Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type Ⅳ

AIM： To investigate the regulation of activin receptor-interacting protein 2 （ARIP2） expression and its possible relationships with collagen type Ⅳ （collagen Ⅳ） in mouse hepatoma cell line Hepal-6 cells. METHODS： The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate （PMA）, forskolin and A23187. After pcDNA3- ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor （ActRⅡ） and collagen Ⅳ expression were evaluated. RESULTS： The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation （2 or 4 h）. The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 （25.3% ± 5.7% vs 48.1% ± 3.6%, P 〈 0.01）. ARIP2 overexpression could remarkably suppress the expression of ActRⅡA mRNA in dose-dependent manner, but has no effect on ActRⅡB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION： These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

RIP2的研究进展

受体相互作用蛋白（receptor—interacting protein．RIP）2属于RIP家族成员，在多种组织中均有分布。最初研究发现RIP2是Toll样受体（toll—lik ereceptors，TLRs）及NOD样受体（NOD—like receptors，NLRs）信号通路的下游因子，

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Receptor-interacting Protein 140 Overexpression Promotes Neuro-2a Neuronal Differentiation by ERK1/2 Signaling

Background：Abnormal neuronal differentiation plays an important role in central nervous system （CNS） development abnormalities such as Down syndrome （DS）,a disorder that results directly from overexpression of genes in trisomic cells.Receptor-interacting protein 140 （RIP 140） is significantly upregulated in DS brains,suggesting its involvement in DS CNS development abnormalities.However,the role of RIP140 in neuronal differentiation is still not clear.The current study aimed to investigate the effect of RIP 140 overexpression on the differentiation of neuro-2a （N2a） neuroblastoma cells,in vitro.Methods：Stably RIP 140-overexpressing N2a （N2a-RIP140） cells were used as a neurodevelopmental model,and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot.Retinoic acid （RA） was used to stimulate N2a differentiation.Combining the expression of Tuj 1 at the mRNA and protein levels,the percentage of cells baring neurites,and the number of neurites per cell body was semi-quantified to determine the effect of RIP 140 on differentiation of N2a cells.Furthermore,western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP 140 induces differentiation of N2a cells.Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.Results：Compared to untransfected N2a cells RIP140 expression in N2a-RIP 140 cells was remarkably upregulated at both the mRNA and protein levels.N2a-RIP 140 cells had a significantly increased percentage of cells baring neurites,and numbers of neurites per cell,as compared to N2a cells,in the absence and presence of RA （P 〈 0.05）.In addition,Tuj l,a neuronal biomarker,was strongly upregulated in N2a-RIP140 cells （P 〈 0.05） and phosphorylated ERK1/2 （p-ERK1/2） levels in N2a-RIP140 cells were dramatically increased,while differentiation was inhibited by the ERK 1/2-specific inhibitor U0126.Conclusions：RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

This study aims to demonstrate that blocking the receptor-interacting protein2(Rip2)expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality.Murine Rip2 small interfering RNA(siRNA)plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Cell proliferation was assayed withMTT.TNF-a concentration was assayed with ELISA and high-mobility group box 1 protein(HMGB1)level with semi-quantitative western blot after lipopolysaccharide(LPS)stimulation.LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated.Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation.Blocking Rip2 could attenuate LPS-induced TNF-a and HMGB1 production.The HMGB1 expression in the liver decreased to(40.21±11.03)pg/g,and serum TNF-a level decreased to(300.43±59.26)ng/L(P<0.05).The survival rate of mice from endotoxemia was also improved(P<0.05).The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2,decrease the production of TNF-a and HMGB1 and protect mice from fatal endotoxemia.

Foot-and-Mouth Disease Virus Inhibits RIP2 Protein Expression to Promote Viral Replication

Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.

乙酰胆碱受体对胞壁酰二肽激活小鼠巨噬细胞NLR2/RIP2通路的调控作用

目的 评价乙酰胆碱受体对胞壁酰二肽（MDP）激活小鼠巨噬细胞Nod样受体2/受体相互作用蛋白2（NLR2/RIP2）通路的调控作用.方法 RAW264.7细胞长至对数生长期时,接种于12孔培养板（细胞密度1＆#215;106个/ml,2 ml/孔）,108个培养孔.采用随机数字表法,将其分为3组（n=36）,正常对照组（C组）常规培养;M组加入MDP,终浓度为10 μg/ml;G组同时加入MDP和α7烟碱型乙酰胆碱受体特异性激动剂GTS-21,终浓度分别为10、50 μg/ml.分别于MDP孵育1、6、24h时取12个培养孔,取细胞悬液,采用实时荧光定量PCR法检测NLR2 mRNA表达,采用Western blot印迹法检测RIP2表达,采用ELISA法检测培养液TNF-α和高迁移率族蛋白-1（HMGB1）的浓度.结果 与C组比较,M组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平升高（P＜0.05）;与M组比较,G组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平降低（P＜0.05）.结论 乙酰胆碱受体可抑制MDP激活小鼠巨噬细胞NLR2/RIP2通路转导.

The contribution of oligodendrocytes and oligodendrocyte progenitor cells to central nervous system repair in multiple sclerosis： perspectives for remyelination therapeutic strategies

Oligodencrocytes（OLs） are the main glial cells of the central nervous system involved in myelination of axons. In multiple sclerosis（MS）, there is an imbalance between demyelination and remyelination processes, the last one performed by oligodendrocyte progenitor cells（OPCs） and OLs, resulting into a permanent demyelination, axonal damage and neuronal loss. In MS lesions, astrocytes and microglias play an important part in permeabilization of blood-brain barrier and initiation of OPCs proliferation. Migration and differentiation of OPCs are influenced by various factors and the process is finalized by insufficient acummulation of OLs into the MS lesion. In relation to all these processes, the author will discuss the potential targets for remyelination strategies.