Abstract: TH-PO360

Constitutive Akt1 Activation in Renal Tubule Cells Ameliorates TGF-β Induced Oxidative Stress and Pro-Fibrotic Signaling by Preserving NF-E2 Expression

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Jin, Shunying, UNIVERSITY OF LOUISVILLE, LOUISVILLE, Kentucky, United States
  • Gupta, Arushi, University of Louisville, Louisville, Kentucky, United States
  • Rane, Sanjana, University of Louisville, Louisville, Kentucky, United States
  • Korte, Erik, None, Louisville, Kentucky, United States
  • Barati, Michelle T., University of Louisville, Louisville, Kentucky, United States
  • Cai, Lu, University of Louisville, Louisville, Kentucky, United States
  • Merchant, Michael, University of Louisville Medicine, Louisville, Kentucky, United States
  • Rane, Madhavi J., University of Louisville, Louisville, Kentucky, United States
Background

TGF-β-induced renal fibrosis is mediated by p38 and JNK MAPK activation. Preliminary laboratory studies found TGF-β (10 ng/ml; 24 h) treatment of human renal proximal tubule (HK-11) cells decreased Akt1 activation and Nuclear Factor-Erythroid derived 2 (NF-E2) protein expression and increased CTGF and PAI-1 expression. Current studies identified signaling mechanisms by which TGF-β regulates NF-E2 and renal fibrosis which may lead to generation of new therapies.

Methods

In vitro Akt kinase assay was performed using active Akt, recombinant NF-E2 and γ-32P-ATP. Real-time NF-E2 PCR was performed on total RNA from control and TGF-β treated HK-11 cells. HK-11 cells were transfected with pUse vector or pUse-NF-E2 or pUse-AktCA (c-myc tagged), or scrambled or NF-E2 siRNA and treated with TGF-β for 24 h. Cell lysates were subjected to immunoblotting for pp38 MAPK, cleaved caspase-3, pJNK, NF-E2, CTGF, PAI-1, c-myc, and GAPDH antisera. Additionally, HK-11 cells were pre-treated (1 h) with DMSO or p38 MAPK inhibitor, 3 µM SB203580, or transfected with pUse or pUse-NF-E2 or pUse-AktCA followed by treatment with/without TGF-β for 24 h. Following day cell were stained with dihydroethidium (DHE) (10 µg/ml) for 30 m. Cells were washed and viewed by confocal microscopy for superoxide generation.

Results

RT-PCR and immunoblotting demonstrated that NF-E2 transcript was significantly induced after TGF-β treatment for 24 h but protein expression was down-regulated. NF-E2 was identified as an Akt substrate in vitro and over-expression of AktCA preserved NF-E2 expression in presence of TGF-β (24 h). NF-E2 overexpression, AktCA overexpression, or SB203580 inhibited TGF-β-induced superoxide in HK-11 cells. AktCA over-expression inhibited TGF-β-induced pp38 activation, pJNK activation, caspase-3 cleavage, CTGF and PAI-1 expression. NF-E2 over-expression inhibited CTGF and PAI-1 expression while silencing NF-E2 expression significantly increased CTGF expression in presence of TGF-β.

Conclusion

Akt1 substrate NF-E2, serves as a putative negative regulator of oxidative stress and pro-fibrotic signaling and its expression is regulated by post-translational modification in TGF-β treated HK-11 cells.

Funding

  • Other NIH Support