The Role Of PTEN In Regulating LPS Induced TLR4 Signaling Pathway And Its Mechanism

Background and Objective:Current wisdom implies that after severe injury or infectious challenge some patients respond by overexpressing inflammatory mediators that result in a systemic inflammatory response culminating in severe shock, multiorgan failure, and death. However, the cellular and molecular mechanisms involved in the initiation and propagation of septic injury remain unclear.Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, is the principal initiator of septic shock. Further insight into LPS-dependent signal transduction will assist the development of novel rational therapy to control LPS-mediated inflammatory diseases.The common LPS signal transduction cascade that is currently believed as the route TLR4→MyD88→IRAK→TRAF6→TAK1→ERK/P38/JNK/IκB followed by the activation of downstream transcription factors NF-κB, AP-1, and Egr-1, which result in production and secretion of cytokines and other inflammatory mediators that constitute the pro-inflammatory response. Meanwhile, another LPS signaling pathway which is involved TRAM-TRIF-RIP-IKKi-IRF3 was also identified, named MyD88 independent pathway.Negative regulation is always concomitant with positive. The balance between them regulates the expression of inflammatory cytokines and mediators, as well as makes the LPS induced signaling under control. However, the complicated intracellular signal network is still not well known up to date for abounding intracellular signal kinases. Hence, more and more labs focus their attention on exploring some other signal pathways associated with LPS.PTEN (phosphatase and tensin homolog deleted on chromosome ten) was discovered as a tumor suppressor gene. It functions as a protein tyrosine phosphatase and as a lipid phosphatase. As a lipid phosphatase, PTEN antagonizes PI3K/Akt signaling by dephosphorylating the D3 position of the inositol ring of phosphatidylinositol 3,4,5-trisphosphate (PIP3), to generate phosphatidylinositol-4,5,-biphosphate(PIP2). On the other hand, as a protein tyrosine phosphatase, PTEN can dephosphorylate itself, focal adhesion kinase (FAK) and the platelet derived growth factor receptor, involves in the migration, adhension of cells.Several researches have illustrated that Phosphatidylinositol-3-kinase (PI3K) is an important negative regulator in LPS-induced signal transduction. Therefore, it is possible that PTEN also involve in the LPS mediated inflammatory response.Cao's groups reported that PTEN was a positive regulator of TLR4 signaling and mediator of macrophage inflammatory responses. However, the detailed molecular mechanism is not well clarified.To identify whether or how PTEN involves in LPS induced TLR4 signal transduction, we used the RAW264.7 macrophage cell lines as the cell model. At first, we detected the effects of LPS on the expression, activation of PTEN as well as the role of PTEN in the LPS induced TNF-αsecretion and try to figure out whether PTEN indeed participates the TLR4 signal transduction. Then, the effects of PTEN on the LPS induced activation of MAPKs and NF-κB were examined to investigate the intracellular molecular mechanism of PTEN on TNF-αproduction. After that, we analysed the effect of PTEN on LPS induced Akt phosphorylation level. Finally, we estimated the relationship between PTEN and TLR4.Methods:1. RAW264.7 cells were stimulated with LPS in different doses for 8, 24, 48 hours respectively, cell lysate was harvested, and the expression of PTEN protein was detected by Western Blot; RAW 264.7 cells were treated with LPS (1μg/ml) for different time course (5, 10, 20, 30, 45, 60, 120min), cell lysate was harvested, the level of phosphorylated PTEN as well as total PTEN protein were detected by Western Blot.2. RAW264.7 cells were seeded in cultured plate and transfected the next day with 0.8μg of siRNA-PTEN or scrambled siRNA, using the Lipofectamine 2000. Cells were cultured for 24 hours, then stimulated with 1μg/ml LPS for another 6 hours, concentrations of TNF-αin the supernatants were measured by ELISA. In another experiment, RAW264.7 cells were treated with PTEN inhibitor for 1 hour or not, followed by the stimulation of LPS for 6 hours, concentrations of TNF-αin the supernatants were measured by ELISA. 3. RAW264.7 cells were seeded in culture flask and transfected the the next day with 8μg of PCMV-GFP vector alone or PCMV vectors containing PTEN cDNA, using the Lipofectamine 2000.About twenty hours after transfection, culture medium was changed to the serum free RPMI 1640 for 8-12 hours, cells were treated with LPS(1μg/ml) for 0,15,30min , cell lysate was harvested, p-ERK1/2, p-P38, p-JNK and the total protein level were detected by Western Blot. In another experiment, RAW264.7 cells were seeded in culture flask, cells were left untreated or treated with the PTEN inhibitor bpv(pic) for 1 hour, followed by stimulation of LPS for 0, 10, 20, 30, 45, 60min, then cell lysate was harvested, p-ERK, p-P38, p-JNK as well as the total protein level were detected by Western Blot.4. RAW264.7 cells were transfected by Lipofectamine 2000 with 0.8μg wild-type PTEN, C124A-PTEN, G129E-PTEN, control empty plasmid(CMV), siRNA-PTEN, scrambled siRNA, 0.2μg NF-κB-Luc plasmid, and 4ng of pRL-CMV-Renilla-luciferase plasmid. Twenty-four hours later, the transfected cells were left untreated or treated with LPS (1μg/ml) for 6 hours. In the other experiment, RAW264.7 cells were transfected by Lipofectamine 2000 with 0.8μg NF-κB-Luc plasmid and 16ng of pRL-CMV- Renilla- luciferase plasmid. Twenty-four hours later, the transfected cells were cultured in the presence of PTEN inhibitor for 1 hour or not, followed by the stimulation of LPS (1μg/ml) for 6 hours. Luciferase activities were measured using Dual-Luciferase Reporter Assay System according to the manufactures'instructions.5. RAW264.7 cells were seeded in culture flask and transfected next day with 8μg of PCMV-GFP vector alone or PCMV vectors containing PTEN cDNA, using the Lipofectamine 2000. About twenty hours after transfection, culture medium were changed to the serum free RPMI 1640 for 8-12 hours, cells were treated with LPS (1μg/ml) for 0, 15, 30min, then cell lysate was harvested. Phosphorylation and the total protein levels of Akt were detected by Western Blot. In the other experiment, RAW264.7 cells were seeded in culture flask, cells were left untreated or treated with the PTEN inhibitor for 1 hour, followed by stimulation of LPS(1μg/ml) for 0, 10, 20, 30, 45, 60min, then cell lysate was harvested, p-Akt and the total protein levels were detected by Western Blot.6. RAW264.7 cells were transfected with wild-type PTEN, mutant PTEN or control empty plasmid (CMV) and cultured for 24 hours, then stimulated with 1μg/ml LPS for another 6 hours. The concentrations of TNF-αin the supernatants were measured by ELISA.7. RAW264.7 cells were stimulated with LPS 1μg/ml for 20 min or not, cell lysate was harvested, followed by immunoprecipitated with TLR4 antibody, then the complex was detected by Western Blot using PTEN antibody.Results:1. The level of PTEN protein in RAW264.7 cells treated for 24 hours with LPS decreased in a dose-dependent manner, however, the level of PTEN protein in the cells increased reversely in a dose-dependent manner at 48 hours treated with LPS. LPS stimulation prompted a decrease in dephosphorylation of PTEN, suggesting PTEN was activated after LPS stimulation.2. Using specific siRNA to down-regulate expression of PTEN, macrophages stimulated with LPS produced lower levels of TNF-αin comparison to macrophages that received scrambled siRNA. Similar results were found after inhibition of PTEN by its specific antagonist.3. LPS-induced phosphorylation of ERK1/2, P38 and JNK was significantly inhibited in the cells transfected with wild-type PTEN, compared with the cells transfected with control empty vector. In contrast, LPS-induced phosphorylation of ERK1/2, P38 and JNK was significantly increased in the presence of PTEN inhibitor.4. Overexpressed PTEN enhanced LPS-induced NF-κB activation. No significant effect was found in mutant PTEN groups and control group. Consistently, down regulating the expression of PTEN or using PTEN inhibitor decreased LPS-induced NF-κB activation compared with the control group.5. LPS-induced phosphorylation of Akt was significantly inhibited in the cells transfected with wild-type PTEN, compared with the cells transfected with control empty vector. On the contrary, LPS-induced phosphorylation of Akt was significantly increased in the presence of PTEN inhibitor.6. Overexpression of wild-type PTEN enhanced LPS-induced TNF-αproduction in macrophages. However, overexpression of lipid phosphatase activity–disrupted mutant PTEN, lipid and protein phosphatases activity–disrupted mutant PTEN, did not show the difference with the control group.7. Co-immunoprecipitation showed PTEN physically associated with TLR4. The association decreased after the stimulation of LPS for 20min.Conclusions:1. The expression of PTEN is regulated by LPS. The level of PTEN protein in the cells treated for 8h with LPS did not show significant changes, however, markedly decreased in a dose-dependent manner after 24h, increased in a dose-dependent manner after 48h. These results show that the level of PTEN is regulated by LPS in different periods. PTEN was dephosphorylated after the LPS stimulation, suggests that PTEN is activated and participating in LPS signaling.2. PTEN enhances LPS-induced cytokine production by activation of NF-κB in macrophages through a lipid phosphatase activity dependent mechanism.3. PTEN inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation. The mechanism and significance remain unclear.4. Interaction between PTEN and TLR4 exists. It is possible that TLR4 plays an important role in the upstream regulation of PTEN.In summary, our results suggest that PTEN physically associates with TLR4. Stimulation of LPS to RAW264.7 cells could cleave the association between PTEN and TLR4, which allows PTEN recruit to its phosphoinositide substrate PI(3,4,5)P3 in membrane and exerts its dephosphorylated function that antagonizes the effects of PI3K,subsequently depresses the activity of downstream molecule Akt, results in the activation of NF-κB, followed by the secretion of inflammatory mediators such as TNF-α. Therefore, a novel signaling pathway involving PTEN is proposed besides classical MyD88 dependent and non-MyD88 dependent manners while LPS stimulates toll-like receptor 4.