; Topsham, ME, USA).

	Acknowledgements

 
This work was supported in part by Research Grants-In-Aid for Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan and University of Occupational and Environmental Health, Japan.

	Footnotes

 
Communicated by: Shigeo Koyasu

^* Correspondence: E-mail: jtsukada{at}med.uoeh-u.ac.jp

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Received: 12 December 2005
Accepted: 22 May 2006

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