TLR4 Signaling Pathway Mediates The LPS/ischemia-induced Expression Of MCPIP1 In Microglia

BackgroundCentral nervous system(CNS)keeps a dynamic equilibrium through an intricate regulating network against slight abnormal inside and outside environmental changes.Once disrupted,neuroinflammation is produced and participates in the pathogenesis of many CNS diseases,including stroke,infection,neurodegenerative diseases,and brain traumatic injury.Consequently,the negative regulatory factors in response to neuroinflammation may provide potential and novel therapeutic targets for the treatment of neuroinflammation-associated diseases.Monocytechemotactic protein-induced protein 1(MCPIP1),also known as Regnase-1 and ZC3H12A,is newly recognized as one of those negative regulatory factors.With a PilT N-terminal(PIN)-like RNase domain(also called NYN domain),MCPIP1 exerts mRNA endonuclease activity that can specifically recognize and effectively destabilize a subset of mRNAs encoding proinflammatory cytokines,such as interleukin(IL)-1β,IL-6,IL-12,tumor necrosis factor(TNF)-a and IL-2.Moreover,MCPIP1 also acts as an deubiquitinase to negatively regulate JNK and NF-κB signalings by targeting TNF receptor-associated factors(TRAFs).In CNS,MCPIP1 is mainly produced by microglia and normally keeps in a low level.However,it can be undergone a robust induction upon a variety of exogenous and endogenous stimulus to microglia,such as lipopolysaccharide(LPS),dangerassociated molecular patterns(DAMPs),monocytechemotactic protein(MCP),IL1β and other cytokines.Despite of the indisputable anti-inflammation functions of MCPIP1 and the rapid induction by diverse conditions,the precise engaged molecular mechanisms of MCPIP1 induction are still unclear.NF-κB signaling pathway and mitogen activated protein kinase(MAPK)cascades are reportedly the most important intracellular signaling pathways to promote MCPIP1 expression after multiple challenges.Exploring the underlying signaling pathways not only provides insight into the pathogenesis of neuroinflammation but also helps to seek for novel therapeutic targets for neuroinflammation-related disorders.Purposes1.To confirm LPS/ischemia induce the expression of MCPIP1 in microglia;2.To explore roles of TLR4,TLR2,and RAGE signaling pathways on the induction of MCPIP1 by LPS and ischemic attack.Methods and materials1.BV2 cells C57/BL6 mice were subjected to LPS stimulation or transient middle cerebral artery occlusion(MCAO)to examine the modulation of MCPIP1;2.Specific inhibitors for TLR4,TLR2 or RAGE were preadministered to explore the mechanisms of MCPIP1 expression;3.The role of NF-κB and MAPK pathways on MCPIP1 expression by LPS is further examined.Results1.LPS increases MCPIP1 expression in BV2 cells in time and dose depend.Treatment of 1 μg/ml LPS increased both mRNA and protein levels of MCPIP1 within 24h and peaked at 4h.4h time course performance showed that mRNA levels of MCPIP1 revealed a substantial increase at 0.5h and peaked at 4h,while protein levels underwent significant decreases at 0.5h and 1h,but increased and peaked at 4h.2.TLR4 inhibition decreases LPS-induced expression of MCPIP1 in BV2 cells.Compared with the vehicle control,TLR4 inhibition markedly suppressed LPSmediated upregulation of MCPIP1 mRNA.Protein level of MCPIP1 was also remarkably suppressed by inactivation of TLR4,evidenced by reduction of intensities of Western blot bands and the immunofluorescent signal.Immunofluorescent staining also indicated the cytoplasmic location of MCPIP1.Whereas,there were no significant changes observed in MCPIP1 expression under inhibition of either TLR2 or RAGE.3.LPS/ischemia increases MCPIP1 expression in mouse brains.MCPIP1 mRNA levels from the LPS-stimulated mouse brains were much higher than those in saline group and revealed a time-dependent increase within 24h,consistent with protein changes after LPS treatment.Mice were also subjected to MCAO to induce brain ischemia for a dynamic examination of MCPIP1 expression within 24h.Expectedly,mRNA and protein levels of MCPIP1 from the MCAO models were much higher than the sham group.Significant increase of both mRNA and protein was detected at 4handmaximal level was sustained up to 24h.4.TLR4 inhibition decreases LPS/ischemia-induced expression of MCPIP1 in mouse brains.Similar to the in vitro data,both mRNA and protein levels of MCPIP1 upregulated by LPS were significantly decreased under TLR4 inhibition,but neither TLR2 nor RAGE had significant effects.Likewise,ischemia-upregulated MCPIP 1 expression was dramatically decreased in mRNA and protein levels by pretreatment of TLR4 inhibitor,whereas,there were no significant changes observed under inhibition of either TLR2 or RAGE.5.TLR4-MyD88-MAPK/NF-κB signaling pathway participates in LPSinduced expression of MCPIP1 in BV2 cells.Protein levels of TLR4,MyD88,phosphorylatedMAPK(p-P38)and IκBα(p-IκBα),as well as the translocation of NF-κB(P65)were detected under TLR4 inactivation.In addition,pretreatment of TLR4 inhibitor restrained LPS-facilitated translocation of NF-κB to nucleus and NF-κB was retained in cytoplasm under TLR4 inactivation.Whereas,the inhibition of TLR2 or RAGE could not obviously change the levels of p-P38 and p-IκBα.ConclusionIn conclusion,we demonstrate that MCPIP 1 can be strongly induced under LPS/ischemia stimulation in microglia,and TLR4 signaling,not TLR2 or RAGE,predominantly mediates this induction.Our discovery is favorable for better understanding the underlying molecular mechanisms for MCPIP1 induction and may be helpful to search new targets to restrain neuroinflammation in CNS diseases.