ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype

Cell Mol Biol Lett. 2017 Apr 4:22:7. doi: 10.1186/s11658-017-0036-2. eCollection 2017.

Abstract

Background: Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype.

Methods: Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype.

Results: To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21Cip p53 independent; and 4) downregulation of lamin A/C.

Conclusions: These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

Keywords: ABCC6; Cell cycle; Reductive stress; Senescence.

MeSH terms

  • Cellular Senescence / genetics*
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • G1 Phase Cell Cycle Checkpoints*
  • Gene Knockout Techniques
  • Hep G2 Cells
  • Humans
  • Multidrug Resistance-Associated Proteins / genetics*
  • Oxidative Stress*
  • Pseudoxanthoma Elasticum / genetics
  • Pseudoxanthoma Elasticum / metabolism*
  • Pseudoxanthoma Elasticum / physiopathology
  • Up-Regulation

Substances

  • ABCC6 protein, human
  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Multidrug Resistance-Associated Proteins