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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 - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.

ABSTRACT
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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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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pone.0119020.g006: 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.

Mentions: As demonstrated by Nimnual et al. [48], cytoskeletal rearrangements based on Rac-1 activation involve the production of reactive oxygen species (ROS) and the consequent oxidation and inhibition of LMWPTP. As mentioned before, our model of sorbitol-induced hyperosmotic stress did not cause alteration in Rac-1 expression, which is in agreement with our results showing increased activity of LMWPTP. In addition, since LMWPTP is able to dephosphorylate Rho-GAP, contributing to the maintenance of active Rho-A, it is possible to suggest that beside the demonstrated increase in Rho expression, the activity of this protein is also increased. Interestingly, our results showed that LMWPTP silencing in HaCaT cells (without exposition to sorbitol) increased Rac-1 expression, suggesting that LMWPTP also influences the expression of this small GTPase (Fig. 5A and B). The same pattern of results was obtained when expression of Rac-1 was analyzed by confocal microscopy (Fig. 6). In addition, the results also showed that when LMWPTP-siRNA transfected cells were treated with sorbitol, a lack of inhibition of Rac-1 expression was observed compared with its expression in Scramble-siRNA transfected cells (Fig. 5A and C). The same pattern of results was obtained when expression of Rac was analysed through confocal microscopy (Fig. 6). These results demonstrated that LMWPTP is involved in the mechanisms associated with Rac-1 expression and that the expression of this phosphatase is involved in the effects of sorbitol treatment in Rac-1 during hyperosmotic stress. How LMWPTP activity inhibits the expression of Rac-1 is still an unknown issue. Moreover, its effect on cytoskeleton was also suggested as these results were followed by changes in F-actin organization, with a different distribution at the leading edge (Fig. 6).


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 - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363792&req=5

pone.0119020.g006: 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.
Mentions: As demonstrated by Nimnual et al. [48], cytoskeletal rearrangements based on Rac-1 activation involve the production of reactive oxygen species (ROS) and the consequent oxidation and inhibition of LMWPTP. As mentioned before, our model of sorbitol-induced hyperosmotic stress did not cause alteration in Rac-1 expression, which is in agreement with our results showing increased activity of LMWPTP. In addition, since LMWPTP is able to dephosphorylate Rho-GAP, contributing to the maintenance of active Rho-A, it is possible to suggest that beside the demonstrated increase in Rho expression, the activity of this protein is also increased. Interestingly, our results showed that LMWPTP silencing in HaCaT cells (without exposition to sorbitol) increased Rac-1 expression, suggesting that LMWPTP also influences the expression of this small GTPase (Fig. 5A and B). The same pattern of results was obtained when expression of Rac-1 was analyzed by confocal microscopy (Fig. 6). In addition, the results also showed that when LMWPTP-siRNA transfected cells were treated with sorbitol, a lack of inhibition of Rac-1 expression was observed compared with its expression in Scramble-siRNA transfected cells (Fig. 5A and C). The same pattern of results was obtained when expression of Rac was analysed through confocal microscopy (Fig. 6). These results demonstrated that LMWPTP is involved in the mechanisms associated with Rac-1 expression and that the expression of this phosphatase is involved in the effects of sorbitol treatment in Rac-1 during hyperosmotic stress. How LMWPTP activity inhibits the expression of Rac-1 is still an unknown issue. Moreover, its effect on cytoskeleton was also suggested as these results were followed by changes in F-actin organization, with a different distribution at the leading edge (Fig. 6).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus