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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 expression is upregulated by LPS in the mouse brain. a mRNA expression of PTP1B in the brain 24 h after LPS injection (5 mg/kg). The expression of PTP-Meg2 and TC-PTP were also measured by RT-PCR along with the inflammatory maker, Lcn2. The band intensity of PTP1B, PTP-Meg2, and TC-PTP mRNA from three to four independent experiments was measured and normalized to β-actin expression. *p < 0.05 versus vehicle control. NS not significant. b Western blot analysis of PTP1B protein expression levels in the brain 6 h or 24 h after LPS injection. α-tubulin was used as a loading control. The graphs show the average band intensity of PTP1B and the error bars show standard error from 5 animals 24 h after LPS injection. *p < 0.05 versus vehicle-injected control, analyzed by Student’s t test. c PTP1B expression (green) in the cortex area. PTP1B is co-localized with Iba-1 (red), a microglia marker, 24 h after LPS injection (5 mg/kg). Arrows indicate colocalization of PTP1B and Iba-1 expression. Nuclei were stained with DAPI (blue)
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Fig1: PTP1B expression is upregulated by LPS in the mouse brain. a mRNA expression of PTP1B in the brain 24 h after LPS injection (5 mg/kg). The expression of PTP-Meg2 and TC-PTP were also measured by RT-PCR along with the inflammatory maker, Lcn2. The band intensity of PTP1B, PTP-Meg2, and TC-PTP mRNA from three to four independent experiments was measured and normalized to β-actin expression. *p < 0.05 versus vehicle control. NS not significant. b Western blot analysis of PTP1B protein expression levels in the brain 6 h or 24 h after LPS injection. α-tubulin was used as a loading control. The graphs show the average band intensity of PTP1B and the error bars show standard error from 5 animals 24 h after LPS injection. *p < 0.05 versus vehicle-injected control, analyzed by Student’s t test. c PTP1B expression (green) in the cortex area. PTP1B is co-localized with Iba-1 (red), a microglia marker, 24 h after LPS injection (5 mg/kg). Arrows indicate colocalization of PTP1B and Iba-1 expression. Nuclei were stained with DAPI (blue)

Mentions: We first investigated whether PTP1B expression is regulated by inflammatory conditions in the mouse brain. For an animal model of neuroinflammation, we used LPS-injected mice. Whole brains were collected 24 h after an i.p. injection of LPS (5 mg/kg). The gene expression levels of PTP1B, TC-PTP (also known as PTPN2, a phosphatase highly homologous to PTP1B) and PTP-Meg2 (also known as PTPN9), members of non-receptor types of PTP family, were assessed by RT-PCR using gene-specific primers. The PTP1B expression was increased after 24 h (Fig. 1a). The inflammatory marker Lcn2 was also highly upregulated by LPS. PTP-Meg2 has an effect on insulin signaling in a manner similar to PTP1B [30, 31]. TC-PTP and PTP-Meg2 expression levels were not increased by LPS, indicating a specific induction of PTP1B under inflammatory conditions. We next tested whether PTP1B protein levels were also increased by LPS. After LPS stimulation, PTP1B protein levels were modestly higher in the total brain lysates 24 h after LPS injection compared to saline-administered brain lysates (Fig. 1b). Because microglia are the resident immune cells in the CNS and participate in the initiation and propagation of an inflammatory response, we examined PTP1B expression in brain microglia using immunostaining. PTP1B protein expression was increased in the cytoplasm of Iba-1-positive microglia after LPS treatment (Fig. 1c).Fig. 1


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 expression is upregulated by LPS in the mouse brain. a mRNA expression of PTP1B in the brain 24 h after LPS injection (5 mg/kg). The expression of PTP-Meg2 and TC-PTP were also measured by RT-PCR along with the inflammatory maker, Lcn2. The band intensity of PTP1B, PTP-Meg2, and TC-PTP mRNA from three to four independent experiments was measured and normalized to β-actin expression. *p < 0.05 versus vehicle control. NS not significant. b Western blot analysis of PTP1B protein expression levels in the brain 6 h or 24 h after LPS injection. α-tubulin was used as a loading control. The graphs show the average band intensity of PTP1B and the error bars show standard error from 5 animals 24 h after LPS injection. *p < 0.05 versus vehicle-injected control, analyzed by Student’s t test. c PTP1B expression (green) in the cortex area. PTP1B is co-localized with Iba-1 (red), a microglia marker, 24 h after LPS injection (5 mg/kg). Arrows indicate colocalization of PTP1B and Iba-1 expression. Nuclei were stained with DAPI (blue)
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Related In: Results  -  Collection

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Fig1: PTP1B expression is upregulated by LPS in the mouse brain. a mRNA expression of PTP1B in the brain 24 h after LPS injection (5 mg/kg). The expression of PTP-Meg2 and TC-PTP were also measured by RT-PCR along with the inflammatory maker, Lcn2. The band intensity of PTP1B, PTP-Meg2, and TC-PTP mRNA from three to four independent experiments was measured and normalized to β-actin expression. *p < 0.05 versus vehicle control. NS not significant. b Western blot analysis of PTP1B protein expression levels in the brain 6 h or 24 h after LPS injection. α-tubulin was used as a loading control. The graphs show the average band intensity of PTP1B and the error bars show standard error from 5 animals 24 h after LPS injection. *p < 0.05 versus vehicle-injected control, analyzed by Student’s t test. c PTP1B expression (green) in the cortex area. PTP1B is co-localized with Iba-1 (red), a microglia marker, 24 h after LPS injection (5 mg/kg). Arrows indicate colocalization of PTP1B and Iba-1 expression. Nuclei were stained with DAPI (blue)
Mentions: We first investigated whether PTP1B expression is regulated by inflammatory conditions in the mouse brain. For an animal model of neuroinflammation, we used LPS-injected mice. Whole brains were collected 24 h after an i.p. injection of LPS (5 mg/kg). The gene expression levels of PTP1B, TC-PTP (also known as PTPN2, a phosphatase highly homologous to PTP1B) and PTP-Meg2 (also known as PTPN9), members of non-receptor types of PTP family, were assessed by RT-PCR using gene-specific primers. The PTP1B expression was increased after 24 h (Fig. 1a). The inflammatory marker Lcn2 was also highly upregulated by LPS. PTP-Meg2 has an effect on insulin signaling in a manner similar to PTP1B [30, 31]. TC-PTP and PTP-Meg2 expression levels were not increased by LPS, indicating a specific induction of PTP1B under inflammatory conditions. We next tested whether PTP1B protein levels were also increased by LPS. After LPS stimulation, PTP1B protein levels were modestly higher in the total brain lysates 24 h after LPS injection compared to saline-administered brain lysates (Fig. 1b). Because microglia are the resident immune cells in the CNS and participate in the initiation and propagation of an inflammatory response, we examined PTP1B expression in brain microglia using immunostaining. PTP1B protein expression was increased in the cytoplasm of Iba-1-positive microglia after LPS treatment (Fig. 1c).Fig. 1

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