Abstract
BackgroundTissue inhibitors of metalloproteinases (TIMPs) are known to be endogenous inhibitors of matrix metalloproteinases (MMPs). Our preliminary study showed that TIMP-2 is constitutively expressed in microglia but significantly inhibited by lipopolysaccharide (LPS) treatment. The current study was undertaken to investigate the role of TIMP-2 in microglia.MethodsThe expression of TIMP-2 was evaluated in the BV2 mouse microglial cell line and rat primary cultured microglia. To investigate the role of TIMP-2, a TIMP-2 expression plasmid or small interfering RNA (siRNA) was introduced into BV2 cells by transient transfection, and their effects on LPS-induced inflammatory reactions were examined. We further analyzed the molecular mechanism underlying the anti-inflammatory effects of TIMP-2 by electrophoretic mobility shift assay (EMSA), a reporter gene assay and Western blot analysis.ResultsOverexpression of TIMP-2 significantly inhibited the production of nitric oxide (NO), TNF-α, IL-1β, and reactive oxygen species (ROS), while increasing anti-inflammatory IL-10 production. On the other hand, knockdown of TIMP-2 augmented the production of pro-inflammatory molecules and downregulated IL-10 in LPS-stimulated BV2 cells. The results suggest that endogenously expressed TIMP-2 plays an anti-inflammatory role. Further mechanistic studies revealed that overexpression of TIMP-2 suppresses microglial activation via inhibition of the activity of mitogen-activated protein kinases (MAPKs) and NF-κB with enhancement of the activity of anti-inflammatory Nrf2 and cAMP-response element binding protein (CREB) transcription factors. TIMP-2 also inhibited the activity and expression of LPS-induced MMP-3, -8, and -9. Finally, we demonstrated that TIMP-2 exerts a neuroprotective effect via the inhibition of microglial activation.ConclusionsEnhancement of TIMP-2 expression may be a potential therapeutic target for neuroinflammatory disorders.
Highlights
Tissue inhibitors of metalloproteinases (TIMPs) are known to be endogenous inhibitors of matrix metalloproteinases (MMPs)
Lipopolysaccharide inhibited tissue inhibitor of metalloproteinase-2 expression in BV2 cells and primary microglia When we measured TIMP-2 mRNA expression in LPStreated BV2 cells at the indicated time points, TIMP-2 was constitutively expressed in resting BV2 cells, and LPS treatment significantly inhibited its expression after 3 h (Figure 1A)
Tissue inhibitor of metalloproteinase-2 abrogated lipopolysaccharide-induced phosphorylation of three types of mitogen-activated protein kinases we examined the effect of TIMP-2 on the activity of MAP kinases, which are crucial signaling molecules involved in inflammatory reactions and MMP gene expression
Summary
Tissue inhibitors of metalloproteinases (TIMPs) are known to be endogenous inhibitors of matrix metalloproteinases (MMPs). Our preliminary study showed that TIMP-2 is constitutively expressed in microglia but significantly inhibited by lipopolysaccharide (LPS) treatment. The current study was undertaken to investigate the role of TIMP-2 in microglia. Resident immune cells of the central nervous system (CNS), play an important role in brain physiology, including sensing and regulating neuronal activities in the intact brain [1,2]. MMP activity is regulated by several mechanisms, including gene transcription, proenzyme activation and inhibition by various endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs) [10,11]. In the adult central nervous system (CNS), the expression of TIMP-2 is enriched with populations of neuronal progenitor cells, suggesting the role of TIMP-2 in neurogenesis [10]. TIMP-2 has been suggested to be a therapeutic candidate for CNS disorders, such as MS, stroke, and Parkinson’s disease
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