TMEM4 Is Highly Expressed in Plasma Cell Neoplasms And
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e sord rs & i Im Chen-Feng et al., Immunol Disord Immunother 2016,1:1 l D a m c u i n g o o l t h o e n Immunological Disorders r u a p m y m I and Immunotherapy ResearchResearch Article Article OpenOpen Access Access TMEM4 is Highly Expressed in Plasma Cell Neoplasms and Associated with B Lymphocyte Differentiation Chen-Feng Qi1*, Shao Xiang1,2, Guifang Ouyang3, Xing-pei Hao1, Min Sun Shin1, Alexander L Kovalchuk1, Peng Liang4, Huabao Xiong4, Herbert C Morse III1, Zhengping Zhuang5, Jeff X Zhou3 and Susan K Pierce1 1Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, USA 2Taussig Cancer Institute, Cleveland Clinic Foundation, Cleveland, USA 3Ningbo University School of Medicine, Ningbo, China 4Immunology Institute, Mount Sinai School of Medicine, New York, USA 5Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institutes of Cancer, National Institutes of Health, Bethesda, USA Abstract Aiming to identify biomarkers for plasma cell neoplasms, we analyzed gene expression profile and proteomic characteristics of mouse models of plasmacytomas along with other types of B-cell neoplasms. We found that transmembrane protein 4 (Tmem4) was highly expressed in plasmacytomas in comparison with early-stage B-cell neoplasms. The serum levels of Tmem4 were also significantly greater in mice with plasmacytoma comparing to those with early-stage B-cell neoplasms (P<0.01). Mechanistically, increased Tmem4 expression in B cells led to increased levels of IL-10, a cytokine that promotes the growth of malignant plasma cells and participates in the terminal differentiation of B cells into plasma cells. In addition, over-expression of Tmem4 promoted B cell terminal differentiation as evidenced by the increase in expression of XBP1, CD38, and CD138. Furthermore, we found that TMEM4 was highly expressed in plasma cells of multiple myeloma patients. These findings suggested that Tmem4 plays an important role in plasma cell differentiation and has a potential to serve as a biomarker for plasma cell neoplasms. Keywords: Neoplasms; Plasma cell; TMEM4; CNPY2; Lymphoma phosphorylation of myosin regulatory light chain (MRLC) in neuron cells [6]. Recently, it has been reported that Tmem4 was a target protein Introduction of the Wnt/GSK3 signaling pathway. Wnt increased the intracellular concentration Tmem4, which was likely through its inhibitory effect Plasma cells, as the terminally differentiated form of B lymphocytes, on GSK3-mediated proteasome degradation, thus stabilizing Tmem4 play a key role in humoral immunity by producing and secreting [7]. Several proteomic profiling studies revealed that Tmem4 was one immunoglobulin (Ig). The development of plasma cells from mature of the prominent proteins expressed in stem cells including mouse B cells is precisely regulated, with a distinct gene expression program consisting of up- and down-regulated expression of genes. The unique embryonic stem cells [8], mouse spleen stem cells [9], and human gene expression program determines the differentiation and proper embryonic stem cells [10]. Furthermore, it was shown that stem-cell function of plasma cells. For instance, during plasma cell differentiation, factors OCT4 and NANOG regulated the expression of TMEM4 via the levels of transcription factor B lymphocyte-induced maturation undetermined mechanisms [11]. Most recently, it was reported that protein 1 (BLIMP1) increase [1]. BLIMP1 inhibits the expression of TMEM4 mediated the regulatory effect of fibroblast growth factor-21 genes required for B cell receptor signaling and germinal center (GC) (FGF21) on low-density lipoprotein receptor (LDLR) [12]. However, B cell function, while it promotes the expression of genes important for the role of TMEM4 in B cell function remains unclear. plasma cell function such as XBP1 [1]. XBP1 encodes X-box binding In this study, we report that Tmem4 was highly expressed in mouse protein 1 (XBP1), a highly expressed transcription factor in plasma plasma cells and plasma cell neoplasms, and increasing levels of Tmem4 cells. XBP1 plays a key role in the unfolded protein response which is drove mature B cell differentiation into plasma cells and enhanced cell critical to the process of Ig production, thus maintaining the survival motility. and function of plasma cells [2,3]. Like other lymphoid neoplasms, the uniquely expressed molecules Methods in plasma cells could serve as biomarkers for plasma cell neoplasms such Animal model as multiple myeloma (MM). MM is an incurable disease largely due to the difficulty in early detection and ineffectiveness of treatment. Recent Plasmacytoma was induced in BALB/c mice by intraperitoneal studies showed that XBP1 could be used as a predictive biomarker for the efficacy of thalidomide-based treatment for MM [4]. Nevertheless, there is still a strong need for additional biomarkers for disease *Corresponding author: Chen-Feng Qi, Laboratory of Immunogenetics, National stratification, drug efficacy prediction, prognoses, and druggable targets Institute of Allergy and Infectious Diseases, National Institutes of Health, 5640 for plasma cell malignancy. Fishers Lane, Rockville, MD, 20852, USA, Tel: (301)761-3986; Fax: (301)402- 0077; E-mail: [email protected] In our effort to identify novel molecules specific for normal and malignant plasma cells, we took advantage of our mouse models which Received: January 14, 2016; Accepted: January 29, 2016; Published: February 01, 2016 develop a variety of B-cell lineage neoplasms [5]. By comparing the gene expression profile between plasmacytomas and neoplasms derived from Citation: Qi C, Xiang S, Ouyang G, Hao X, Shin MS, et al. (2016) TMEM4 is earlier-stage B cells, we found that Tmem4 expression was significantly Highly Expressed in Plasma Cell Neoplasms and Associated with B Lymphocyte Differentiation. Immunol Disord Immunother 1: 102. increased in plasmacytomas. Tmem4 encodes transmembrane protein 4 (Tmem4), also called MIR-interacting saposin-like protein (Msap) Copyright: © 2016 Qi C, et al. This is an open-access article distributed under the or canopy FGF signaling regulator 2 (CNPY2). Studies showed that terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and TMEM4 enhanced cell spreading and motility through increasing the source are credited. Immunol Disord Immunother Volume 1 • Issue 1 • 1000102 Citation: Qi C, Xiang S, Ouyang G, Hao X, Shin MS, et al. (2016) TMEM4 is Highly Expressed in Plasma Cell Neoplasms and Associated with B Lymphocyte Differentiation. Immunol Disord Immunother 1: 102. Page 2 of 6 injection of pristane which caused chronic granulomatous peritonitis, ampholytes), IPG buffer, and bromophenol blue, and subsequently as described previously [13]. The tumors developed in granulomas rehydrated overnight with Immobiline Drystrips (pH 4/7, 11cm) and were dependent on IL-6. In majority of cases, the tumors had on a Reswelling Tray (all from Amersham Biosciences, NJ, USA). spontaneous IgH-Myc translocation t(12;15) involving Myc on The first dimension electrophoresis of 2DGE was performed using a chromosome 15 and IgH on chromosome 12 [13]. All animal studies Multiphore II Electrophoresis System, and the second dimension was were performed under NIAID IACUC approved protocol LIP6. performed using precast ExcelGel SDS gels on a Multiphor II Flated System (Amersham Biosciences, NJ, USA). Protein gels were stained Human bone marrow biopsy using a silver staining kit (Amersham Biosciences). The protein Bone marrow was aspirated from patients with MM or idiopathic spots were detected, quantified, and matched using Proteomweaver thrombocytopenic purpura (ITP) during diagnosis process. The software (Definiens, Munich, Germany). Protein spots of interest were protocol was approved by the Ethics Committee of the First Municipal excised from the gel and subjected to in-gel digestion. Peptides from Hospital of Ningbo. Written informed consent was obtained from each in-gel digests were analyzed using a ProteomeX LC-MS/MS system patient. (ThermoElectron, San Jose, CA, USA). MS/MS spectra were searched against database utilizing the BioWorks and SEQUEST programs Cell culture and gene transfection (Thermo Fisher, Waltham, MA, USA). A protein ID was assigned when Murine B-cell lymphoma cell lines were created from the mouse MS/MS spectra of at least two peptides from the same protein exhibited models as previously described [14]. The B-cell leukemia line BCL1- at a minimum the default Xcorr vs. charge values set by the program. 3B3 was obtained from American Type Culture Collection (ATCC, VA, Western blot analysis USA) and the plasmacytoma cell line PCT-AP was kindly provided by Dr. M. Potter (National Cancer Institute, NIH, Bethesda, MD, USA). Selective tissue dissection was performed prior to protein analysis Mouse plasma cells were prepared from the spleen using magnetic beads to avoid procurement of normal tissue or areas of necrosis, hemorrhage, conjugated with CD138 antibody (Miltenyi Biotec, CA, USA). The cells or inflammation, as previously described[13] . Tissues for western blot were maintained at 37°C with 5% CO2 in RPMI 1640 (Invitrogen Life analysis were homogenized in T-PER tissue protein extraction reagent Technologies, CA, USA) with 10% fetal bovine serum (FBS) (Hyclone, (Pierce, IL, USA) with protease inhibitor cocktail (Roche Molecular IL), 10 mM sodium pyruvate, 2 mM glutamine, 1x