Nan Mu

A novel NF-κB/YY1/microRNA-10a regulatory circuit in fibroblast-like synoviocytes regulates inflammation in rheumatoid arthritis

The main etiopathogenesis of rheumatoid arthritis (RA) is overexpressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target gene expression and play important roles in various diseases. Here, we explored the mechanisms of microRNAs in RA. We found that microRNA (miR)-10a was downregulated in the fibroblast-like synoviocytes (FLSs) of RA patients compared with osteoarthritis (OA) controls, and this downregulation could be triggered by TNF-α and IL-1β in an NF-κB-dependent manner through promoting the expression of the YingYang 1 (YY1) transcription factor. Downregulated miR-10a could accelerate IκB degradation and NF-κB activation by targeting IRAK4, TAK1 and BTRC. This miR-10a-mediated NF-κB activation then significantly promoted the production of various inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, and MCP-1, and matrix metalloproteinase (MMP)-1 and MMP-13. In addition, transfection of a miR-10a inhibitor accelerated the proliferation and migration of FLSs. Collectively, our data demonstrates the existence of a novel NF-κB/YY1/miR-10a/NF-κB regulatory circuit that promotes the excessive secretion of NF-κB-mediated inflammatory cytokines and the proliferation and migration of RA FLSs. Thus, miR-10a acts as a switch to control this regulatory circuit and may serve as a diagnostic and therapeutic target for RA treatment.

Body protective compound-157 enhances alkali-burn wound healing in vivo and promotes proliferation, migration, and angiogenesis in vitro

Chemical burns take up a high proportion of burns admissions and can penetrate deep into tissues. Various reagents have been applied in the treatment of skin chemical burns; however, no optimal reagent for skin chemical burns currently exists. The present study investigated the effect of topical body protective compound (BPC)-157 treatment on skin wound healing, using an alkali burn rat model. Topical treatment with BPC-157 was shown to accelerate wound closure following an alkali burn. Histological examination of skin sections with hematoxylin–eosin and Masson staining showed better granulation tissue formation, reepithelialization, dermal remodeling, and a higher extent of collagen deposition when compared to the model control group on the 18th day postwounding. BPC-157 could promote vascular endothelial growth factor expression in wounded skin tissues. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell cycle analysis demonstrated that BPC-157 enhanced the proliferation of human umbilical vein endothelial cells (HUVECs). Transwell assay and wound healing assay showed that BPC-157 significantly promoted migration of HUVECs. We also observed that BPC-157 upregulated the expression of VEGF-a and accelerated vascular tube formation in vitro. Moreover, further studies suggested that BPC-157 regulated the phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as its downstream targets, including c-Fos, c-Jun, and Egr-1, which are key molecules involved in cell growth, migration, and angiogenesis. Altogether, our results indicated that BPC-157 treatment may accelerate wound healing in a model of alkali burn-induced skin injury. The therapeutic mechanism may be associated with accelerated granulation tissue formation, reepithelialization, dermal remodeling, and collagen deposition through ERK1/2 signaling pathway.

The discoidin domain receptor 2/annexin A2/matrix metalloproteinase 13 loop promotes joint destruction in arthritis through promoting migration and invasion of fibroblast-like synoviocytes.

Discoidin domain receptor 2 (DDR‐2)/matrix metalloproteinase (MMP) signaling is an important pathway involved in cartilage destruction in rheumatoid arthritis (RA). However, the molecular mechanisms of this pathway have not been clearly identified. This study was undertaken to screen key molecules involved in this pathway and evaluate their biologic functions in synovium invasion of RA.

Therapeutic Effects of PADRE-BAFF Autovaccine on Rat Adjuvant Arthritis

B cell activating factor (BAFF) is a cytokine of tumor necrosis factor family mainly produced by monocytes and dendritic cells. BAFF can regulate the proliferation, differentiation, and survival of B lymphocytes by binding with BAFF-R on B cell membrane. Accumulating evidences showed that BAFF played crucial roles and was overexpressed in various autoimmune diseases such as systemic lupus erythematous (SLE) and rheumatoid arthritis (RA). This suggests that BAFF may be a therapeutic target for these diseases. In the present study, we developed a BAFF therapeutic vaccine by coupling a T helper cell epitope AKFVAAWTLKAA (PADRE) to the N terminus of BAFF extracellular domains (PADRE-BAFF) and expressed this fusion protein in Escherichia coli. The purified vaccine can induce high titer of neutralizing BAFF antibodies and ameliorate the syndrome of complete Freunds adjuvant (CFA) induced rheumatoid arthritis in rats. Our data indicated that the BAFF autovaccine may be a useful candidate for the treatment of some autoimmune diseases associated with high level of BAFF.

DDR2–CYR61–MMP1 Signaling Pathway Promotes Bone Erosion in Rheumatoid Arthritis through Regulating Migration and Invasion of Fibroblast‐Like Synoviocytes

Regulation of matrix metalloproteinases (MMPs) by collagen in the fibroblast‐like synoviocytes (FLSs) plays a critical role in joint destruction in rheumatoid arthritis (RA). Our previous study indicated that discoidin receptor 2 (DDR2) mediated collagen upregulation of MMPs. However, the precise underlying mechanism remains unclear. We report here that CYR61, a secreted, extracellular matrix–associated signaling protein which is capable of regulating a broad range of cellular activities, including cell adhesion, migration, proliferation, and apoptosis, is significantly upregulated in collagen II–stimulated RA FLS. Further studies found that collagen II–activated phosphorylated‐DDR2 induces CYR61 through activation of transcription factor activator protein 1 (AP‐1). The elevated CYR61, in turn, accelerates MMP1 production via ETS1 (ETS proto‐oncogene 1). In addition, CYR61 significantly promotes FLS invasion and migration. Blockade of CYR61 by an adenovirus expressing CYR61 shRNA (Ad‐shCYR61) in vivo remarkably ameliorated the severity of arthritis, reduced inflammatory cytokine secretion, and attenuated bone erosion as detected by micro–computed tomography (μCT), in collagen‐induced arthritis (CIA) rats. Taken together, we uncovered the Collagen II–DDR2–AP‐1–CYR61–ETS1–MMP1 loop in RA FLS. In which, CYR61 acts as a hinge to promote cartilage damage through regulating FLS invasion, migration, and MMP1 production and the inflammatory cascade in RA. Thus, CYR61 may be a promising diagnostic and therapeutic target for RA treatment.

A DPP-IV-resistant glucagon-like peptide-2 dimer with enhanced activity against radiation-induced intestinal injury

Abstract Although radiotherapy is a highly effective treatment for abdominal or pelvic cancer patients, it can increase the incidence of severe gastrointestinal (GI) toxicity. As an intestinal growth factor, glucagon‐like peptide 2 (GLP‐2) has been shown to improve the preclinical models of both short bowel syndrome and inflammatory bowel disease by stimulating intestinal growth. Teduglutide ([Gly2]GLP‐2), a recombinant human GLP‐2 variant, has a prolonged half‐life and stability as compared to the native GLP‐2 peptide, but still requires daily application in the clinic. Here, we designed and prepared a new degradation‐resistant GLP‐2 analogue dimer, designated GLP‐2&U2461;, with biotechnological techniques. The purity of GLP‐2&U2461;reached 97% after ammonium sulphate precipitation and anion exchange chromatography purification, and the purification process was simple and cost‐effective. We next confirmed that the GLP‐2&U2461; exhibited enhanced activities compared with [Gly2]GLP‐2, the long‐acting, degradation‐resistant analogue. Notably, GLP‐2&U2461; offers a pharmacokinetic and therapeutic advantage in the treatment of radiation‐induced intestinal injury over [Gly2]GLP‐2. We further demonstrated that GLP‐2&U2461; rapidly activates divergent intracellular signaling pathways involved in cell survival and apoptosis. Taken together, our data revealed a potential novel and safe peptide drug for limiting the adverse effect of radiotherapy on the gastrointestinal system. Graphical abstract Figure. No Caption available.

Inhibition of mouse SP2/0 myeloma cell growth by the B7-H4 protein vaccine.

B7-H4 is a member of B7 family of co-inhibitory molecules and B7-H4 protein is found to be overexpressed in many human cancers and which is usually associated with poor survival. In this study, we developed a therapeutic vaccine made from a fusion protein composed of a tetanus toxoid (TT) T-helper cell epitope and human B7-H4IgV domain (TT-rhB7-H4IgV). We investigated the anti-tumor effect of the TT-rhB7-H4IgV vaccine in BALB/c mice and SP2/0 myeloma growth was significantly suppressed in mice. The TT-rhB7-H4IgV vaccine induced high-titer specific antibodies in mice. Further, the antibodies induced by TT-rhB7-H4IgV vaccine were capable of depleting SP2/0 cells through complement-dependent cytotoxicity (CDC) in vitro. On the other hand, the poor cellular immune response was irrelevant to the therapeutic efficacy. These results indicate that the recombinant TT-rhB7-H4IgV vaccine might be a useful candidate of immunotherapy for the treatment of some tumors associated with abnormal expression of B7-H4. [BMB Reports 2014; 47(7): 399-404]

Expression, purification and characterization of galectin-1 in Escherichia coli

As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1 and Gal-1② were expressed in E. coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal.

PRL-3 is a potential glioblastoma prognostic marker and promotes glioblastoma progression by enhancing MMP7 through the ERK and JNK pathways

Purpose: Glioblastoma is the most common and aggressive type of primary brain malignancy and is associated with a poor prognosis. Previously, we found that phosphatase of regenerating liver-3 (PRL-3) was significantly up-regulated in glioblastoma as determined by a microarray analysis. However, the function of PRL-3 in glioblastoma remains unknown. We aimed to investigate the clinical relationship between PRL-3 and glioblastoma, and uncover the mechanisms of PRL-3 in the process of glioblastoma. Methods: PRL-3 expression was evaluated in 61 glioblastoma samples and 4 cell lines by RT-qPCR and immunohistochemistry. Kaplan-Meier analysis was performed to evaluate the prognostic value of PRL-3 for overall survival (OS) and progression-free survival (PFS) for glioblastoma patients. Proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and EdU proliferation assay, migration and invasion by wound-closure/Transwell assays, and qRT-PCR/immunoblotting/IHC were used for both in vivo and in vitro investigations. Result: A high PRL-3 expression level was closely correlated with unfavorable OS and PFS for glioblastoma patients, and was also significantly correlated with Ki-67 expression. Down-regulation of PRL-3 inhibited glioma cell proliferation, invasion and migration through ERK/JNK/matrix metalloproteinase 7 (MMP7) in vitro and in vivo. Conclusions: PRL-3 expression enhances the invasion and proliferation of glioma cells, highlighting this phosphatase as a novel prognostic candidate and an attractive target for future therapy in glioblastoma.

Synergistic tumoricidal effect of combined hPD-L1 vaccine and HER2 gene vaccine

Immunotherapy is gathering momentum as a kind of important therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only in a small subset of patients. Combination therapies targeting multiple pathways often can augment an immune response to improve survival further. Here, the tumoricidal effects of the dual hPD-L1(human programmed cell death ligand 1) vaccination/HER2(human epidermal growth factor receptor 2) gene vaccination immunotherapy against the established HER2-expressed cancers were observed. Animals treated with combination therapy using hPD-L1 vaccine and HER2 gene vaccine had significantly improved survival in a mammary carcinoma model. We observed an increase in tumor growth inhibition following treatment. The percentage of the tumor-free mice (%) was much higher in the combined PD-L1/HER2 group. Furthermore, under the tumor-burden condition, hPD-L1 vaccine enhanced humoral immunity of HER2 gene vaccine. And the combination treatment increased the IFN-γ-producing effector T cells. Additionally, splenocytes from the combined PD-L1/HER2 group immunized mice possessed higher CTL activity. Notably, vaccination with combination therapy induced a significant decrease in the percentage of CD4+CD25+ Treg cells. Collectively, these data demonstrate that PD-L1/HER2 gene vaccine combination therapy synergistically generates marked tumoricidal effects against established HER2-expressing cancers.