Ren-Peng Zhou

Functions of interleukin-34 and its emerging association with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic, synovial inflammation affecting multiple joints, finally leading to extra‐articular lesions for which limited effective treatment options are currently available. Interleukin‐34 (IL‐34), recently discovered as the second colony‐stimulating factor‐1 receptor (CSF‐1R) ligand, is a newly discovered cytokine. Accumulating evidence has disclosed crucial roles of IL‐34 in the proliferation and differentiation of mononuclear phagocyte lineage cells, osteoclastogenesis and inflammation. Recently, IL‐34 was detected at high levels in patients with active RA and in experimental models of inflammatory arthritis. Blockade of functional IL‐34 with a specific monoclonal antibody can reduce the severity of inflammatory arthritis, suggesting that targeting IL‐34 or its receptors may constitute a novel therapeutic strategy for autoimmune diseases such as RA. Here, we have comprehensively discussed the structure and biological functions of IL‐34, and reviewed recent advances in our understanding of the emerging role of IL‐34 in the development of RA as well as its potential utility as a therapeutic target.

Novel Insights into Acid-Sensing Ion Channels: Implications for Degenerative Diseases

Degenerative diseases often strike older adults and are characterized by progressive deterioration of cells, eventually leading to tissue and organ degeneration for which limited effective treatment options are currently available. Acid-sensing ion channels (ASICs), a family of extracellular H(+)-activated ligand-gated ion channels, play critical roles in physiological and pathological conditions. Aberrant activation of ASICs is reported to regulate cell apoptosis, differentiation and autophagy. Accumulating evidence has highlighted a dramatic increase and activation of ASICs in degenerative disorders, including multiple sclerosis, Parkinsons disease, Huntingtons disease, intervertebral disc degeneration and arthritis. In this review, we have comprehensively discussed the critical roles of ASICs and their potential utility as therapeutic targets in degenerative diseases.

Interleukin-6 enhances acid-induced apoptosis via upregulating acid-sensing ion channel 1a expression and function in rat articular chondrocytes.

The inflammatory cytokine interleukin-6 (IL-6) is a causative agent of rheumatoid arthritis (RA), a chronic inflammatory disease complicated with degenerative arthritic cartilage. However, the precise mechanism of IL-6 on chondrocyte apoptosis is largely unclear. Acid-sensing ion channels (ASICs), a family of extracellular H(+)-activated cation channels, can be transiently activated by extracellular acid and play a pivotal role in acid-induced cell injury. In the present study, to investigate the role of IL-6 in regulating acid-induced articular chondrocyte apoptosis, primary rat articular chondrocytes were subjected to different treatments with or without IL-6 in the presence of acid. The results showed that the mRNA and protein expressions of ASIC1a were significantly increased in articular cartilage and chondrocytes of adjuvant arthritis (AA) rats. IL-6 could dramatically upregulate the level of ASIC1a in a time- and dose-dependent manner, and induce the activation of JAK2, STAT3, ERK, JNK and NF-κB in articular chondrocytes. Moreover, both the respective inhibitors of these signaling pathways and the specific antibody against IL-6 receptor (tocilizumab) could partially abrogate the ASIC1a upregulation induced by IL-6. Furthermore, IL-6 inhibited the cell viability and enhanced LDH release, [Ca(2+)]i elevation, and apoptosis in acid-induced articular chondrocytes, and these changes could be reversed by using psalmotoxin 1(PcTX1), which is the specific antagonist of ASIC1a. In addition, pretreatment with PcTX1 could inhibit the downregulated expression of Bcl-2 and the upregulated expression of Bax induced by IL-6 in acid-induced articular chondrocytes. Taken together, these results indicated that IL-6 could enhance acid-induced articular chondrocyte apoptosis, the mechanism of which might partially be involved with its ability of regulating the activation of ASIC1a-dependent JAK2/STAT3 and MAPK/NF-κB signaling pathways.

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca2+ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.

Autophagy contributes to 4-Amino-2-Trifluoromethyl-Phenyl Retinate-induced differentiation in human acute promyelocytic leukemia NB4 cells

ABSTRACT As a classic differentiation agent, all‐trans retinoic acid (ATRA) has been widely used in treatment of acute promyelocytic leukemia (APL). However, clinical application of ATRA has limitations. Our previous studies suggested that 4‐Amino‐2‐Trifluoromethyl‐Phenyl Retinate (ATPR), a novel all‐trans retinoic acid (ATRA) derivative designed and synthesized by our team, could induce differentiation of APL cells in vivo and in vitro. To explore the underlying mechanism of ATPR, the effect of ATPR on autophagy of APL cells was observed in the present study. The results showed that the differentiation effect of ATPR on APL cells was accompanied with autophagy induction and PML‐RAR&agr; degradation via activating Notch1 signaling pathway. Moreover, inhibition of autophagy using 3‐methyladenine (3‐MA) or small interfering RNA (siRNA) that targets essential autophagy gene ATG5 abrogated the ATPR‐induced cell differentiation. Furthermore, when pretreated with DAPT, a &ggr;‐secretase inhibitor, the Notch1 signaling pathway was blocked in APL cells, followed by the reduction of ATPR‐induced autophagy and differentiation. Taken together, these results suggested that autophagy play an important role in ATPR‐induced cell differentiation, which may provide a novel approach to cure APL patients. HIGHLIGHTSATPR induces autophagy in APL cell line NB4 cells.Autophagy induction is essential for cell differentiation in NB4 cells.Notch1 signaling is involved in ATPR‐induced autophagy and differentiation in NB4 cells.

ASIC1a Promotes Acid-Induced Autophagy in Rat Articular Chondrocytes through the AMPK/FoxO3a Pathway

Acid-sensing ion channel 1a (ASIC1a) is a member of the extracellular H+-activated cation channels family. Our previous studies suggested that ASIC1a contributed to acid-induced rat articular chondrocytes autophagy. However, its potential mechanisms remain unclear. The present study demonstrated the effect of ASIC1a on rat articular chondrocytes autophagy and explored the underlying molecular mechanisms. The results demonstrated that ASIC1a contributed to acid-induced autophagy in rat articular chondrocytes, and which was associated with an increase in (Ca2+)i, as indicated that acid-induced increases in mRNA and protein expression of LC3B-II and other autophagy-related markers were inhibited by ASIC1a-specific blocker, PcTx1 and calcium chelating agent, BAPTA-AM. Furthermore, the results showed that extracellular acid increased level of Forkhead box O (FoxO) 3a, but was reversed by inhibition of ASIC1a and Ca2+ influx. Moreover, gene ablation of FoxO3a prevented acid-induced increases in mRNA and protein expression of LC3B-II, Beclin1 and the formation of autophagosome. Finally, it also showed that ASIC1a activated adenine nucleotide (AMP)-activated protein kinase (AMPK). In addition, suppression of AMPK by Compound C and its small interfering RNA (siRNA) prevented acid-induced upregulation of total and nuclear FoxO3a and increases in mRNA and protein expression of LC3B-II, Beclin1, and ATG5. Taken together, these findings suggested that AMPK/FoxO3a axis plays an important role in ASIC1a-mediated autophagy in rat articular chondrocytes, which may provide novel mechanistic insight into ASIC1a effects on autophagy.

Effects of autophagy on acid-sensing ion channel 1a-mediated apoptosis in rat articular chondrocytes

Rheumatoid arthritis (RA) is a degenerative joint disease that is caused by multiple pathogenic factors. However, the precise etiology of RA is still unknown. Our previous studies demonstrated that acid-sensing ion channel 1a (ASIC1a)-mediated articular chondrocyte apoptosis played a key role in the progression of RA. In this study, we aim to explore whether ASIC1a mediates autophagy or not and the effect of autophagy on ASIC1a-mediated apoptosis. Primary articular chondrocytes, extracted from rat knee joints, were exposed to different concentrations of concentrated hydrochloric acid for different time intervals in vitro. The results indicated that extracellular acid treatment induced autophagy of rat articular chondrocytes. Moreover, inhibition of ASIC1a with either psalmotoxin 1 or ASIC1a short hairpin RNA reduced the autophagy flux. The results suggested that ASIC1a mediated acid-induced autophagy. Pretreatment with autophagy antagonist 3-methyladenine decreased the autophagy, but increased the apoptosis mediated by ASIC1a. Furthermore, knockdown of Beclin 1 by small interfering RNA attenuated autophagy but potentiated ASIC1a-mediated apoptosis of rat articular chondrocytes. Taken together, these findings suggested that both inhibition and silencing of autophagy could enhance ASIC1a-mediated apoptosis in rat articular chondrocytes, and therefore, autophagy is likely to be a new mechanism involved in ASIC1a-mediated apoptosis of articular chondrocytes during the pathogenesis of RA.

ASIC2a overexpression enhances the protective effect of PcTx1 and APETx2 against acidosis-induced articular chondrocyte apoptosis and cytotoxicity

Acid hydrarthrosis is another important pathological character in rheumatoid arthritis (RA), and acid-sensing ion channel 1a (ASIC1a) plays a destructive role in acidosis-induced articular chondrocyte cytotoxicity. Recently, ASIC2a has been reported to possess neuroprotective effect on acidosis-induced injury of neuronal cells. However, whether ASIC2a has an enhanced effect on the protective effect of blocking ASIC1a and ASIC3 against acid-induced chondrocyte apoptosis is still unclear. The aim of present study was to investigate the chondroprotective effect of ASIC2a with PcTx1 (ASIC1a specific blocker) and APETx2 (ASIC3 specific blocker) on acidosis-induced chondrocyte apoptosis. Our results revealed that acid (pH 6.0) decreased the cell viability and induced apoptosis of articular chondrocytes. PcTx1 and APETx2 combination significantly attenuated acidosis-induced chondrocyte cytotoxicity due to inhibit apoptosis, and this role could be enhanced by ASIC2a overexpression compared with the PcTx1 and APETx2 combination alone group. Moreover, both the [Ca2+]i levels and the levels of phosphorylated ERK1/2 as well as p38 were further reduced in acidosis-induced chondrocytes after ASIC2a overexpression in the presence of PcTx1 and APETx2. Furthermore, ASIC2a overexpression also reduced acid-induced the expression of ASIC1a. In addition, ASIC2a overexpression further promoted the PcTx1 and APETx2-increased levels of type II collagen in acidosis-induced chondrocytes. Taken together, the current data suggested that ASIC2a overexpression might enhance the anti-apoptotic and protective role of PcTx1 and APETx2 against acid-induced rat articular chondrocyte apoptosis by regulating ASIC1a expression and the [Ca2+]i levels and at least in part, suppressing p38 and ERK1/2 MAPK signaling pathways.

Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells

4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been demonstrated its anti-tumor effect through inducing differentiation and inhibiting proliferation. Eukaryotic initiation factor 3a (eIF3a) plays a critical role in affecting tumor cell proliferation and differentiation. However, whether eIF3a is implicated in chronic myeloid leukemia cells differentiation remains unclear. Our results demonstrated that eIF3a could be suppressed by ATPR in K562 cells. The results also confirmed that ATPR could arrest cell cycle in G0/G1 phase and induced differentiation. Moreover, over-expression of eIF3a promoted not only protein expression of c-myc and cyclin D1, but also prevented the expression of p-Raf-1, p-ERK and the myeloid differentiation markers CD11b and CD14 and had an influence on inducing the morphologic mature. However, silencing eIF3a expression by small interfering RNA could have an adverse effect on K562 cells. In addition, PD98059 (a MEK inhibitor) could block cell differentiation of CML cells and contributed to the expression of c-myc and cyclin D1. In conclusion, these results indicated that eIF3a played an important role in ATPR-induced cell differentiation in K562 cells, its mechanism might be related to its ability in regulating the activation of ERK1/2 signaling pathway in vitro.