Heat Shock Protein 27 Is the Major Differentially Phosphorylated Protein Involved in Renal Epithelial Cellular Stress Response and Controls Focal Adhesion Organization and Apoptosis

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 α-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. 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 α-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. Acute renal failure, caused by either ischemia/reperfusion injury or exposure to nephrotoxic xenobiotics, is still an important clinical problem. Renal proximal tubular epithelial cells (PTC) 1The abbreviations used are: PTC, proximal tubular cells; DCVC, 1,2-dichloro-vinyl-l-cysteine; 2D-DIGE, two-dimensional difference gel electrophoresis; Hsp27, heat shock protein 27; FA, focal adhesion; MALDI-TOF, matrix assisted laser desorption ionization-time-of-flight; DMEM, Dulbecco's modified Eagle's medium; c-Jun N-terminal kinase; FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AMC, aminomethylcoumarin; TFEC, S-(1,1,2,2)-tetrafluoroethyl-l-cysteine; DPPD, N,N-9-diphenyl-p-phenylenediamine; ERKs, extracellular signal-regulated kinases; MS, mass spectrometry. are the primary target, and injury to these cells results in either the onset of necrosis or apoptosis, depending on the severity of the injury. There is increasing evidence that sublethal PTC injury results in changes in cell adhesion at the level of cellular to extracellular matrix interactions at focal adhesions (1Molitoris B.A. Marrs J. Am. J. Med. 1999; 106: 583-592Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These interactions are under the tight of changes of the changes in the cellular interactions after sublethal PTC injury are associated with altered is important to the involved in the of the and cell adhesion after sublethal PTC injury. adhesions are the cells and the extracellular adhesions are by a of and These both as and and as focal adhesion and as well as the mitogen-activated protein JNK and These a the cell adhesion and the by the focal adhesion protein is in cellular cell and In in as well as primary renal PTC and renal cell an of focal in the of PTC which of the focal adhesions and a reorganization of the Am. J. Full Text Full Text PDF PubMed Scopus Google Scholar). stress results in activation of these at focal adhesions, the that the in cell adhesions after PTC injury are in of activation of these a of not The of of extracellular signal-regulated which activated of the c-Jun N-terminal and p38 that are to cellular stress as heat and PubMed Scopus Google Scholar). JNK and p38 in the cellular stress in the after renal cell injury. Renal ischemia/reperfusion activation of and J. J. Am. J. Google J. PubMed Scopus Google J. J. Full Text PDF PubMed Google as a of stress J. J. Am. J. Google J. PubMed Scopus Google Scholar). a of the activation of JNK and p38 J. J. Full Text Full Text PDF PubMed Scopus Google J. Am. J. PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google Scholar). In as well as in either or that activation of JNK after PTC injury cell J. PubMed Scopus Google PubMed Scopus Google Scholar). PTC injury that results in the activation of JNK and p38 after an ischemia/reperfusion J. Full Text Full Text PDF PubMed Scopus Google J. Full Text Full Text PDF PubMed Scopus Google under these activation was J. Full Text Full Text PDF PubMed Scopus Google Scholar). Although these data that JNK activation cell the of p38 activation in the of PTC apoptosis is the by which JNK and p38 PTC cell is JNK and p38 and the protein J. J. PubMed Scopus Google J. J. PubMed Scopus Google J. PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google Scholar). JNK and p38 of cellular proteins, which the of the cellular stress JNK the protein cells for apoptosis J. Full Text Full Text PDF PubMed Scopus Google 1999; PubMed Scopus Google Scholar). localization of JNK at focal adhesion in the phosphorylation of as focal adhesion and cell adhesion PubMed Scopus Google Scholar). In p38 the which heat shock as Hsp27. Hsp27 and related are to the as well as cell in cell is as PTC which to the of nephrotoxic J. 1999; Full Text Full Text PDF PubMed Scopus Google Am. J. Google Am. J. PubMed Google PubMed Scopus Google PubMed Scopus Google Scholar). of DCVC an model to of DCVC is by renal proximal tubular cells both in and in is by the renal in the of that cell injury PubMed Scopus Google J. Full Text PDF PubMed Google Scholar). PTC injury is associated with of focal adhesions and cell adhesion, which is by activation and the onset of apoptosis J. 1999; Full Text Full Text PDF PubMed Scopus Google J. J. Am. J. Google Am. J. Google Scholar). to an important in the of DCVC-induced apoptosis, DCVC the of phosphorylation and and the of focal adhesion J. Full Text Full Text PDF PubMed Scopus Google Scholar). JNK and p38 are activated DCVC to the and of these stress in the of cellular of PTC adhesion in to the onset of apoptosis. In the used a two-dimensional difference gel electrophoresis PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google J. J. PubMed Scopus Google and mass to that are either or modified in a after DCVC-induced injury of Hsp27 was as the most prominent early modified protein in the cellular stress after DCVC treatment of renal epithelial cells. is related to a phosphorylation of Hsp27. p38 and the phosphorylation of Hsp27 are in the protection of renal epithelial cells against a reorganization of focal adhesions, activation of and the onset of apoptosis caused by nephrotoxic cellular modified Eagle's PBS, and were The and were by for was was SP600125 and SB203580 were was DCVC was as Google Scholar). and Treatment renal epithelial cell LLC-PK1 cells were maintained in with serum and at in a of and and J. Full Text Full Text PDF PubMed Scopus Google Scholar). of Hsp27 cell LLC-PK1 cells were with of of or to as Am. J. 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In JNK and p38 and the of that by the cell of a stress the early DCVC-induced in used to the changes in the level and of in cell caused by DCVC in renal epithelial cells. used cells that were to DCVC for the protein were of both and LLC-PK1 cells. cells with and DCVC cells with were on the and was in protein were and the of and are as in and in the and gel were and with in a protein and cells at a difference found on were and the data were to changes in protein a difference protein after DCVC treatment of LLC-PK1 of which were were with and were for by mass the protein were a of 9 heat shock protein pyruvate dehydrogenase, cofilin, aconitase and the proteins, Hsp27 was the most protein and was in Hsp27 linked to renal in J. Full Text Full Text PDF PubMed Scopus Google on protein for as well as in the of of the results of difference of and after DCVC of and for the shock protein shock protein shock protein shock protein shock protein of and after DCVC of and for the in a of Hsp27 after DCVC to protein were as Hsp27 the of these two-dimensional for Hsp27 for Hsp27 shock are after exposure to nephrotoxic which is related to activation of heat shock J. Full Text PDF PubMed Google PubMed Scopus Google Scholar). of DCVC was on the level of Hsp27 In heat shock proteins, and were not not Treatment of the LLC-PK1 cells resulted in the activation of the stress p38 and JNK Hsp27 is to on serine and by a of the protein J. J. 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J. Am. J. Google Scholar). Treatment of LLC-PK1 cells with DCVC caused a in of cell adhesion after SB203580 caused a of the of cells that In contrast, SP600125 DCVC-induced cell of inhibition of either p38 or JNK on changes in cell adhesion were in primary proximal tubular cells with DCVC not these data an for p38 not JNK in the of Hsp27 phosphorylation in association with the of cell adhesion after cellular of JNK at in with of the extracellular matrix and the cell adhesion resulted in of focal adhesions the cells and the extracellular are protein that the to the cytoskeletal cytoskeletal proteins, proteins, and are at that a of cell as cell and JNK is associated with and activated cell adhesions PubMed Scopus Google J. 1999; PubMed Scopus Google Scholar). Inhibition of Hsp27 phosphorylation with an of p38 DCVC-induced cell under these JNK was maintained and inhibition of JNK of cell adhesion, that an of JNK at is to involved in the cell caused by not only an in of JNK after DCVC as on data and of at that co-localized with the protein the that the of at was related to the of the with the activated of cells were with a of an of phosphorylation did not JNK phosphorylation or the of at as by and not active JNK is associated with that JNK involved in the reorganization of and the of DCVC inhibition of JNK with SP600125 resulted in association of with and not a for JNK in after cellular stress related to p38 We the of inhibition of JNK and p38 on reorganization after DCVC treatment by the on and DCVC caused a reorganization of as well as after of which the onset of apoptosis and cell is in with in primary proximal tubular cells J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). reorganization was prominent cells were with DCVC in the of a of p38 in the of focal adhesion of SP600125 on the DCVC-induced reorganization of focal adhesions was is an important in the of and which is on phosphorylation at residue DCVC the of residue to onset of apoptosis J. 1999; Full Text Full Text PDF PubMed Scopus Google a of SB203580 and SP600125 on protection against by DCVC was after inhibition of either p38 or JNK not data the association and protein protein with JNK an protein PubMed Scopus Google Scholar). that an important in the activation of JNK and activation of used LLC-PK1 cells that a dominant mutant of that the Overexpression of which for the localization of at focal adhesion J. 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Overexpression of Hsp27 did not the of apoptosis with cells In contrast, of LLC-PK1 cells DCVC apoptosis. determine was related to activation of Although DCVC caused activation of in cells to a as in a in was in cells. in cells was in cell The data that activation of p38 is in the maintenance of focal adhesion after cellular stress and that p38 inhibition apoptosis and Hsp27 is a of the Hsp27 phosphorylation focal adhesion after DCVC the of DCVC on Treatment of and cells resulted in of of as by We the of focal adhesions in these cells. FAK, which at focal adhesions, was at focal adhesion in the cell in cell DCVC caused a reorganization of focal adhesions to the of the and cells; these focal adhesions were still in cells DCVC caused a reorganization of focal localization was and localization was the cell these data an for Hsp27 phosphorylation in the of cellular apoptosis in renal epithelial cells in association with maintenance of matrix adhesion of Hsp27 in heat shock with to of heat shock to changes in the in which are for of the and which as against cellular stress PubMed Scopus Google J. J. 1999; Full Text Full Text PDF PubMed Scopus Google J. 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The most prominent changes after cellular stress in LLC-PK1 cells were in Hsp27, which to the of heat shock that difference in the cellular level of Hsp27 was after DCVC changes in the two-dimensional protein of Hsp27 were to phosphorylation of Hsp27, which a pI shift to the mass phosphorylation of Hsp27 on Although phosphorylation of residue was by both and against did not the Hsp27 that were by Hsp27 is on and in LLC-PK1 which for the in the two-dimensional of the of Hsp27 by phosphorylation, in LLC-PK1 cells of a mutant accelerated the onset of apoptosis caused by DCVC, the of these Hsp27 in the of renal cell Although of Hsp27 cell of LLC-PK1 cells caused by either stress or J. 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Although the of Hsp27 did not and cells the phosphorylation status The Hsp27 in the cells is not of and protection of focal adhesion reorganization in cell of Hsp27 on is by which is activated by In the phosphorylation of Hsp27 was on p38 SB203580 Hsp27 still phosphorylation of Hsp27 to activation of SB203580 not prevent activation of in LLC-PK1 cells J. Am. J. PubMed Scopus Google Scholar). protein activated of DCVC treatment Hsp27 J. Am. J. PubMed Scopus Google Scholar). the by inhibition of JNK with which resulted in of p38 the phosphorylation of Hsp27 the the a of changes in the of p38 activation Hsp27 phosphorylation The and the of protein and Inhibition of p38 with SB203580 did not p38 phosphorylation, which is most to the that the phosphorylation is by protein that are not by Inhibition of p38 with SB203580 accelerated the reorganization of focal adhesions after DCVC was associated with a rapid cell and onset of apoptosis. In a of the Hsp27 phosphorylation mutant in LLC-PK1 cells the reorganization of focal adhesions caused by DCVC as well as the of apoptosis. these data fit with a model whereby cellular stress activation of p38 in phosphorylation of Hsp27, which in against the cellular Although data a for Hsp27 phosphorylation in protection against the reorganization of focal adhesions, of mutant Hsp27 not against a of not the by which Hsp27 focal adhesion that Hsp27 phosphorylation that are for the onset of apoptosis. that Hsp27 apoptosis by maintenance of the the of PubMed Scopus Google Scholar). as a of cell injury the of the that the of apoptosis PubMed Scopus Google a is to Hsp27 Hsp27 which is an for the of the and activation of and PubMed Scopus Google Scholar). DCVC caused the activation of which was associated with focal Inhibition of JNK with SP600125 cell and apoptosis. JNK localization at focal adhesions is J. 1999; PubMed Scopus Google Scholar). Overexpression of in LLC-PK1 cells the localization of at focal adhesions, was an accelerated after DCVC In cells active JNK was associated with focal in cells DCVC caused an c-Jun phosphorylation as well as a Hsp27 phosphorylation, thus a for localization of active JNK is these stress Although inhibition of JNK cell detachment, protection against DCVC-induced focal adhesion reorganization was inhibition of JNK did not focal adhesion reorganization in either or cells not In contrast, SP600125 the maintenance of as by for not a of the at focal adhesions, is a of which related to the of focal In LLC-PK1 is at is that JNK at these adhesion a of to of JNK these Although data a of cell adhesion and the onset of apoptosis, that DCVC-induced JNK activation results in the phosphorylation of cellular that the onset of apoptosis. resulted in the of that either as of nephrotoxic or as after renal Hsp27 phosphorylation a in the of the onset of cell in renal cells. are to the of the modified in the of We for Hsp27 and for with gel and of the of of the for for and