TRPC3 May Contribute To LPS-induced Astrocyte Activation

Astrocyte is one of the most numerous cell types in the central nervous system (CNS). Traditionally, astrocyte has been relegated primarily to a supportive or structural role in the brain. Astrocyte exerts functions in maintaining local ion, pH, transmitter homeostasis. However, this view has changed radically in recent years with some discoveries. In the past decade or so, emerging evidence has provided new insights into astrocyte activities such as synapse formation, communication, cerebrovascular tone regulation, immune regulation and adult neurogenesis. These findings suggest that impairment of astrocytes even at minor levels may contribute to neurodegenerative disorders. In fact, astrocyte has been associated with many neurodegenerative disorders. So, astrocyte has recently become focus.Astrocyte becomes active in response to CNS injury. This reaction, called reactive astrogliosis is characterized by increased astrocyte proliferation, astrocyte hypertrophy and an increased expression of GFAP, accompanying secretion of a variety of cytokines, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-lbeta (IL-1 beta). Both beneficial and detrimental effects have been attributed to reactive astrocytes after CNS injury. Therefore, selective targeting of astrocyte function to minimize the damaging effects is most ideal for the development of astrocyte-specific therapies for CNS diseases. However, the conflicting dual roles contribute to the difficult task of therapy design.Calcium is an essential intracellular messenger and serves critical cellular functions in both excitable and non-excitable cells. Astrocytes exhibit increase in intracellular calcium concentration ([Ca2+]i that represents a form of astrocyte excitability. Alterations in Ca2+ homeostasis have been suggested in the onset/progression of neurological diseases, such as Parkinson's, Alzheimer's and Huntington's disease. Imbalance of calcium homeostasis and abnormal calcium signaling may be a key factor in astrocyte activation. Canonical transient receptor potential (TRPC) subfamily comprises a group of non-selective cation channels(TRPC 1-7)which open in response to a wide range of stimuli including intra-and extracellular messengers, chemical, mechanical and osmotic stress. Available data on TRPC protein indicate that these proteins initiate Ca2+ entry and are essential in maintaining cytosolic, ER, and mitochondrial Ca2+ levels. TRPC channels not only contribute towards normal physiological processes such as cell growth and survival, neurosecretion, but also are implicated in several pathological conditions. It is suggested that these channels could be used as potential therapeutic targets for many of neurological diseases. Studies have confirmed the existence of TRPC 1-7 channels in astrocytes, but their functions are not clear. Thus, In this study, we observed the roles of TRPC3 on astrocyte activation induced by LPS in vitro.LPS is a component of the bacterial cell wall of gram negative bacteria that activates toll-like receptor (TLR4) in mammals. TLR4 activation results in CNS inflammation. So, we treated cultured rat neonatal cortical astrocytes with LPS to establish a model of astrocyte activation. The proliferation of astrocytes was examined by MTS assay, bromodeoxyuridine (BrdU) incorporation and flow cytometry amalysis. The secretion of IL-6 was assessed by ELISA and GFAP expression was determined by immunofluorescence and western blot. Then, we observed LPS-induced the expression of TRPC3 by real-time PCR, Western blot, immunofluorescence and the involvement of TRPC in LPS-mediated Ca2+ entry. Pharmacological characterizations of TRPC were observed with two kinds of TRPC blockers.To determine the relationship between astrocyte activation and LPS, we first examined the effect of LPS on proliferation of cultured rat astrocytes. MTS assay and BrdU incorporation showed that LPS promoted astrocytes proliferation in a dose dependent manner. LPS (0.25-10μg/ml,48 hours) significantly increased cell survival (by 32.00%,44.69%,48.17%,58.45% and 55.20%, respectively) and BrdU incorporation (by 33.88%,37.71%,49.10%,53.66% and 39.62%, respectively) compared with control. Then we used 0.5μg/ml LPS to treat cells for 2 h,6 h,12 h, 24 h and 48 h, An increase of survival was observed in the presence LPS for 24 h and 48 h (46.37 and 55.36%, respectively)than in the absence of LPS for 24 and 48 h (19.62 and 24.08%, respectively). The subsequent proliferation experiments were therefore done in media supplemented with 0.5μg/ml LPSe for 48h.GFAP is a kind of intermediate filament protein. Its expression can be regarded as a sensitive and reliable marker of astrocyte activation. The level of GFAP in astrocytes after LPS (0.5μg/ml) traeatment for various time periods was detected using Western bot. The level of GFAP was greatly enhanced (2.16-fold,2.03-fold, 2.36-fold,2.31-fold and 2.30-fold, respectively) for 2-48 h compared with control. A similar result was obtained by fluorescence method. In unstimulated astrocytes, low level of IL-6 was detected in culture medium (1.90 pg/ml).IL-6 level was elevated to 32.02 pg/ml at 2 h after LPS stimulation, and the maximum level of IL-6 production (242.58 pg/ml) was observed at 48 h.These results suggested that LPS could induce astrocytes activation shown as cell proliferation, increased GFAP expression and IL-6 secretion. Next, we determined whether mRNA and protein expression of TRPC3 were increased by LPS treatment with real time PCR, Western blot and confocol microscopy. LPS treatment with 0.5μg/ml for 2 h-48 h significantly enhanced the TRPC3 transcript level (3.47-fold,6.24-fold,6.24-fold,7.44-fold and 3.33-fold, respectively), and protein expression level (1.84-fold,2.25-fold,2.32-fold,2.45-fold and 1.91-fold, respectively). Western blot analysis was according with RT-PCR results showed the up-regulation of TRPC3 expression. Immuno fluorescence assay showed that TRPC3 expressed in the cytoplasm and nucleus, and higher levels in nucleus were observed in unstimulated cell. LPS could upregulate TRPC3 expression in the cytoplasm and nucleus.Several agents have been widely employed as TRPC inhibitor, including SKF96365,2-APB, although none are specific. So, we observed the effcets of 2-APB and SKF96365 on LPS-induced astrocytic activation.2-APB and SKF96365 inhibited LPS-induced cell proliferation in a dose-dependent fashion. BrdU results showed tha astrocyte proliferation was significantly blocked by 2-APB (5μM and 10μM) or SKF96365 (5μM). Flow cytometric analysis revealed that treatment with 2-APB (10μM) or SKF96365 (5μM) induced G0/G1 cell cycle arrest. Treatment with 2-APB (10μM) or SKF-96365 (5μM) significantly inhibited the elevation of GFAP expression levels induced by LPS. The levels of IL-6 in 2-APB group and SKF-96365 group were 142.73 pg/ml and 224.34 pg/ml, respectively. It was significantly reduced than that of LPS group (362.05 pg/ml).We investigated the relationship between Ca2+ influx through TRPC channels and astrocyte activation induced by LPS. [Ca2+]i changes in astrocytes were monitored by laser scanning confocal microscopy with the Ca2+ sensitive fluorescent probe fluo-4. LPS induced a rapid transient increase of [Ca2+]i followed by a sustained influx in the presence of calcium medium, while LPS induced a rapid transient increase of [Ca2+]i in the calcium free medium. The elevation of [Ca2+]i induced by LPS was from intracellular stores and calcium influx.2-APB inhibited the calcium elavation both from intracellular stores and calcium influx, while SKF96365 only inhibited calcium influx. The above results indicated that calcium entry induced by LPS might associate with TRPC.In summary, LPS could increase astrocyte activation, the expression of TRPC3 and the concentration of calcium. Moreover, TRPC channels inhibitors could block the effects of LPS on astrocytes. These results suggested that TRPC channels, especially TRPC3, might play important roles in the process of astrocyte activation.