A proinvasive role for the Ca2+‐activated K+ channel KCa3.1 in malignant glioma

Glioblastoma multiforme are highly motile primary brain tumors. Diffuse tissue invasion hampers surgical resection leading to poor patient prognosis. Recent studies suggest that intracellular Ca²⁺ acts as a master regulator for cell motility and engages a number of downstream signals including Ca²⁺‐activated ion channels. Querying the REepository of Molecular BRAin Neoplasia DaTa (REMBRANDT), an annotated patient gene database maintained by the National Cancer Institute, we identified the intermediate conductance Ca²⁺‐activated K⁺ channels, KCa3.1, being overexpressed in 32% of glioma patients where protein expression significantly correlated with poor patient survival. To mechanistically link KCa3.1 expression to glioma invasion, we selected patient gliomas that, when propagated as xenolines in vivo, present with either high or low KCa3.1 expression. In addition, we generated U251 glioma cells that stably express an inducible knockdown shRNA to experimentally eliminate KCa3.1 expression. Subjecting these cells to a combination of in vitro and in situ invasion assays, we demonstrate that KCa3.1 expression significantly enhances glioma invasion and that either specific pharmacological inhibition with TRAM‐34 or elimination of the channel impairs invasion. Importantly, after intracranial implantation into SCID mice, ablation of KCa3.1 with inducible shRNA resulted in a significant reduction in tumor invasion into surrounding brain in vivo. These results show that KCa3.1 confers an invasive phenotype that significantly worsens a patient's outlook, and suggests that KCa3.1 represents a viable therapeutic target to reduce glioma invasion. GLIA 2014;62:971–981     

Canonical transient receptor potential channel subtype 3‐mediated hair cell Ca2+ entry regulates sound transduction and auditory neurotransmission

The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca²⁺ entry pathway, with dual activation via G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca²⁺ releases Ca²⁺–calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca²⁺ homeostasis. Confocal microfluorimetry with the Ca²⁺ indicator Fluo‐4 acetoxymethylester showed that, when cytosolic Ca²⁺ was depleted, Ca²⁺ re‐entry was significantly impaired in mature TRPC3^?/? inner and outer hair cells. The impact of this disrupted Ca²⁺ homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3^?/? mice showed significantly stronger DPOAE (2f₁ ? f₂) growth functions than wild‐type (WT) littermates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3^?/? and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca²⁺ set‐point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback‐regulated Ca²⁺ entry pathway. This TPRC3‐regulated Ca²⁺ homeostasis shapes the sound transduction input–output function and auditory neurotransmission.     

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca2+‐permeable TRPC6 channels

The standardized extract of the St. John's wort plant (Hypericum perforatum ) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na⁺ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca²⁺‐permeable, resulting in intracellular Ca²⁺ elevations. Indeed, hyperforin activates TRPC6‐mediated currents and Ca²⁺ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca²⁺ transients and depolarizing inward currents sensitive to the TRPC channel blocker La³⁺, thus resembling the actions of the neurotrophin brain‐derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF. © 2012 Wiley Periodicals, Inc.     

DAG‐sensitive and Ca2+ permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study, we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu_1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6‐expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed‐forward and feed‐back inhibitory circuits in this brain region. © 2012 Wiley Periodicals, Inc.     

Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes

Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+-dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells.     

Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell‐autonomous fashion

Ca²⁺ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha‐1C subunit (i.e., Ca_v1.2) of the voltage‐dependent l ‐type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Ca_v1.2 channels located on astrocyte‐like stem cells and their descendants in the development of new granule neurons, we created Tg^{GLAST‐CreERT2}/Cacna1c^fl/fl/RCE:loxP mice, a transgenic tool that allows cell‐type‐specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type‐1 cells. FACS‐sorted Cacna1c‐deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c‐deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type‐1 cells decreased type‐1 cell proliferation and reduced the neuronal fate‐choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type‐1 cells, indicating that there must be Ca_v1.2‐independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L‐type 1.2 channels on type‐1 cells. Ca_v1.2 channels promote type‐1 cell proliferation and push the glia‐to‐neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Ca_v1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell‐autonomous fashion.     

Loss of NECL1, a novel tumor suppressor,can be restored in glioma by HDAC inhibitor‐Trichostatin A through Sp1 binding site

Nectin‐like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue‐specific immunoglobulin‐like cell–cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re‐expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at ?271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor‐Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co‐immunoprecipitation (co‐IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation. © 2008 Wiley‐Liss, Inc.     

TRPC channels are not required for graded persistent activity in entorhinal cortex neurons

Adaptive behavior requires the transient storage of information beyond the physical presence of external stimuli. This short‐lasting form of memory involves sustained (“persistent”) neuronal firing which may be generated by cell‐autonomous biophysical properties of neurons or/and neural circuit dynamics. A number of studies from brain slices reports intrinsically generated persistent firing in cortical excitatory neurons following suprathreshold depolarization by intracellular current injection. In layer V (LV) neurons of the medial entorhinal cortex (mEC) persistent firing depends on the activation of cholinergic muscarinic receptors and is mediated by a calcium‐activated nonselective cation current (I_CAN). The molecular identity of this conductance remains, however, unknown. Recently, it has been suggested that the underlying ion channels belong to the canonical transient receptor potential (TRPC) channel family and include heterotetramers of TRPC1/5, TRPC1/4, and/or TRPC1/4/5 channels. While this suggestion was based on pharmacological experiments and on effects of TRP‐interacting peptides, an unambiguous proof based on TRPC channel‐depleted animals is pending. Here, we used two different lines of TRPC channel knockout mice, either lacking TRPC1‐, TRPC4‐, and TRPC5‐containing channels or lacking all seven members of the TRPC family. We report unchanged persistent activity in mEC LV neurons in these animals, ruling out that muscarinic‐dependent persistent activity depends on TRPC channels.     

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2‐APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I_tail) and the associated dendritic Ca²⁺ influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I_tail. In addition, pretreatment with the calmodulin (CaM) inhibitor W‐7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I_tail) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3‐kinase (PI₃K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I_tail and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy. © 2010 Wiley‐Liss, Inc.     

Role of Transient Receptor Potential Channel 1 (TRPC1) in Glutamate-Induced Cell Death in the Hippocampal Cell Line HT22

Transient receptor potential channel 1 (TRPC1; a cation channel activated by store depletion and/or through an intracellular messenger) is expressed in a variety of tissues, including the brain. To study the physiological function of TRPC1, we investigated the role of endogenously expressed TRPC1 in glutamate-induced cell death, using the murine hippocampal cell line HT22. Knocking down TRPC1 mRNA using TRPC1-shRNA or blocking of TRPC channels using 2-APB (≥200 μM) robustly attenuated glutamate-induced cell death after 24 h of incubation with 5 mM glutamate. Glutamate toxicity in HT22 cells seems to involve metabotropic glutamate receptor mGluR5 since MPEP (2-methyl-6-(phenylethynyl)-pyridine), an mGluR5 antagonist (≥100 μM), abrogated glutamate toxicity. Furthermore, a direct activation of mGluR5 by CHPG [(RS)-chloro-5-hydroxyphenylglycine; 100 μM or 300 μM] promoted HT22 cell death. TRPC1 knock-down markedly reduced CHPG-induced cell death. These observations suggest that glutamate-induced cell death in HT22 cells activates mGluR5 receptors, which significantly increases Ca²⁺ influx through TRPC1 channels. TRPC1 knock-down prevented glutamate- and CHPG-induced cell death, suggesting that glutamate-induced toxicity in HT22 cells is mediated through TRPC1 channels and an mGluR5-dependent pathway. Together, this work provides evidence for a novel receptor activation pathway of TRPC1 in glutamate-induced toxicity.     

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin‐1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem‐like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133⁺‐C6) or by infection of C6 cells with control lentivirus (mock‐C6). Stable hCD133 expression promoted the self‐renewal ability of C6‐formed spheres with an increase in the expression of the stemness markers, Bmi‐1 and SOX2. Akt phosphorylation, Notch‐1 activation, and Notch‐1 target gene expression (Hes‐1, Hey1 and Hey2) were increased in hCD133⁺‐C6 when compared to mock‐C6. The inhibition of Akt phosphorylation, Notch‐1 activation, and Hes‐1 in hCD133⁺‐C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133⁺‐C6. An elevated expression of GTPase‐activating protein 27 (Arhgap27) was detected in hCD133⁺‐C6. A decline in the invasion of hCD133⁺‐C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133⁺‐C6. In vivo study further showed that hCD133⁺‐C6 formed aggressive tumors in vivo compared to mock‐C6. Exposure of hCD133⁺‐C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch‐1 activation and Hes‐1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch‐1/Hes‐1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133⁺‐C6 in vitro, as well as progressive tumor formation in vivo.     

Bimodal anti‐glioma mechanisms of cilengitide demonstrated by novel invasive glioma models

Integrins are expressed in tumor cells and tumor endothelial cells, and likely play important roles in glioma angiogenesis and invasion. We investigated the anti‐glioma mechanisms of cilengitide (EMD121974), an αvβ3 integrin inhibitor, utilizing the novel invasive glioma models, J3T‐1 and J3T‐2. Immunohistochemical staining of cells in culture and brain tumors in rats revealed positive αvβ3 integrin expression in J3T‐2 cells and tumor endothelial cells, but not in J3T‐1 cells. Established J3T‐1 and J3T‐2 orthotopic gliomas in athymic rats were treated with cilengitide or solvent. J3T‐1 gliomas showed perivascular tumor cluster formation and angiogenesis, while J3T‐2 gliomas showed diffuse single‐cell infiltration without obvious angiogenesis. Cilengitide treatment resulted in a significantly decreased diameter of the J3T‐1 tumor vessel clusters and its core vessels when compared with controls, while an anti‐invasive effect was shown in the J3T‐2 glioma with a significant reduction of diffuse cell infiltration around the tumor center. The survival of cilengitide‐treated mice harboring J3T‐1 tumors was significantly longer than that of control animals (median survival: 57.5 days and 31.8 days, respectively, P < 0.005), while cilengitide had no effect on the survival of mice with J3T‐2 tumors (median survival: 48.9 days and 48.5, P = 0.69). Our results indicate that cilengitide exerts a phenotypic anti‐tumor effect by inhibiting angiogenesis and glioma cell invasion. These two mechanisms are clearly shown by the experimental treatment of two different animal invasive glioma models.     

Calcium ion influx in microglial cells: Physiological and therapeutic significance

Microglial cells, the immunocompetent cells of the central nervous system (CNS), exhibit a resting phenotype under healthy conditions. In response to injury, however, they transform into an activated state, which is a hallmark feature of many CNS diseases. Factors or agents released from the neurons, blood vessels, and/or astrocytes could activate these cells, leading to their functional and structural modifications. Microglial cells are well equipped to sense environmental changes within the brain under both physiological and pathological conditions. Entry of calcium ions (Ca²⁺) plays a critical role in the process of microglial transformation; several channels and receptors have been identified on the surface of microglial cells. These include store‐operated channel, Orai1, and its sensor protein, stromal interaction molecule 1 (STIM1), in microglial cells, and their functions are modulated under pathological stimulations. Transient receptor potential (TRP) channels and voltage‐ and ligand‐gated channels (ionotropic and metabotropic receptors) are also responsible for Ca²⁺ influx into the microglial cells. An elevation of intracellular Ca²⁺ concentration subsequently regulates microglial cell functions by activating a diverse array of Ca²⁺‐sensitive signaling cascades. Perturbed Ca²⁺ homeostasis contributes to the progression of a number of CNS disorders. Thus, regulation of Ca²⁺ entry into microglial cells could be a pharmacological target for several CNS‐related pathological conditions. This Review addresses the recent insights into microglial cell Ca²⁺ influx mechanisms, their roles in the regulation of functions, and alterations of Ca²⁺ entry in specific CNS disorders. © 2014 Wiley Periodicals, Inc.     

Establishment and partial characterization of five malignant glioma cell lines

Five malignant glioma cell lines (YMG1, 2, 3, 4, and 5) were established from surgical specimens obtained from patients with glioblastoma or anaplastic astrocytoma, and these lines were partially characterized. Three glioma cell lines (YMG1, 3, and 5) were weakly positive for GFAP by Western blot analysis and two cell lines were negative. S‐100 protein was positive in all glioma cell lines. The expression of p53, p16, p15, cyclin‐dependent kinase 4 (CDK4), and EGF receptor (EGFR) proteins was examined by Western blotting. YMG1 and 2 cell lines showed accumulation of p53 protein and loss of p16 and p15 expression. YMG3 and 4 showed accumulation of p53 protein and expression of p16 and p15 proteins. YMG5 revealed weak expression of p53 protein, suggesting wild‐type p53, and loss of p16 and p15 expression. All cell lines expressed various levels of CDK4 protein. YMG1, 2, and 3 showed higher EGFR protein expression and YMG4 and 5 showed lower EGFR expression compared to U251 glioblastoma cells, which express high levels of EGFR. Fluorescence in situ hybridization analysis for EGFR gene expression did not show any amplification in the glioma cell lines. Immunohistochemical studies revealed that the patterns of p53 and EGFR expressions in the original tumor tissues were mostly correlated with those in the malignant glioma cell lines. These results suggest that the characteristics of p53 and EGFR expression in the malignant glioma cell lines were passed over from the original tumor tissues. These newly established malignant glioma cell lines can be used for further analysis of the mechanisms of tumor growth and progression.     

Alterations in Potassium Currents May Trigger Neurodegeneration in Murine Scrapie

Conventional electrophysiological intracellular recording techniques were used to test the hypothesis that enhanced calcium entry via voltage-gated calcium channels or theN-methyl-d-aspartate (NMDA) subtype of glutamate receptor–channel complex may be a primary pathological mechanism triggering neurodegeneration in scrapie and related diseases. This study was carried out at a time when cell loss is known to occur and when hippocampal pyramidal cells in area CA1 are rendered hyperexcitable following scrapie infection. There was no change to the NMDA receptor-mediated component of the Schäffer collateral evoked excitatory postsynaptic potential (EPSP) or the level of spontaneous firing activity of CA1 cells following addition of the specific NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV, 20 μM), to the perfusate in scrapie-infected mice, indicating that the NMDA receptor–channel complex is not compromised by scrapie. There was also no change seen in the non-NMDA mediated component of the EPSP. The calcium spike of CA1 pyramidal cells was not significantly altered by scrapie infection, indicating that high threshold voltage-gated Ca²⁺channel function is not compromised by scrapie. By contrast, cells from scrapie-infected mice fired calcium spikes repetitively and the long, slow AHP, which in control cells inhibited repetitive firing, was absent. Cells from scrapie-infected mice showed more depolarized membrane potentials than controls but this difference in potential was no longer observed after exposure to TEA. These data indicate a loss of TEA-insensitive and TEA-sensitive potassium conductances. We suggest that altered potassium currents rather than increased calcium entry via voltage-sensitive calcium channels or the NMDA receptor complex may be the primary pathological mechanism triggering neurodegeneration in scrapie and related diseases.     

Sphingosine‐1‐phosphate induces Ca2+ signaling and CXCL1 release via TRPC6 channel in astrocytes

A biologically active lipid, sphingosine‐1‐phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein‐coupled receptors, some of which generates Ca²⁺ mobilization. In astrocytes, S1P is reported to evoke Ca²⁺ signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca²⁺‐permeable cation channels expressed in astrocytes and involved in Ca²⁺ influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P‐induced cellular responses. In Ca²⁺ imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca²⁺ in astrocytes, followed by sustained elevation. The sustained Ca²⁺ response was markedly suppressed by S1P₂ receptor antagonist JTE013, S1P₃ receptor antagonist CAY10444, or non‐selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca²⁺ mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca²⁺ signaling in astrocytes via G_q‐coupled receptors S1P₂ and S1P₃, followed by Ca²⁺ influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.