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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 inhibitor suppressed expression of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS (100 ng/ml) in the presence or absence of 10 μM PTP1Bi for 6 h for RT-PCR analysis (a) and 24 h for TNF-α ELISA analysis (b). After treatment, total RNA was isolated and specific mRNA levels were determined by real-time RT-PCR. Levels of iNOS, IL-1β, and TNF-α were normalized to GAPDH levels and expressed as percent value (n = 3). Levels of LPS-only treated cells were set to 100 %. b. The culture media of BV-2 cells after treatment was collected and subjected to a TNF-α sandwich ELISA. The data were expressed as the mean ± SEM and are representative of the results obtained from four independent experiments. *p < 0.05 versus LPS-only treatment; one-way ANOVA with Tukey’s multiple comparison test
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Fig5: PTP1B inhibitor suppressed expression of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS (100 ng/ml) in the presence or absence of 10 μM PTP1Bi for 6 h for RT-PCR analysis (a) and 24 h for TNF-α ELISA analysis (b). After treatment, total RNA was isolated and specific mRNA levels were determined by real-time RT-PCR. Levels of iNOS, IL-1β, and TNF-α were normalized to GAPDH levels and expressed as percent value (n = 3). Levels of LPS-only treated cells were set to 100 %. b. The culture media of BV-2 cells after treatment was collected and subjected to a TNF-α sandwich ELISA. The data were expressed as the mean ± SEM and are representative of the results obtained from four independent experiments. *p < 0.05 versus LPS-only treatment; one-way ANOVA with Tukey’s multiple comparison test

Mentions: PTP1B overexpression potentiated microglial production of NO and proinflammatory cytokines following LPS treatment. These results led us to hypothesize that PTP1B inhibition may inhibit microglial activation. To test this hypothesis, we used PTP1Bi, a PTP1B specific inhibitor, which we previously developed [25, 34]. PTPs share a conserved catalytic domain for the phosphatase enzyme activity. Nevertheless, PTP1B inhibitor (indicated as PTP1Bi in this study), originally called compound 2, has been shown to be highly specific for PTP1B [24, 25]. Firstly, we investigated the effect of PTP1Bi on NO production in LPS-stimulated BV-2 microglial cells. The BV-2 cells were pretreated with different concentrations of PTP1Bi before LPS stimulation. LPS-induced NO levels were decreased by PTP1Bi in a dose-dependent manner (IC50 value of 10.27 μM) (Fig. 4a). PTP1Bi itself did not alter the basal levels of NO production. No significant cytotoxicity was observed with PTP1Bi at the concentrations tested as determined by the MTT assay (Fig. 4a right). The inhibitory effect of PTP1Bi on NO production was also observed in mouse primary microglial cells (Fig. 4b) and in HAPI cells, a rat microglial cell line (Fig. 4c). TNF-α-induced NO production was also inhibited by PTP1Bi (Fig. 4d). Next, we examined whether PTP1Bi could also inhibit the production of proinflammatory cytokines. A pretreatment with PTP1Bi inhibited LPS-induced proinflammatory molecules, including iNOS, IL-1β, TNF-α, and COX-2 (Fig. 5a), as measured by RT-PCR. Moreover, PTP1Bi significantly inhibited LPS-induced TNF-α protein release in the microglial culture media, as measured by ELISA (Fig. 5b). We obtained similar findings with commercially available PTP1B inhibitor, CinnGel (Additional file 1: Figure S1).Fig. 4


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 inhibitor suppressed expression of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS (100 ng/ml) in the presence or absence of 10 μM PTP1Bi for 6 h for RT-PCR analysis (a) and 24 h for TNF-α ELISA analysis (b). After treatment, total RNA was isolated and specific mRNA levels were determined by real-time RT-PCR. Levels of iNOS, IL-1β, and TNF-α were normalized to GAPDH levels and expressed as percent value (n = 3). Levels of LPS-only treated cells were set to 100 %. b. The culture media of BV-2 cells after treatment was collected and subjected to a TNF-α sandwich ELISA. The data were expressed as the mean ± SEM and are representative of the results obtained from four independent experiments. *p < 0.05 versus LPS-only treatment; one-way ANOVA with Tukey’s multiple comparison test
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Fig5: PTP1B inhibitor suppressed expression of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS (100 ng/ml) in the presence or absence of 10 μM PTP1Bi for 6 h for RT-PCR analysis (a) and 24 h for TNF-α ELISA analysis (b). After treatment, total RNA was isolated and specific mRNA levels were determined by real-time RT-PCR. Levels of iNOS, IL-1β, and TNF-α were normalized to GAPDH levels and expressed as percent value (n = 3). Levels of LPS-only treated cells were set to 100 %. b. The culture media of BV-2 cells after treatment was collected and subjected to a TNF-α sandwich ELISA. The data were expressed as the mean ± SEM and are representative of the results obtained from four independent experiments. *p < 0.05 versus LPS-only treatment; one-way ANOVA with Tukey’s multiple comparison test
Mentions: PTP1B overexpression potentiated microglial production of NO and proinflammatory cytokines following LPS treatment. These results led us to hypothesize that PTP1B inhibition may inhibit microglial activation. To test this hypothesis, we used PTP1Bi, a PTP1B specific inhibitor, which we previously developed [25, 34]. PTPs share a conserved catalytic domain for the phosphatase enzyme activity. Nevertheless, PTP1B inhibitor (indicated as PTP1Bi in this study), originally called compound 2, has been shown to be highly specific for PTP1B [24, 25]. Firstly, we investigated the effect of PTP1Bi on NO production in LPS-stimulated BV-2 microglial cells. The BV-2 cells were pretreated with different concentrations of PTP1Bi before LPS stimulation. LPS-induced NO levels were decreased by PTP1Bi in a dose-dependent manner (IC50 value of 10.27 μM) (Fig. 4a). PTP1Bi itself did not alter the basal levels of NO production. No significant cytotoxicity was observed with PTP1Bi at the concentrations tested as determined by the MTT assay (Fig. 4a right). The inhibitory effect of PTP1Bi on NO production was also observed in mouse primary microglial cells (Fig. 4b) and in HAPI cells, a rat microglial cell line (Fig. 4c). TNF-α-induced NO production was also inhibited by PTP1Bi (Fig. 4d). Next, we examined whether PTP1Bi could also inhibit the production of proinflammatory cytokines. A pretreatment with PTP1Bi inhibited LPS-induced proinflammatory molecules, including iNOS, IL-1β, TNF-α, and COX-2 (Fig. 5a), as measured by RT-PCR. Moreover, PTP1Bi significantly inhibited LPS-induced TNF-α protein release in the microglial culture media, as measured by ELISA (Fig. 5b). We obtained similar findings with commercially available PTP1B inhibitor, CinnGel (Additional file 1: Figure S1).Fig. 4

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