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. 2017 Sep;21(9):2036-2045.
doi: 10.1111/jcmm.13123. Epub 2017 Mar 8.

The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta

Zhanfei Chen  1 , Nanhong Tang  1   2 , Xiaoqian Wang  1 , Yanling Chen  1   2
Affiliations

The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta

Zhanfei Chen et al. J Cell Mol Med. 2017 Sep.

Abstract

Carbamoyl phosphate synthase 1 (CPS1) is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver. However, CPS1 epigenetic regulation involves promoter analysis and the role of liver-enriched transcription factors (LETFs), which is not fully elucidated. In this work, the promoter region of hCPS1 gene was cloned, and its activity was investigated. An LETF, hepatocyte nuclear factor 3-beta (HNF3β), was found to promote the transcriptional expression of CPS1 in liver-derived cell lines. In addition, dual-luciferase reporter assay shows that the essential binding sites of the HNF3β may exist in the oligonucleotide -70 nt to +73 nt. Two putative binding sites are available for HNF3β. Mutation analysis results show that the binding site 2 of HNF3β was effective, and the transcriptional activity of CPS1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay (EMSA) and ChIP assay confirmed that HNF3β can interact with the binding site in the CPS1 promoter region of -70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS1. Moreover, HNF3β overexpression enhanced the transcription of CPS1 and consequently improved the mRNA and protein levels of CPS1, whereas the knockdown of HNF3β showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF3β. HNF3β plays a vital role in regulation of CPS1 gene and could promote the metabolism of ammonia by regulating CPS1 expression.

Keywords: ammonia detoxification; carbamoyl phosphate synthetase 1; hepatocyte nuclear factor 3-beta; liver-derived cell; promoter.

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Figures

Figure 1
Figure 1
The regulatory effect of HNF3β on CPS1 promoter transcription. (A) and (B), The plasmid pGL4‐2086 (1 μg) was transfected transiently with LETFs (HNF1α, HNF3β, HNF4α, HNF6, C/EBPα and C/EBPβ) (500 ng), respectively, into the HepG2 and BEL‐7404 cells. Luciferase activities were measured 48 hrs after transfection, and the plasmid pGL4‐2086 served as the negative control. (C), Activity analysis of CPS1 promoter. BEL‐7404 cells were transfected with 1 μg each of the CPS1 promoter constructs (reporter plasmid); 100 ng of the renilla luciferase expression vector pRL‐TK was used for normalization, and the promoterless vector pGL4Basic served as the negative control. Luciferase activities were measured 48 hrs after transfection. (D), 1 μg each of the CPS1 promoter constructs was cotransfected with 500 ng HNF3β, respectively, into the BEL‐7404 cells. In each recombinant plasmid, fluorescent activity of non transfected with HNF3β served as the negative control. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments (*P < 0.05).
Figure 2
Figure 2
Binding sites of HNF3β in the CPS1 promoter. (A), Nucleotide sequence of −70 nt to +73 nt region of CPS1 promoter is shown. The translation initiation site (+1) is indicated by the arrow. The putative HNF3β binding sites in the nucleotide region −38 nt~−27 nt (site 1) and −21 nt~−10 nt (site 2) are underlined. In the binding site 1, TT is mutation into AC, named mut1. Similarly, TA is mutation into AG named mut2 in the binding site 2. (B), Effect of mutation of the HNF3β binding sites on the activity of the CPS1 promoter. HNF3β binding sites were subjected to site‐directed mutagenesis. A total of 1 μg of mutants together with 100 ng of pRL‐TK was cotransfected into HepG2 cells. pGL4Basic served as the negative control. Luciferase activities were measured 48 hrs after transfection. The relative luciferase units (RLU) were obtained (right) by comparison with the wild‐type of pGL4‐70, which was set to 1. (C), Western blot analysis of CPS1 and HNF3β expression in the various cell lines; 30 μg of cellular proteins was used in the Western blot. β‐Actin serves as an internal control. (D), EMSA of HNF3β. Competition assays with unlabelled cold probe at a concentration of 50 (lane 3)‐ or 100 (lane 5)‐fold molar excess over that of the biotin‐labelled probe (lane 2). In the lane 6, supershift assays were carried out with 4 μg specific antibody raised against HNF3β and the quality of poly (dI‐dC) is 0.8 μg. An arrowhead indicates the shift band (SB, left) and the supershift band (SS, right). (E), ChIP assay. Chromatin from HepG2 cells was immunoprecipitated with the anti‐HNF3β. The total extracted DNA (Input DNA, 5%) prior to immunoprecipitation and the immunoprecipitated samples were PCR‐amplified using primers specific to a region that spanned −70 nt to +73 nt (containing the HNF3β binding sites) of the CPS1 promoter. The normal rabbit IgG or no antibody control was also performed for control purpose. Band signals were quantified using the densitometric software, and the relative intensities to the input which was set to 1.00 were calculated. ND: None detected (*P < 0.05).
Figure 3
Figure 3
Transcription regulation of CPS1 promoter by HNF3β. (A), Overexpression of HNF3β enhances CPS1 promoter activities. HepG2 and BEL‐7404 cells were cotransfected with 0.2 μg of pGL4‐70 and increasing amounts (0, 0.5, 1, 1.5 or 2.0 μg) of expression vectors (pFLAG‐HNF3β or empty vector pFLAG‐CMV‐2); 10 ng of pRL‐TK was used to normalize the transfection efficiency. Cells were harvested 48 hrs after transfection. The relative luciferase units (RLU) were obtained by comparison with the pFLAG‐CMV‐2, which was set to 1. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments. (B), Knockdown of endogenous HNF3β decreased CPS1 promoter activity. HepG2 and BEL‐7404 cells were cotransfected with HNF3β siRNA and 0.2 μg of pGL4‐70. At 48 hrs after transfection, the relative luciferase units (RLU) were obtained by comparison with the negative control (NC), which was set to 1. Each transfection was performed in duplicate, and the data were expressed as the mean ± SD of three separate experiments. *P < 0.05 versus NC. (C) and (D), Western blot analysis in BEL‐7404 cells after overexpression of HNF3β or interference of HNF3β siRNA. (C): BEL‐7404 cells were transfected with 0.5 μg of pFLAG‐HNF3β or pFLAG‐CMV‐2 (empty vector); (D): BEL‐7404 cells were treated with 100 pmol of HNF3β siRNA or negative control (NC). Cells were harvested 48 hrs after transfection; 30 μg of cellular proteins was used in the Western blot and β‐actin served as a loading control. (E) and (F), Influences of HNF3β or HNF3β siRNA on CPS1 gene mRNA level in HepG2 cells. Overexpression of HNF3β increased CPS1 gene transcription, HepG2 cells were transfected with 0.5 μg of pFLAG‐HNF3β or empty control pFLAG‐CMV‐2 (E); knockdown of endogenous HNF3β decreased CPS1 gene transcription, HepG2 cells were treated with 100 pmol of HNF3β siRNA or negative control (NC) (F). Cells were harvested 48 hrs after transfection, 3 μg of the total RNA was used to detect the CPS1 mRNA level by real‐time RT‐PCR (*P < 0.05).
Figure 4
Figure 4
Urea production in cells with different concentrations of ammonia. With increasing ammonia concentration, (A) urea production in HepG2 and HepG2/0.25μgHNF3β cells increased. *denotes a significant difference compared to HepG2 (P < 0.05, n = 3). (B) While in BEL‐7404/0.1μgHNF3β and BEL‐7404/0.25μgHNF3β cells, urea production increased at the beginning and decreased later. The median lethal dose (LD50) of NH4Cl in each cell line was different. The cell viability and function gradually declined with increasing ammonia concentration; thus, the amount of urea products decreased. *denotes a significant difference compared to BEL‐7404 cells (P < 0.05, n = 3).
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