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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

PTP1B potentiates LPS-induced NO production. a PTP1B expression in BV-2 cells overexpressing HA-PTP1B. Western blot analysis showed a 2.59-fold increase in PTP1B expression in cells stably expressing HA-PTP1B. β-actin was used as a loading control. b LPS-induced NO production in BV-2 cells with or without PTP1B overexpression. BV-2 cells were treated with LPS at the indicated concentration (0–1000 ng/ml) for 24 h. Nitrite accumulation was measured using the Griess reaction. Real-time RT-PCR was performed to determine mRNA expression of TNF-α (c), iNOS (d), and IL-6 (e) in either control or HA-PTP1B-transfected BV-2 cells after LPS (100 ng/ml) treatment for 6 h. *p < 0.05 versus control BV-2 with LPS; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f PTP1B expression in BV-2 cells transfected with PTP1B siRNA (siPTP1B). Real-time RT-PCR was performed to determine mRNA expression of PTP1B. g LPS-induced NO production in BV-2 cells after knockdown of PTP1B expression. The data were expressed as the mean ± SEM (n = 3). *p < 0.05 versus control BV-2 cells transfected with control siRNA (siCont)
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Fig3: PTP1B potentiates LPS-induced NO production. a PTP1B expression in BV-2 cells overexpressing HA-PTP1B. Western blot analysis showed a 2.59-fold increase in PTP1B expression in cells stably expressing HA-PTP1B. β-actin was used as a loading control. b LPS-induced NO production in BV-2 cells with or without PTP1B overexpression. BV-2 cells were treated with LPS at the indicated concentration (0–1000 ng/ml) for 24 h. Nitrite accumulation was measured using the Griess reaction. Real-time RT-PCR was performed to determine mRNA expression of TNF-α (c), iNOS (d), and IL-6 (e) in either control or HA-PTP1B-transfected BV-2 cells after LPS (100 ng/ml) treatment for 6 h. *p < 0.05 versus control BV-2 with LPS; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f PTP1B expression in BV-2 cells transfected with PTP1B siRNA (siPTP1B). Real-time RT-PCR was performed to determine mRNA expression of PTP1B. g LPS-induced NO production in BV-2 cells after knockdown of PTP1B expression. The data were expressed as the mean ± SEM (n = 3). *p < 0.05 versus control BV-2 cells transfected with control siRNA (siCont)

Mentions: To investigate the functional role of increased PTP1B expression in microglia under inflammatory conditions, we established a line of BV-2 microglial cells stably overexpressing HA-PTP1B. The enhanced PTP1B protein expression in the stable HA-PTP1B transfectants was confirmed by western blot analysis (Fig. 3a). Since NO production is an indicator of microglial inflammatory activation, we investigated the effect of a forced upregulation of PTP1B on LPS-induced NO production. Parental BV-2 cells and the stable HA-PTP1B transfectants were stimulated with LPS for 24 h. Subsequently, the accumulated nitrite in the culture media was estimated using Griess reaction as an index for NO synthesis. NO production was increased by LPS in a dose-dependent manner (Fig. 3b). PTP1B overexpression potentiated LPS-induced NO production at all LPS concentrations. As increased inflammatory cytokine levels are an indicator of hyperactivated microglia [32, 33], the effects of PTP1B overexpression on the production of proinflammatory cytokines was also determined in microglial cells by RT-PCR. Indeed, PTP1B overexpression potentiated the LPS-induced expression of TNF-α, iNOS, and IL-6 mRNA (Fig. 3c–e). To knockdown PTP1B expression, BV-2 cells were transfected with siRNA against PTP1B. We obtained 60 % downregulation of PTP1B expression (Fig. 3f), and the PTP1B knocking down reduced LPS-induced NO production (Fig. 3g).Fig. 3


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)

PTP1B potentiates LPS-induced NO production. a PTP1B expression in BV-2 cells overexpressing HA-PTP1B. Western blot analysis showed a 2.59-fold increase in PTP1B expression in cells stably expressing HA-PTP1B. β-actin was used as a loading control. b LPS-induced NO production in BV-2 cells with or without PTP1B overexpression. BV-2 cells were treated with LPS at the indicated concentration (0–1000 ng/ml) for 24 h. Nitrite accumulation was measured using the Griess reaction. Real-time RT-PCR was performed to determine mRNA expression of TNF-α (c), iNOS (d), and IL-6 (e) in either control or HA-PTP1B-transfected BV-2 cells after LPS (100 ng/ml) treatment for 6 h. *p < 0.05 versus control BV-2 with LPS; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f PTP1B expression in BV-2 cells transfected with PTP1B siRNA (siPTP1B). Real-time RT-PCR was performed to determine mRNA expression of PTP1B. g LPS-induced NO production in BV-2 cells after knockdown of PTP1B expression. The data were expressed as the mean ± SEM (n = 3). *p < 0.05 versus control BV-2 cells transfected with control siRNA (siCont)
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Fig3: PTP1B potentiates LPS-induced NO production. a PTP1B expression in BV-2 cells overexpressing HA-PTP1B. Western blot analysis showed a 2.59-fold increase in PTP1B expression in cells stably expressing HA-PTP1B. β-actin was used as a loading control. b LPS-induced NO production in BV-2 cells with or without PTP1B overexpression. BV-2 cells were treated with LPS at the indicated concentration (0–1000 ng/ml) for 24 h. Nitrite accumulation was measured using the Griess reaction. Real-time RT-PCR was performed to determine mRNA expression of TNF-α (c), iNOS (d), and IL-6 (e) in either control or HA-PTP1B-transfected BV-2 cells after LPS (100 ng/ml) treatment for 6 h. *p < 0.05 versus control BV-2 with LPS; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f PTP1B expression in BV-2 cells transfected with PTP1B siRNA (siPTP1B). Real-time RT-PCR was performed to determine mRNA expression of PTP1B. g LPS-induced NO production in BV-2 cells after knockdown of PTP1B expression. The data were expressed as the mean ± SEM (n = 3). *p < 0.05 versus control BV-2 cells transfected with control siRNA (siCont)
Mentions: To investigate the functional role of increased PTP1B expression in microglia under inflammatory conditions, we established a line of BV-2 microglial cells stably overexpressing HA-PTP1B. The enhanced PTP1B protein expression in the stable HA-PTP1B transfectants was confirmed by western blot analysis (Fig. 3a). Since NO production is an indicator of microglial inflammatory activation, we investigated the effect of a forced upregulation of PTP1B on LPS-induced NO production. Parental BV-2 cells and the stable HA-PTP1B transfectants were stimulated with LPS for 24 h. Subsequently, the accumulated nitrite in the culture media was estimated using Griess reaction as an index for NO synthesis. NO production was increased by LPS in a dose-dependent manner (Fig. 3b). PTP1B overexpression potentiated LPS-induced NO production at all LPS concentrations. As increased inflammatory cytokine levels are an indicator of hyperactivated microglia [32, 33], the effects of PTP1B overexpression on the production of proinflammatory cytokines was also determined in microglial cells by RT-PCR. Indeed, PTP1B overexpression potentiated the LPS-induced expression of TNF-α, iNOS, and IL-6 mRNA (Fig. 3c–e). To knockdown PTP1B expression, BV-2 cells were transfected with siRNA against PTP1B. We obtained 60 % downregulation of PTP1B expression (Fig. 3f), and the PTP1B knocking down reduced LPS-induced NO production (Fig. 3g).Fig. 3

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