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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 microglial activation in a mouse neuroinflammation model. a C57BL/6 mice were injected i.c.v. with vehicle (saline containing 0.5 % DMSO and 5 % propylene glycol) or PTP1B inhibitor (diluted in saline containing 5 % propylene glycol). At 30 min after the injection of PTP1B inhibitor, mice were injected i.p. with LPS (5 mg/kg). The mice were anesthetized and transcardially perfused with ice-cold saline 48 h after the LPS injection. The expression of PTP1B in the brain 48 h after the LPS injection was measured by RT-PCR. GAPDH was used for the loading control. b The brains were removed and the sections were stained with Iba-1 (a marker for microglia). Iba-1-positive cells were observed in the cortex, hippocampus (hippo), and thalamus region of mouse brains. Scale bar, 50 μm. c. The graph shows activated microglial cell number per square millimeter. NS not significant. The expression levels of proinflammatory genes were determined by real-time RT-PCR 48 h after the LPS injection. Levels of TNF-α (d) and IL-1β mRNA (e) were normalized to β-actin levels and expressed as fold increase. *p < 0.05 versus LPS + vehicle-injected animals; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Phosphorylation of Y527 Src in brain 48 h after LPS i.p. injection with or without PTP1Bi i.c.v. administration. Phospho (Y527)- and total Src protein levels were determined by western blot analysis. β-actin levels were used as loading controls. Lcn2 was used as a neuroinflammatory marker
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Fig7: PTP1B inhibitor suppressed microglial activation in a mouse neuroinflammation model. a C57BL/6 mice were injected i.c.v. with vehicle (saline containing 0.5 % DMSO and 5 % propylene glycol) or PTP1B inhibitor (diluted in saline containing 5 % propylene glycol). At 30 min after the injection of PTP1B inhibitor, mice were injected i.p. with LPS (5 mg/kg). The mice were anesthetized and transcardially perfused with ice-cold saline 48 h after the LPS injection. The expression of PTP1B in the brain 48 h after the LPS injection was measured by RT-PCR. GAPDH was used for the loading control. b The brains were removed and the sections were stained with Iba-1 (a marker for microglia). Iba-1-positive cells were observed in the cortex, hippocampus (hippo), and thalamus region of mouse brains. Scale bar, 50 μm. c. The graph shows activated microglial cell number per square millimeter. NS not significant. The expression levels of proinflammatory genes were determined by real-time RT-PCR 48 h after the LPS injection. Levels of TNF-α (d) and IL-1β mRNA (e) were normalized to β-actin levels and expressed as fold increase. *p < 0.05 versus LPS + vehicle-injected animals; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Phosphorylation of Y527 Src in brain 48 h after LPS i.p. injection with or without PTP1Bi i.c.v. administration. Phospho (Y527)- and total Src protein levels were determined by western blot analysis. β-actin levels were used as loading controls. Lcn2 was used as a neuroinflammatory marker

Mentions: Finally, we examined whether the PTP1B inhibitor limited neuroinflammation. Microglia activation is a hallmark of neuroinflammation [33, 35–38]. Therefore, the brain tissues were collected and stained with anti-Iba-1 antibody, a microglia marker, to evaluate the intensity of Iba-1 staining and microglial morphological changes 48 h after LPS i.p. injection, when the PTP1B expression in the brain remained elevated (Fig. 1a). LPS significantly increased the number of Iba-1-positive cells and hypertrophic microglia (Fig. 7b, c). Interestingly, the inhibition of PTP1B activity via PTP1Bi i.c.v. injection significantly reduced LPS-induced microglial activation 48 h after LPS injection. To confirm the anti-inflammatory effect of PTP1Bi in vivo, proinflammatory cytokine expression levels were also measured in brain tissues after LPS and PTP1Bi injection. The expression levels of TNF-α and IL-1β mRNA were significantly diminished by PTP1B inhibition in the inflammatory brain as measured by real-time RT-PCR (Fig. 7d, e). PTP1Bi injection increased Src phosphorylation at Y527, further confirming PTP1B’s effects on Src phosphorylation at Y527 in vivo (Fig. 7f). Taken together, inflammatory stimuli increased PTP1B expression to induce microglial activation in the brain. Inhibiting PTP1B activity under inflammatory conditions prevented microglial inflammatory activation in vitro and in vivo.Fig. 7


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 microglial activation in a mouse neuroinflammation model. a C57BL/6 mice were injected i.c.v. with vehicle (saline containing 0.5 % DMSO and 5 % propylene glycol) or PTP1B inhibitor (diluted in saline containing 5 % propylene glycol). At 30 min after the injection of PTP1B inhibitor, mice were injected i.p. with LPS (5 mg/kg). The mice were anesthetized and transcardially perfused with ice-cold saline 48 h after the LPS injection. The expression of PTP1B in the brain 48 h after the LPS injection was measured by RT-PCR. GAPDH was used for the loading control. b The brains were removed and the sections were stained with Iba-1 (a marker for microglia). Iba-1-positive cells were observed in the cortex, hippocampus (hippo), and thalamus region of mouse brains. Scale bar, 50 μm. c. The graph shows activated microglial cell number per square millimeter. NS not significant. The expression levels of proinflammatory genes were determined by real-time RT-PCR 48 h after the LPS injection. Levels of TNF-α (d) and IL-1β mRNA (e) were normalized to β-actin levels and expressed as fold increase. *p < 0.05 versus LPS + vehicle-injected animals; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Phosphorylation of Y527 Src in brain 48 h after LPS i.p. injection with or without PTP1Bi i.c.v. administration. Phospho (Y527)- and total Src protein levels were determined by western blot analysis. β-actin levels were used as loading controls. Lcn2 was used as a neuroinflammatory marker
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Related In: Results  -  Collection

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Fig7: PTP1B inhibitor suppressed microglial activation in a mouse neuroinflammation model. a C57BL/6 mice were injected i.c.v. with vehicle (saline containing 0.5 % DMSO and 5 % propylene glycol) or PTP1B inhibitor (diluted in saline containing 5 % propylene glycol). At 30 min after the injection of PTP1B inhibitor, mice were injected i.p. with LPS (5 mg/kg). The mice were anesthetized and transcardially perfused with ice-cold saline 48 h after the LPS injection. The expression of PTP1B in the brain 48 h after the LPS injection was measured by RT-PCR. GAPDH was used for the loading control. b The brains were removed and the sections were stained with Iba-1 (a marker for microglia). Iba-1-positive cells were observed in the cortex, hippocampus (hippo), and thalamus region of mouse brains. Scale bar, 50 μm. c. The graph shows activated microglial cell number per square millimeter. NS not significant. The expression levels of proinflammatory genes were determined by real-time RT-PCR 48 h after the LPS injection. Levels of TNF-α (d) and IL-1β mRNA (e) were normalized to β-actin levels and expressed as fold increase. *p < 0.05 versus LPS + vehicle-injected animals; analyzed by one-way ANOVA with Tukey’s multiple comparison test. f Phosphorylation of Y527 Src in brain 48 h after LPS i.p. injection with or without PTP1Bi i.c.v. administration. Phospho (Y527)- and total Src protein levels were determined by western blot analysis. β-actin levels were used as loading controls. Lcn2 was used as a neuroinflammatory marker
Mentions: Finally, we examined whether the PTP1B inhibitor limited neuroinflammation. Microglia activation is a hallmark of neuroinflammation [33, 35–38]. Therefore, the brain tissues were collected and stained with anti-Iba-1 antibody, a microglia marker, to evaluate the intensity of Iba-1 staining and microglial morphological changes 48 h after LPS i.p. injection, when the PTP1B expression in the brain remained elevated (Fig. 1a). LPS significantly increased the number of Iba-1-positive cells and hypertrophic microglia (Fig. 7b, c). Interestingly, the inhibition of PTP1B activity via PTP1Bi i.c.v. injection significantly reduced LPS-induced microglial activation 48 h after LPS injection. To confirm the anti-inflammatory effect of PTP1Bi in vivo, proinflammatory cytokine expression levels were also measured in brain tissues after LPS and PTP1Bi injection. The expression levels of TNF-α and IL-1β mRNA were significantly diminished by PTP1B inhibition in the inflammatory brain as measured by real-time RT-PCR (Fig. 7d, e). PTP1Bi injection increased Src phosphorylation at Y527, further confirming PTP1B’s effects on Src phosphorylation at Y527 in vivo (Fig. 7f). Taken together, inflammatory stimuli increased PTP1B expression to induce microglial activation in the brain. Inhibiting PTP1B activity under inflammatory conditions prevented microglial inflammatory activation in vitro and in vivo.Fig. 7

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