Transforming Growth Factor β1-induced Astrocyte Response Is Mediated By Cysteinyl Leukotrienes And CysLT1Receptor

BackgroundsInflammation after cerebral ischemia plays an important role in the pathophysiological process. After ischemia, inflammatory responses are activated in the central nervous system, pro-inflammatory molecules are expressed and secreted, and intracerebral inflammation occurs. One of the consequences is activation of astrocytes that gradually form a glial scar. Study of activation of astrocytes by inflammatory factors will contribute to understanding of the mechanism of glial scar formation and the relevant interventions. The inflammatory factors induced by erebral ischemia include transforming growth factor beta1(TGF-β1) and cysteinyl leukotrienes (CysLTs). TGF-β1is widely considered as an injury-related cytokine or growth factor, and CysLTs are a kind of important inflammatory mediators. Both of them play important roles in ischemia and traumatic brain injury as well as in other central nervous system diseases.In normal brain tissue, TGF-β1expression level is low. After cerebral ischemia, TGF-β1mRNA expression in the brain is increased in early phase (6h after ischemia), which reaches peak levels in days2to4, continues for14-26days. In the early phase of ischemia, its expression is diffusive overall whole brain, and mainly expressed in astrocytes at7-14days. This is associated with glial scar formation after brain injury. The in vitro experiments indicate that TGF-β1is able to induce astrocyte hypertrophy and promote astrocyte migration.CysLTs are5-lipoxygenase (5-LOX) products generated from arachidonic acis, and their actions are mediated by cysteinyl leukotriene receptors (CysLT receptors), mainly including two subtypes, i.e. CysLT1and CysLT2receptors. CysLT1receptor expression level is low in normal brain tissue. In rats with focal cerebral ischemia, its expression is significantly up-regulated in the ischemic core at3,6,12h and7-14days after reperfusion. In the boundary zone, CysLT1receptor expression does not significantly change during3days, but significantly increases7-14days after reperfusion, which is primarily localized in proliferated astrocytes. Pharmacological study indicates that the CysLT1receptor antagonist pranlukast can inhibit glial scar formation after focal cerebral ischemia in mice.In the primary cultures of rat astrocytes, mild ischemic injury induced by1h of oxygen-glucose deprivation (OGD) enhances CysLT1receptor expression, which mediates astrocyte proliferation and activation. Whereas, CysLT2receptor mediates astrocyte injury induced by moderate OGD (4h). The mechanisms underlying CysLT1receptor-mediated astrocyte activation remains to be clarified. Because astrocyte activation is regulated by a variety of factors, it is possible that the effects of CysLT1receptor may result from interactions with other factors.In the peripheral tissues, TGF-β1up-regulates the expression of CysLT1receptor in bronchial smooth muscle cells, and enhanced the effect of LTD4on cell proliferation. TGF-β1also up-regulates CysLT1receptor expression in fetal lung fibroblasts. Furthermore, TGF-β1increases the expression of5-LOX and5-LOX activating protein (FLAP) as well as increases leukotriene synthesis in dendritic cells. In the central nervous system, whether and how TGF-β1interacts with CysLTs and its receptors in regulation of astrocyte activation remains to be elucidated. Especially, the interaction between TGF-β1and CysLT1receptor is the key point that will be investigated in this study.AimsIn the present study, we aimed to investigate the interactions of TGF-β1with CysLTs and CysLT1receptor in regulation of astrocyte activation in following aspects.1. At first, to preliminarily reveal the possible interaction, we determined the effects of TGF-β1as well as the drugs related to CysLT receptors on astrocyte viability; these drugs include the5-LOX inhibitor zileuton, and the agonists and antagonist of CysLT1and CysLT2receptors.2. Next, to further clarify the interaction beween TGF-β1and CysLT1receptor, we determined astrocyte activation, including proliferation and migration, induced by TGF-β1as well as CysLT receptor agonists, antagonists and specific siRNA.3. Then, to explore the properties of the interaction beween TGF-β1and CysLT1receptor, we analyzed the following two aspects. On one hand, we analyzed the actions of TGF-β1on the activation of CysLT1receptor and5-LOX, the key synthetic enzyme of the endogenous ligands CysLTs. On the other hand, we analyzed the actions of CysLTi receptor agonist on TGF-β1production.4. Finally, to elucidate the implications of the interaction beween TGF-β1and CysLT1receptor, we assessed whether mild OGD induces TGF-β1release based on previously reported findings that mild OGD enhances the expression and effects of CysLT1receptor. MethodsIn primarily cultured of rat cortical astrocytes, cell scratching was performed as a model of cell migration; MTT reduction assay was done to detect cell viability; flow cytometry was to assess cell proliferation; immunohistochemistry was to detect5-LOX nuclear translocation; ELISA was to detect CysLTs release. RT-PCR, Western blotting analysis and cellular immunofluorescence assay were performed to detect the expression of5-LOX, CysLT1receptor and CysLT2receptor. The non-selective CysLT receptor agonist LTD4, the CysLT2receptor selective agonist NMLTC4, the TGF-β1signaling inhibitor repsox, the5-LOX inhibitor zileuton, the CysLT1receptor selective antagonist montelukast and the CysLT2receptor selective antagonist Bay cysLT2were used in pharmacological treatments; and CysLT1receptor siRNA was used for gene interference of CysLT1receptor.In primary cultures of rat cortical astrocytes, ischemic injury was induced by OGD for1or4h. MTT reduction assay and LDH release were used to detect the viability and damage of astrocytes after OGD/recovery (OGD/R). ELISA was assessed to analyze the release of TGF-β1.Results1. Effects of TGF-β1and CysLT receptors on astrocyte viabilityIn the primary cultures of rat astrocytes, the results of MTT reduction assay showed that TGF-β1and the non-selective CysLT receptor agonist LTD4increased astrocyte viability, and they potentiated the effects each other. However, the CysLT2receptor agonist NMLTC4did not show any effect. Moreover, the effect of TGF-β1was attenuated by the5-LOX inhibitor zileuton and the CysLT1receptor antagonist montelukast, but not by the CysLT2receptor antagonist Bay cysLT2. These results suggested a possible interaction between TGF-β1and CysLT1receptor. 2. Effects of TGF-β1and CysLT1receptor on proliferation and migration of astrocytesTo further clarify the interaction between TGF-β1and CysLT1receptor in astrocyte activation, we assessed the effects of TGF-β1and the drugs related to CysLT1receptor on the proliferation and migration of astrocytes. Flow cytometric results showed that TGF-β1and LTD4at various concentrations did not affect astrocyte proliferation24h after exposure. Cell healing test showed that TGF-(31promoted astrocyte migration24h after scratching in a time and concentration-dependent manner. LTD4at lower concentrations (0.1-10nM) also promoted the migration, and potentiated the effect of TGF-β1on astrocyte migration. The TGF-(31signaling inhibitor repsox completely reversed the effect of TGF-β1. The5-LOX inhibitor zileuton and the CysLT1receptor antagonist montelukast as well as CysLT1receptor siRNA attenuated the effect of TGF-β1. However, the CysLT2receptor antagonist Bay cysLT2did not affect the effect of TGF-β1. These results indicated that the effect of TGF-β1on astrocyte activation (migration) can be mediated by activation of CysLT1receptor and5-LOX, the synthetic enzyme of endogenous CysLTs.3. The regulatory interactions between TGF-β1and5-LOX/CysLT1receptorTo characterize the interactions between TGF-β1and CysLT1receptor, we analyzed the regulatory interactions between them. RT-PCR, Western blotting analysis and cell immunofluorescence showed that TGF-β1up-regulated the expression of5-LOX and CysLT1receptor, but not that of CysLT2receptor. Cell immunofluorescence showed that5-LOX translocation from the cytosol to the nuclear membrane6h after exposure to TGF-β1, and the translocation disappeared24h after the exposure. ELISA results showed that TGF-β1increased the content of CysLTs in the culture media from1.5h, reached a peak at12h, and maintained until24h after exposure. On the other hand, RT-PCR and ELISA results showed that LTD4and NMLTC4at1-100nM did not affect the expression and release of TGF-β1. These results suggested that the interactions between TGF-β1and CysLT1receptor might be characterized by that TGF-β1potentiates the production of endogenous ligands of CysLT1receptor and the expression of CysLT1receptor, but not by potentiation of TGF-β1by CysLT1receptor.4. Mild OGD induces TGF-β1releaseIt has previously reported that mild OGD enhances CysLT1receptor expression and potentiates its effects. Here, we confirmed the relation of this phenomenon to TGF-β1. We found that following recovery for48and72h after1-h OGD, astrocyte viability was increased but LDH release did not change. At12h of recovery after1-h OGD, TGF-β1release was significantly increased. However, moderate OGD (4h) induced astrocyte injury, and did not affect TGF-β1release. Thses results suggested that astrocyte activation induced by mild ischemic injury might be associated with the regulation by TGF-β1/CysLT1receptor system.Conclusions:1. The pharmacological experiments of astrocyte viability preliminarily show the possible interactions of TGF-β1with CysLT1receptor and5-LOX activation.2. In the experiments of astrocyte activation (proliferation and migration), the effect of TGF-β1on astrocyte migration is mediated by activation of CysLT1receptor and5-LOX, a key synthetic enzyme of endogenous CysLTs. However, these agents do not affect astrocyte proliferation.3. The production of endogenous ligands due to5-LOX activation and the expression of CysLT1receptor are enhanced by TGF-β1, but TGF-β1is not regulated by CysLTi receptor. Namely, the interactions are characterized by potentiation of the signaling system of CysLT1receptor by TGF-β1, in other wards, the effect of TGF-β1is mediated by the CysLT1receptor signaling..4. Mild OGD induces TGF-β1release, suggesting that mild ischemic injury may enhance astrocyte activation through regualting TGF-β1/CysLT1receptor system.