Effects Of Low Level Lead Exposure On Brain Barriers: Role Of Activation Of Nonreceptor Tyrosine Kinase Src

Background Lead(Pb),as a widespread environmental pollutant,is known to cause a wide range of neurotoxic effects,including decreased intelligence quotient,cognitive deficits,poor attention span,and increased aggression.Pb has long been recognized as a neurodevelopmental toxin.Developing blood-brain barrier(BBB)and blood-cerebrospinal fluid barrier(BCB)are known to be targets of Pb neurotoxicity.Brain capillary endothelial cells with tight junctions(TJs)between adjacent cells,astrocyte end feet,and pericytes within the capillary basement constitute the structural basis of the BBB.Choroid plexus epithelial cells with tight junctions(TJs)between adjacent cells constitute the structural basis of the BCB.However,the underlying mechanisms of Pb-induced reduction of TJ proteins of BBB and BCB are still unclear.The way Pb enters into the brain parenchyma or cerebrospinal fluid can be classified into two categories: intercellular regions after breakdown TJ proteins and Pb transporter in the endothelial/epithelial cell bodies.Recently,hemichannels of Connexin 43(Cx43),the most ubiquitously expressed gap junction proteins in the BBB and BCB,were found to be important pathways for many substances.In previous study,Cx43 expression was enhanced in the hippocampus,a major target of Pb-induced neurotoxicity,implying that Pb may accumulate in the hippocampus via Cx43 hemichannel.Src is nonreceptor protein tyrosine kinases that mediates the integrin signaling on the focal adhesion complex at the cell-toextracellular matrix interface.Src kinase activity is required for both assembly and disassembly of TJ proteins in different epithelial cells.In addition,Src has long been known to interact with and phosphorylate Cx43 to promote downregulation of GJ communication and cause GJ disassembly,even channel closure.Src phosphorylation was found to increase greater than other kinases in brain microvessels after Pb exposure by using a phosphoprotein antibody array.Aims This study was designed to investigate(1)whether Pb acted on the Src-mediated cascade event leading to an altered TJ proteins expression at BBB and BCB;(2)the roles of Cx43 in Pb uptake in the brain capillary endothelial cells and choroid plexus epithelial cells;(3)involvement of Src phosphorylation in regulation of Cx43 expression and cellular Pb absorption.Methods Rat brain microvascular endothelial RBE4 cells were employed as BBB in vitro models.Choroidal epithelial Z310 cell lines were employed as BCB in vitro models.Western blot was utilized to detect protein levels of P-Src,C-Src,GRP78 or Cx43 in RBE4 and Z310 cells after Pb exposure.RT-PCR was used to analyze m RNA of GRP78,Cx43.GRP78 si RNA and Src-specific inhibitor dasanitib were used to explore the association of GRP78 and Src,and their roles in regulation of occludin or ZO-1.Then,distribution of GRP78 or Cx43 in RBE4 and Z310 cells were detected with immunocytochemistry.Further,ultra-localization of GRP78 of RBE4 cells were examined by immunogold-silver staining.We used biotin-streptavidin precipitation to quantify cell-surface-located GRP78 with or without Pb treatment.Cellular Pb accumulation were analyzed by atomic absorption spectroscopy.Eth Bruptake was employed to measure the function of connexin hemichannel.To overcome the shortcoming of transient transfections,we constructed a Z310 cell-based doxycyclineinducible Cx43 expression cell line(i ZCx43)to further examine the role of Cx43 in Pb uptake.Finally,Src-specific inhibitor was used to analyze the relationship of Src and Cx43 including cellular Pb uptake.Results When cultured RBE4 or Z310 cells were exposed to 10 ?M Pb for 24 h,both expressions of phosphor-Src were significantly increased,but an upstream regulator GRP78 were only increased in RBE4 cells.Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin,but not ZO-1;small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction.Furthermore,Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member.However,the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure,suggesting that the cytosolic GRP78,rather than cell-surface GRP78,was responsible to Pb-induced Src activation and ensuing occludin reduction.When treated with 10 ?M Pb for 24 h,the expression level of Cx43 was up-regulated in RBE4 cells,but down-regulated in Z310 cells.Changes of Cx43 protein levels caused by Pb exposure paralleled cellular Pb concentrations respectively.Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations.Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor,carbenoxolone.Overexpression of Cx43 with transient plasmids transfections before Pb exposure increased the Pb accumulation.In the i ZCx43,doxycycline induced a significant increase(3-fold)in Pb uptake,corresponding to the increased Cx43 levels.Inactivation of Src reversed Pb-induced upregulation of Cx43 in RBE4 cells,accelerated Pb-induced downregulation of Cx43 in Z310 cells.Inactivation of Src decreased/increased Pb uptake in RBE4/Z310 cells,respectively,suggesting that other substrates regulated by Src phosphorylationmay be also involved in Pb transport in Z310 cells.Conclusion Taken together,this study provides the evidence of a novel linkage of Src activation to downregulation of occludin,and BBB/BCB disruption during Pb exposure.Further,these data establish that Pb can accumulate in the BBB/BCB,and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Src phosphorylation.