Activation Of The Low Molecular Weight Protein Tyrosine Phosphatase In Keratinocytes Exposed To Hyperosmotic Stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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.103Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2006/07315-3]CNPq [PQ-2

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Campinas, Dept Bioquim, Inst Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniv Estadual Paulista, Dept Quim & Bioquim, IBB, São Paulo, BrazilUniv São Paulo, Dept Genet & Biol Evolut, São Paulo, SP, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilFAPESP: 2006/07315-3CNPq: PQ-2Web of Scienc

Effects of hyperosmotic stress on regulatory enzymes associated with cytoskeletal remodeling.

<p>HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119020#sec002" target="_blank">Materials and Methods</a>. 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. <b>(A)</b> 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>(B)</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). <b>(C)</b> Hyperosmotic stress causes significant increases in the expression of RhoA and Rac-1, while levels of PKAα remained unchanged. <b>(D)</b> Hyperosmotic stress is associated with STAT5 activation. <b>(E)</b> Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSTAT5/STAT5 ratios normalized to the protein ratio of controls (1). <b>(F)</b> 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.</p

Rac-1 and F-actin staining in LMWPTP silenced HaCaT cells.

<p>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.</p

LMWPTP is markedly activated in stressed keratinocytes.

<p>HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed. <b>(A)</b> Hyperosmotic stress increases the activity of immunoprecipitated LMWPTP (IP-LMWPTP). *P < 0.001 compared with control. <b>(B)</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. <b>(C)</b> 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. <b>(D)</b> 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.</p

Effect of LMWPTP knockdown with siACP1-CV on expression/activity of Rac-1, Src, FAK and STAT5.

<p>HaCaT cells were transfected with siACP1-CV 40 nM, exposed to 1 M sorbitol for 2 h, harvested and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119020#sec002" target="_blank">Materials and Methods</a>. 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 <i>ACP1</i> knockdown in Rac-1, Src, FAK and STAT5 proteins were evaluated in HaCaT cells before and after exposition to sorbitol-induced stress <b>(A)</b>. <b>(B-E)</b> 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.</p