Activity Of The Human Arginase-1 Promoter In Liver-derived Cells Is Dependent On CCAAT/enhancer-binding Protein Beta

Objective: The Arginase-1(Arg1) gene in live-derived cell lines plays an important role in urea cycle. In recent years, studies have shown that the liver-enriched transcription factors(LETFs) play important roles in the regulation of liver-enriched gene in transcription level. However, the study on the regulation of Arg1 gene expression in liver cells is rarely reported. In this study, we investigated the molecular mechanisms that LETFs affect the expression of Arg1, aim to providing a suitable target for the construction of a new type of liver cell with better solution of ammonia detoxification.Methods: Full length(-2050 nt to +28nt) and a series of 5'-deletion plasmid of the Arg1 promoter were constructed. The promoter activity was detected by dual-luciferase reporter assay and the crucial region was determined. TFSEARCH and TFBIND programs were used to predict potential transcription factors in the promoter. Site-directed mutants of the C/EBP and HNF3? binding sites were then generated to confirm their regulation to Arg1 gene. At the mean time, western blot and RT-q PCR were adopted to detect the change of m RNA and protein level of Arg1 gene caused by overexpression and knockdown of C/EBP?. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation assay were performed to demonstrate that the transcription factor binding to the Arg1 promoter specifically. In addition, the expression of m RNA and protein of Arg1 and promoter activity were detected respectively in liver and non-liver derived cell lines.Results: Dual-luciferase reporter assay revealed that the-160 nt to +28nt region remained the full activity of Arg1 promoter. Two live-enriched transcription factors including C/EBP and HNF3? were identified in this region by TFSEARCH and TFBIND. Furthermore, the C/EBP and HNF3? mutation constructs p GL4B-160-HNF3?mut and p GL4B-160-C/EBPmut were established and luciferase activity was then detected. The result demonstrated that the luciferase activity of pGL4B-160-C/EBPmut was reduced, but that of the p GL4B-160-HNF3?mut was unaffected. Hep G2 cells were co-transfected with Arg1 promoter and pc DNA3.1-C/EBP?, pc DNA3.1-C/EBP? plasmid, the increasement of Arg1 activity was observed in overexpression of C/EBP? but not in C/EBP?. Western blot and RT-q PCR were performed respectively to detect the effect of pc DNA3.1-C/EBP? overexpression vector and C/EBP?-si RNA in protein and m RNA level. The expression of Arg1 protein was increased with the induction of the Arg1 transcription when C/EBP? was overexpressed, in contrast, it was reduced when C/EBP? was interfered. After that, EMSA and CHIP assay confirmed that Arg1 gene was regulated by C/EBP? in a combination pattern. In addition, both the m RNA and protein levels of Arg1 paralleled with the promoter activities were dramatically higher in liver-derived Hep G2 and Huh7 cells compared with AGS cells from stomach and 293 A cells from kidney.Conclusion: The Arg1 promoter displayed highest activities in liver-derived cell lines. C/EBP? is a specific transcription factor in the up-regulation of Arg1 expression. Furthermore, Arg1 is involved in the urea cycle, targeting C/EBP? may contribute to provide a suitable target for the construction of a new type of liver cell with better solution of ammonia detoxification.