The Molecular Mechanism Of Bacteria Lipopeptide Pam3CSK4Up-regulation Of MKP-1by Activation Of Toll-like Receptor2(TLR2) In HUVEC

ObjectivesToll-like receptors activate MAPK signal pathways to elicit inflammatoryresponse, resulting in the onset and progress of cardiovascular diseases.Mitogen-activated protein kinase phosphatase-1(MKP-1) inhibits inflammatoryresponse by negatively regulates MAPK signaling. This study analyzed the effect ofTLR2activation on MKP-1expression, elucidated the molecular mechanism forlipopeptide Pam3CSK4-induced controlled inflammation by TLR2activation, andfinally to provide clues and experimental data for clinical prevention ofcardiovascular diseases.MethodsHuman umbilical vein endothelial cells (HUVEC) and human monocytic THP1cells were culture in DMEM and RPMI1640medium by use of usual cell culturemethods. The cells grew in exponential phase for the subsequent experiments. Theconcentration for analysis of Pam3CSK4dose response was0,0.01,0.05,0.1,0.51μg/ml. The concentration for time course analysis was1μg/ml, and the time periodswere0,1,3,6,12,24hours. The mRNA levels were detected by RT-PCR afterPam3CSK4treatment. The protein levels were detected by western blot. Theconcentration of the inhibitors for signal transduction was10μM. The protein levelsof phosphorylated signal transduction molecules were detected by western blot aswell. The results with gray scale scanning to half quantitative analysis，the statisticalmethods used to use t test, P values <0.05as statistically significant. Results1. HUVEC cells were treated with various concentration of Pam3CSK4for1hour. The RT-PCR results showed that mRNA levels of TLR2were up-regulated in adose-dependent manner.2. HUVEC cells were treated with1μg/ml Pam3CSK4for1-24hours, TheRT-PCR results showed that mRNA levels of TLR2were up-regulated in atime-dependent manner.3. HUVEC cells were treated with various concentration of Pam3CSK4for1hour. The RT-PCR results showed that mRNA levels of proinflammatory cytokinessuch as TNF-α, IL-6and Cox-2were up-regulated in a dose-dependent manner.4. HUVEC cells were treated with1μg/ml Pam3CSK4for1-24hours, theRT-PCR results showed that mRNA levels of proinflammatory cytokines such as IL-6,TNF-α, MCP-1and Cox-2were up-regulated in a time-dependent manner.5. HUVEC cells were treated with various concentration of Pam3CSK4for1hour. Both RT-PCR and Western blot results showed that MKP-1levels of bothmRNA and protein were up-regulated in a dose-dependent manner.6. HUVEC cells were treated with1μg/ml Pam3CSK4for1-24hours, theRT-PCR results showed that mRNA levels of MKP-1, the western blot results showedthat protein levels of MKP-1, were up-regulated in a time-dependent manner.7. HUVEC cells were treated with various concentration of Pam3CSK4for0.5hour. The western blot results showed that the phosphorylated protein levels of ERK,JNK and P38were up-regulated in a dose-dependent manner.8. HUVEC cells were treated with various concentration of Pam3CSK4for0.5hour. The western blot results showed that the protein levels of IκB-α weredown-regulated in a dose-dependent manner.9. HUVEC cells were treated with10μM U0126, or SP600125, or SB203580, orBay117082for30minutes, followed by the treatment with1μg/ml Pam3CSK4for1hour. The RT-PCR results showed U0126, SB203580and Bay117082were down-regulated Pam3CSK4-induced MKP-1expression significantly.10. HUVEC cell were treated with various SB203580for30minutes, followedby the treatment with1μg/ml Pam3CSK4for1hour. RT-PCR results showed thatMKP-1was down-regulated in a dose-dependent manner.Conclusions1. Vascular endothelial cell HUVEC expresses TLR2;2. Synthetic bacterial lipopeptide Pam3CSK4induces the expression ofproinflammatory cytokines by activation of TLR2;3. Pam3CSK4activation of TLR2induces the expression of MKP-1;4. The up-regulation of MKP-1is mainly related to P38signaling pathway;5. MKP-1may regulate TLR2-induced cardiovascular diseases.