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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 microglia. a mRNA expression of PTP1B in BV-2, a mouse microglial cell line 6 and 24 h after LPS treatment (100 ng/ml). b PTP1B expression in primary microglia as measured by real-time RT-PCR. Data from triplicate determination are shown (mean and standard error). *p < 0.05 versus vehicle-treated control, analyzed by Student’s t test. c Immunostaining of PTP1B (green) in BV-2 microglial cell line with or without LPS (100 ng/ml) treatment for 24 h. Nuclei were stained with DAPI (blue)
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Fig2: PTP1B expression is upregulated by LPS in microglia. a mRNA expression of PTP1B in BV-2, a mouse microglial cell line 6 and 24 h after LPS treatment (100 ng/ml). b PTP1B expression in primary microglia as measured by real-time RT-PCR. Data from triplicate determination are shown (mean and standard error). *p < 0.05 versus vehicle-treated control, analyzed by Student’s t test. c Immunostaining of PTP1B (green) in BV-2 microglial cell line with or without LPS (100 ng/ml) treatment for 24 h. Nuclei were stained with DAPI (blue)

Mentions: Having shown LPS-induced PTP1B upregulation in mouse brain and the localization of PTP1B expression in brain microglia, we next utilized the BV-2 mouse microglial cell line to further investigate the regulation of PTP1B expression. PTP1B, but not PTP-Meg, mRNA levels were increased after 24-h stimulation with LPS (100 ng/ml) (Fig. 2a). LPS-induced PTP1B mRNA expression was similarly observed in primary microglial cultures (Fig. 2b). Immunostaining using anti-PTP1B antibody revealed a cytoplasmic expression of PTP1B protein in the BV-2 microglial cells as well as its upregulation after LPS treatment (Fig. 2c). Taken together, our results indicate that inflammatory stimuli increased PTP1B expression levels in brain microglia.Fig. 2


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 microglia. a mRNA expression of PTP1B in BV-2, a mouse microglial cell line 6 and 24 h after LPS treatment (100 ng/ml). b PTP1B expression in primary microglia as measured by real-time RT-PCR. Data from triplicate determination are shown (mean and standard error). *p < 0.05 versus vehicle-treated control, analyzed by Student’s t test. c Immunostaining of PTP1B (green) in BV-2 microglial cell line with or without LPS (100 ng/ml) treatment for 24 h. Nuclei were stained with DAPI (blue)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837589&req=5

Fig2: PTP1B expression is upregulated by LPS in microglia. a mRNA expression of PTP1B in BV-2, a mouse microglial cell line 6 and 24 h after LPS treatment (100 ng/ml). b PTP1B expression in primary microglia as measured by real-time RT-PCR. Data from triplicate determination are shown (mean and standard error). *p < 0.05 versus vehicle-treated control, analyzed by Student’s t test. c Immunostaining of PTP1B (green) in BV-2 microglial cell line with or without LPS (100 ng/ml) treatment for 24 h. Nuclei were stained with DAPI (blue)
Mentions: Having shown LPS-induced PTP1B upregulation in mouse brain and the localization of PTP1B expression in brain microglia, we next utilized the BV-2 mouse microglial cell line to further investigate the regulation of PTP1B expression. PTP1B, but not PTP-Meg, mRNA levels were increased after 24-h stimulation with LPS (100 ng/ml) (Fig. 2a). LPS-induced PTP1B mRNA expression was similarly observed in primary microglial cultures (Fig. 2b). Immunostaining using anti-PTP1B antibody revealed a cytoplasmic expression of PTP1B protein in the BV-2 microglial cells as well as its upregulation after LPS treatment (Fig. 2c). Taken together, our results indicate that inflammatory stimuli increased PTP1B expression levels in brain microglia.Fig. 2

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