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Epidermal Growth Factor (EGF) Suppresses Chaperone‐Mediated Autophagy (CMA) Via FoxO1 Transcription Factors in Kidney Cells
Author(s) -
Franch Harold A.,
Ding Changlin,
Zoromsky Sara
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.906.1
Subject(s) - foxo1 , protein kinase b , epidermal growth factor , chemistry , transcription factor , luciferase , growth factor , phosphorylation , endocrinology , medicine , microbiology and biotechnology , biology , receptor , transfection , biochemistry , gene
Diabetic kidney growth suppresses CMA via Akt, but oxidative stress (OS), which increases CMA, also increases Akt signaling. To examine how Akt suppresses CMA when OS activates Akt, we looked at isoform differences in EGF and OS Akt‐mediated phosphorylation (P) of FoxOs 1,3a, and 4. At 48 hrs in NRK‐52E tubular cells, 10 nM EGF reduced proteolysis, increased CMA marker protein pax2 (2.7 fold), and decreased the CMA receptor, LAMP2a (54%, p<0.05, n=4), consistent with reduced CMA. OS (H 2 O 2 100μM) did not reduce proteolysis, increase pax2, or decrease LAMP2a. EGF and OS both increased Akt P equally and, using a FoxO response element luciferase reporter, reduced FoxO transcription. Akt regulates FoxO abundance and thus activity by P on T and S sites. Using phospho‐specific antibodies, EGF for one hour induced FOXO1 T34 P(255%, p<0.05) and FoxO3a T24 P(>10‐fold), while OS had little effect (40% & −4%, NS, n=3). OS increased FoxO4 S193 P much more than EGF(>10 fold vs 220% p<0.05). EGF decreased FoxO1 and FoxO4 abundance by 40% and 31% (p<0.05) at one hour, but did not change FoxO3a. OS decreased FoxO4 abundance by 69% (p<0.05), but not FoxO3a or FoxO1. Adenoviral expression of constitutively active (CA) FoxO1 activated FoxO luciferase 3‐fold, reversed EGF‐induced Pax2 and GAPDH accumulation, and increased the expression of the LAMP2a consistent with increased CMA. CA FoxO1 accelerated lysosomal proteolysis by 21 & −4% (n=12, p<0.01) and FoxO1 DN reduced proteolysis. siRNA against FoxO1 or 3a reduced these proteins by >60% or >80% and FoxO luciferase activity 50% or 20% respectively. EGF or FoxO1 siRNA, but not OS, scrambled or FoxO3a siRNA, reduced CMA as measured by peri‐nuclear LAMP2a aggregates. In conclusion, EGF reduces FoxO1 abundance which regulates CMA, while OS reduces FoxO3a and FoxO4 abundance. FoxO isoform specificity may allow for growth factor suppression of CMA in the presence of OS during diabetes.