Glucose-6-Phosphate Isomerase Promotes the Proliferation And

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Glucose-6-Phosphate Isomerase Promotes the Proliferation And Zong et al. Arthritis Research & Therapy (2015) 17:100 DOI 10.1186/s13075-015-0619-0 RESEARCH ARTICLE Open Access Glucose-6-phosphate isomerase promotes the proliferation and inhibits the apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis Ming Zong, Tianbao Lu, Shasha Fan, Hui Zhang, Ruhan Gong, Lishan Sun, Zhiyan Fu and Lieying Fan* Abstract Introduction: Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to investigate the role of glucose 6-phosphate isomerase (GPI) in the proliferation of RA-FLS. Methods: The distribution of GPI in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical analysis. FLS were isolated and cultured, cellular GPI level was detected by real-time polymerase chain reaction (PCR) and Western blot analysis, and secreted GPI was detected by Western blot and enzyme-linked immunosorbent assay (ELISA). Doxorubicin (Adriamycin, ADR) was used to induce apoptosis. Cell proliferation was determined by MTS assay. Flow cytometry was used to detect cell cycle and apoptosis. Secreted pro-inflammatory cytokines were measured by ELISA. Results: GPI was abundant in RA-FLS and was an autocrine factor of FLS. The proliferation of both RA and OA FLS was increased after GPI overexpression, but was decreased after GPI knockdown. Meanwhile, exogenous GPI stimulated, while GPI antibody inhibited, FLS proliferation. GPI positively regulated its receptor glycoprotein 78 and promoted G1/S phase transition via extracellular regulated protein kinases activation and Cyclin D1 upregulation. GPI inhibited ADR-induced apoptosis accompanied by decreased Fas and increased Survivin in RA FLS. Furthermore, GPI increased the secretion of tumor necrosis factor-α and interleukin-1β by FLS. Conclusions: GPI plays a pathophysiologic role in RA by stimulating the proliferation, inhibiting the apoptosis, and increasing pro-inflammatory cytokine secretion of FLS. Introduction microenvironment through directly producing pro- Rheumatoid arthritis (RA) is a chronic inflammatory inflammatory factors or indirectly activating or recruit- joint disease that eventually leads to the destruction of ing other immune cells [1,2]. Therefore, FLS play a the joint architecture. Synovial hyperplasia is considered critical role in RA pathogenesis, and targeting FLS might a hallmark of RA, in which fibroblast-like synoviocytes improve clinical outcomes of inflammatory arthritis (FLS) and immune cells communicate in a unique in- without suppressing systemic immunity [3]. flammatory microenvironment. The hyperplasia of the Glucose-6-phosphate isomerase (GPI; EC 5.3.1.9), also synovial lining is largely composed of increased numbers known as phosphoglucose isomerase and phosphohexose of FLS and macrophages. Other inflammatory cells, such isomerase, catalyzes the interconversion of D-glucose-6- as mast cells, dendritic cells, macrophages, and lympho- phosphate and D-fructose-6-phosphate, a crucial step in cytes, are also recruited and accumulate in the sub lin- glycolysis and gluconeogenesis [4]. In addition to its en- ing. As they accumulate to form pannus tissue, RA-FLS zymatic activity, GPI acts as a cytokine and growth factor exhibit local tumor-like destructive and invasive charac- in a wide variety of extracellular processes [5-8]. GPI has teristics. Moreover, FLS contribute to the inflammatory been identified as a motility factor: autocrine motility fac- tor (AMF) [5], neuroleukin [7,8] or maturation factors [9]. * Correspondence: [email protected] AMF/GPI is a multifunctional cytokine that exhibits multi- Department of Clinical Laboratory, Shanghai East Hospital, School of functional growth factor-like activity via a unique cognate Medicine, Tong Ji University, 150 Ji Mo Road, Shanghai 200120, People’s Republic of China 78 kDa (glycoprotein 78, gp78) seven-transmembrane © 2015 Zong et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zong et al. Arthritis Research & Therapy (2015) 17:100 Page 2 of 12 glycoprotein receptor (autocrine motility factor receptor, incubated with complete RPMI 1640 medium supple- AMFR)[10].ManystudieshaveshownthatAMFnotonly mented with 10% fetal calf serum in a humidified atmos- stimulates AMF-producing tumor cell motility in an auto- phere containing 5% CO2. The medium was changed every crine manner, but also acts as a paracrine factor for vein three to five days and non-adherent tissue pieces were endothelial cells. AMF induces angiogenesis by stimulating carefully removed. FLS were grown further over four to six cell motility and up-regulating vascular endothelial growth passages. To characterize the cytological phenotype of syn- factor receptor (VEGFR) expression [11]. Overexpression ovial cultures, the third passage cells were stained with of AMF/GPI and AMFR has been found in a wide mouse monoclonal antibodies (mAb) to human fluorescein spectrum of malignancies, and is associated with cancer isothiocyanate (FITC)-CD14 and phycoerythrin (PE)-CD90 progression, metastasis and angiogenesis [12-16]. (eBioscience, San Diego, CA, USA), and showed 2.8% The autoantibodies against glucose-6-phosphate isom- CD14 and 97.0% CD90 expression, as measured by flow erase (anti-GPI Abs) were present in the K/BxN T-cell cytometry. For cell treatment, FLS were treated with hu- receptor (TCR)-transgenic inflammatory arthritis mouse man GPI (Abcam product number: ab87625, endotoxin: model [17]. Furthermore, recombinant human GPI was <1 EU/μg protein, Cambridge, UK). shown to have the ability to induce chronic arthritis in the mice, which was major histocompatibility complex RNA interference and plasmid construction (MHC) associated and B-cell dependent [18]. The total To design specific small interfering RNA (siRNA) targeting GPI protein level, in both enzymatically active and in- GPI and gp78 (named siGPI and sigp78), several sequences active forms, was significantly higher in the sera of RA from the human GPI gene were selected using BLOCK-iT™ patients compared with patients with other immune-based RNAi Designer (Life Technologies, Carlsbad, CA, USA). or non-immune-based inflammatory arthritis [19]. We pre- The target sequences for GPI siRNA (siGPI: 5′-CCATACG viously demonstrated that 76.1% of patients with RA, but GAAGGGTCTGCATCACAATT-3′), gp78 siRNA (sigp78: not controls, had increased concentration of soluble GPI in 5′-GTCG GCACAAGAACTATCTTT-3′) and negative their sera and synovial fluid (SF), and serum GPI concen- control siRNA (si-NC: 5′-TTCTCCGAA-CGTGTCAC tration was higher in active RA patients than in non-active GT-3′) were synthesized by Genepharma Inc (Shanghai, RA patients [20]. However, it remains unclear where exces- China). SiRNA transfection was performed using Lipofec- sive GPI comes from in RA joints, and whether it is associ- tamine® 2000 (Invitrogen, Carlsbad, CA, USA). According ated with joint tissue pathological changes of RA. In this to the sequence of GPI cDNA (NM_001184722.1), we de- study, we aimed to characterize the features of autocrine signed the primers 5′-CGGAATTCCA TGGTAGCTCTC GPIfromRA-FLS,andclarifytheroleofGPIintheregula- TGCAGCCT-3′ (forward) and 5′-CCCTCGAGGTTAT tion of FLS proliferation and apoptosis. TGGACTCTGG CCTCG C-3′ (reverse), and obtained the full length of GPI cDNA by PCR. GPI cDNA was Methods inserted into pcDNA3.1-Flag (Invitrogen) vector and plas- Patients and controls mid DNA transfection was performed using Lipofecta- Synovial tissues were obtained from eight RA patients mine -LTX (Invitrogen). PCI-Neo-gp78/JM20 (plasmid and eight osteoarthritis (OA) patients who underwent 13303) was purchased from Addgen (Teddington, UK). knee arthroscopic or replacement surgery at Shanghai East Hospital. Serum samples were taken before the sur- Immunohistochemical analysis gery from all 16 patients. All the subjects fulfilled the The tissues were fixed in 10% neutral buffered formalin 2010 American College of Rheumatology (ACR) criteria and embedded in paraffin. Next, the tissues were cut for the diagnosis of RA and OA [21]. Informed consent into 5-μm thick sections, deparaffinized and rehydrated. was obtained from all patients and the study protocol The sections were then subjected to hematoxylin and was approved by the Ethics Committee of Shanghai East eosin (H&E) or immunohistochemical staining. For immu- Hospital (2012-df-043). There were no differences in eth- nostaining, the sections were heated at 95°C for 20 minutes nicity among the groups and no patient with RA or OA with Dako Target Retrieval Solution (Dako, Copenhagen, had tumor and renal disease. RA patients were divided Denmark). Sections were incubated with mouse anti-GPI into two groups according to serum GPI level: GPI (+) RA mAb (1:500, Abcam Cambridge, UK) or mouse anti-gp78 and GPI (−) RA, with 0.2 mg/L as the cut-off value. mAb (1:100, Santa-Cruz Dallas, TX, USA) at 4°C overnight, and then incubated with second antibody (Envision™ Isolation and culture of fibroblast-like synoviocytes Detection Kit, Dako) for 30 minutes at room temperature. Synovial tissues were minced into pieces
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