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. 2009 Mar;296(3):C463-75.
doi: 10.1152/ajpcell.00467.2008. Epub 2008 Dec 24.

Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

Ana C P Thirone  1 , Pam Speight, Matthew Zulys, Ori D Rotstein, Katalin Szászi, Stine F Pedersen, András Kapus
Affiliations

Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

Ana C P Thirone et al. Am J Physiol Cell Physiol. 2009 Mar.

Abstract

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

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Figures

Fig. 1
Fig. 1
Hyperosmolarity induces rapid and reversible cofilin phosphorylation in kidney tubular cells. A: confluent layers of LLC-PK1 proximal tubule cells grown in 6-well plates were preincubated in isotonic (Iso) medium for 10 min and then exposed to iso- or hyperosmolarity (Hyper = isotonic medium supplemented with 300 mM sucrose) for the indicated times. Cells were then lysed, and aliquots of cell lysates containing equal amounts of protein were subjected to Western blot analysis using an anti-phospho-cofilin (pcof) antibody. To check for total cofilin expression and the equality of protein loading, the blots were reprobed using antibodies against cofilin (cof) and actin. Blot is not shown for the isotonic full time course. Routinely a 5-min isotonic treatment was used as control (labeled as Iso), since maximal responses (plateau) to hyperosmolarity were usually attained at this time point. Bottom: densitometric analysis of the hyperosmolarity-induced cof phosphorylation. Data are expressed as fold change, normalized to the pcof-to-cof ratio of the isotonic sample as 1. Means ± SE for n = 12 separate experiments are shown. B: reversibility of the osmotically provoked cof phosphorylation. Cells were treated iso- or hypertonically for 5 min as in A and then isotonicity was reestablished for the indicated times (min). Subsequently cells were lysed and processed as in A. C: dependence of the hypertonicity-induced cof phosphorylation on the osmotic concentration of the medium. Osmolarity was set to the indicated values using various concentrations of sucrose added to the isotonic medium. After 10 min preincubation in the isotonic medium, cells were challenged with the corresponding level of osmolarity for 5 min and then processed as in A. Changes in pcof levels are expressed compared with isotonic control. D: Madin-Darby canine kidney (MDCK) distal tubular cells exhibit similar cof phosphorylation upon hyperosmotic treatment. Similar conditions were used as in A. E: hyperosmolarity does not increase cof phosphorylation in Chinese hamster ovary (CHO) cells.
Fig. 2
Fig. 2
The effect of hyperosmolarity on Rac and PAK in LLC-PK1 cells. A: confluent layers of LLC-PK1 cells in 10-cm dishes were challenged with a hypertonic solution for the indicated time. Cells were then lysed and processed for the Rac activity assay as described under MATERIALS AND METHODS. Before the pull-down assay, an aliquot was taken from each lysate to measure total Rac. Note that hyperosmolarity induced a rapid, small, but significant increase in the amount of captured (GTP-bound) Rac, followed by a substantial decrease (n = 4, *P < 0.05 and *** P < 0.001). B: effect of hyperosmolarity on the intracellular localization of Rac. LLC-PK1 cells, grown to confluence on glass coverslips were treated iso- or hypertonically as indicated. Cells were then fixed and stained for Rac using a primary and a Cy-3-labeled secondary antibody. Note that under isotonic condition Rac distribution is primarily cytosolic, exhibiting some vesicular pattern; after exposure to hypertonicity, the most prominent Rac labeling occurs at the cell periphery, whereas the overall cytosolic staining is reduced. We often observed enhanced and polarized Rac labeling in the perinuclear area. C: hyperosmolarity induces transient PAK phosphorylation. Cells were treated as indicated, and cellular lysates were processed for Western blot analysis using a phospho-specific (Ser 142/144) PAK1/2 antibody. Results of densitometric analyses obtained from 3 independent experiments are shown.
Fig. 3
Fig. 3
Constitutively active (CA) Rho and ROCK but not Rac and PAK induce cof phosphorylation in tubular cells. A and B: LLC-PK1 cells grown on coverslips were transfected with one of the indicated constructs (1 μg DNA) encoding for the Myc epitope-tagged constitutively active form of Rho, Rac, ROCK, and PAK. Forty-eight hours later, the cells were briefly washed, fixed, and doubly stained for phosphorylated cof (red) and the Myc epitope (green) to identify the successfully transfected cells. Identical cells on the corresponding images are marked with identical symbols (arrows or asterisks). C: cells grown in 10-cm dishes were transfected with empty vector (Mock), CA-Rho, or CA-Rac and lysed 48 h later. Proteins of the whole cell lysates were separated by SDS-PAGE and subjected to Western blotting using anti-phospho-cof. The blots were reprobed with anti-Rho and anti-Myc antibodies to detect all and endogenous Rho proteins, respectively. Despite the fact that the transfection efficiency was only a few percent (< 10%), a highly significant (P < 0.001) 1.44-fold increase was detected in the phospho-cof content of the CA-Rho-transfected monolayers. This increase corresponds to a 5- to 10-fold increase in the CA-Rho expressing cells. D: cells were transfected with empty vector or Myc-tagged CA-ROCK or CA-PAK1 and then processed as in C. Tubulin was used as loading control. Note that CA-ROCK caused a highly significant increase in the phospho-cof content of the entire cell population (with a few % transfection) originating from a large increase present in the ROCK-expressing cells. CA-PAK failed to increase cof phosphorylation despite the fact that PAK1 expression was stronger than ROCK expression, as verified by the Myc signal.
Fig. 4
Fig. 4
Dominant negative (DN) Rho but not DN-Rac prevents the hypertonicity-induced phosphorylation of cof. Cells were transfected with Myc-tagged DN-Rho (A) or DN-Rac (B) constructs, as described under Fig. 3, treated iso- or hypertonically (as indicated), and stained for phospho-cofilin (pcof, red) and the Myc epitope (green). The presence of cells was visualized by the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) (blue). To help identification of the transfected cells on the red image, the area of the corresponding cell(s) was traced with a dashed line. Note that the expressions of DN-Rho almost completely suppressed the hyperosmolarity-triggered increase in pcof staining, whereas the pcof content of DN-Rac-expressing cells is indistinguishable from their nontransfected neighbors. In DN-Rho expressors, we occasionally observed some pcof labeling in nuclear/perinuclear areas.
Fig. 5
Fig. 5
DN-ROCK but not DN-PAK prevents the hypertonicity-induced phosphorylation of cof. Cells were transfected with Myc-tagged DN-ROCK (A) or DN-PAK1 (B) constructs, treated iso- or hypertonically, and stained for pcof (red) and the Myc epitope (green). *Identical cells on the red and green images. In A the contours of the transfected cell clusters are also shown. Note that DN-ROCK strongly reduces the hypertonically provoked rise in pcof labeling, whereas DN-PAK had no effect.
Fig. 6
Fig. 6
Hyperosmolarity induces ROCK-mediated LIM kinase and cof phosphorylation. A and B: cells grown in 6-well plates were preincubated for 10 min with vehicle or 10 μM Y-27632 in isotonic medium and then challenged with hyperosmolarity (300 mM sucrose) in the presence or absence of Y-27632 for the indicated times. Subsequently cells were lysed and the lysates were subjected to Western blot anaylsis using pcof and cof antibodies. Cumulated data for n = 6. B: similar experiments were performed as in A, and the lysates were probed with phospho-LIMK and LIMK2 antibodies (n = 4). Blotting with anti-LIMK1 did not provide clear, specific labeling (not shown). C: cells were preincubated with vehicle or 10 μM PAK18, a selective PAK inhibitor, for 20 min under isotonoic condition and then challenged with hyperosmolarity for 5 min in the presence or absence of the drug. Cof and pcof content were determined by Western blots. GADPH was used as a loading control. D, top: cells were pretreated in isotonic medium with or without Y-27632, treated iso-or hypertonically for 5 min (as indicated) in the absence or presence of 10 μM Y-27632, and then processed for Western blotting using an anti-phospho-p38 antibody. The blots were reprobed with anti-p38. Bottom: cells were preincubated with 10 μM SB-203580, treated in the absence or presence of the drug, and processed as stated above. Cell lysates were probed for pcof and reprobed for cof. E: similar experiments were performed as in C, and the lysates were probed with anti-p38 and anti-phospho-p38 antibodies.
Fig. 7
Fig. 7
Role of cof and its phosphorylation in the regulation of basal F-actin levels and osmotically induced changes in F-actin content. A: cells were transfected with 20 nM nonrelated small interfering RNA (siNR) or siRNA against cof (siCof) for 48 h as described under MATERIALS METHODS. The efficiency of siCof was evaluated by Western blots, using tubulin as a loading control (n = 9). B: cells grown in 6-well plates were transfected with siNR or siCof, and 48 h later they were serum-deprived, preincubated in isotonic medium for 10 min, and exposed to iso- (I) or hypertonicity (H, 300 mM sucrose) for an additional 5 min. Subsequently, cells were fixed, permeabilized, incubated with 0.33 μM rhodmine-labeled phalloidin for 1 h, and extensively washed. Bound phalloidin (as a measure of total F-actin) was extracted with methanol and quantified fluorimetrically as described under MATERIALS AND METHODS (n = 9). Both visual inspection and parallel protein determinations verified that the confluent layers remained intact during the extraction procedure. C: wild-type (WT) LLC-PK1 cells were incubated with vehicle or 10 μM Y-27632 for 10 min and then challenged with iso- or hypertonic medium (300 mM sucrose) for 5 min. F-actin content was measured as above (n = 5). D: LLC-PK1 cells, stably expressing AA-MLC, a nonphosphorylatable, dominant negative form of the myosin light chain, were treated as in C (n = 6). Bottom: hyperosmolarity induced cof phosphorylation in AA-MLC cells as well is shown. E: expression of AA-MLC inhibits the osmotically triggered phosphorylation of MLC. Wild-type and AA-MLC cells were treated iso- or hypertonically for 5 min. Lysates obtained from these cells were probed with antibodies against Myc (to detect Myc-tagged AA-MLC), phospho-MLC and MLC, as indicated. F: effect of hyperosmolarity and Y-27632 on F-actin content was investigated in ELA cells, under iso- and hypertonic conditions (n = 4).
Fig. 8
Fig. 8
Effect of long-term, strong hyperosmolarity on cofilin expression and phosphorylation. LLC-PK1 cells were exposed to iso-osmolarity or challenged with strong hyperosmolarity (isotonic Na+ medium supplemented with 600 mM sucrose) for 3 h. The levels of total and pcof were detected by Western blotting and are expressed as percentage compared with the isotonic control (n = 5).
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