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. 2017 Dec;21(12):3214-3223.
doi: 10.1111/jcmm.13225. Epub 2017 May 30.

Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism

Yi Wang  1 , Le Chang  1 , Jiahui Zhai  1 , Qiao Wu  2 , Donggen Wang  1 , Yunfang Wang  1
Affiliations

Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism

Yi Wang et al. J Cell Mol Med. 2017 Dec.

Abstract

Both primary hepatocytes and stem cells-derived hepatocyte-like cells (HLCs) are major sources for bioartificial liver (BAL). Maintenance of hepatocellular functions and induction of functional maturity of HLCs are critical for BAL's support effect. It remains difficult to assess and improve detoxification functions inherent to hepatocytes, including ammonia clearance. Here, we aim to assess ammonia metabolism and identify ammonia detoxification enhancer by developing an imaging strategy. In hepatoma cell line HepG2, and immortalized hepatic cell line LO2, carbamoyl phosphate synthetase 1 (CPS1) gene, the first enzyme of ammonia-eliminating urea cycle, was labelled with fluorescence protein via CRISPR/Cas9 system. With the reporter-based screening approach, cellular detoxification enhancers were selected among a collection of 182 small molecules. In both CPS1 reporter cell lines, the fluorescence intensity is positively correlated with cellular CPS1 mRNA expression, ammonia elimination and secreted urea, and reflected ammonia detoxification in a dose-dependent manner. Surprisingly, high-level CPS1 reporter clones also reserved many other critical hepatocellular functions, for example albumin secretion and cytochrome 450 metabolic functions. Sodium phenylbutyrate and resveratrol were identified to enhance metabolism-related gene expression and liver-enriched transcription factors C/EBPα, HNF4α. In conclusion, the CPS1-reporter system provides an economic and effective platform for assessment of cellular metabolic function and high-throughput identification of chemical compounds that improve detoxification activities in hepatic lineage cells.

Keywords: Ammonia; CRISPR/CAS9; Carbamoyl phosphate synthetase 1; Hepatocellular functions; Resveratrol; Sodium phenylbutyrate.

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Figures

Figure 1
Figure 1
Targeting of tdTomato fluorescence gene into CPS1 loci in liver cell line. (A) Diagram of the knockin strategy. (B) CPS1 expression in original liver cell lines and general fluorescent imaging of reporter cells HepG2‐CPS1‐tdTm (HCT) and LO2‐CPS1‐tdTm (LCT) derived from HepG2 and LO2 cells. Scale bar: 100 μm. (C) Fluorescence histograms of reporter cells HCT and LCT using flow cytometry analysis. P3→P5: subpopulation of reporter cells with decreasing intensity. (D) Identification of the reporter clones with the primer set a+b. To facilitate the description of these clones, each clone was named ‘parent cell name‐intensity‐clone number'. For instance, ‘HM1' stands for HepG2 derived clone 1 with medium intensity. (E) Colocalization of tdTomato fluorescence signal and mitochondria indicated with MitoTracker® Green FM® in reporter cells. (F) Colocalization of tdTomato fluorescence signal and CPS1 protein detected with immunostaining in reporter cells. Scale bar: 50 μm.
Figure 2
Figure 2
Characterization and quantization of CPS1‐tdTomato reporter liver cell clones. (A) qRT‐PCR analysis of different CPS1‐tdTomato reporter clones derived from HepG2 and LO2. Relative mRNA levels of each gene in HH2 were shown in the right panel as a bar histogram. (B) Quantitative analysis of human albumin secreted by each clone over 24 hrs. (C) CYP3A4 activity from each clone detected by P450‐Glo™ assays.
Figure 3
Figure 3
Correlation among fluorescent intensity, CPS1 expression, ammonia elimination and synthesize urea in liver cells. (A‐E) Cells of different HCT and LCT clones were seeded into 96‐well clear bottom black plate. Cell images were captured and fluorescent intensity of each cell was analysed with GE 2000 automated high‐content cell imaging system. The cells from each clone were collected for analysis of CPS1 mRNA level. The supernatant were collected for ammonia elimination and urea concentration determination. Each point accounts for cells from one clone. The continuous line represents the least square fit linear regression, and the related goodness of fit coefficient (R 2) is indicated on each graph. (F) Representative image of HepG2 reporter cells M4 transfected with different siRNA targeting CPS1.Scale bar: 50 μm.
Figure 4
Figure 4
Small compounds screening targeting CPS1 regulation with reporter cells. (A) A schematic of the chemical screening platform. (B) A plot indicating the results from assays on 180 small molecules screened for their activity of CPS1 regulation. Highlighted dots filled with red were labelled as representative compounds that showed increase of CPS1 expression. Highlighted dots filled with black were labelled as representative compounds suppressing CPS1 expression. The dotted line showed the signal intensity of control.
Figure 5
Figure 5
The dosage effects of NaPB, resveratrol and NaB on cellular ammonia metabolism. (A‐C) The dosage effects of NaPB and resveratrol on CPS1 expression, ammonia elimination, urea concentration, and cell viability as assessed by cell counting, of HCT and LCT cells 24 hrs after the addition of different doses of NaPB and resveratrol.(D) Ammonia metabolism in HCT and LCT cells treated with NaB and NaPB for 24 hrs. The concentration of compounds was 2 μM for HCT cells, 4 μM for LCT cells. ***P < 0.001.
Figure 6
Figure 6
Improved functional gene expression and transcription factors by NaPB and resveratrol in liver cell lines. (A‐B) qRT‐PCR analysis of representative functional genes and transcription factors in HCT and LCT cells treated with different doses of NaPB and resveratrol.
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