Estradiol-induced Nongenomic Calcium Signaling Regulates Genotropic Signaling in Macrophages*

Estradiol (E 2 ) exerts not only genotropic but also non- genomic actions through nuclear estrogen receptors (ER). Here, we provide a novel paradigm for nongenomic E 2 signaling independent of nuclear ER. E 2 in- duces a rapid rise in the intracellular free Ca 2 (cid:1) concentration ([Ca 2 (cid:1) ] i ) through membrane estrogen receptors in murine RAW 264.7 macrophages. This E 2 -induced Ca 2 (cid:1) signaling is not prevented by different ER blockers and cannot directly activate stably transfected c- fos promoter or the mitogen-activated protein kinases p38, ERK1/2, and SAPK/JNK, or NO production. However, the E 2 -induced rise in [Ca 2 (cid:1) ] i specifically down-regulates the serum-stimulated activation of c- fos promoter and ERK1/2, and conversely, it specifically up-regulates li-popolysaccharide-stimulated activation of c- fos promoter, p38, and NO production. The E 2 -changed activa- tion of c- fos promoter can be prevented by an intracellular Ca 2 (cid:1) chelator. Our data indicate that E 2 induced nongenomic Ca 2 (cid:1) signaling through membrane ER is able to specifically modulate genotropic signaling pathways with impact on macrophage activation.

Estrogens with its most active 17␤-estradiol (E 2 ) 1 are required not only for development of the female phenotype but also exert a series of both beneficial and adverse effects on human health. For instance, estrogens have been described to be protective in diverse neurodegenerative diseases (1,2) including Alzheimer's disease (3), osteoporosis (5), heart disease (6), arteriosclerosis (7) and stroke (4). Adverse effects are ascribed to the cell proliferative activity of E 2 , promoting diverse cancer forms in breast (8) and endometrium (9). Moreover, E 2 is known to increase risk for autoimmune disorders such as lupus erythematosus and multiple sclerosis (10). All these activities of E 2 are considered to be mediated at the cellular level through estrogen receptor (ER) ␣ or ER␤ belonging to the nuclear receptor superfamily (11). For a long time, it has been thought that the ER, after binding of E 2 and translocation into the nucleus, acts exclusively as a ligand-inducible transcription factor causing E 2 -specific gene expression and repression, respectively (12).
In recent years, however, the action spectrum of nuclear ER is turning out to be much more complex than originally anticipated. Indeed, evidence is accumulating that ER does mediate not only genotropic but also a series of nongenomic actions of E 2 (13,14). For instance, the E 2 ⅐ER complex has been described to directly activate mitogen-activated protein kinase (MAPK) (15)(16)(17)(18)(19) and endothelial nitric-oxide synthase (20,21) and to increase cAMP (22), inositol 1,4,5-trisphosphate (23,24), and the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) (15). However, there is also information available that nongenomic E 2 actions can be independent of the classical nuclear ER. In particular, the E 2 -induced rise in [Ca 2ϩ ] i has been reported to be mediated through membrane ER (mER) on the cell surface (25)(26)(27)(28). The molecular nature of mER is currently under controversial debate. Some authors suggest the mER to be largely identical with the classical ER (23,29), whereas other authors emphasize the views that the mER belongs to the class of G-protein-coupled receptors (GPCR) (26,27,30) or that the mER even represents larger complexes containing GPCR linked somehow to nuclear ER (31).
At present, however, the key question is whether the E 2induced nongenomic rise in [Ca 2ϩ ] i through mER, irregardless of its molecular nature, is able to exert any effects on cell functioning, in particular on gene expression at all. Indeed, there is information available that Ca 2ϩ signaling has a profound influence on expression of especially immediate early genes such as c-fos (32)(33)(34). In general, however, the E 2 -induced rises in [Ca 2ϩ ] i are characterized by low amplitude and short duration, and they are therefore widely regarded as meaningless for affecting gene expression and cell functions. Here, however, we demonstrate that E 2 -induced Ca 2ϩ signaling through surface receptors, not inhibitable by ER blockers, exerts specific regulatory effects on genotropic signaling pathways induced by serum and lipopolysaccharide (LPS) in murine macrophages.

EXPERIMENTAL PROCEDURES
Construction of Cell Line RAW-fos13-The c-fos promoter from Ϫ520 to ϩ109 was amplified from mouse DNA, cloned into pSEAP2 Basic Vector (CLONTECH, Heidelberg, Germany), and stably transfected with FuGENE 6 (Roche Molecular Biochemicals) in combination with pEGFPN3 (CLONTECH) into the macrophage cell line RAW 264.7 (ATCC TIB-71). The cells were grown in phenol red-free Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 5% heat-inactivated low endotoxin fetal calf serum (FCS) (PAA Laboratories, Cölbe, Germany). Clones were selected and maintained in the presence of 250 g/ml G418. The clone RAW-fos13, which did not express any detectable enhanced green fluorescent protein, was used throughout all experiments. Cell stocks were preserved in liquid nitrogen, and the cells used were passaged not more than eight times. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Antibody Labeling-Both intact cells and permeabilized cells were labeled with the anti-ER antibodies ER␣(MC-20) and ER␣(H-184) (Santa Cruz Biotechnology, Heidelberg, Germany) and with the secondary antibodies goat anti-rabbit IgG FITC conjugate (Sigma) and donkey anti-goat-FITC antibody (a gift from P. Traub, MPI Ladenburg, Germany) as detailed elsewhere (27).
Flow Cytometry-Aliquots of 1.5 ϫ 10 6 cells labeled with antibodies or E 2 -BSA-FITC as above were postfixed with 1% paraformaldehyde, analyzed in a FACSCalibur sorter (Becton Dickinson, Sunnyvale, CA) with 10,000 gated cells per sample, and evaluated with the FACScan software as described previously (36).
Confocal Laser Scanning Microscopy (CLSM)-Cells grown overnight on poly-L-lysine-coated glass coverslips were labeled with antibodies or E 2 -BSA-FITC or BSA-FITC as above. The cells were postfixed with 1% paraformaldehyde, mounted on slides, and analyzed with a Leica TCS NT confocal laser-scanning microscope version 1.5.451 (Leica Lasertechnik, Heidelberg, Germany) using a 488-nm argon laser line for excitation of FITC fluorescence and a 568-nm krypton laser line for rhodamine fluorescence. Evaluation was described previously (37).
Ca 2ϩ Fluorescence Spectroscopy-Cells at a concentration of 10 7 /ml were suspended in 20 mM HEPES buffer (pH 7.2) containing 130 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 1 mM Na 2 HPO 4 , and 1 mg/ml glucose before loading with 1 M Fura-2/acetoxymethylester (Sigma) for 1 h. The Ca 2ϩ measurements were performed in a LS 55 luminescence spectrometer (PerkinElmer Life Sciences) measuring Fura-2 fluorescence at 340 and 380 nm for excitation and 509 nm for emission with a dissociation constant of Fura-2-Ca 2ϩ complex taken as 224 nM. The [Ca 2ϩ ] i was computed from the ratio of 340/380-nm fluorescence values as described previously (27,38).
Stimulation of Cells-After serum starvation for 18 h, cells were stimulated with 10% (v/v) FCS for various periods. The FCS used was stripped by incubating twice with 1 g of charcoal and 0.1 g of dextran for 500 ml of FCS for 30 min at 56 and 37°C, respectively, with final filtration through 0.22-m pore-sized membranes (Nunc, Wiesbaden, Germany). For stimulation with LPS, the cells were incubated in Iscove's modified Dulbecco's medium with 5% stripped FCS for 18 h before adding 1 g/ml LPS.
NO Determination-NO in the supernatants of cells was measured as nitrite using Griess reagent as described previously (35).
Assay for c-fos Promoter Activity-Aliquots of 5 ϫ 10 5 cells in 1 ml were cultured in 24-well plates overnight. Just before stimulation, 100 l of culture fluid from the wells were collected as controls (RLU 0 h ) and another 100 l after 3 h stimulation (RLU 3 h ). Secreted alkaline phosphatase activity was determined using a Phospha-Light kit (Tropix, Weiterstadt, Germany) with a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) as described previously (35). The relative activity of c-fos promoter was calculated by subtraction of 0.9 ϫ RLU 0 h from RLU 3 h and normalizing to controls.

Expression of Nuclear and Membrane ERs-Macrophages,
which play a key role in immunity, have been reported to express both nuclear ER (39) and mER (27). The presence of both classical nuclear ERs and mERs is confirmed in the murine macrophage cell line RAW 264.7, which we have stably transfected with the reporter gene human secreted alkaline phosphatase driven by the c-fos promoter. These RAW-fos13 macrophages express ER␣, but not ER␤, as revealed by reverse transcriptase-PCR (Fig. 1A). Expression of ER␣ can be also detected, but only in permeabilized cells, by flow cytometry using the antibody ER␣(H-180) binding to the N-terminal steroid binding domain of ER␣ (Fig. 1B) and the antibody ER␣(MC-20), probing the C-terminal part of ER␣ (data not shown). The majority of ER␣ was localized in the cytoplasm of cells, as revealed by CLSM (Fig. 1C). However, these two anti-ER␣ antibodies did not react with any ER␣ on the surface of intact RAW-fos13 cells. Nevertheless, the RAW-fos13 cells exhibit binding sites for E 2 on their surface. When intact RAW-fos13 cells were incubated with the plasma membrane-impermeable E 2 -BSA-FITC for 1 min, the cells revealed an increased fluorescence intensity, as determined by flow cytometry ( Fig.  2A). CLSM localized this bound fluorescence exclusively on the cell surface (Fig. 2B). By contrast, the cells did not bind any BSA-FITC under the same experimental conditions (Fig. 2, A  and B). Moreover, the red fluorescence of concanavalin A-rhodamine was localized on the surface of intact cells and was colocalized with E 2 -BSA-FITC (Fig. 2B).
] i -At the physiological concentration of 1 nM, E 2 , but not 17␣-E 2, induced a rise in [Ca 2ϩ ] i , as determined in Fura-2-loaded RAW-fos13 cells by Ca 2ϩ fluorescence spectroscopy (Fig. 3A). The amount of E 2 -raised Ca 2ϩ varied from experiment to experiment between about 50 to 90 nM Ca 2ϩ . About the same increase in [Ca 2ϩ ] i can be induced with the plasma membrane-impermeable E 2 -BSA, whereas BSA alone was ineffective in affecting [Ca 2ϩ ] i (Fig. 3B). Moreover, the E 2 -induced rise in [Ca 2ϩ ] i cannot be prevented upon pre-incubation of the cells with different ER blockers such as ICI 182,780, tamoxifen, and raloxifene (Fig. 3, C and D). The rise in [Ca 2ϩ ] i could be due to an influx of extracellular Ca 2ϩ and/or to the release of Ca 2ϩ from intracellular Ca 2ϩ stores such as endoplasmic reticulum. When extracellular Ca 2ϩ was removed by 2 mM EGTA, E 2 was still able to induce a slight increase in [Ca 2ϩ ] i (Fig. 3E). In addition, the phospholipase C inhibitor U-73122 prevented most of the E 2 -induced rise in [Ca 2ϩ ] i , whereas the ineffective analog compound U-73343 did not abrogate the E 2 -induced increase in [Ca 2ϩ ] i (Fig. 3F). This indicates that E 2 induces both influx of extracellular Ca 2ϩ and release of Ca 2ϩ from intracellular Ca 2ϩ stores in RAW-fos13 cells. Furthermore, preincubation of cells with pertussis toxin (PTX) abolished most of the E 2 -induced rise in Ca 2ϩ , which suggests participation of G-proteins in the E 2 -induced rise in [Ca 2ϩ ] i (Fig. 3G).
Inability of E 2 to Activate MAPK and c-fos Promoter-It has been reported that E 2 can directly activate MAPKs (15)(16)(17)(18)(19). We therefore examined the effect of E 2 on the activation of the three MAPK family extracellular signal-regulated kinases (ERK1/2) p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). When the cells were incubated with 1 nM E 2 for various periods up to 180 min, none of the three MAPKs became activated, as revealed by immunodetection (data not shown). Moreover, E 2 is reported to induce expression of the Ca 2ϩ -sensitive immediate early gene c-fos (17). However, 1 nM E 2 was not able to induce any significant activation of the stably transfected c-fos promoter in RAW-fos13 cells (Fig. 4A). In line with these negative results, E 2 cannot induce any detectable NO production in cells.
E 2 -induced Up-regulation of LPS Signaling-LPS is known to activate macrophages including induction of the immediate early gene c-fos (40,41). In accordance, we can show that LPS also stimulates the c-fos promoter by about 4-fold in RAW-fos13 cells (Fig. 4A). E 2 even augmented this LPS effect, whereas 17␣-E 2 was ineffective (Fig. 4B). The up-regulatory E 2 effect cannot be prevented by the ER inhibitors ICI 182,780, tamoxifen, and raloxifene (Fig. 4B). E 2 exerts its augmentation of LPS stimulation on the c-fos promoter through the rise of [Ca 2ϩ ] i . Indeed, 1,2-bis(O-aminophenoxy)ethane N,N,NЈ,NЈtetraacetic acid tetra(acetoxymethyl) ester (BAPTA), when used under conditions blocking intracellular free Ca 2ϩ accumulation (Fig. 3H), also prevented the E 2 -induced augmentation of LPS-stimulated c-fos promoter (Fig. 4C). There was also no increase of LPS stimulation by E 2 when Ca 2ϩ rise was inhibited with the phospholipase C inhibitor U-73122, in contrast to the ineffective compound U-73343 (Fig. 4C). Moreover, the inhibitor GF109203X of the Ca 2ϩ -sensitive protein kinase C prevented the E 2 -induced up-regulation of LPS-activated c-fos promoter (Fig. 4C). Because PTX blocks this E 2 activation, G-proteins are obviously involved in the E 2 -induced augmentation of c-fos promoter activity (Fig. 4C).
Activation of macrophages by LPS is also associated with activation of the three MAPK families ERK1/2, p38, and SAPK/ JNK (42,43). Indeed, when RAW-fos13 cells were stimulated with LPS for various periods up to 180 min, all of the three MAPK families were mostly activated at 15 min, and thereafter, activation was declined but still detectable after 180 min (Fig. 5). E 2 selectively interfered with activation of MAPKs induced by LPS. E 2 did not affect the activities of ERK1/2 and SAPK/JNK but only the activation of p38 became up-regulated at 15 min (Fig. 5).
Activation of p38 MAPK is known to be involved in the control of NO production of macrophages (44). We have there-  fore examined the effect of E 2 on LPS-stimulated NO production. In RAW-fos13 cells, stimulation with LPS for 24 h caused a dramatic increase in NO production to ϳ30 M nitrite (Fig.  6A). Moreover, co-incubation with E 2 resulted even in an increase of NO production by about 50%. This stimulatory E 2 effect cannot be inhibited by the ER blockers tamoxifen and raloxifene (Fig. 6A). However, the E 2 effect can be abrogated by preincubation of cells with the intracellular Ca 2ϩ chelator BAPTA (Fig. 6A). The view that Ca 2ϩ is important for this E 2  up-regulatory activity is further supported by the fact that phospholipase C inhibitor U-73122 blocked the E 2 -stimulated LPS activation of NO production but not the ineffective analog compound U-73343 (Fig. 6B). Moreover, the inhibitor GF109203X of the Ca 2ϩ -sensitive protein kinase C prevented the E 2 -induced up-regulation of LPS-stimulated NO production just as did PTX (Fig. 6B). E 2 -induced Down-regulation of FCS Signaling-In contrast to its effects on LPS signaling, we found the opposite effect of E 2 on charcoal-stripped FCS-induced signaling. FCS stimulated the activation of the c-fos promoter by about 5-fold, whereas E 2 , but not 17␣-E 2 , down-regulated FCS-stimulated activity of c-fos promoter to about 2-3-fold (Fig. 7A). This down-regulation cannot be prevented by tamoxifen or raloxifene (Fig. 7A). The down-regulatory effect of E 2 is mediated through mobilization of intracellular Ca 2ϩ , as substantiated by experiments using BAPTA, U-73122, U-73343, and GF109203X (Fig. 7B). Also, PTX prevented the E 2 -induced down-regulation of FCS-stimulated c-fos promoter (Fig. 7B). FCS stimulation of RAW-fos13 cells activated ERK1/2 ( Fig. 8) but not p38 and SAPK/JNK (data not shown). The presence of E 2 during FCS stimulation down-regulated ERK1/2 activation to a similar extent as it did in c-fos promoter activation (Fig. 8).

DISCUSSION
Our data provide evidence for a novel paradigm of E 2 action at the cellular level: E 2 triggers nongenomic signaling, inde-pendent of the classical nuclear ER, and this nongenomic signaling specifically regulates genotropic signaling induced by FCS or LPS in murine macrophages.
Nongenomic E 2 signaling manifests itself as an E 2 -induced rapid rise in [Ca 2ϩ ] i of RAW-fos13 cells. This rise is due to both influx of extracellular Ca 2ϩ and release of Ca 2ϩ from intracellular Ca 2ϩ stores, as we have recently also found in mouse IC-21 macrophages (27). E 2 -induced nongenomic Ca 2ϩ signaling has been described to be independent of the classical nuclear ER (25,27) as well as to be dependent on ER (15). Although the RAW-fos13 cells express ER␣, the E 2 -induced Ca 2ϩ signaling is not mediated through ER␣ but rather through mER. This view is supported by the following findings. First, the E 2 -induced rise in [Ca 2ϩ ] i cannot be prevented by the ER blockers ICI 182,780, tamoxifen, and raloxifene. Second, E 2 -BSA, which is not able to enter the cells and which binds to neither ER␣ nor ER␤ (45), is also capable of rapidly raising [Ca 2ϩ ] i , just as free E 2 . Third, the RAW-fos13 cells exhibit distinct binding sites for E 2 -BSA-FITC on their surfaces that are not accessible for anti-ER antibodies on intact cells. Fourth, FIG. 5. Effects of E 2 on activation of three different MAPKs induced by LPS. Cells were stimulated with 1 g/ml LPS and in combination with 1 nM E 2 for different periods, and protein extracts were subjected to immuno-determination. The upper panels for p-ERK1/2, p-JNKp46/p54, or p-p38 MAPK represent the phosphorylated forms, and the lower panels represent the corresponding total kinases. The relative activation of p38 MAPK was expressed in terms of the ratios of the phosphorylated forms to total kinase protein, setting the ratio of the negative unstimulated control (C) as 1. As a positive control for MAPK activation, the cells were stimulated with 10 g/ml anisomycin (AN) for 30 min. Representative blots are shown, and the results were verified in at least two independent experiments.
FIG. 6. Influence of E 2 on LPS-induced NO production of RAW-fos13 cells. A, cells were cultured in Iscove's modified Dulbecco's medium containing 5% stripped FCS for 18 h, then stimulated for 24 h with 1 nM E 2 and 1 g/ml LPS with or without 1 h of preincubation with 0.5 M tamoxifen (tam) or 0.5 M raloxifene (ral) or 10 min of preincubation with 10 M BAPTA. N.D., not detectable. The results shown are the means Ϯ S.E. from at least three independent experiments performed in triplicate. B, cells were stimulated with 1 g/ml LPS or LPS plus 1 nM E 2 . Before stimulation, the cells were pretreated with U-73122, U-73343, PTX, GF109203X as described in Fig. 4. our finding that the E 2 -induced increase in [Ca 2ϩ ] i via phospholipase C is insensitive to ICI 182,780 contrasts to recent data showing inhibition by ICI 182,780 of E 2 -induced activation of phospholipase C via transfected ER␣ and ER␤, which also are expressed in the plasma membranes of Chinese hamster ovary cells (23). This strongly supports the view that the E 2 -induced nongenomic Ca 2ϩ signaling in RAW-fos13 cells does not rely on the classical ER linked somehow to the plasma membrane. Fifth, inhibition of the E 2 -induced Ca 2ϩ signaling by PTX indicates that the mER is not identical with the classical nuclear ER, but rather the mER belongs to the class of G-protein-coupled receptors. As with numerous other G-protein-coupled receptors, the mER is agonist-sequestrable, as recently characterized in detail in mouse IC-21 macrophages (27).
There is accumulating evidence that classical nuclear ERs mediate nongenomic E 2 effects such as activation of MAPK (15)(16)(17)(18)(19), activation of endothelial nitric oxide synthase (20, 46 -48), and activation of c-fos (17) as well as activation of NO production (46,48). Although RAW-fos13 cells express ER␣, E 2 is not able to directly induce any activation of the c-fos promoter or the NO production or one of the three MAPK families p38, ERK1/2, SAPK/JNK. Nonetheless, all these parameters except SAPK/JNK are responsive to E 2 , which becomes apparent only when cells are activated by LPS or FCS. Indeed, E 2 down-regulates FCS-stimulated c-fos promoter activation, whereas it up-regulates LPS-induced c-fos promoter activation in RAW-fos13 cells. These E 2 effects are not mediated through ER␣ but rather through nongenomic Ca 2ϩ signaling. This view is substantiated by our findings that (i) these E 2 effects are not prevented by the ER blockers ICI 182,780, tamoxifen, and raloxifene, (ii) they can be abolished by BAPTA under conditions capturing the intracellular free Ca 2ϩ ions generated by E 2 , and (iii) they are prevented by PTX and inhibitors of phospholipase C, which both inhibit an E 2 -induced rise in [Ca 2ϩ ] i . The down-and up-regulation of genotropic FCS and LPS signaling, respectively, by an E 2 -induced rise in [Ca 2ϩ ] i are not simply due to a general dampening or stimulation of all cell activities but, rather, are highly specific. Indeed, although LPS activates all of the three MAPK families, p38, ERK1/2, and SAPK/JNK, only the p38 MAPK is up-regulated by the E 2induced Ca 2ϩ signaling. Finally, the specific up-regulatory effect of E 2 -induced Ca 2ϩ signaling on LPS-stimulated p38 and c-fos promoter results in increased activation of macrophage, which becomes evident as increased NO production. Again, this up-regulatory effect of E 2 on LPS-stimulated NO production of macrophages is independent of ER␣.
Collectively, our data provide the first evidence for a linkage of E 2 -induced nongenomic Ca 2ϩ signaling through mER, independent of the classical nuclear ER, with cell functions. We are aware of the fact that our results are in conflict to numerous other findings showing ER-mediated nongenomic E 2 effect (15, 17, 20, 46 -48). At present, a plausible explanation for this difference is that nongenomic E 2 actions vary among different cell types and that such a nongenomic E 2 signaling as we described in RAW-fos13 macrophages is possibly restricted to macrophages. But even if this type of nongenomic E 2 signaling were restricted to macrophages, it is presumably of importance FIG. 7. E 2 -attenuated activation of c-fos promoter induced by charcoal-stripped FCS. A, RAW-fos13 cells were serum-starved for 18 h and then stimulated for 3 h with 10% (v/v) FCS or in combination with 1 nM E 2 , 1 nM 17␣-E 2 , 0.5 M tamoxifen (tam) and 0.5 M raloxifene (ral). The c-fos promoter activity of each group was determined relative to the vehicle-treated control. The values represent the means Ϯ S.E. from at least three independent experiments performed in triplicate. B, cells were stimulated for 3 h with 10% (v/v) FCS or FCS plus 1 nM E 2 . Inhibitors were used as indicated in Fig. 4. The c-fos promoter activity from each group was expressed relative to the c-fos promoter activity induced by FCS.

FIG. 8.
Effects of E 2 on activation of ERK1/2 induced by charcoal-stripped FCS. Cells were stimulated with 10% (v/v) FCS and 1 nM E 2 plus FCS for different periods, with 10 g/ml anisomycin (AN) as a positive control of ERK1/2 activation. Cell lysates were subjected to immunodetection, and the relative activation of ERK1 and ERK2 from each group was expressed relative to the activation level at 0 min of FCS stimulation. Representative blots are shown, and the results were verified in at least two independent experiments. for human health, for example, with respect to the outcome of infectious diseases. Activation of macrophages, induced by components of infectious agents such as the bacterial LPS, is a prerequisite to effectively recognize and kill microorganisms as well as even malignant tumor cells (49). By contrast, overactivation of macrophages is believed to be responsible for deleterious clinical manifestations of infectious diseases, such as hypercoagulation, hypotension, cachexia, somnolence, multiple organ failure, and even death (50).