Phosphoproteomic analysis of FAC overload-triggered human hepatic cells reveals G2/M phase arrest

• Published in: Biochemical and biophysical research communications 2022-09, Vol.619, p.62-67
• Main Authors: Li, Shiwei, Wang, Yingna, Dun, Weipeng, Han, Wanqing, Zhang, Xinxin, Guo, Ning, Wang, Mulian
• Format: Article
• Language: English
• Subjects: 14-3-3 sigma; Cdk1; G2/M arrest; Iron overload; Phosphoproteomics
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2022.06.027

Hepatic iron overload is a universal phenomenon in patients with myelodysplastic syndromes (MDS) who undergo bone marrow transplantation and may experience the toxicity of peri- and post-bone marrow transplantation. To clarify the mechanisms of iron overload-triggered liver injury, we determined the effects of iron overload on changes in protein phosphorylation in human hepatocyte cell line HH4 in vitro by using a phosphoproteomics approach. The hepatocytes were exposed to high concentrations of ferric ammonium citrate (FAC) to build up an iron overload model in vitro. Changes in protein phosphorylation initiated by iron overloading were studied by 2D-LC/MS. We identified 335 differentially expressed phosphorylated proteins under the condition of excess hepatocyte iron, 11% of which were related to cell cycle progression. The results of phosphoproteomics showed that iron overload induced 10.9 times increase in Thr 14/Tyr 15-phosphorylated Cdk1 in HH4 cells. Flow cytometry analysis revealed that FAC-treated HH4 cells showed significant G2/M phase arrest. Our subsequent RT-PCR and Western blot experiments indicated that FAC-induced G2/M phase arrest was related to the activation of p53-p21-Cdk1, p53-14-3-3 sigma-Cdk1, and 14-3-3 gamma pathway. Our findings demonstrate the first evidence that iron overload causes G2/M arrest in HH4 hepatocytes. •The protein levels of 14-3-3 sigma and p-Cdk1 (Tyr15) are up-regulated in FAC-treated HH4 cells.•FAC overload induced HH4 G2/M arrest possibly via activation of p53-p21-CDK1, p53-14-3-3 σ-Cdk1 and 14-3-3 γ pathway.•The p53-p21-CDK1, p53-14-3-3 σ-Cdk1 and 14-3-3 γ pathway may trigger G2/M phase arrest in FAC overload induced HH4 cells.