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NERVOUS SYSTEM CELL BIOLOGY
-induced Ca2+ signals in mouse cortical astrocytes: roles of store- and receptor-operated Ca2+ entryDepartment of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
Submitted 12 June 2007 ; accepted in final form 25 July 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. IL-1 disrupts Ca2+ homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1 induces Ca2+ dysregulation are not completely defined. Here, we examined how acute and chronic (24–48 h) treatment with IL-1 affect Ca2+ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured with fura-2 using digital imaging. An acute application of 10 ng/ml IL-1 induced Ca2+ mobilization from intracellular stores and activated store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1 for 24 and 48 h elevated resting [Ca2+]cyt, decreased Ca2+ store content [associated with sarco(endo)plasmic reticulum Ca2+-ATPase 2b downregulation], and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca2+ channels are Ba2+ permeable, Ba2+ entry was used to distinguish receptor-operated Ca2+ channels from store-operated Ca2+ channels. ROCE was activated by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba2+, OAG-induced elevations of cytosolic Ba2+ (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1. These changes in IL-1-treated astrocytes correlate with augmented expression of transient receptor potential cation channel (TRPC)6 protein, which likely mediates ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1-induced dysregulation of Ca2+ homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca2+ homeostasis appears to be an upstream component in the cascade of IL-1-activated pathways leading to neurodegeneration. transient receptor potential cation channel proteins
IL-1 is one of the most critical inflammatory cytokines in neurodegeneration (14, 51). IL-1 activates astrocytes and promotes the synthesis and release of additional cytokines and neuroactive molecules such as IL-6, TNF-
, amyloid -protein, arachidonic acid, and nitric oxide (29, 33). Neuronal injury arising from IL-1-induced insults can activate microglia with further overexpression of IL-1, thus producing feedback amplification and self-propagation of this cycle (22).
IL-1 receptor (IL-1R) stimulation is associated with elevation of the intracellular free Ca2+ concentration ([Ca2+]cyt) (26, 44). IL-1 also modulates Ca2+ wave propagation between astrocytes in culture (28) and alters Ca2+ responses to quisqualate, serotonin, and ATP (26, 31, 61). Thus, inflammatory conditions that chronically elevate the IL-1 level can alter astrocytic responses to normal physiological stimuli. Elevated [Ca2+]cyt is a universal signal that controls many cell processes (6). The mechanisms by which IL-1 induces Ca2+ dysregulation, although not completely defined, may be causally implicated in reactive functional changes of astrocytes leading to neurodegeneration. They may involve Ca2+ mobilization from intracellular stores as well as activation of Ca2+ influx across the plasma membrane (PM).
Recent findings indicate that Ca2+ entry through store-operated Ca2+ channels (SOCs) and receptor-operated Ca2+ channels (ROCs) in the PM plays an important role in shaping cytoplasmic Ca2+ signals in astrocytes (20, 45, 50). Some evidence indicates that mammalian SOCs and ROCs are homo- or heterotetramers and formed by members of a family of seven proteins [transient receptor potential cation channels (TRPC1–TRPC7)] that are homologous to the Drosophila transient receptor potential channel involved in phototransduction (52, 55). In particular, TRPC1 and TRPC4 may form, or be part of the endogenous SOCs activated by endoplasmic reticulum (ER) Ca2+ store depletion, whereas TRPC3 and TRPC6 can be activated by inositol (1,4,5)-trisphosphate (IP3) and/or diacylglycerol and may not be store dependent (52). The effect of IL-1 on the expression of TRPCs in astrocytes is unknown. Information is also lacking about the contributions of store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) to IL-1-induced [Ca2+]cyt changes in astrocytes. These issues are addressed in the present study.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, cells grown on coverslips and culture dishes were first synchronized by serum deprivation (0.5%) for 24 h. The medium was then aspirated and replaced with DMEM-F-12 medium plus 0.5% FBS, supplemented with IL-1 (10 ng/ml), or DMEM-F-12 (0.5% FBS) (control) for a further 24 and 48 h. Experiments were performed on subconfluent cultures on days 8–12 in vitro. Freshly dissociated astrocytes. Astrocytes were prepared from the brains of fetal mice (embryonic days 16–17) using a modification of previously published methods (64). Cerebral cortices were separated from the meninges and hippocampus. Cortical slices were cut using a manual tissue slicer and incubated for 1 h at room temperature in Ca2+-free solution containing (in mM) 124 NaCl, 5 KCl, 2 MgSO4, 26 NaHCO3, 1 Na-pyruvate, and 10 D-glucose, aerated with 95% O2-5% CO2 to maintain pH at 7.35. Slices were then transferred to artificial cerebrospinal fluid containing (in mM) 124 NaCl, 5 KCl, 1.3 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 D-glucose, supplemented with papain (24 U/ml) and L-cysteine (0.24 mg/ml) and bubbled with 95% O2-5% CO2 for 30 min (22°C). The enzymatic digestion was halted by washing of the slices in Ca2+-free solution. Slices were stored in this solution at room temperature for at least 1 h for recovery. They were then transferred to standard physiological solution containing 1 mM kynurenic acid and 0.1 mM leupeptin, a Ca2+-activated protease inhibitor. Slices were dispersed by trituration with a fire-polished Pasteur pipette. Cells were then directly deposited on poly-L-lysine-coated glass coverslips. Astrocytes were allowed to settle on the coverslips for 30–40 min before being loaded with fura-2. At the conclusion of each Ca2+-imaging experiment, the same cells were labeled for glial fibrillary acidic protein (GFAP; Boehringer Mannheim) to characterize glial cells (5). In these experiments, nuclei also were identified by labeling for 5 min with a 50 µM solution of 4',6'-diamidino-2-phenylindole (DAPI) (20). Most of the cells (96%) were GFAP positive.
Ca2+ imaging. [Ca2+]cyt was measured with fura-2 using digital imaging. Details of fluorescence imaging and analysis techniques have been previously published (18). Astrocytes grown on coverslips were loaded with fura-2 by an incubation for 35 min in culture medium containing 3.3 µM fura-2 AM (20–22°C, 5% CO2-95% O2). After the dye loading, coverslips were transferred to a tissue chamber mounted on a microscope stage, where cells were superfused for 15–20 min (35–36°C) with standard physiological salt solution (PSS) to wash away extracellular dye. The PSS contained (in mM) 140 NaCl, 5.9 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (pH 7.4). Cells were studied for 40–60 min during continuous superfusion with PSS (35°C).
The imaging system was designed around a Zeiss Axiovert 100 microscope (Carl Zeiss, Thornwood, NY) optimized for UV transmission. Fura-2-loaded cells were illuminated with a diffraction grating-based system (Polychrome II, Applied Scientific Instruments, Eugene, OR) (18). Fluorescent images were recorded using a Gen III ultrablue intensified charge-coupled device camera (Stanford Photonics, Palo Alto, CA). Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Universal Imaging, West Chester, PA). [Ca2+]cyt was calculated by determining the ratio of fura-2 fluorescence excited at 380 and 360 nm as previously described (20). Intracellular Sr2+ and Ba2+ measurements are shown as the fura-2 340-to-380-nm excitation ratio with fluorescent emission at 510 nm (57).
Immunocytochemistry. Freshly isolated astrocytes were fixed and cross-reacted with polyclonal antibodies raised against GFAP and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). This enabled us to visualize the distribution of the primary label with a fluorescence microscope (Zeiss Axiovert 100, Carl Zeiss). Details have been previously published (5).
Western immunoblot analysis.
Membrane proteins were separated by 7.5% SDS-PAGE as previously described (19) and transferred electrophoretically to a nitrocellulose membrane (Amersham BioSciences). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at room temperature with appropriate primary antibodies. The following antibodies were used: rabbit polyclonal anti-TRPC1, anti-TRPC3, anti-TRPC4, anti-TRPC5, and anti-TRPC6 (Allomone Laboratories, Israel); rabbit polyclonal anti-TRPC3 and anti-TRPC7 [gifts from Dr. W. Schilling, Case Western Reserve University (17)]; monoclonal anti-Na+/Ca2+ enchanger 1 (NCX1; clone R3F1) (Swant, Switzerland); polyclonal anti-sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b; a gift from Dr. F. Wuytak, Katholieke University, Leuven, Belgium); and rabbit polyclonal anti-PM Ca2+-ATPase 1 (Affinity BioReagents, Golden, CO). Gel loading was controlled with polyclonal or monoclonal anti--actin antibodies (Sigma-Aldrich, St. Louis, MO). After being washed, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h at room temperature. The immune complexes on the membranes were detected by enhanced chemiluminescence-plus (Amersham BioSciences) and exposure to X-ray film (Eastman Kodak, Rochester, NY). Quantitative analysis of immunoblots was performed by using a Kodak DC120 digital camera and 1D Image Analysis Software (Eastman Kodak).
Short interfering RNA knockdown. Primary cultured mouse cortical astrocytes were transfected with short interfering (si)RNA ON-Target plus Smart pool (20 µM) designed against TRPC6 or siCONTRL (Dharmacon, Lafayette, CO). The sequences of the TRPC6/siRNA duplexes were as follows: 5'-UCUAACGACAGUCUCUCCCUU-3', 5'-UCUUCCAAGUGAAAUCUGCUU-3', 5'-AAUCCGUACAUAACCUUUAUU-3', and 5'-GAUUAGCUAACCUUCUUCCUU-3'. Twenty-four hours before treatment, cells were placed in the culture medium (DMEM-F-12) without antibiotics and further transfected with siRNA using Lipofectamine 2000 reagent in Opti-MEM (Invitrogen). Following 24 h of incubation, the medium was aspirated and replaced with DMEM-F-12 without siRNA for 77 h before Ca2+ measurements or Western blot analysis were performed.
Materials.
IL-1 and the IL-1 type 1 receptor (IL-1RI) antagonist (IL-RA) were purchased from R&D Systems (Minneapolis, MN). FBS was obtained from Atlanta Biologicals (Lawrenceville, GA). All other tissue culture reagents were obtained from GIBCO-BRL (Grand Island, NY). Fura-2 AM was obtained from TefLabs (Austin, TX). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) was purchased from Calbiochem (San Diego, CA). Cyclopiazonic acid (CPA), papain, L-cysteine, kynurenic acid, leupeptin, DMSO, poly-L-lysine, DAPI, -actin, penicillin G, and streptomycin were purchased from Sigma-Aldrich. All other reagents were analytic grade or the highest purity available.
Statistical analysis. Numerical data presented in the RESULTS are means ± SE from n single cells (1 value/cell). Numbers of different animals and different litters are also presented where appropriate. Data from four to five litters were obtained for most protocols and were consistent from litter to litter. Statistical significance was determined using Student's t-test and ANOVA.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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induces complex [Ca2+]cyt responses in cultured mouse cortical astrocytes.
The effect of 10 ng/ml IL-1 was examined on [Ca2+]cyt as this concentration causes reactive functional changes in astrocytes (28). The application of IL- for 10 min induced either a rapid initial Ca2+ transient increase often followed by low-amplitude [Ca2+]cyt oscillations or fluctuations (Fig. 1A; 37% of cells) or only a small, slow [Ca2+]cyt rise without an initial Ca2+ transient (Fig. 1B; 25% of cells). [Ca2+]cyt returned to the resting level 7–10 min after washout of IL-1. Approximately 38% of the 122 cells did not response to IL-1. The absence of the Ca2+ response to IL-1 in some cultured as well as freshly dissociated cells was likely due to low expression of IL-1R under normal physiological conditions (58).
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-induced Ca2+ response in the presence of extracellular Ca2+ could be attributed to both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular fluid. Therefore, to eliminate the contribution of extracellular Ca2+, these experiments were repeated with Ca2+-free medium. Under these circumstances, the IL-1-induced Ca2+ transients persisted (Fig. 1C), although their peak amplitude was significantly smaller (323 vs. 433 nM, P < 0.05, n = 85; Fig. 1D). Restoration of external Ca2+ induced an additional small rise in [Ca2+]cyt apparently associated with SOCE (Fig. 1C). The effects of IL-1 were completely reversible. During prolonged application of IL-1, the slow rise in [Ca2+]cyt seen in the presence of extracellular Ca2+ (Fig. 1, A and B) was not observed in Ca2+-free medium (not shown). Thus, the IL-1 response depends on both Ca2+ mobilization from intracellular stores and extracellular Ca2+ influx. To determine whether the IL-1-induced Ca2+ signals observed in cultured cells prevail in the native tissue, experiments were repeated in freshly dissociated astrocytes.
IL-1 releases intracellular Ca2+ and activates SOCE and ROCE in freshly dissociated astrocytes.
The resting [Ca2+]cyt level in freshly dissociated astrocytes was significantly smaller than in cultured cells (74 vs. 135 nM, P < 0.001, n = 257; Figs. 1D and 2B). In the presence of extracellular Ca2+, IL-1 evoked a rapid rise in [Ca2+]cyt in 76% of the 125 freshly dissociated cells (Fig. 2A). [Ca2+]cyt then declined, approaching the basal level due to Ca2+ buffering and extrusion. Approximately 14% of the freshly dissociated astrocytes showed only a slow, progressive rise in [Ca2+]cyt (data not shown), similar to that in cultured astrocytes (Fig. 1B), and 10% of the cells had no response to IL-1. The variety of IL-1-induced Ca2+ responses did not correlate with heterogeneity in astrocyte morphology. Immediately after Ca2+ measurements, cells were stained with anti-GFAP antibody and DAPI (Fig. 2C), as described in MATERIALS AND METHODS. A large majority of the cells were GFAP positive but were quite heterogeneous in morphology (Fig. 2C,a). Most cells were round with fine processes, whereas 
10% of the astrocytes were larger and exhibited a flat protoplasmic form with occasional filopodial extensions (Fig. 2C,a). Nevertheless, both types of freshly dissociated astrocytes exhibited similar IL-1-induced Ca2+ responses, as shown in Fig. 2A.
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-induced Ca2+ transients in Ca2+-free medium was significantly smaller than that in the presence of external Ca2+ (289 vs. 378 nM, P < 0.001, n = 95; Fig. 2B). The transient in Ca2+-free medium is a manifestation of the unloading of the intracellular Ca2+ store. Subsequent restoration of external Ca2+ in the presence of IL-1 evoked a secondary rise in [Ca2+]cyt, mediated by Ca2+ entry (Fig. 2A). A similar profile of Ca2+ responses was observed when Ca2+ stores were depleted with CPA, a specific inhibitor of SERCA (Fig. 3A).
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induces not only Ca2+ release from the ER store but also Ca2+ entry from the extracellular fluid in both freshly dissociated and cultured astrocytes. Some of this "signal Ca2+" may enter through SOCs and/or ROCs. SOCs have high Ca2+ selectivity and, unlike ROCs, are virtually impermeable to other alkaline-earth divalent cations, such as Sr2+ and Ba2+ (42, 56). Therefore, to distinguish ROCs from SOCs, we used Sr2+ entry. Figure 4 shows that after depletion of the Ca2+ store with IL-1 in the absence of external Ca2+ in freshly dissociated astrocytes, the addition of Sr2+ increased the fura-2 340-to-380-nm ratio, presumably due to ROCE. Subsequent restoration of extracellular Ca2+ induced a large Ca2+ entry, which is typical of SOC activation. To eliminate the contribution of voltage-gated Ca2+ channels to IL-1-induced Ca2+ entry, all solutions in these experiments contained 10 µM nifedipine, a L-type Ca2+ channel blocker. The data indicate that the acute application of IL-1 activates both ROCE and SOCE.
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occurs through activation of IL-1RI, freshly dissociated (Fig. 5) and cultured astrocytes (not shown) were treated with IL-1RA. This agent blocked IL-1-induced Ca2+ responses in both cell types. Thus, IL-1 acts through IL-1RI.
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treatment of cultured astrocytes elevates resting [Ca2+]cyt, increases ROCE, and upregulates expression of TRPC6 protein.
Treatment of cultured astrocytes with 10 ng/ml IL-1 for 24 h altered cell morphology (Fig. 6A, a'). It induced the development of multiple processes, which is associated with reactive gliosis (34). These morphological changes were accompanied by increased resting [Ca2+]cyt (Fig. 6, A,b' and C). SOCE, activated by Ca2+ store depletion with 10 µM CPA, was not significantly changed under these conditions (Fig. 6, B and C). The stored Ca2+, as evaluated by measuring peak amplitudes of CPA-induced Ca2+ transients in Ca2+-free medium, was, however, smaller in cells treated with IL-1 for either 24 h (227 ± 15 vs. 270 ± 18 nM, n = 258) or 48 h (305 ± 17 vs. 370 ± 22 nM, n = 218). Reduction of CPA-releasable ER Ca2+ stores after IL-1 treatment correlated with a significant decrease of SERCA2b protein expression, which was, however, increased during cell culture (Fig. 7, A and B).
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for 24 and 48 h (data not shown). On the other hand, the expression of TRPC6, which may form ROCs, was significantly augmented under these conditions (Fig. 7, C and D). IL-1 did not affect the expressions of TRPC3 and TRPC7 (data not shown), which have also been implicated in ROCE in different cell types (23).
To determine whether chronic IL-1 treatment increases ROCE, astrocytes were stimulated with the cell-permeable diacylglycerol analog OAG, which opens TRPC6 and TRPC3 channels in a PKC-independent manner (24). Based on evidence that ROCs, but not SOCs, are Ba2+ permeable, Ba2+ entry was used to distinguish ROCs from SOCs in these experiments. In contrast to Sr2+, Ba2+ is not transported by SERCA or PM Ca2+ pumps (32). In the presence of extracellular Ba2+, OAG (40 µM)-induced elevations of cytosolic Ba2+ (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1 for 24 and 48 h (P < 0.001, n = 425; Fig. 8). These changes in IL-1-treated astrocytes correlated with augmented expression of TRPC6 protein (Fig. 7, C and D), which is an obligatory component of endogenous ROCs in a variety of cell types (24, 55). To determine whether TRPC6 is involved in ROCE in astrocytes, a RNA interference strategy was employed. Selective inhibition of TRPC6 protein expression, using siRNA targeted to the TRPC6 gene (Fig. 9, A and B) significantly attenuated OAG (40 µM)-induced elevations of cytosolic Ba2+ (fura-2 340-to-380-nm ratio, P < 0.001, n = 93; Fig. 9, C and D). The results strongly suggest that channels formed by TRPC6 mediate ROCE in mouse cortical astrocytes. Acute application of IL-1 did not activate receptor-operated Sr2+ entry in cells treated with TRPC6/siRNA (not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B, cAMP, tyrosine kinase, and PKC (31, 53). IL-1 also promotes astrocytic synthesis and the release of neuroactive molecules, such as nitric oxide, arachidonic acid, and amyloid -protein, that can result in neuron and astrocyte death (8, 29, 33). Many, if not all, of these signaling pathways are regulated by intracellular Ca2+ (11, 25). Therefore, early disruption of Ca2+ homeostasis might be critical for regulating IL-1-induced downstream events. How does IL-1 disrupt Ca2+ homeostasis? Here, we show that acute application of IL-1 induces Ca2+ mobilization from intracellular stores as well as Ca2+ entry via SOCs and ROCs in freshly dissociated and primary cultured mouse cortical astrocytes. IL-1 treatment of cultured astrocytes for 24–48 h increases [Ca2+]cyt levels. This is apparently due to reduced Ca2+ sequestration in the ER (associated with SERCA2b downregulation) and augmented ROCE; the later correlates with upregulated expression of TRPC6.
Freshly dissociated and primary cultured mouse cortical astrocytes express IL-1R linked to Ca2+ signaling pathways.
The biological effects of IL-1 are mediated by IL-1RI, which requires association with an accessory protein for signal transduction (21, 49). The IL-1 type II receptor (IL-1RII) also binds IL-1 but lacks an intracellular domain and does not initiate signal transduction (49). IL-1RI is expressed by both glia and neurons (35), albeit at low levels in the healthy, adult CNS (58). There are two types of IL-1, IL-1 and IL-1, both of which bind to IL-1RI (15). IL-1 is the most rapidly expressed, initially by microglia, in response to brain damage (12). The IL-1 system is unique in that, to date, it is the only cytokine that has an endogenous, highly selective, competitive receptor antagonist (IL-1RA), which interacts with the receptor without transmitting a signal (2).
Studies of IL-1 action on Ca2+ signals in astrocytes have been limited to cell culture systems (26, 44). In C6 rat glioma cells, IL-1 produced a slow rise in [Ca2+]cyt that was blocked by IL-1RA, indicating that IL-1 influences Ca2+ signaling via IL-1RI (44). Holliday and Gruol (26) demonstrated that IL-1 induced a large Ca2+ transient response followed by Ca2+ oscillations in cultured rat astrocytes pretreated with forskolin (conditions that may upregulate the expression of IL-1RI). In untreated cells, IL-1 evoked only a small, slow rise in [Ca2+]cyt.
We observed similar patterns of Ca2+ responses to IL-1 not only in cultured but also in freshly dissociated astrocytes under normal conditions (without any cell pretreatment). The acute application of IL-1 induced rapid, large Ca2+ transients or small, slowly developing rises in [Ca2+]cyt (Figs. 1 and 2). Pretreatment with IL-1RA eliminated IL-1-induced Ca2+ responses (Fig. 5). Thus, not only cultured cells, but also freshly dissociated astrocytes, express IL-1RI, which is linked to intracellular Ca2+ signaling. The apparent diversity of IL-1-induced Ca2+ responses was not associated with heterogeneity in astrocyte morphology (Fig. 2C,a). The different responses might reflect differences in the expression of IL-1RI and/or it's coupling to Ca2+ signaling pathways in cells isolated from different regions of the cortex. The presence of endogenous IL-1RI in the native tissue indicates that the IL-1 system may be functional in the embryonic brain. Several studies (16, 37) have implicated IL-1 in CNS development, but few specifics are known.
Responses to IL- involve ER Ca2+ release and ROCE and SOCE.
It is important to identify the primary pathways for Ca2+ delivery into the cytoplasm that initiate and shape Ca2+ responses to IL-1. The complex nature of Ca2+ responses reflects Ca2+ mobilization from intracellular stores and/or Ca2+ influx from the extracellular fluid. The initial, IL-1-induced fast transient rise in [Ca2+]cyt is due to Ca2+ release from ER stores, mediated largely by IP3, as it persists in Ca2+-free medium (Figs. 1 and 2). There are reports in other types of cells that IL-1 activates tyrosine kinase and phospholipase C, which, in turn, stimulate the formation of IP3 (13, 60). IL-1-induced low-amplitude [Ca2+]cyt oscillations or fluctuations, as well as the small slowly developing Ca2+ signals (Fig. 1), depend mainly on extracellular Ca2+ entry, likely through SOCs and/or ROCs. Mobilization of Ca2+ from ER stores activates SOCs in a large variety of cell types (43), including cultured astrocytes (19, 20, 45, 50, 62) and freshly dissociated astrocytes (this report). When ER Ca2+ stores are depleted by IL-1 or CPA in Ca2+-free medium, restoration of Ca2+ induces a Ca2+ influx through SOCs (Figs. 2 and 3). In addition, when extracellular Ca2+ is replaced by Sr2+, which enters through ROCs, but not SOCs (42), IL-1 induces Sr2+ entry. Thus, acutely applied IL-1 activates both ROCE and SOCE (Fig. 4).
Chronic IL-1-induced astrocyte activation elevates resting [Ca2+]cyt, augments TRPC6 expression, and increases ROCE.
Astrocytes treated with IL-1 for 24–48 h exhibit structural changes characterized by multiple highly branched processes (stellation) (Fig. 6A). There is reorganization of the actin cytoskeleton, accompanied by disruption of focal adhesion complexes and loss of their signaling function (30). We show that these morphological changes are accompanied by a significant increase in resting [Ca2+]cyt (Fig. 6). Disruption of Ca2+ homeostasis might be one of the principal mechanisms by which IL-1 manifests its neurotoxicity. Indeed, there have been several reports (11, 36) showing that Ca2+ disregulation plays an important role in the production of IL-1 and nitric oxide synthase in cytokine-stimulated astrocytes.
Elevated [Ca2+]cyt may result from reduced Ca2+ sequestration in the ER, decreased Ca2+ extrusion from the cytosol by the PM Ca2+ pump and/or NCX, and/or increased extracellular Ca2+ entry, largely through SOCs and ROCs. Here, we demonstrate that IL-1-treatment dramatically reduces the expression of SERCA2b (Fig. 7, A and B). This can explain the significant decrease in CPA-releasable ER Ca2+ store content (Fig. 6, B and C). IL-1-induced downregulation of SERCA and depletion of the ER Ca2+ store have also been described in cultured pancreatic -cells (7) and cardiomyocytes (10), although the expression of SERCA2b was increased in response to IL-1 in human myometrical smooth muscle cells (54). We did not observe any change in PM Ca2+ pump or NCX expression in astrocytes treated with IL-1 for 24 or 48 h (not shown). Cell treatment with IL-1 also did not affect SOCE (Fig. 6, B and C). IL-1 treatment did, however, significantly augment ROCE, which was measured as extracellular Ba2+ influx activated by the cell-permeable diacylglycerol analog OAG (Fig. 8).
TRPC family proteins may be responsible for SOCE and ROCE in a variety of cell types (9, 23, 38, 52), including astrocytes (20). Recently, the mammalian proteins Orai1 and stromal interacting molecule 1 (STIM1) have been shown to be essential for the activation of SOCE (27, 41, 63). Orai1 may form the Ca2+ selectivity filter of the Ca2+ release-activated Ca2+ (CRAC) channel (63), which is probably only one species of SOC (43). STIM1, the putative Ca2+ sensor in the ER, regulates SOCs and CRAC channels, but not ROCs (27).
Our data demonstrate that enhanced ROCE in IL-1-treated astrocytes is correlated with upregulated expression of TRPC6 (Figs. 7, C and D, and 8), an essential component of the ROCs that mediate ROCE in mouse cortical astrocytes. Cell transfection with TRPC6/siRNA significantly reduced both the expression of TRPC6 protein (Fig. 9, A and B) and ROCE (Fig. 9, C and D). TRPC6 channels are activated by diacylglycerols in a store depletion-independent manner (24). Particularly noteworthy is the fact that IL-1RI activation leads to generation of diacylglycerol (48, 59). Therefore, it is not surprising that IL-1 treatment increases extracellular Ca2+ influx. We do not exclude, however, other Ca2+ entry pathways that might also be activated by IL-1 treatment. In human astrocytes, for example, IL-1 upregulates not only the expression of purinergic metabotropic P2Y receptors, which are involved in Ca2+ wave propagation between astrocytes (28), but also the expression of ionotropic P2X7 receptors, which form pores in response to ligand stimulation. This greatly increases membrane permeability (40).
In summary, this report demonstrates that astrocytes freshly dissociated from the normal, healthy embryonic mouse brain express functionally active IL-1RI. Furthermore, we identified mechanisms of IL-1-induced Ca2+ signals in freshly dissociated astrocytes that include not only Ca2+ mobilization from the ER but also the activation of SOCE and ROCE. Finally, chronic (24–48 h) IL-1 treatment of cultured astrocytes downregulates SERCA2b, upregulates TRPC6 expression, increases ROCE, and elevates resting [Ca2+]cyt. The disruption of Ca2+ homeostasis is likely to be an upstream component in the cascade of IL-1-activated pathways leading to neurodegeneration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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