Int J Hematol (2014) 99:750–757 DOI 10.1007/s12185-014-1586-y

ORIGINAL ARTICLE

SCF/C-KIT signaling modulates tryptase expression in acute myeloid leukemia cells Jingchun Jin • Yan Li • Yue Wang Pingping Wang • Yazhu Wang

•

Received: 9 January 2014 / Revised: 17 April 2014 / Accepted: 18 April 2014 / Published online: 8 May 2014 Ó The Japanese Society of Hematology 2014

Abstract Tryptase is a serine protease with a variety of biological functions. Recently, elevated serum tryptase has been detected in certain patients with acute myeloid leukemia (AML). However, the underlying mechanism for the regulation of tryptase expression remains elusive. In this study, we aimed to investigate the role of stem cell factor (SCF)/C-KIT signaling in regulating the expression of tryptase in AML cells. We found a significant positive correlation between tryptase and C-KIT expression levels in AML patients. Furthermore, real-time PCR, Western blot and ELISA analysis showed that SCF upregulated tryptase mRNA and protein expression in U937 cells, and that this effect was abolished by pretreatment with PD98059 and SB230580. In addition, levels of phosphorylated ERK1/2 and p38MAPK correlated with tryptase levels. Taken together, these data suggest that the expression of tryptase is regulated by SCF/C-KIT signaling via the ERK1/2 and p38MAPK pathways. Keywords Acute myeloid leukemia  Tryptase  Stem cell factor  KIT  p38MAPK  ERK1/2

Introduction Acute myeloid leukemia (AML) is a malignant disease, with outcomes that depend on the specific genetic mutations driving the abnormal differentiation of hematopoietic progenitor cells [1, 2]. Tryptase is a serine protease abundantly expressed in certain leukemia cell lines such as U937, Mono Mac 6, as well

as human mast cells [3, 4]. Recently, it was reported that elevated tryptase levels were detected in AML patients, especially those with the M2 and M4Eo subtypes of AML, and the outcomes of these patients are poor [5]. These studies suggest that tryptase may participate in the formation of new blood vessels in AML through the upregulation of vascular endothelial growth factor [6]. However, the mechanism that regulates tryptase expression in AML precursor cells is elusive. Recently, it is found that the elevation of serum tryptase is associated with KIT mutations, that is, levels of serum tryptase are much higher in AML patients with the KIT mutation compared with those without the mutation [7]. The KIT mutations result in ligand-independent autophosphorylation of the KIT receptor protein, thereby activating the downstream mitogen-activated protein kinase (MAPK) pathway and promoting cell proliferation and survival [8, 9]. Stem cell factor (SCF) ligand binding leads to the phosphorylation and activation of the KIT receptor and its downstream signaling proteins. It has been reported that KIT receptor was overexpressed and activated in the presence of SCF [8, 10]. In addition, tryptase was co-expressed with CD117 in AML [11]. These observations prompted us to investigate the role of SCF/C-KIT signaling in tryptase expression and the underlying mechanism. We aimed to detect the tryptase level when C-KIT is activated by SCF in AML cells. Furthermore, we explored whether MAPK pathway is involved in SCF/C-KIT mediated tryptase expression.

Materials and methods J. Jin  Y. Li (&)  Y. Wang  P. Wang  Y. Wang Department of Hematology, The First Affiliated Hospital of China Medical University, No. 155, Nanjing North Street, Shenyang 110001, China e-mail: [email protected]

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Patients In total, 75 patients with primary AML (median age 60 years, range 16–89 years, female-to-male ratio 1:1) and

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Table 1 Characteristics of AML patients with high and low tryptase mRNA levels Relative tryptase mRNA B1

Relative tryptase mRNA [1

Patients, no.

35

40

Female/male

17/18

21/19

Median age (years)

50.9

44.3

Median WBC(10*109/L)

41.2

33.9

0.62

Median HB (g/L)

81.5

71.3

0.06

Median PLT (10*10 /L)

51.1

53.8

0.89

Median blasts (%)

70.8

73.8

0.59

9

P

Tryptase F

50 -GCAAAATACCACCTTGGCGCCTACACGG-30

Tryptase R

50 -GTGACACGGGTGTAGATGCCAGGC-30

C-KIT F

50 -TGGGTGCTGGAGCTTTCG-30

C-KIT R

50 -AATATTCTGTAATGACCAGGGTG-30

ABL F

50 -CCT TCA GCG GCC AGT AGC-30

ABL R

50 -GGA CACAGG CCC ATG GTA C-30

0.108

Cytogenetics, no. (total no.) t(8;21)

2(11)

7(10)

0.03

t(15;17)

0(1)

7(9)

0.22

inv(16)

0(0)

1(1)

11q23/MLL

1(4)

2(2)

0.4

7(10)

4(15)

0.049

Complete remission*

Table 2 Primers for quantitative RT-PCR

*excluding M3 subtype

30 healthy controls were examined. According to the classification of the French–American–British (FAB) cooperative study group [12, 13], patients were diagnosed as AML M1 (n = 1), M2 (n = 32), M3 (n = 19), M4eo (n = 1), M5 (n = 20), and M6 (n = 1). 25 of 75 AML patients were hospitalized patients, and they were treated with standard-dose cytarabine 100 mg/m2 continuous infusion 9 7 days with idarubicine 8 mg/m2 9 3 days or daunorubicin 60 mg/m2 9 3 days and assessed the complete remission (CR) rate at the first chemotherapy cycle. The characteristics of AML patients are shown in Table 1. The Ethics Committee of China Medical University approved this study. All patients signed informed consent before bone marrow puncture. Cell line and cell culture The human myeloid leukemia cell line U937 was provided by Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. The cells were cultured in PMI1640 (Gibco, Carlsbad, CA) supplemented with 10 % fetal bovine serum (Hyclone, Logan, USA) and 1 % penicillin/ streptomycin, at 37 °C in a 5 % CO2 humidified incubator. Real-time quantitative polymerase chain reaction analysis

(Applied Biosystems, USA) using SYBR Green PCR Mix (TaKaRa, Dalian, China). The mRNA expression of the ABL gene was used as an internal standard. Primer sequences used for real-time analysis are shown in Table 2. KIT mutation analysis KIT mutations were detected by PCR and direct sequencing. Genomic DNA was obtained with a DNA extraction kit (TaKaRa, Dalian, China). To amplify exons 8 and 17 of KIT, the following primers were used: 50 -CTCCCTGAAA GCAGAAAC-30 (8F), 50 -CAGAAAGATAACACCAAAA TAG-30 (8R), 50 -GCAAAGGCATATTAGGAACTC-30 (17F), and 50 -GTTGTAGTAATGTTCAGCATA-30 (17R) (SangonBiotech, Shanghai, China). Aliquots (1 lL) of genomic DNA were used for PCR amplification in a final volume of 10 lL containing 1 lL 109 PCR buffer, 0.2 U KOD Plus DNA polymerase (Toyobo, Japan), 1 lL dNTP Mix, 0.6 lL MgSO4, and 25 pM of each upstream and downstream primers. PCR products were directly sequenced on an ABI3730xl Genetic Analyzer (Applied Biosystems). Sequencing data were analyzed by Sequencing Analysis software using GenBank NM 000222.2 as reference. Flow cytometry Approximately 2 9 105 U937 cells were treated with human SCF for 24 h, collected by centrifugation at 1,000 rpm for 5 min, and then single cells were washed twice with phosphate-buffered saline (PBS) containing 2 % human serum and subjected to surface marker profiling analysis. Single cells were stained with fluorescein isothiocyanate-conjugated mouse anti-human CD117 antibody (BD Biosciences, CA, USA) for 30 min on ice in the dark in PBS containing 1 % human serum. The cells were analyzed on a BD FACSCanto. Sample analysis was performed using BD FACS Diva software. Enzyme-linked immunosorbent assay (ELISA)

Total RNA was isolated with Trizol reagent (TaKaRa, Dalian, China), and cDNA was synthesized with M-MLV Reverse Transcriptase (Promega, Madison, WI) in accordance with the manufacturer’s instructions. Quantitative RT-PCR was run on an ABI 7500 real-time PCR system

U937 cells were incubated with or without 100 ng/mL human SCF (Cell Signaling Technology, MA, USA) for 24 h at 37 °C. Tryptase secreted by U937 cells in culture medium was measured by an ELISA kit (Biosamite,

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Shanghai, China) in accordance with the instructions of the manufacturer. The tryptase concentrations were evaluated by absorbance at 450 nm. Western blot analysis Total protein was extracted using RIPA lysis buffer (50 mM Tris, 150 mM sodium chloride, 1 % NP-40, 0.25 % deoxycholate) supplemented with inhibitors aprotinin, leupeptin and pepstatin. Cell lysates (30 lg protein per lane) were resolved via 10 % SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5 % skimmed milk for 2 h, the membranes were incubated serially with anti-tryptase (Abcam, MA, USA), anti-p38MAPK, antiphospho-specific p38MAPK (Cell Signaling Technology, MA, USA), anti-ERK1/2 and anti-phospho-specific ERK1/ 2 (ImmunoWay Biotechnology, USA) overnight at 4 °C, washed with 19 Tris-buffered saline containing 0.1 % Tween 20, and incubated with horseradish peroxidaseconjugated secondary antibody (Bioss, Beijing, China) for 1 h. Finally, the signals were visualized using enhanced chemiluminescence. Statistical analyses All the variables were first checked to verify their distribution using the Kolmogorov–Smirnov test. For analysis of differences between two groups, Student’s t test, Chisquare test or Wilcoxon Rank-Sum test were performed. To analyze the correlation between two groups, a linear correlation was applied. All data were expressed as mean ± standard deviation (SD). The level of statistical significance was set at P \ 0.05.

The mRNA levels of tryptase and C-KIT were higher in AML blasts

Fig. 1 Real-time PCR analysis of tryptase and KIT mRNA levels in AML patients. a Tryptase and KIT mRNA expression in AML patients and healthy controls (* P \ 0.001, AML patients vs. healthy controls). b Tryptase mRNA levels in FAB subtypes of AML and controls. The median tryptase mRNA level in each subtype is indicated by a horizontal bar. c Correlation between tryptase and KIT mRNA levels. Tryptase and KIT mRNA levels were measured at the time of diagnosis (r = 0.554, P \ 0.001)

We detected the mRNA expression of KIT and tryptase by real-time PCR in 75 AML patients and 30 controls. The results showed that the mRNA levels of tryptase and KIT were higher in AML patients than in controls (P \ 0.05, Fig. 1a). The mRNA levels of tryptase in AML patients stratified by FAB subtypes and controls are shown in Fig. 1b. A significant correlation between tryptase and KIT mRNA levels was found (r = 0.554, Fig. 1c). The relative tryptase mRNA in controls was 0.5 and we divided AML patients into two groups at the margin of twice of the value (Table 1). We detected karyotypes of

t(8;21), t(15;17), inv(16) and 11q23/MLL in M2, M3, M4 and M5, respectively. Furthermore, we analyzed the tryptase mRNA level in the groups with or without t(8;21). The relative mRNA levels was 3.7 ± 4.0 and 1.3 ± 2.5, respectively (Wilcoxon Rank-Sum test: P = 0.012). We also analyzed the correlation between the clinical response to chemotherapy at the first chemotherapy cycle and tryptase mRNA level in AML excluding M3. 7 of 10 (70 %) patients with lower tryptase achieved CR, whereas only 4 of 15 (26.6 %) patients with high tryptase obtained CR (P = 0.049). There were no significant correlations between tryptase levels and other laboratory parameters,

Results

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Fig. 2 KIT mutational analysis in AML patients. a Six patients had exon 17 mutations. Five patients involved codon 816 (D816V A1 n = 2, D816H A2 n = 1, D816Y A3 n = 2) and one patient involved codon 822 (n = 1 N822K A4). b Different mRNA levels of tryptase and KIT in patients with or without KIT exon 17 mutations and with FAB classification M2 (*Ptryptase = 0.038, *Pkit = 0.034, mut-kit with mutated KIT, wild-kit without mutated KIT)

including white blood cell count, hemoglobin, platelet, and the percentage of blasts. The mRNA levels of tryptase and C-KIT were higher in AML-M2 patients with mutated KIT KIT mutational analysis (for exon 8 and exon 17) was performed in 54 AML patients. The KIT exon 17 mutation was found in 6 of the 54 patients (11 %), while the exon 8 mutation was not detected in all patients. All of the 6 KIT exon 17 mutations were found in 27 AML-M2 patients (22 %; Fig. 2a). In AML-M2 patients, the mRNA levels of tryptase and KIT were higher in patients with mutated KIT (n = 6) than in patients without mutated KIT (n = 21; Fig. 2b). Human SCF stimulated KIT expression in U937 cells Flow cytometry was used to estimate CD117 expression in U937 cells. CD117 expression in U937 cells was obviously

increased when treated with human SCF for 24 h (Fig. 3a). After incubation with 100 ng/mL human SCF for 24, 36, or 48 h, the KIT mRNA levels were significantly upregulated (Fig. 3b).

SCF/C-KIT signaling upregulated tryptase expression in U937 cells To confirm that human SCF regulates tryptase expression in U937 cells, we incubated U937 cells with human SCF 100 ng/mL for 24, 36, or 48 h. Real-time PCR showed a significant elevation in tryptase mRNA after human SCF treatment (Figs. 4, 5a). In addition, Western blot and ELISA were performed to measure the protein levels of tryptase in both U937 cells and culture medium after treatment with 100 ng/mL human SCF for 24 h. Compared to the control group, the tryptase levels were significantly higher in both the U937 cells and medium (Fig. 5b, c).

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Fig. 3 Human SCF stimulated CD117 and KIT mRNA expression in U937 cells. a Expression of CD117 is elevated from 2.7 % (A-left) to 12.2 %(A-right) when stimulated with SCF 100 ng/mL for 24 h in U937 cells. b KIT mRNA expression in U937 cells treated with 100 ng/mL human SCF for 24, 36, or 48 h. The data were expressed as mean ± SD of three independent experiments (*P24h = 0.012, P36h = 0.018 vs. control)

Fig. 4 Human SCF was associated with increased tryptase mRNA levels in U937 cells. U937 cells were treated by 100 ng/mL human SCF for 24, 36, or 48 h. Each bar showed the mean ± SD of tryptase mRNA expression values in three independent experiments (*P24h \ 0.001, P36h = 0.001 vs. control)

SCF/C-KIT activated ERK1/2 and p38MAPK signaling pathways To determine whether the ERK1/2 and p38MAPK pathways were involved in SCF/C-KIT-mediated upregulation of tryptase expression in U937 cells, we pretreated U937 cells with 20 mM PD98059 (ERK1/2 inhibitor) or 10 mM SB230580 (p38MAPK inhibitor) for 30 min, and then treated the cells with 100 ng/mL human SCF for 24 h.

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Real-time PCR, Western blot and ELISA showed that the upregulation of tryptase stimulated by human SCF in U937 cells was inhibited by both PD98059 and SB230580 (Fig. 5). Furthermore, the levels of ERK1/2, p38MAPK, and phosphorylated ERK1/2 and p38MAPK in U937 cells were detected with Western blot. The results showed that ERK1/ 2 and p38MAPK were phosphorylated after stimulation with human SCF. However, after pre-incubation with PD98059 and SB230580, the levels of phosphorylated ERK1/2 and p38MAPK proteins were reduced (Fig. 6).

Discussion Our results indicate that there is a significant correlation between tryptase and KIT mRNA levels in AML cells. SCF/C-KIT signaling may contribute to the upregulation of tryptase expression in AML cells and MAPK pathway may participate in the regulation. In this study, we detected the expression of KIT and tryptase in AML blasts. The data showed that mRNA levels of tryptase and KIT were correlated in AML patients. Furthermore, higher levels of tryptase and KIT mRNA were

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Fig. 5 ERK1/2 and p38MAPK inhibitors abrogate tryptase expression induced by SCF in U937 cells. U937 cells were pretreatment with 20 mM PD98059 or 10 mM SB230580 for 30 min, and then treated with 100 ng/mL huSCF for 24 h. Tryptase mRNA expression in U937 cells was detected by real-time PCR (a *P \ 0.001 vs. control. #PPD = 0.026, PSB = 0.014 vs. huSCF treatment alone), tryptase secretion into the medium was detected by ELISA

(b *P = 0.008 vs. control. #PPD = 0.01, PSB = 0.011 vs. huSCF treatment alone), and tryptase protein expression in U937 cells was detected by Western blot analysis (c *P = 0.011 vs. control. #PPD = 0.02, PSB = 0.007 vs. huSCF treatment alone). The data were expressed as mean values ± SD of three independent experiments

found in patients with mutated KIT compared with AML patients without the KIT mutation. These results indicate that elevation of tryptase expression is significantly associated with KIT expression. Our findings are similar to a previous report of a correlation between elevated serum tryptase levels and the presence of KIT mutation [7]. Our data showed that patients with t(8;21) had higher mRNA level of tryptase and patients with high tryptase occurred with high frequency of t(8;21). Previous studies also showed that the mutation of KIT was frequently found in core-binding factor AML [14, 15]. In addition, the elevation of tryptase has been coincidentally detected in M2 and M4Eo patients, whereas no elevation was found in acute lymphoid leukemia patients [5]. We chose U937 cells as a model because tryptase was expressed in U937 cells [3]. In our initial study, we also chose kasumi-1 cells as an experimental subject which is characterized by KIT Asn822Lys mutation and C-KIT overexpression. The level of tryptase mRNA in kasumi-1 cells was almost 300 times lower than U937 cells (data not shown). The proto-oncogene KIT, which is expressed in bone marrow-derived endothelial stem/progenitor cells, belongs to the tyrosine kinase family [16, 17]. CD117 is a transmembrane receptor tyrosine kinase that is encoded by KIT, which was expressed mainly in most of AML, especially in a large proportion of core-binding factor AML, and rarely in acute lymphoid leukemia [18]. In tryptase-positive

AML, leukemia cells of the bone marrow were found to coexpress CD117 [11]. Either the overexpression of KIT or the mutation of KIT results in the activation of KIT receptor and downstream signaling proteins. SCF, as a kit ligand, contributes to the proliferation, differentiation, and survival in hematopoietic stem cell via activating KIT receptor and downstream signaling pathways [8, 9, 19, 20]. Ligand-independent activation of the KIT receptor due to KIT mutation was detected in gastrointestinal, stromal tumors, AML, systemic mastocytosis, and germ cell tumors [21–23]. Non-mutated KIT has a low level of autophosphorylation, which is increased by treatment with its ligand, SCF [24]. We investigated the activation of SCF/C-KIT signaling in AML cells in vitro. The results showed that SCF/C-KIT signaling markedly induced the expression of tryptase. A recent study showed that the density of tryptase in SCF-positive specimens was significantly higher than in SCF-negative specimens [25]. In the present study, a distinct upregulation of KIT mRNA was observed in U937 cells in the presence of human SCF. A previous report showed that the expression of CD117 was slightly increased when treated with human SCF [26]. The data also showed that SCF induced antigen/IgE-mediated degranulation of mast cells and the effects disappeared in the absence of KIT expression [26]. These results suggest that the expression of KIT is essential to mediate the effects of SCF.

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Fig. 6 SCF/C-KIT mediated activation of ERK1/2 and p38MAPK in U937 cells. a U937 cells were treated with 100 ng/mL human SCF for 24 h with or without pretreatment with 20 mM PD98059 for 30 min. Total ERK1/2 and p-ERK1/2 levels were detected. (*P = 0.042 vs. control. #P = 0.009 vs. human SCF treatment alone). b U937 cells

were treated with 100 ng/mL human SCF for 24 h with or without pretreatment with 10 mM SB230580 for 30 min. Total p38MAPK and p-p38MAPK levels were detected. The data are expressed as mean values ± SD of three independent experiments. (*P = 0.04 vs. control. #P = 0.032 vs. human SCF treatment alone)

A previous study reported that KIT mRNA was detected in U937 cells, but KIT protein was too low to be detected by Western blot analysis [4]. Although in our study, total and phosphorylated KIT were undetectable in U937 cells using Western blot, CD117 could be measured through flow cytometry. The expression of CD117 and KIT was increased apparently after treatment with human SCF. Moreover, a previous report suggested that bone marrow fibroblasts stimulated by tryptase expressed higher levels of SCF mRNA [27]. Thus, the interactions of tryptase and SCF may be complex, as SCF is regulated by tryptase and SCF/C-KIT signaling modulates tryptase expression in AML cells. Further studies will be necessary to characterize the interactions between tryptase and SCF/C-KIT. Several downstream signaling pathways are involved in SCF/C-KIT signaling. The Ras-Raf-MAP kinase pathway was the first shown to be activated by non-mutated KIT [28, 29]. SCF treatment resulted in the activation of ERK-2 but not ERK-1 or p38MAPK in melanocytes [30]. Furthermore, human SCF induced the tyrosine phosphorylation of Erk1 and Erk2 in both M-07e cells with non-

mutated KIT and kasumi-1 cells with the KIT Asn822Lys mutation [9]. An earlier study showed that the level of phospho-p38 MAPK was significantly increased in SCFtreated cardiac stem cells, while the total protein level of p38 MAPK was not markedly altered [31]. Following binding human SCF and the initial autophosphorylation of KIT, KIT elicits a number of crucial signaling events such as p38MAPK, and ERK1/2 [26]. Our data show that phosphorylations of ERK1/2 and p38MAPK are strongly activated by SCF, suggesting that different downstream signaling is stimulated by SCF/C-KIT in a variety of cells. Notably, we found that the upregulation of tryptase by SCF/C-KIT signaling was significantly reduced when U937 cells were pretreated with PD98059 or SB230580. These data suggest that SCF/C-KIT signaling contributes to the expression of tryptase by the activation of ERK1/2 and p38MAPK pathways. SCF/C-KIT signaling can also activate PI3K/AKT signaling in mouse mast cells [26], which suggests that C-KIT activation is associated with diverse signaling pathways. Mechanisms other than MAPK pathway may be also involved in the regulation of tryptase expression.

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In conclusion, our study found that in vitro treatment with human SCF led to increased expression of tryptase in AML cells, which was mediated by the stimulation of KIT and the activation of ERK1/2 and p38 MAPK. These results indicate that SCF/C-KIT may be a potential therapeutic target for the treatment of AML. Acknowledgments The Ethics Committee of China Medical University approved this study. This study was supported by the Natural Science Foundation of Liaoning Province (20082117) and the Fund Project of Liaoning Province Education Office (2008782). The authors are grateful to all study participants. We also thank Institute of Basic Medical Sciences Chinese Academy of Medical Sciences for providing the U937 cells. Conflict of interest interests.

The authors declare no competing financial

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