Abstract
We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.
Highlights
Acute renal failure, caused by either ischemia/reperfusion injury or exposure to nephrotoxic xenobiotics, is still an important clinical problem
Activation of Stress Kinases p38 and Jun N-terminal kinases (JNK) Precedes Nephrotoxicant-induced Apoptosis of LLC-PK1 Cells—To investigate the activation of p38 and JNK in relation to chemically induced injury of renal proximal tubular epithelial cells, LLC-PK1 cells were treated with the model nephrotoxicant DCVC that causes apoptosis of renal epithelial cells in a -lyase-dependent manner [22, 34]
Because amino acids present in complete medium are known to compete for uptake of DCVC and to alter the kinetics of DCVC-induced apoptosis [40], we compared the cytotoxicity of DCVC in both culture medium and our standard buffer for toxicant treatment, Hanks’/HEPES
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, penicillin/streptomycin, PBS, and trypsin/EDTA were from Invitrogen. Fresh cell lysates were labeled for 30 min on ice, in the dark, with N-hydroxysuccinimide ester derivatives of the cyanine dyes Cy-3 and Cy-5 (4 pmol/g protein) for DIGE analysis. Protein Separation by Two-dimensional Gel Electrophoresis, Gel Imaging, and Two-dimensional Blotting—For isoelectric focusing, immobilized pH gradient strips pH 3–10 (GE Healthcare) were rehydrated with the cyanine dye-labeled samples at room temperature in the dark overnight. Matched spots from triplicate gels, which showed a statistically significant difference (p Ͻ 0.05) in spot volume after exposure of the cells to DCVC, had a Ͼ1.5 average fold difference in spot volume and could be detected by SyproRuby post-staining, were selected and excised from the DIGE gel using a Syprot spot picker (GE Healthcare). Bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.4 kDa)
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