Cloning And Identification Of New TPA Response Element-binding Proteins Of NGAL Gene In Esophageal Carcinoma Cells

Recently,many researches suggested that neutrophil gelatinase-associated lipocalin(NGAL) gene,which was up-expressed in numerous human cancers such as colon cancer and breast cancer,was an important tumor related gene.Our previous studies have shown that NGAL gene was over-expressed in the progression of human esophageal squamous cell carcinoma cells and a novel 12-O-tetradecanoyl phorbol-13-acetate(TPA) Response Elements(TRE) was found on the -152～- 60 position of the 5' flanking region of the promoter.However,the nucleic proteins bound to this element have not been identified.For this purpose,we have purified the TRE binding-proteins from EC109 cells following TPA induction by Oligo-nucleotide trapping DNA affinity chromatograph and MALDI-TOF-MS.The aim of this study is to further identify the TRE-binding proteins and related signal transduction pathways which were involved in the regulation of TPA-induced NGAL expression by using the molecular cloning technology, RT-PCR,Dual-Luciferase Reporter Assay System,Western blotting and bioinformatics.First,we analyzed the results of the MALDI-TOF-MS according to analysis method of biological data,and eight proteins(C19,KIAA1949,TDRD1,RXRβ,FAM54A,KLF15,KLF10 and YY-1) were identified according to their function,localization and molecular mass.Then, RT-PCR was performed to determine the TPA responsiveness of these nucleic proteins.Results showed that C19,KIAA1949,TDRD1,RXRβand KLF15 proteins have a obvious TPA responsiveness in the transcription level,indicating that these proteins might respond the stimulation of TPA and regulate the expression of NGAL in EC 109 cells.To further explore the relationship between the selected TRE-binding proteins and new TPA Response Elements,the full-length of C19,KLF10,KLF15,KIAA1949 and RXRβwere amplified from the esophageal cancer cell line EC109 by using RT-PCR.The PCR products were sequentially subcloned into pcDNA3 vector and a serious of pcDNA3 expression vectors were constructed.Then EC109 cells were transfected with these expression plasmids,and cell lines with high expression of C19,KLF10,KLF15,KIAA1949 and RXRβwere acquired. Finally,Dual-Luciferase Reporter Assay System(DLR) was performed and results demonstrated that the promoter activity of NGAL gene was significantly increased by C19,KLF10,KLF15, KIAA1949 and RXRβ.Then,some experiments were performed to verify the signal transduction pathways for regulation of NGAL gene.First,the NGAL protein expression levels in EC109 cells treated by TPA were detected by Western blotting.We found that the TPA-induced NGAL expression showed a time-dependent manner and the NGAL expression level was the most strong after being induced by TPA for 12 hours.This result furtherly confirmed that NGAL expression had TPA responsiveness.Second,we study the signal pathway by the use of with PKC (Myristoylated protern kinase C peptide Inhibitor) and mitogen-activated protein kinases (MAPK) specific inhibitors(MEK inhibitor U0126 and PD98059,SB203580,JNK Inhibitor SP600125).Results of DLR showed that,1) the promoter activity of NGAL gene was mainly mediated by MEK pathway;2) PKC inhibitor had no effect on the activity of NGAL promoter. Moreover,the MAPK signal pathway related proteins were detected by Western blotting.We found that,1) The expression of ERK1/2 were not associated with TPA,but p-ERK1/2 varied with treatment time of TPA.When EC109 cells were treated for 12 hours,the protein signal was the most strong;2) The p-ERK1/2 were obviously inhibited by U0126 in a time-dependent mode, but the ERK1/2 level did not changed;3) The p-JNK was not affected by JNK Inhibitor SP600125.We proposed that MEK specific inhibitor may block TPA signal from passing to the next kinase cascades,and then decrease the NGAL gene expression.In addition,to further investigate whether TRE-binding proteins were the target genes of MEK→ERK signal transduction pathway,we detected the expression levels of these genes in the U0126 and PD98059 treated EC109 cells.Results of RT-PCR showed that their mRNA expressions were not affect by inhibitor.These revealed that the expression of those above-mentioned genes were associated with other signal transduction pathway.Finally,we found there are some serine,threonine,tyrosine phosphorylation sites in C 19, RXRβ,KLF10,KLF15 and KIAA1949 proteins after prediction by analysis method of NetPhos 2.0 Server,indicating that these proteins may regulate NGAL gene expression by the change of phosphorylation pattern.In conclusion,our results suggested C19,KLF10,KLF15,KIAA1949 and RXRβmay respond to the stimulation of TPA and regulate the expression of NGAL in EC 109 cells,and the phosphorylation of TRE-binding proteins mediated by MEK→ERK signal transduction pathway might play a major role in the TPA-induced NGAL gene expression.This study will be helpful to approve that NGAL is a new target gene for TPA induction,illustrate the signal transduction pathway induced by TPA,reveal the molecular regulation mechanism of NGAL gene in Esophageal squamous cell carcinoma.