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PLoS One
2015 Jan 01;103:e0119020. doi: 10.1371/journal.pone.0119020.
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Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.
Silva RA
,
Palladino MV
,
Cavalheiro RP
,
Machado D
,
Cruz BL
,
Paredes-Gamero EJ
,
Gomes-Marcondes MC
,
Zambuzzi WF
,
Vasques L
,
Nader HB
,
Souza AC
,
Justo GZ
.
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Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.
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Fig 1. Sorbitol cytotoxicity in HaCaT cells involves induction of apoptosis.
(A) Confluent layers of HaCaT cells were challenged with different concentrations of sorbitol (0.2–2 M in serum-free culture medium) for 2 h, and cellular viability was assessed by three different endpoints assays [MTT reduction (MTT), neutral red uptake (NRU) and nucleic acid content (NAC)]. The results were expressed as percentage of control cell viability (100%) and represented as mean ± SD of three independent experiments run in quadruplicate. HaCaT cells exposed to 1 M sorbitol for 2 h exhibited typical signs of apoptosis. (B) Increased fluorescence intensity of the large fragment (17/19 kDa) of activated caspase-3 detected by flow cytometry in stressed (open histogram) relative to control (black histogram) cells. One representative histogram of three independent experiments for each sample is presented. (C) Increased levels of the small fragment of cleaved PARP-1 by immunoblotting. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. (D) Increased phosphatidylserine exposition on the extracellular face of the membranes of stressed compared to control cells, as shown by confocal microscopy analysis of membrane labeled with WGA594 (red) and phosphatidylserine exposure labeled with annexin V-APC (blue). Images are representative of three independent experiments at the end of treatment (2 h). Bars = 20 μm. (E) Time-dependent increase in the relative fluorescence intensity of annexin V/WGA was quantified from temporal images captured with intervals of 10 min in experiment depicted in (D), using the Examiner 4.2 software. The results were expressed as mean ± standard deviation of the relative fluorescence intensities of each cell per field of view. At least three different fields were analyzed in three independent experiments.
Fig 2. Effects of hyperosmotic stress on regulatory enzymes associated with cytoskeletal remodeling.HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin or GAPDH was used as loading control. (A) Hyperosmotic stress induces cofilin phosphorylation and PP2A inhibition. To check for total cofilin expression and equality of protein loading, antibodies against cofilin and β-actin were used. (B) Densitometric analysis of the hyperosmolarity-induced cofilin phosphorylation. Data are expressed as phospho-cofilin/cofilin ratio normalized to the protein ratio of controls (1). (C) Hyperosmotic stress causes significant increases in the expression of RhoA and Rac-1, while levels of PKAα remained unchanged. (D) Hyperosmotic stress is associated with STAT5 activation. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSTAT5/STAT5 ratios normalized to the protein ratio of controls (1). (F) Sorbitol promotes inactivation of Src kinase. Despite the increase in protein levels of total Src kinase, a significant decrease in the phosphorylation of Y416 located on the activation loop of the kinase, and an increase of Y527, which corresponds to its inhibitory site, occur. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSrc/total Src ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. *P < 0.05 and **P < 0.001 compared with control.
Fig 3. LMWPTP is markedly activated in stressed keratinocytes.HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed. (A) Hyperosmotic stress increases the activity of immunoprecipitated LMWPTP (IP-LMWPTP). *P < 0.001 compared with control. (B) LMWPTP was immunoprecipitated and an anti-phosphotyrosine immunoblotting was performed. The blot was then stripped and reprobed with anti-LMWPTP antibody for normalization by densitometric analysis. (C) Hyperosmolarity increases the relative phosphorylated/non-phosphorylated LMWPTP ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. *P < 0.001 compared with control. (D) Sorbitol does not affect LMWPTP expression. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with anti-LMWPTP antibody. β-actin was used as loading control. One representative immunoblot of three independent experiments is presented.
Fig 4. Changes in the cellular redox status induced by hyperosmotic stress.
(A) Hyperosmotic stress dramatically increases GSH concentration in HaCaT cells, especially considering the reduced number of viable cells after stress. Viability was determined by the trypan blue exclusion method. All the results were expressed as percentage of control (100%) and represented as mean ± SD of three independent experiments run in triplicate. *P < 0.05 compared with control. (B) Hyperosmotic stress causes significant increases in the expression of glutathione peroxidase (GPX) and glutathione reductase (GR), while levels of catalase remained unchanged. After hyperosmotic stress, cells were harvested and lysed. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin was used as loading control. (C) The influence of hyperomostic stress on the malondialdehyde content (nM.ug prot.-1) and (D) glutathione-S-transferase and catalase activity (nmol.ug prot.-1.min-1). Results were represented as mean ± standard deviation of three independent experiments. *P < 0.05 compared with control.
Fig 5. Effect of LMWPTP knockdown with siACP1-CV on expression/activity of Rac-1, Src, FAK and STAT5.HaCaT cells were transfected with siACP1-CV 40 nM, exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. GAPDH was used as loading control. The effects of ACP1 knockdown in Rac-1, Src, FAK and STAT5 proteins were evaluated in HaCaT cells before and after exposition to sorbitol-induced stress (A). (B-E) Densitometric analysis of the results normalized to the protein content of LMWPTP-expressing cells (value equal to 1) comparing stressed and non-stressed cells. Results were represented as mean ± standard deviation of three independent experiments. *P < 0.001 compared with control.
Fig 6. Rac-1 and F-actin staining in LMWPTP silenced HaCaT cells.HaCaT cells were transfected with siACP1-CV 40 nM and exposed to 1 M sorbitol for 2 h. Rac-1 distribution and the organization of the actin filaments (F-actin) were evaluated by laser confocal microscopy after incubation of the cells with specific antibody for Rac-1, followed by staining with Alexa Fluor 594 goat anti-rabbit IgG antibody (red), and Alexa Fluor 488-conjugated phalloidin (green). The nuclei were stained with DAPI (blue). Bar = 20 μm. Representative results of 3 independent experiments.
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