C-Jun Signaling Pathway Of Benzo(a)pyrene-Induced Alternations Of The Cell Cycle And G₁ Regulatory Proteins

Benzo(a)pyrene(B(a)P) exposure is associated with an increased risk of human cancers.With the advance of the researches on the mechanisms of cell cycle control, it is gradually recognized that misregulation of the cell cycle plays an important role during carcinogenesis.Although numerous studies demonstrated that B(a)P could induce cell cycle alternations,the molecular basis of this is still obscure so far.Our previous studies showed that phosphatidylinositol-3 kinase(PI-3K) and mitogen activated protein kinase(MAPK) medicate B(a)P-induced activator protein 1(AP-1) transactivation and cell cycle alternations.Transcription factor c-Jun,acting as a primary member of AP-1,could be intiated by a various of signaling pathways, mediating cell cycle progression through cell cycle regulators,but the role of PI-3K, MAPK and p53 pathways in B(a)P-induced c-Jun activation,the relationship of these signaling pathway,and the mechanisms of c-Jun involvement in cell cycle control are largely unknown.The present study investigated the likely interaction of several pathways in B(a)P-induced c-Jun activation as well as the potential effect of c-Jun in B(a)P-induced alternations of the cell cycle and G₁ regulatory proteins in human embryonic lung fibroblast(HELF),to dissect the mechanisms of cell cycle alternations induced by B(a)P at the level of signaling transduction,and provide useful information for future research.The cellular distribution of phosphorylative and total c-Jun was analyzed by immunofluorescence assay.The expression levels and activity of MAPK,Akt,p53 and c-Jun were determined by Western blot.p53 molecular/chemical inhibitors as well as dominant negative mutants of MAPK and PI-3K pathway were applied to detect the upstream or downstream relationship of related signaling molecules.Further,we established a stably transfectant expressing dominant negative mutant of c-Jun (TAM67).Cell proliferation was analyzed by MTT assay.Flow cytometry was employed to detect the distributions of cell cycle,cyclin D1 luciferase activity was determined by Luciferase reporter gene assay.Western blot was performed to analyze the expression levels of cyclin D1,pRB and E2F1.Results are as followed:1) Exposure of HELF cells to B(a)P caused c-Jun activation,and increased the activity of MAPK,PI-3K,p53 and cyclin D1 pathway.2) B(a)P-induced c-Jun activation was inhibited by dominant negative mutants of extracellular signal-regulated protein kinase(ERK) or c-Jun NH₂-terminal kinase (JNK),but not by p38,impling that JNK and ERK pathways medicate c-Jun activation induced by B(a)P.3) Overexpression of dominant-negative mutants PI-3K and Akt potently blocked phosphorylations of c-Jun and ERK,but not JNK in response to B(a)P,suggesting that PI-3K/Akt pathway positively regulates B(a)P-induced c-Jun activation through ERK.4) Inhibition of p53 by its chemical or molecular inhibitor markedly increased the phosphorylation levels of c-Jun,Akt and ERK upon B(a)P stimulation,indicating that p53 negatively medicates B(a)P-induced c-Jun activation through PI-3K/Akt/ERK pathway.5) The cell lines expressed TAM67 exhibits no significant affecting normal cell growth properties.6) TAM67 was able to significantly block G₁-S transition and subsequent cell proliferation,suggesting that c-Jun is essential for cell cycle alternations elicited by B(a)P.7) Overexpression of TAM67 impaired B(a)P-induced cyclin D1 activation,decreasing expression of E2F1 and pRb,indicating that c-Jun participates in the modulation of B(a)P-induced activation of cyclin D1/pRb/E2F1 pathway.8) Stably expression of TAM67 led to the increases in the expression levels of p53 and p21,elevating phosphorylation level of p53,clearly indicating that c-Jun regulates p53/p21 pathway activation induced by B(a)RCollectively,PI3K/Akt/ERK pathway mediated B(a)P-induced c-Jun activation through p53-dependent mechanism.Activation of c-Jun modulates B(a)P-induced cell cycle alternations through cyclin D1/Rb/E2F1 and p53/p21 signaling pathways.These findings will help us to understand the signal transduction mechanisms involved in the carcinogenic effects of B(a)P at the cell cycle level.