Involvement Of Src Tyrosine Kinase In Escherichia Coli K1 Invasion Of Human Brain Microvascular Endothelial Cells

PurposeNeonatal bacteria meningitis is one of the most severe diseases of CNS infection with incidence about 1‰.Although making advances in antimicrobial chemotherapy and supportive care,5%～40%of children suffering bacterial meningitis are dead, moreover 30- 50%of the survivors suffer neurological sequelae,such as falling sickness,mental retardation or dyskinesis.Escherichia coli K1(E.coli K1) is the most common gram-negative pathogen of neonatal meningitis.Most cases of E.coli K1 develop as a result of hematogenous spread to cross the blood-brain barrier.Invasion of human brain microvascular endothelial cell(HBMEC) is essential to the crossing over the blood-brain barrier by E.coli K1,but the molecular mechanism of E.coli crossing the blood-brain barrier is not clear.Therefore,elucidating the molecular mechanism involved in E.coli crossing the blood-brain barrier may provide targeting point for the prevention of neonatal meningitis.The blood-brain barrier(BBB) is a structural and functional barrier that is formed by brain microvascular endothelial cells,astrocytes and pericytes.The blood-brain barrier restricts the free passage of cells and molecules from systemic compartment into the central nervous system(CNS).It maintains the neural microenvironment by regulating the passage of molecules into and out of the brain and providing nutritional ingredient into the brain.It strictly controls the stability of the central nervous system by restricting the diffusion between cells and decreasing endocytosis and the appearance of special vehicle such as ion,opypeptide and nutritional ingredient. HBMEC is the main structure of BBB and maintains the relative stability of the internal milieu in the nervous system by inhibiting many materials into brain but nutritive materials and metabolic products can pass through successfully. Src is the first reported oncogene and has been a prototype in identifying many characteristics of follow on oncogenes,c-Src and its retroviral form(v-Src) are both non-receptor tyrosine kinases,encoding a 60kDa membrane-associated protein tyrosine kinase(PTK).Src play a key role in cell morphology,motility,proliferation,survival, differentiation,signal transduction and cell cycle regulation.Src substrates are proteins that become tyrosine phosphorylated as a result of src gene function and are direct or indirect targets of Src.The Src substrates are found in transformed cells.Most of them found at focal adhesions,are key components in the integrin-mediated signal transduction and bound to actin or integrin,for example,vinculin,cortactin,talin, paxillin,FAK,tensin,ezrin and p130cas.Furthermore,junctional proteins,such asβ-andγ-catenin,ZO-1,occludin,p120ctn,connexin 43,nectin-2 delta are identified as major sites of tyrosine phosphorylation by Src kinases.Multiple factors are involved in E.coli K1 invasion of HBMEC.Our previous research have shown that E.coli K1 invades HBMECs by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase(PI3K).PI3K phosphorylation was induced following by activation of PI3K→p-Akt→LIMK→Cofilin pathway in response to the E.coli K1 invasion,leading to reorganization of the cytoskeleton,which eventually promote E.coli K1 invasion of HBMECs.In addition,PI3K may inhibit invasion through PI3K→Rock→LIMK→p-Cofilin pathway.However,upstream signaling that locally activates PI3K in E.coli K1 invasion still remains obscure.Recent study shows that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and Src may regulate activation of Akt,perhaps by a Src→c-Cbl→PI3K→Akt pathway.Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.Therefore,we infer that Src tyrosine kinase may be involved in E.coli K1 invasion of HBMEC;Src family kinase may be the upstream molecule of the PI3K/Akt pathway. In this study,we use HBMEC as the model of the blood-brain barrier in vitro.We investigated the role of Src in the process of E.coli K1 invasion of HBMEC and the change of cytoskeleton in the invasion process.This research hopes to investigate the mechanism of Src in the process of E.coli K1 invasion of the blood-brain barrier,and to provide efficient methods to treat inflammation of CNS concerning on future works.Methods1.The activation of Src in E.coli K1 invasion of HBMECs(1)Cell culture:HBMEC is cultured in RPMI1640,supplemented with 10%of Nu-serum and 10%of FBS.(2)Western Blot:To explore the phosphorylation of Src in E.coli K1 invasion of HBMECs.(3)Bacteria invasion assay:To investigate the invasion of E.coli K1 after the HBMEC cells were pretreated with the Src inhibitor(PP1/PP2).(4)Bacteria association assay:To investigate the association of E.coli K1 after the HBMEC cells were pretreated with the Src inhibitor(PP1/PP2).(5)Fluorescent staining of actin:To explore the cellular localization of actin in HBMEC cells treated with src inhibitor prior to invasion by E.coli K1.(6)Transfection:Transfect HBMEC with pEGFP-Srcdn by lipoFectamine2000. Select positive clone with G418.Establish the stable Srcdn cell line.(7)Western Blot:To explore the phosphorylation of Src in E.coli K1 invasion of HBMECs transfected with Srcdn.(8)Bacteria invasion and association assay:To investigate the invasion and association of E.coli K1 after HBMECs were transfected with Srcdn. 2.Src-mediated signal pathways in E.coli K1 invasion of HBMECs(1)Western Blot:To explore the phosphorylation of Akt after Src inhibitor pretreated and the phosphorylation of Src after PI3K inhibitor pretreated in E.coli K1 invasion of HBMECs.To explore the phosphorylation of Src and Akt in E.coli K1 invasion of HBMEC transfected byΔp110(dominant-negative PI3K) or Srcdn.(2)Immunoprecipitation:To explore the interaction between Src and p85 in E.coli K1 invasion of HBMECs.(3)Immunofluorescence:To explore the co-localization of Src and p85 in E.coli K1 invasion of HBMEC by confocal microscopy.3.Flt-1 receptor may be involved in Src-mediated E.coli K1 invasion of HBMECs(1)Immunoprecipitation:To explore the interaction among Src,p85 and Flt-1 in E.coli K1 invasion of HBMECs.(2)Bacteria invasion assay:To investigate the E.coli K1 invasion of HBMEC pretreated by VEGFR inhibitor(SU5416 or KRN633) or KDR inhibitor.(3)Bacteria invasion assay:To explore the E.coli K1 invasion of HBMEC transfected by Flt1-RNAi.(4)Western Blot:To explore the phosphorylation of Akt in E.coli K1 invasion of HBMEC transfected by Flt1-RNAi.Results1.The activation of Src in E.coli K1 invasion of HBMECs(1)Src is phosphorylated in E.coli K1 invasion of HBMECs and the highest expression is at 15min.(2)Src family tyrosine kinase selective inhibiter PP1/PP2 inhibited E.coli K1 invasion of HBMEC in a dose-dependent manner,but Src inhibitor analog PP3 did not. There is no difference in Bacteria Association Assay in E.coli K1 invasion of HBMEC pretreated by PP1/PP2/PP3.(3)PP1/PP2 abolished the phosphorylation of Src in E.coli K1 invasion of HBMEC,but PP3 did not.(4)The expression of Srcdn inhibited the E.coli K1 invasion of HBMEC,but had no influence in association.(5)The expression of Srcdn abolished the phosphorylation of Src in E.coli K1 invasion of HBMECs.(6)E.coli K1 invasion of HBMECs could result in the actin rearrangement.The results of fluorescent staining showed that the percent of actin rearrangement decreased when E.coli K1 invaded HBMECs pretreated by PP1 or PP2.However,we could still observe the actin rearrangement when E.coli K1 invaded HBMECs pretreated by PP3.(7)The results of fluorescent staining showed that the percent of actin rearrangement decreased after the E.coli K1 invasion of HBMECs transfected by Srcdn.2.Src-mediated signal pathways in E.coli K1 invasion of HBMECs(1)Src selective inhihitor inhibited the phosphorylation of Akt in E.coli K1 invasion of HBMECs.(2)Akt was not phosphorylated in E.coli K1 invasion of HBMECs transfected with Srcdn.(3)PI3K inhibitor did not inhibit the phosphorylation of Src in E.coli K1 invasion of HBMECs.(4)Src was still phosphorylated in E.coli K1 invasion of HBMECs transfected with dominant-negative PI3K(Δp110).(5)The interaction of Src and p85 was increased at 15min in E.coli K1 invasion of HBMECs.3.Flt-1 receptor may be involved in Src-mediated E.coli K1 invasion of HBMECs (1)Src,PI3K and Flt-1 composed complex in E.coli K1 invasion of HBMECs.(2)Flt-1 inhibitor(SU5416 or KRN633) inhibited E.coli K1 invasion of HBMEC in a dose-dependent manner,but KDR inhibitor did not.(3)The invasion ability was reduced in E.coli K1 invasion of HBMEC transfected by Flt-1-RNAi.(4)The phosphorylation of Src and Akt were both inhibited in E.coli K1 invasion of transfected by Flt-1-RNAi.ConclusionsSrc tyrosine kinase is involved in E.coli K1 invasion of human brain microvascular endothelial cells.Src mediated actin rearrangement in the process of E.coli K1 invasion.Src is an upstream signal molecule of PI3K in E.coli K1 invasion of HBMECs.Flt-1 receptor may be involved in this process.