Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways
John Cijiang He, … , Ravi Iyengar, Paul E. Klotman
John Cijiang He, … , Ravi Iyengar, Paul E. Klotman
Published September 1, 2004
Citation Information: J Clin Invest. 2004;114(5):643-651. https://doi.org/10.1172/JCI21004.
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Article Nephrology

Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways

Abstract

In collapsing focal segmental glomerulosclerosis (FSGS) of HIV-associated nephropathy (HIVAN), podocytes exhibit a high proliferation rate and loss of differentiation markers. We have found previously that the nef gene of HIV-1 is responsible for these changes. Here, we investigated the signaling pathways induced by Nef and its role in the pathogenesis of HIVAN. Using conditionally immortalized podocytes after differentiation, we found that infection of podocytes with nef increased Src kinase activity and signal transducer and activator of transcription 3 (Stat3) phosphorylation and activated the Ras–c-Raf–MAPK1,2 pathway. A dominant negative mutant of Src abolished the Nef effect, whereas inhibition of MAPK1,2 or dominant negative Stat3 reduced Nef effects partially. Reducing the expression of Nef with small interference RNA reversed the Nef effect. Mutation of Nef in the PxxP or R105R106 motifs diminished Nef signaling and the phenotypic changes in podocytes. Both phospho-MAPK1,2 and phospho-Stat3 staining increased in podocytes of kidneys from HIV-1 transgenic mice compared with their littermates and in podocytes of kidneys from HIVAN patients compared with HIV patients with non-HIVAN kidney diseases or non-HIV patients with idiopathic FSGS, classic FSGS, or minimal-change disease. These data suggest that Nef-induced activation of Stat3 and Ras-MAPK1,2 via Src-dependent pathways is responsible for podocyte proliferation and dedifferentiation, a characteristic finding in collapsing FSGS of HIVAN.

Authors

John Cijiang He, Mohammad Husain, Masaaki Sunamoto, Vivette D. D’Agati, Mary E. Klotman, Ravi Iyengar, Paul E. Klotman

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Nef activates the Src-Stat3 pathway. Podocytes were infected with nef-co...
Nef activates the Src-Stat3 pathway. Podocytes were infected with nef-containing vector (Nef) or control vectors (V) as described, and differentiation was induced at 37°C on collagen I for 10 days. (A) Src kinase activity was determined by an in vitro phosphorylation assay as described in Methods. Densitometric data: Src activity/Src protein ratio: V = 0.15 ± 0.02, Nef = 0.45 ± 0.05, P < 0.05, n = 3. (B) Both phosphorylated and total Src and Stat3 were detected by Western blot as described in Methods. Densitometric data: phospho-Src/total Src ratio: V = 0.13 ± 0.02, Nef = 0.40 ± 0.04, V + PP2 = 0.14 ± 0.05, Nef + PP2 = 0.22 ± 0.07, P < 0.05 when Nef is compared with Nef + PP2, n = 4; phospho-Stat3/total Stat3 ratio: V = 0.24 ± 0.05, Nef = 0.65 ± 0.07, V + PP2 = 0.14 ± 0.06, Nef + PP2 = 0.18 ± 0.07, P < 0.01 when Nef is compared with V or with Nef + PP2, n = 4. (C) Phosphorylation of Hck and Fyn was also detected using the same method as that described for Src. Densitometric data: phospho-Hck/total Hck ratio: V = 0.93 ± 0.25, Nef = 0.98 ± 0.32; phospho-Fyn/total Fyn ratio: V = 0.94 ± 0.30, Nef = 1.05 ± 0.35; P = NS, n = 3.