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A novel role for protein tyrosine phosphatase 1B as a positive regulator of neuroinflammation.

Song GJ, Jung M, Kim JH, Park H, Rahman MH, Zhang S, Zhang ZY, Park DH, Kook H, Lee IK, Suk K - J Neuroinflammation (2016)

Bottom Line: To confirm the role of PTP1B in neuroinflammation, we employed a highly potent and selective inhibitor of PTP1B (PTP1Bi).PTP1B-mediated Src activation led to an enhanced proinflammatory response in the microglial cells.This study demonstrates that PTP1B is an important positive regulator of neuroinflammation and is a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Brain Science and Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Republic of Korea.

ABSTRACT

Background: Protein tyrosine phosphatase 1B (PTP1B) is a member of the non-transmembrane phosphotyrosine phosphatase family. Recently, PTP1B has been proposed to be a novel target of anti-cancer and anti-diabetic drugs. However, the role of PTP1B in the central nervous system is not clearly understood. Therefore, in this study, we sought to define PTP1B's role in brain inflammation.

Methods: PTP1B messenger RNA (mRNA) and protein expression levels were examined in mouse brain and microglial cells after LPS treatment using RT-PCR and western blotting. Pharmacological inhibitors of PTP1B, NF-κB, and Src kinase were used to analyze these signal transduction pathways in microglia. A Griess reaction protocol was used to determine nitric oxide (NO) concentrations in primary microglia cultures and microglial cell lines. Proinflammatory cytokine production was measured by RT-PCR. Western blotting was used to assess Src phosphorylation levels. Immunostaining for Iba-1 was used to determine microglial activation in the mouse brain.

Results: PTP1B expression levels were significantly increased in the brain 24 h after LPS injection, suggesting a functional role for PTP1B in brain inflammation. Microglial cells overexpressing PTP1B exhibited an enhanced production of NO and gene expression levels of TNF-α, iNOS, and IL-6 following LPS exposure, suggesting that PTP1B potentiates the microglial proinflammatory response. To confirm the role of PTP1B in neuroinflammation, we employed a highly potent and selective inhibitor of PTP1B (PTP1Bi). In LPS- or TNF-α-stimulated microglial cells, in vitro blockade of PTP1B activity using PTP1Bi markedly attenuated NO production. PTP1Bi also suppressed the expression levels of iNOS, COX-2, TNF-α, and IL-1β. PTP1B activated Src by dephosphorylating the Src protein at a negative regulatory site. PTP1B-mediated Src activation led to an enhanced proinflammatory response in the microglial cells. An intracerebroventricular injection of PTP1Bi significantly attenuated microglial activation in the hippocampus and cortex of LPS-injected mice compared to vehicle-injected mice. The gene expression levels of proinflammatory cytokines were also significantly suppressed in the brain by a PTP1Bi injection. Together, these data suggest that PTP1Bi has an anti-inflammatory effect in a mouse model of neuroinflammation.

Conclusions: This study demonstrates that PTP1B is an important positive regulator of neuroinflammation and is a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

The role of PTP1B in Src-dependent microglial activation. a PTP1B overexpression dephosphorylated Src at Y527, a negative regulatory site. The phosphorylation of Src Y527 was quantified and normalized to total Src in either control or HA-PTP1B-transfected BV-2 cells. The graph shows the average value of phosphorylation of Y527/β-actin from four independent experiments. *p < 0.05 control versus HA-PTP1B-transfected BV-2, Student t test. b The phosphorylation of Src Y416 and p38 was quantified and normalized to total Src levels following treatment of BV-2 cells with LPS (100 ng/ml) or PTP1Bi (10 μM) for 30 min. The graph shows the average value of phosphorylation of Y416/total Src expression from four independent experiments. *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. c LPS (100 ng/ml)-induced nitrite production was measured in BV-2 after PP2 (Src kinase inhibitor, 5 μM) or PDTC (ammonium pyrrolidinedithiocarbamate, NF-κB inhibitor, 20 μM) treatment for 24 h. d BV-2 cells were pretreated with 10 μM PTP1Bi or 10 μM PP2 for 1 h and then treated with LPS for 24 h as indicated. Nitrite levels were measured by Griess solution. e BV-2 cells were pretreated with PTP1Bi for 1 h and then treated with LPS (100 ng/ml) for 30 min. IκB degradation by LPS was measured by western blotting. IκB intensity was measured from four independent experiments and normalized to α-tubulin. The data were expressed as the mean ± SEM (n = 4). *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Diagram depicting a mechanism by which PTP1B may promote proinflammatory cytokine production. PTP1B activates Src through dephosphorylation of Y527. Src may activate NF-κB and increase the production of proinflammatory molecules. NS not significant
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Fig6: The role of PTP1B in Src-dependent microglial activation. a PTP1B overexpression dephosphorylated Src at Y527, a negative regulatory site. The phosphorylation of Src Y527 was quantified and normalized to total Src in either control or HA-PTP1B-transfected BV-2 cells. The graph shows the average value of phosphorylation of Y527/β-actin from four independent experiments. *p < 0.05 control versus HA-PTP1B-transfected BV-2, Student t test. b The phosphorylation of Src Y416 and p38 was quantified and normalized to total Src levels following treatment of BV-2 cells with LPS (100 ng/ml) or PTP1Bi (10 μM) for 30 min. The graph shows the average value of phosphorylation of Y416/total Src expression from four independent experiments. *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. c LPS (100 ng/ml)-induced nitrite production was measured in BV-2 after PP2 (Src kinase inhibitor, 5 μM) or PDTC (ammonium pyrrolidinedithiocarbamate, NF-κB inhibitor, 20 μM) treatment for 24 h. d BV-2 cells were pretreated with 10 μM PTP1Bi or 10 μM PP2 for 1 h and then treated with LPS for 24 h as indicated. Nitrite levels were measured by Griess solution. e BV-2 cells were pretreated with PTP1Bi for 1 h and then treated with LPS (100 ng/ml) for 30 min. IκB degradation by LPS was measured by western blotting. IκB intensity was measured from four independent experiments and normalized to α-tubulin. The data were expressed as the mean ± SEM (n = 4). *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Diagram depicting a mechanism by which PTP1B may promote proinflammatory cytokine production. PTP1B activates Src through dephosphorylation of Y527. Src may activate NF-κB and increase the production of proinflammatory molecules. NS not significant

Mentions: We next investigated the mechanisms by which PTP1B potentiated the LPS-induced inflammatory activation of microglia. Based on literature search, we hypothesized that Src tyrosine kinase may be a potential substrate of PTP1B in microglia because Src has a negative regulatory phosphorylation site (tyrosine 527, Y527). PTP1B may dephosphorylate Src at this negative regulatory site, leading to Src kinase activation, as previously reported in breast cancer cell line [7]. This possibility was tested using the BV-2 microglial cells overexpressing PTP1B and PTP1Bi. The overexpression of PTP1B in BV-2 cells reduced Src phosphorylation at Y527 (45.7 % reduction) (Fig. 6a), consistent with previous observations in a colon cancer cell line [6]. Src activation was increased by LPS, as measured by Y416 phosphorylation (the kinase active site) of Src. This Src activity was significantly inhibited by PTP1Bi pretreatment (Fig. 6b). The PTP1Bi did not alter the levels of tyrosine phosphorylation of p38, demonstrating a specific effect of PTP1B on Src phosphorylation. Because PTP1B overexpression enhanced NO production in LPS-stimulated microglial cells (Fig. 3b), we next determined whether Src was involved in LPS-induced microglial activation. For this, BV-2 cells were treated with LPS in the presence or absence of PP2, a Src inhibitor; subsequently, NO production was measured. PP2 significantly inhibited LPS-induced NO production in microglia, to a similar extent as PDTC, an NF-κB inhibitor (Fig. 6c). The inhibition of LPS-induced NO production by PP2 pretreatment was dose-dependent (Additional file 1: Figure S2). Next, we asked whether PTP1B-mediated microglial activation was dependent on Src activity by examining the anti-inflammatory effects of PTP1Bi in microglial cells pretreated with a Src inhibitor. PP2 treatment abolished anti-inflammatory effect of PTP1Bi in microglia (Fig. 6d). These data suggest that PTP1B-mediated microglial activation is dependent on Src activity. NF-κB plays an important role in the transcriptional regulation of proinflammatory mediators. The blockade of NF-κB transcriptional activity can suppress iNOS and proinflammatory cytokines, such as IL-1β and TNF-α. We therefore investigated the effect of PTP1Bi on NF-κB activity. PTP1Bi decreased LPS-induced NF-κB activity through the suppression of IκB degradation (Fig. 6e). These data suggest that PTP1B can act as a proinflammatory factor via dephosphorylation of Src at Y527 and NF-κB activation in microglia (schematically summarized in Fig. 6f).Fig. 6


A novel role for protein tyrosine phosphatase 1B as a positive regulator of neuroinflammation.

Song GJ, Jung M, Kim JH, Park H, Rahman MH, Zhang S, Zhang ZY, Park DH, Kook H, Lee IK, Suk K - J Neuroinflammation (2016)

The role of PTP1B in Src-dependent microglial activation. a PTP1B overexpression dephosphorylated Src at Y527, a negative regulatory site. The phosphorylation of Src Y527 was quantified and normalized to total Src in either control or HA-PTP1B-transfected BV-2 cells. The graph shows the average value of phosphorylation of Y527/β-actin from four independent experiments. *p < 0.05 control versus HA-PTP1B-transfected BV-2, Student t test. b The phosphorylation of Src Y416 and p38 was quantified and normalized to total Src levels following treatment of BV-2 cells with LPS (100 ng/ml) or PTP1Bi (10 μM) for 30 min. The graph shows the average value of phosphorylation of Y416/total Src expression from four independent experiments. *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. c LPS (100 ng/ml)-induced nitrite production was measured in BV-2 after PP2 (Src kinase inhibitor, 5 μM) or PDTC (ammonium pyrrolidinedithiocarbamate, NF-κB inhibitor, 20 μM) treatment for 24 h. d BV-2 cells were pretreated with 10 μM PTP1Bi or 10 μM PP2 for 1 h and then treated with LPS for 24 h as indicated. Nitrite levels were measured by Griess solution. e BV-2 cells were pretreated with PTP1Bi for 1 h and then treated with LPS (100 ng/ml) for 30 min. IκB degradation by LPS was measured by western blotting. IκB intensity was measured from four independent experiments and normalized to α-tubulin. The data were expressed as the mean ± SEM (n = 4). *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Diagram depicting a mechanism by which PTP1B may promote proinflammatory cytokine production. PTP1B activates Src through dephosphorylation of Y527. Src may activate NF-κB and increase the production of proinflammatory molecules. NS not significant
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Fig6: The role of PTP1B in Src-dependent microglial activation. a PTP1B overexpression dephosphorylated Src at Y527, a negative regulatory site. The phosphorylation of Src Y527 was quantified and normalized to total Src in either control or HA-PTP1B-transfected BV-2 cells. The graph shows the average value of phosphorylation of Y527/β-actin from four independent experiments. *p < 0.05 control versus HA-PTP1B-transfected BV-2, Student t test. b The phosphorylation of Src Y416 and p38 was quantified and normalized to total Src levels following treatment of BV-2 cells with LPS (100 ng/ml) or PTP1Bi (10 μM) for 30 min. The graph shows the average value of phosphorylation of Y416/total Src expression from four independent experiments. *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. c LPS (100 ng/ml)-induced nitrite production was measured in BV-2 after PP2 (Src kinase inhibitor, 5 μM) or PDTC (ammonium pyrrolidinedithiocarbamate, NF-κB inhibitor, 20 μM) treatment for 24 h. d BV-2 cells were pretreated with 10 μM PTP1Bi or 10 μM PP2 for 1 h and then treated with LPS for 24 h as indicated. Nitrite levels were measured by Griess solution. e BV-2 cells were pretreated with PTP1Bi for 1 h and then treated with LPS (100 ng/ml) for 30 min. IκB degradation by LPS was measured by western blotting. IκB intensity was measured from four independent experiments and normalized to α-tubulin. The data were expressed as the mean ± SEM (n = 4). *p < 0.05 versus LPS only; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Diagram depicting a mechanism by which PTP1B may promote proinflammatory cytokine production. PTP1B activates Src through dephosphorylation of Y527. Src may activate NF-κB and increase the production of proinflammatory molecules. NS not significant
Mentions: We next investigated the mechanisms by which PTP1B potentiated the LPS-induced inflammatory activation of microglia. Based on literature search, we hypothesized that Src tyrosine kinase may be a potential substrate of PTP1B in microglia because Src has a negative regulatory phosphorylation site (tyrosine 527, Y527). PTP1B may dephosphorylate Src at this negative regulatory site, leading to Src kinase activation, as previously reported in breast cancer cell line [7]. This possibility was tested using the BV-2 microglial cells overexpressing PTP1B and PTP1Bi. The overexpression of PTP1B in BV-2 cells reduced Src phosphorylation at Y527 (45.7 % reduction) (Fig. 6a), consistent with previous observations in a colon cancer cell line [6]. Src activation was increased by LPS, as measured by Y416 phosphorylation (the kinase active site) of Src. This Src activity was significantly inhibited by PTP1Bi pretreatment (Fig. 6b). The PTP1Bi did not alter the levels of tyrosine phosphorylation of p38, demonstrating a specific effect of PTP1B on Src phosphorylation. Because PTP1B overexpression enhanced NO production in LPS-stimulated microglial cells (Fig. 3b), we next determined whether Src was involved in LPS-induced microglial activation. For this, BV-2 cells were treated with LPS in the presence or absence of PP2, a Src inhibitor; subsequently, NO production was measured. PP2 significantly inhibited LPS-induced NO production in microglia, to a similar extent as PDTC, an NF-κB inhibitor (Fig. 6c). The inhibition of LPS-induced NO production by PP2 pretreatment was dose-dependent (Additional file 1: Figure S2). Next, we asked whether PTP1B-mediated microglial activation was dependent on Src activity by examining the anti-inflammatory effects of PTP1Bi in microglial cells pretreated with a Src inhibitor. PP2 treatment abolished anti-inflammatory effect of PTP1Bi in microglia (Fig. 6d). These data suggest that PTP1B-mediated microglial activation is dependent on Src activity. NF-κB plays an important role in the transcriptional regulation of proinflammatory mediators. The blockade of NF-κB transcriptional activity can suppress iNOS and proinflammatory cytokines, such as IL-1β and TNF-α. We therefore investigated the effect of PTP1Bi on NF-κB activity. PTP1Bi decreased LPS-induced NF-κB activity through the suppression of IκB degradation (Fig. 6e). These data suggest that PTP1B can act as a proinflammatory factor via dephosphorylation of Src at Y527 and NF-κB activation in microglia (schematically summarized in Fig. 6f).Fig. 6

Bottom Line: To confirm the role of PTP1B in neuroinflammation, we employed a highly potent and selective inhibitor of PTP1B (PTP1Bi).PTP1B-mediated Src activation led to an enhanced proinflammatory response in the microglial cells.This study demonstrates that PTP1B is an important positive regulator of neuroinflammation and is a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Brain Science and Engineering Institute, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University School of Medicine, Daegu, Republic of Korea.

ABSTRACT

Background: Protein tyrosine phosphatase 1B (PTP1B) is a member of the non-transmembrane phosphotyrosine phosphatase family. Recently, PTP1B has been proposed to be a novel target of anti-cancer and anti-diabetic drugs. However, the role of PTP1B in the central nervous system is not clearly understood. Therefore, in this study, we sought to define PTP1B's role in brain inflammation.

Methods: PTP1B messenger RNA (mRNA) and protein expression levels were examined in mouse brain and microglial cells after LPS treatment using RT-PCR and western blotting. Pharmacological inhibitors of PTP1B, NF-κB, and Src kinase were used to analyze these signal transduction pathways in microglia. A Griess reaction protocol was used to determine nitric oxide (NO) concentrations in primary microglia cultures and microglial cell lines. Proinflammatory cytokine production was measured by RT-PCR. Western blotting was used to assess Src phosphorylation levels. Immunostaining for Iba-1 was used to determine microglial activation in the mouse brain.

Results: PTP1B expression levels were significantly increased in the brain 24 h after LPS injection, suggesting a functional role for PTP1B in brain inflammation. Microglial cells overexpressing PTP1B exhibited an enhanced production of NO and gene expression levels of TNF-α, iNOS, and IL-6 following LPS exposure, suggesting that PTP1B potentiates the microglial proinflammatory response. To confirm the role of PTP1B in neuroinflammation, we employed a highly potent and selective inhibitor of PTP1B (PTP1Bi). In LPS- or TNF-α-stimulated microglial cells, in vitro blockade of PTP1B activity using PTP1Bi markedly attenuated NO production. PTP1Bi also suppressed the expression levels of iNOS, COX-2, TNF-α, and IL-1β. PTP1B activated Src by dephosphorylating the Src protein at a negative regulatory site. PTP1B-mediated Src activation led to an enhanced proinflammatory response in the microglial cells. An intracerebroventricular injection of PTP1Bi significantly attenuated microglial activation in the hippocampus and cortex of LPS-injected mice compared to vehicle-injected mice. The gene expression levels of proinflammatory cytokines were also significantly suppressed in the brain by a PTP1Bi injection. Together, these data suggest that PTP1Bi has an anti-inflammatory effect in a mouse model of neuroinflammation.

Conclusions: This study demonstrates that PTP1B is an important positive regulator of neuroinflammation and is a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus