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Introduction

Leukocyte differentiation antigens are a series of glycoproteins and glycolipids attached to or inserted into the membrane of leukocytes, which are the major cells of the immune system and malignant cells that derive from them (1). These antigens are able to transmit membrane signals, regulate cell activation and differentiation as well as mediate numerous interactions between the immune system and antigens, other physiological systems or other components of the immune system (1–3). Numerous leukocyte differentiation antigens were reported to be involved in the pathogenesis and development of hematopoietic malignancies; in addition, certain antigens may be used as markers for diagnosis, classification, risk stratification and eventually as treatment targets in these malignant diseases (4,5).

In previous unpublished experiments conducted in 2008, we aimed to identify novel potential leukocyte differentiation antigens using an integrated bioinformatics analysis at a genome-wide level to select proteins matching the following criteria: 1, No reported functional association with the immune system; 2, predominant expression in cells of the immune system; 3, high expression in certain specific subsets of immune cells; and 4, type I and type II membrane proteins preferred. V-set and transmembrane domain-containing 1 (VSTM1) was identified by the data mining as a candidate gene. VSTM1 was found to be located at human chromosome 19q13.4 and to encode two primary splicing forms, VSTM1-v1 and VSTM1-v2. VSTM1-v1 contains 236 amino acids and is a type I membrane molecule, which has an immunoglobulin (Ig)V-like domain in its extracellular region and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail. It has been suggested that VSTM1-v1 may be a novel ITIM-bearing inhibitory immune receptor involved in the regulation of phagocytes (6). VSTM1-v2 is composed of 205 amino acids and was reported to be a classical secretory glycoprotein, which does not contain a transmembrane domain. It was also reported that recombinant VSTM1-v2 promoted the differentiation and activation of human Th17 cells (7).

Genetic instabilities are widely accepted to be one of the major causes of cancer pathogenesis in humans. Chromosome 19q13 has been reported to be involved in numerous types of cancer, including hematopoietic malignancies, due to its instable genomic region. These instabilities include chromosome translocations, single nucleotide polymorphisms, amplifications, mutations and deletions, which occur frequently in certain types of myeloid and lymphoid leukemia and lymphoma (8–12). Furthermore, epigenetic modifications such as DNA methylation were reported to be involed in the dysregulation of genes located on 19q13.3–4 (13–15).

DNA methylation is an epigenetic code that controls the expression of lineage- and development-specific genes. It has been reported that the aberrant methylation of genes regulating the differentiation of hematopoietic lineages induced leukemic transformations (16–19). The aim of the present study was to examine the expression of VSTM1 in normal human peripheral blood leukocytes and hematopoietic tumor cell lines.

Materials and methods

Isolation of peripheral blood granulocytes, monocytes and lymphocytes

Peripheral blood mononuclear cells (PBMCs) were isolated from granulocytes and red blood cells in the buffy coats of healthy donors [obtained at the Beijing Red Cross Blood Center (Beijing, China) between December 2012 and April 2013] using Ficoll/Hypaque density-gradient centrifugation, as described previously (20), and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Inc, Logan, UT, USA) in petri dishes to enable monocyte adhesion. Non-adherent cells, predominantly peripheral blood lymphocytes (PBLs), and adherent monocytes were collected separately (20). Granulocytes were isolated through lysing red blood cells with a hypotonic buffer as previously described (21). Protocols performed in the present study were approved by the Ethics Committee of Peking University Health Science Center (Bejing, China). Written informed consent was obtained from all healthy donors.

Cell lines

KG-1, HL-60, HuT-78, Jurkat, K562, MOLT-4, Raji, Thp-1, U937 and RPMI 8866 cells, purchased from the American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI 1640 medium supplemented with 10% FCS at 37°C in a humidified atmosphere with 5% CO2. Jurkat cells were treated with 2 μmol/l 5-aza-2′-deoxycytidine (Sigma-Aldrich, St. Louis, MO, USA) for 3 days, and a proportion of these cells were then further treated with 100 nmol/l trichostatin A (Cell Signaling Technology, Inc., Danvers, MA, USA) for 16 h as previously described (22,23). Genomic DNA samples were extracted from Jurkat cells using a ZR Genomic DNA II Kit™ (Zymo Research. Irvine, CA, USA) according to the manufacturer’s instructions. Jurkat cell transfection was performed using electroporation; in brief, 25 μg plasmid was mixed with 2×107 cells in 350 μl serum-free medium and electroporated at a single pulse (135 V; 20 ms) in a 2-mm gap cuvette using an ECM 830 Square Wave Electroporation System (BTX Harvard Apparatus, Holliston, MA, USA).

Semiquantitative reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from the cells using TRIzol reagent (Life Technologies Inc., Carlsbad, CA, USA). Reverse transcription was performed according to standard protocols using the RevertAid™ II First Strand complementary (c)DNA synthesis kit (Fermentas, Waltham, MA, USA). Human Multiple Tissue and Immune System MTC™ panels containing cDNA from a pool of donors were purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA). Semiquantitative PCR was performed as previously described (7). GAPDH was then amplified and used as an internal control. qPCR was performed as previously described (23) using the primers 5′-ACTTGCAGCTGGTGGTCACA-3′ and 5′-CCGGAAGTTTGGAATGGCT-3′. Samples with unspecific amplification according to the dissolving curve and electrophoresis were regarded as zero. All samples were normalized to GAPDH using the comparative Ct method (ΔΔCt).

Cytosine-phosphate-guanine (CpG) island screening, DNA bisulfite treatment and methylation analysis

CpG island searcher (http://cpgislands.usc.edu/) was used to screen for CpG islands in the promoter region of VSTM1. Based on the search results, primers for the detection of methylated or unmethylated alleles of the VSTM1 promoter and for bisulfite genomic sequencing (BGS) were designed. Bisulfite modification of DNA, methylation-specific PCR (MSP) and BGS were then performed as previously described (22,23). The bisulfite-treated DNA was amplified using the methylation-specific primer set m3, 5′-TTGTATATTTTGGGGACGAATC-3′ and m4, 5′-AAAAAAAATTCTACGATCATAACG-3′ or the unmethylation-specific primer set u3, 5′-TTTGTATAT TTTGGGGATGAATT-3′ and u4, 5′-AAAAAAAAA TTCTACAATCATAACA-3′ (Beijing AuGCT DNA-SYN Biotechnology Co Ltd., Beijing, China). MSP was performed for 40 cycles using Ampli-Taq Gold (Perkin Elmer Applied Systems, Foster City, CA, USA) and hot start. MSP primers were tested in order to confirm that they did not amplify unbisulfited DNA; in addition, the specificity of MSP was confirmed by direct sequencing of PCR products. For BGS, bisulfite-treated DNA was amplified using the following primers: b1, 5′-AGAGTGGGGTAGAGAATTAGAGTGTTA-3′ and b2, 5′-ACCTAAACTACCACTCCCACATAAAT-3′. The PCR products were cloned into the pGEM-T easy vector (Promega Corp., Maddison, WI, USA) and sequenced using ten randomly selected colonies.

Flow cytometric analysis

Cells were blocked using fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) containing 2% FCS] on ice for 30 min. A total of 1×106 cells were then incubated on ice for 1 h with rabbit anti-VSTM1 polyclonal antibody (prepared by authors as previously described; 2 μg/sample in 100 μl FACS buffer) (24). Cells were then washed in PBS and stained with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Ig; dilution, 1:100 in FACS buffer; Biolegend, Inc., San Diego, CA, USA) in the dark at 4°C for 30 min. Cells were susequently washed in PBS and analyzed using a BD FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

Protein extraction and western blot analysis

Cells were harvested and lysed using 20 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton X-100, and 1% protease inhibitor cocktail (Pierce, Rockford, IL, USA). Proteins were applied to 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Hybond® Inc., Escondido, CA, USA). VSTM1 was detected using rabbit anti-VSTM1 polyclonal antibodies (pAb; 2 μg/ml) and secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (dilution, 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA). Signals were visualized using enhanced chemiluminescence western blot detection reagents (GE Healthcare, Little Chalfont, UK) on an ImageQuant LAS 500 imaging device (GE Healthcare). β-actin was detected using mouse anti-β-actin monoclonal antibody (mAb; dilution, 1:3,000; Clone AC-74; Sigma-Aldrich Shanghai Trading Co., Ltd., Shanghai, China) and used as an internal control to determine lysate input.

Capacity of rabbit anti-VSTM1 polyclonal antibody (pAb) to cross-link VSTM1-v1

In order to analyze whether the rabbit anti-VSTM1 pAb influenced the functional cross-linking of VSTM1, a reporter construct (pcDB-VSTM1-v1-CD3ζ) was generated that expressed a chimeric protein consisting of the extracellular domain of VSTM1-v1 as well as the transmembrane and intracellular domains of CD3ζ through overlapping PCRs as previously described (25). Dual-luciferase reporter assays were then performed. A total of 2×107 Jurkat cells were cotransfected with 20 μg pcDB-VSTM1-v1-CD3ζ or empty vector, 8 μg p-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-Luc plasmid (a construct encoding the firefly luciferase reporter gene under the influence of NFκB binding) and 0.4 μg p-Renilla luciferase (RL)-thymadine kinase (TK) plasmid (a construct encoding the RL gene; Promega Corporation, Madison, WI, USA) as the internal control. 12 h post-transfection, cells were stimulated with anti-CD28 (2 μg/ml; clone 15E8) in combination with different concentrations (1, 3 or 5 μg/ml) of rabbit anti-VSTM1 pAb or normal rabbit IgG (5 μg/ml) (BD Biosciences, San Jose, CA, USA). Plate-bound anti-CD3 (1 μg/ml; clone OKT3) and tumor necrosis factor-α (20 ng/ml; eBioscience, San Diego, CA, USA) were used as positive controls. Following 20 h of stimulation, cells were collected and lysed using Passive Lysis Buffer (Promega Corp.). Firefly and RL activities were measured with the dual-luciferase reporter (DLR) assay system (Promega Corp.) according to the manufacturer’s instructions using a Veritas™ Microplated Luminometer (Promega Corp.).

Cell proliferation assay

Jurkat cells transfected with pCDNA3.1/Myc-His(-)B-VSTM1-v1 (pcDB-VSTM1-v1), or pCDNA3.1/Myc-His(-)B (pcDB) vector plasmids were seeded in 96-well plates at a density of 20,000 per well in the presence or absence of 5 μg/ml anti-VSTM1 pAb or normal rabbit IgG and then incubated at 37°C in a humidified atmosphere with 5% CO2. Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MA, USA). At 0, 24, 48 and 72 h post-transfection, 10 μl CCK-8 solution was added into each well and incubated for 2 h. Absorbance was measured with a Thermo Scientific Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 450 nm in order to assess the number of viable cells (background absorbance was measured at 630 nm). Results were obtained from at least three independent experiments.

Statistical analysis

Values are presented as the mean ± standard error of the mean/standard deviation. Statistical analysis was performed using the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference between values.

Results

VSTM1 is expressed in peripheral blood leukocytes

A previous study reported that VSTM1 expression was higher in cells of the immune system (7). In the present study qPCR was performed in order to quantify the expression levels of VSTM1. As shown in Fig. 1A, VSTM1 was primarily expressed in immune tissues and cells, including bone marrow and peripheral blood leukocytes. In order to further investigate the expression profile of VSTM1 in leukocytes, RT-qPCR was performed on PBMCs and granulocytes of ten healthy donors. The results showed that VSTM1 was detected in each of the samples (Fig. 1B), and according to the molecular size of the spliced variants, it was found that VSTM1-v1 was the predominantly expressed form. PBLs were then separated from monocytes of the PBMCs in samples from a further three individuals (numbered 1–3) and qPCR was performed in order to quantify the expression levels of VSTM1 in each cell population. As shown in Fig. 1C, VSTM1 was highly expressed in granulocytes of each sample; however, the expression levels of VSTM1 in monocytes and PBL were variable. These results were not consistent with a previous study by Steevels et al (6), in which the monoclonal antibody 1A5 was used to detect VSTM1 expression. This previous study reported that VSTM1 was expressed exclusively by myeloid cells and was absent in lymphoid cells. In order to confirm the existence of VSTM1 in PBLs in the present study, flow cytometric and western blot analyses were performed at the protein level with samples from a further nine donors (numbered 4–12). These results revealed a considerable individual difference in expression levels of VSTM1 among donors. Flow cytometric analysis demonstrated that the proportion of surface VSTM1-v1-positive cells in PBL varied from 4.05 to 50.05% (Fig. 1D). The representative data from three donors (4, 6 and 11) are shown in Fig. 1E and F. In samples 4 and 11, surface VSTM1-v1 expression was detected with the positive percentages of 50.05% and 21.72% respectively; however, in sample 6, VSTM1-v1 was detected in markedly fewer cells (6.78%) (Fig. 1E). Consistent results were obtained from representative western blots, which demonstrated a ~60-kDa band of VSTM1 in PBL of donors 4 and 11, which was, however, not observed in sample 6. This band was identical to that of the band observed in VSTM1 overexpressing Jurkat cells; however, the pattern of bands (~55, ~40 and ~37 kDa) in granulocytes was markedly different from that in PBLs. In addition, these results were not comparable to those of a previous study in VSTM1-overexpressing HEK293T cells, which reported bands at ~55 and ~45 kDa (21). This therefore indicated that VSTM1 was diversely modified in cells of different origin.

Figure 1 Expression profile of VSTM1. (A) qPCR analysis of VSTM1 expression in numerous tissue type using human MTC™ panels (I and II; nos. 636742 and 636743, respectively) and human immune system MTC™ panel (III; no. 636748). Expression levels of VSTM1 were measured relative to levels in the kidneys. (B) RT-qPCR of VSTM1 in peripheral blood mononuclear cells and granulocytes from ten healthy donors. GAPDH was used as the internal control. (C) VSTM1 expression in granulocytes, monocytes and PBLs from three individuals (1–3) was analyzed using qPCR. GAPDH was used as the internal control and the expression levels of VSTM1 were measured relative to those of PBLs from donor two. (D) Flow cytometric analysis of surface VSTM1-v1 expression in PBLs from nine donors (4–12). Quadrants were set relative to RbIgG stainings and positive percentages of VSTM1-v1-expressing cells were counted. (E) Flow cytometric analysis of PBLs of three donors (4, 6 and 11). Images are representative results of the nine independent experiments shown in (D). (F) Western blot analysis of VSTM1-v1 expression in PBLs from donors 4,6 and 11. VSTM1-v1-transfected Jurkat and HEK293T cells and granulocytes were examined as positive controls. Loading volumes of lysates were: PBL, 150 μg; granulocyte, 150 μg; Jurkat cells, 100 μg; and HEK293T, 1 μg. RbIgG was used as the control antibody and β-actin was used as the internal control. Arrows indicate specific bands of VSTM1. Values are presented as the mean ± standard deviation. VSTM1, V-set and transmembrane domain-containing 1; qPCR, quantitative polymerase chain reaction; MTC, mutiple tissue complementary DNA; RT-qPCR, reverse transcription-qPCR; VSTM1-v1, VSTM1 spliced variant 1; PBL, peripheral blood lymphocyte; HEK293T, human embryonic kidney 293T; RbIgG, normal rabbit immunoglobulin G.

VSTM1 is silenced following promoter methylation in numerous hematopoietic tumor cell lines

VSTM1 was found to be broadly expressed in normal human PBLs; therefore, RT-qPCR was performed in order to further investigate whether VSTM1 was also expressed in hematopoietic tumor cell lines. The results showed that VSTM1 was silenced in the majority of the examined cell lines (Fig. 2A). The aberrant promoter CpG methylation was previously reported to be associated with gene silencing (16); therefore, in the present study, the VSTM1 promoter was screened for CpG islands using the CpG Island Searcher. The results indicated that the VSTM1 promoter contained a typical CpG island (Fig. 2B). VSTM1 promoter methylation was then analyzed using MSP and it was demonstrated that the promoters of VSTM1-silenced cell lines were methylated to varying degrees (Fig. 2C). This therefore indicated that there was an association between the loss of VSTM1 and CpG hypermethylation in the promoter region.

Figure 2 VSTM1 is silenced in numerous hematopoietic tumor cell lines with promoter methylation. (A) RT-PCR of VSTM1 in hematopoietic tumor cell lines. PBMCs were used as a positive control. (B) A CpG island covers the promoter and exon 1 of VSTM1. Horizontal bars, CpG sites; primers for methylation analysis, MSP primers (m3/u3 and m4/u4) and bisulfite genomic sequencing primers (b1 and b2) are indicated; curved arrow, transcription start site. (C) MSP analysis of VSTM1 showed complete methylation of the sites where the MSP primers were located in Jurkat, K562, MOLT4, Raji and U937 cells and partial methylation in HL60 and RPMI 8866 cells, as well as in PBMCs. M, methylated; U, unmethylated; VSTM1, V-set and transmembrane domain-containing 1; RT-PCR, reverse transcription polymerase chain reaction; PBMC, peripheral blood mononuclear cell; MSP, methylation-specific PCR; CpG, cytosine-phosphate-guanine.

Expression of VSTM1 was restored following pharmacological demethylation

Promoter hypermethylation was performed in Jurkat cells in order to silence VSTM1; cells were then treated with the methyltransferase inhibitor 5-aza-2′-deoxycytidine alone or combination with trichostatin A, a histone deacetylase inhibitor. This treatment was shown to restore mRNA and protein expression levels of VSTM1 (Fig. 3A and B); in addition, MSP analysis demonstrated that this treatment resulted in promoter demethylation (Fig. 3C) and BGS revealed specific demethylation of individual CpG sites (Fig. 3D). These results indicated that VSTM1 silencing in Jurkat cells was due to epigenetic regulation.

Figure 3 Pharmacological demethylation restores VSTM1 expression in Jurkat cells. (A) Semiquantitative RT-PCR and qPCR analyses using WT Jurkat cells and Jurkat cells treated with the methyltransferase inhibitor A alone or in combination with the histone deacetylase inhibitor T. GAPDH was used as an internal control. (B) Flow cytometric analysis confirmed the restoration of surface VSTM1-v1 expression in Jurkat cells following pharmacological demethylation. Representative results and the mean fluorescence intensity of VSTM1 expression for three independent experiments. Normal rabbit immunoglobulin G was used for control staining. Values are presented as the mean ± standard error of the mean. *P<0.05 vs. WT cells. (C) MSP verified the pharmacological demethylation of the VSTM1 promoter. (D) Detailed bisulfite genomic sequencing analysis of the VSTM1 promoter. Circles, CpG sites analyzed; row of circles, an individual promoter allele that was cloned, randomly selected and sequenced; filled circle, methylated CpG site; open circle, unmethylated CpG site. A, 5-aza-2′-deoxycytidine; T, trichostatin A; WT, wild-type; VSTM1, V-set and transmembrane domain-containing 1; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative PCR; VSTM1-v1, VSTM1 spliced variant 1; MSP, methylation-specific PCR; CpG, cytosine-phosphate-guanine.

Overexpression of VSTM1-v1 inhibits Jurkat cell growth

Results of the present study showed that VSTM1 was expressed in normal human leukocytes and silenced in hematopoietic tumor cells; therefore, subsequent experiments were performed in order to determine effects of exogenous VSTM1-v1 expression on the growth of Jurkat cells following VSTM1 promoter hypermethylation and gene silencing. As shown in Fig. 1E, VSTM1-v1 was successfully expressed in Jurkat cells following transfection. CCK-8 assays revealed that VSTM1-v1 inhibited the growth of Jurkat cells compared with that of vector-transfected control cells (Fig. 4A). VSTM1-v1 is a type I transmembrane protein, which contains two cytoplasmic ITIMs. Therefore, it was hypothesized that ligand interaction with the extracellular region may induce the transmission of an inhibitory signal into the cell; however, ligands for VSTM1-v1 have not yet been identified. Therefore, in the present study, the VSTM1 antibody was used as an agonist to bind to the extracellular region of VSTM1. The influence of rabbit anti-VSTM1 pAb on the functional cross-linking of VSTM1 was determined using NFκB-firefly luciferase reporter assays in Jurkat cells with the construct expressing the VSTM1-v1-CD3ζ chimera (25). Incubation with the anti-VSTM1 pAb instead of normal rabbit IgG and in the presence of anti-CD28 resulted in NFκB activation and consequently increased firefly luciferase activity. In addition, firefly luciferase activity was enhanced following increased doses of the anti-VSTM1 pAb (Fig. 4B), which indicated that this antibody was capable of cross-linking VSTM1-v1. VSTM1-v1- or vector-transfected Jurkat cells were then treated with the anti-VSTM1 pAb or normal rabbit IgG (Fig. 4C). A CCK8 assay showed that incubation with the anti-VSTM1 pAb further augmented the inhibitory effect of VSTM1-v1 on Jurkat cell growth.

Figure 4 Overexpression of VSTM1-v1 inhibits Jurkat cell growth. (A) Representative results of ≥five independent CCK-8 assays in WT, pcDB- and VSTM1-v1-transfected cells. Values are presented as the difference between absorbance at 450 and 630 nm (OD450-OD630). (B) NFκB-firefly luciferase reporter assays in Jurkat cells demonstrated the capacity of rabbit anti-VSTM1 pAb to cross-link VSTM1-v1. Representative results from three individual experiments are shown. Relative NFκB activity was the mean of the ratio of firefly to renilla luciferase activities ± SD. TNF-α and plate-bound anti-CD3 were used as positive controls for activating NFκB. (C) CCK-8 assays of VSTM1-v1- and pcDB-transfected Jurkat cells following interaction with anti-VSTM1 pAb. RbIgG control was used as the internal control. Anti-VSTM1 pAb was shown to further inhibit the growth of VSTM1-v1-transfected cells. Representative result of three independent experiments. Values are presented as the mean ± SD. *P<0.05 vs. controls at each time-point. VSTM1, V-set and transmembrane domain-containing 1; VSTM1-v1, VSTM1 spliced variant 1; CCK-8, cell counting kit 8; WT, wild-type; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; pAb, polyclonal antibody; TNF-α, tumor necrosis factor-α; pcDB, pcDB-VSTM1-v1-CD3ζ reporter construct; RbIgG, normal rabbit immunoglobulin G; SD, standard deviation.

Discussion

VSTM1 is a potential leukocyte differentiation antigen gene that was previously identified using immunogenomics. VSTM1 encodes numerous splicing isoforms, of which VSTM1-v1 is the most predominant and widely expressed form (7). VSTM1-v1 is a type I transmembrane molecule that contains an extracellular IgV-like domain and two ITIMs in its cytoplasmic region. VSTM1 was previously reported to be expressed exclusively in myeloid cells and absent in lymphoid cells as indicated by detection using the monoclonal antibody 1A5 (6). However, in the present study, VSTM1 mRNA expression was observed not only in granulocytes and monocytes but also in PBLs from several healthy donors using RT-qPCR; in addition, there were considerable differences in VSTM1 expression levels among individuals. Flow cytometric analysis using rabbit anti-VSTM1 pAb confirmed the expression patterns at the protein level. However, the results of the western blot analysis may provide a possible explanation for the contradictory results from the present and previous study about the expression of VSTM1 in PBLs. The present study determined that the molecular size of VSTM1 detected in PBLs was consistent with that of the overexpressed VSTM1-v1 in Jurkat cells, which was different from that of endogenous VSTM1 in granulocytes and overexpressed VSTM1-v1 in HEK293T cells (21). This indicated that VSTM1 may be diversely modified in cells of different origins, with different molecular sizes and conformations; therefore, the application of monoclonal antibodies for the detection of this molecule is likely to be limited as a monoclonal antibody only recognizes a single epitope of a molecule (26).

VSTM1 is broadly expressed in normal human PBLs; however, it was found to be silenced in multiple leukemia cell lines. VSTM1 is located on chromosome 19q13.4, a genomic region widely reported to be prone to genetic and epigenetic modifications in numerous hematopoietic malignancies (8–12). In the present study, a bioinformatic search tool was used, which indicated that a typical CpG island was present in the promoter of VSTM1. RT-PCR and MSP analyses revealed that there was a link between the expression of silenced VSTM1 in hematopoietic tumor cell lines and CpG hypermethylation in the promoter region. This therefore confirmed that epigenetic modifications were an important mechanism in the regulation of VSTM1, which may be of use as a promising novel diagnostic and prognostic genetic marker for hematopoietic malignancies (27). However, the involvement of genetic alterations in this process remains to be elucidated.

Previous studies have identified numerous inhibitory receptors containing ITIMs, which prevent excessive immune reactions and autoimmunity, thereby maintaining the balance of the immune system (28–30). These studies may therefore indicate the function of the two ITIMs in the cytoplasmic tail of VSTM1-v1. Following interaction with the extracellular portion of the receptor by ligands, cytoplasmic ITIMs bind to the sarcoma homology 2 domain of phosphatases (SHP), resulting in the inactivation of different kinases and downregulation of cell activation (28–30). ITIMs in VSTM1-v1 were also reported to recruit SHP-1 and SHP-2, which inhibited the fragment, crystallizable epsilon receptor I-mediated signaling involved in reactive oxygen species production and the microbicidal activity of phagocytes (6,31). Further studies are required to determine whether the inhibitory effect of VSTM1-v1 on cell growth may also be achieved via SHP recruitment through its cytoplasmic ITIMs.

In conclusion, the results of the present study demonstrated that VSTM1 was expressed in myeloid cells as well as lymphocytes, which were of different molecular size and exhibited considerable variation in their expression levels among donors. VSTM1 was found to be silenced in numerous hematopoietic malignancy cell lines, with CpG hypermethylation within the promoter; however, pharmacological demethylation in Jurkat cells was shown to be able to restore VSTM1 expression. In addition, overexpression of VSTM1-v1 in Jurkat cells inhibited cell growth, which was further restricted following the interaction with the extracellular portion of VSTM1-v1 using anti-VSTM1 antibodies. These results therefore elucidated the expression regulation and functional roles of VSTM1 in the inhibition of hematopoietic tumor cells, which may contribute towards the development of novel diagnostic and treatment strategies for hematopoietic malignancies.

Acknowledgements

The present study was supported by grants from the Specialized Research Fund for the Doctoral Program of Higher Education of China (no. 20110001110016) and the National Natural Science Foundation of China (no. 31400736).

References