ERK and p38 MAPK inhibition controls NF-E2 degradation and profibrotic signaling in renal proximal tubule cells

Abstract

Aims: Transforming growth factor-β (TGF-β) mediates fibrotic manifestations of diabetic nephropathy. We demonstrated proteasomal degradation of anti-fibrotic protein, nuclear factor-erythroid derived 2 (NF-E2), in TGF-β treated human renal proximal tubule (HK-11) cells and in diabetic mouse kidneys. The current study examined the role of mitogen-activated protein kinase (MAPK) pathways in mediating NF-E2 proteasomal degradation and stimulating profibrotic signaling in HK-11 cells.

Main methods: HK-11 cells were pretreated with vehicle or appropriate proteasome and MAPK inhibitors, MG132 (0.5 μM), SB203580 (1 μM), PD98059 (25 μM) and SP600125 (10 μM), respectively, followed by treatment with/without TGF-β (10 ng/ml, 24 h). Cell lysates and kidney homogenates from FVB and OVE26 mice treated with/without MG132 were immunoblotted with appropriate antibodies. pUse vector and pUse-NF-E2 cDNA were transfected in HK-11 cells and effects of TGF-β on JNK MAPK phosphorylation (pJNK) was examined.

Key findings: We demonstrated activation of p38, ERK, and JNK MAPK pathways in TGF-β treated HK-11 cells. Dual p38 and ERK MAPK blockade prevented TGF-β-induced pSer⁸²Hsp27, fibronectin and connective tissue growth factor (CTGF) expression while preserving NF-E2 expression. Blockade of JNK MAPK inhibited TGF-β-induced CTGF expression without preserving NF-E2 expression. MG132 treatment prevented TGF-β-induced pJNK in HK-11 cells and in type 1 diabetic OVE26 mouse kidneys, demonstrating that TGF-β- and diabetes-induced pJNK occurs downstream of proteasome activation. A direct role for NF-E2 in modulating pJNK activation was demonstrated by NF-E2 over-expression.

Significance: ERK and p38 MAPK promotes NF-E2 proteasomal degradation while proteasome activation promotes pJNK and profibrotic signaling in renal proximal tubule cells.

Keywords: Diabetic nephropathy; Nuclear factor-erythroid derived 2; Renal fibrosis; Transforming growth factor-β; Type 1 diabetes.