INTRODUCTION

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The hydrolysis of phosphatidylcholine by phospholipase D produces phosphatidic acid (PA)¹ in various cell types stimulated with agonists (1, 2). PA has several physiological functions as a second messenger in cell regulations such as protein phosphorylation (3), expression of c-fos and c-myc (4), stimulation of DNA synthesis (4, 5), activation of stress fiber formation (6-8), and the production of diacylglycerol (2, 9). It is well known that PA is converted into diacylglycerol by the action of PA phosphohydrolase (2, 9), which contributes to the activation of Ca²⁺-independent protein kinase C (10). PA is also known to activate phospholipase C-1 and raf-1 in A431 and MDCK cells, respectively (11, 12). It has also been reported that PA was involved in the activation of superoxide anion production by activating NADPH oxidase (13-15).

Another important role of PA in cell signalings is the increase of intracellular Ca²⁺ ([Ca²⁺]_i) (4). It is known that [Ca²⁺]_i is regulated by the release of Ca²⁺ from intracellular stores and influx from extracellular sources (16). PA has been known to increase [Ca²⁺]_i by the activation of Ca²⁺ efflux from internal stores (4, 17), even though there have been reports suggesting the activation of Ca²⁺ influx by PA (18, 19). However, there have been contradictory reports on the mechanisms by which PA triggers Ca²⁺ release from internal stores (4, 17). In A431 cells, PA was shown to elevate [Ca²⁺]_i by stimulating the hydrolysis of phosphoinositides (4). Contrary to this report, PA could increase [Ca²⁺]_i in the presence of heparin in Jurkat cells, suggesting that PA stimulated Ca²⁺ efflux from inositol 1,4,5-trisphosphate (IP₃)-insensitive stores (17). However, the detailed mechanisms by which PA increases [Ca²⁺]_i are not still known.

In this report, we present a new possible mechanism of [Ca²⁺]_i increase in response to PA in Rat-2 fibroblasts. The roles of RhoA in the elevation of intracellular H₂O₂ and [Ca²⁺]_i were studied by scrape loading of C3 transferase into the cells. C3 transferase is known to regulate the activity of RhoA by ADP-ribosylation in several cell functions, including stress fiber formation, focal adhesions, and cell motility (20, 21). The introduction of C3 transferase into the Rat-2 cells inhibited the increase of [Ca²⁺]_i and intracellular H₂O₂ stimulated by PA. Furthermore, H₂O₂ scavengers, catalase and N-acetylcysteine (NAC), blocked the PA-induced elevation of [Ca²⁺]_i. These observations support the idea that PA increases [Ca²⁺]_i by activating RhoA and the production of intracellular H₂O₂.

	MATERIALS AND METHODS

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Chemicals and Reagents-- LPA, NAC, EGTA, and catalase from Aspergillus niger were purchased from Sigma. PA (dioleoyl, C18:1, [cis]-9) was obtained from Sigma and further purified by using a solvent system of ethyl acetate/iso-octane/acetic acid/water (130:20:30:100, v/v) to remove any possible effect of LPA. Fetal bovine serum, bovine serum albumin, penicillin/streptomycin solution, and Dulbecco's modified Eagle's medium were from Life Technologies, Inc. H₂O₂ was obtained from Junsei (Tokyo, Japan). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) phallacidin and fluo-3,AM were from Molecular Probes (Eugene, OR). Monoclonal anti-RhoA antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemical agents were of analytical grade.

Cell Culture-- Rat-2 fibroblasts, obtained from American Type Culture Collection (ATCC CRL 1764), were cultured for 2 days at 37 °C in Dulbecco's modified Eagle's medium containing 25 mM HEPES, 10% (v/v) fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin (culture medium). Then, the cells were incubated for 2 days at 37 °C with Dulbecco's modified Eagle's medium supplemented with 5 µg/ml apotransferrin (Sigma), 1 mg/ml bovine serum albumin, 25 mM HEPES, pH 7.4, 2 mM glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin (serum-free medium).

Confocal Microscopic Observation of F-actin-- F-actin was observed by the procedures of Jung et al. (22). Briefly, Rat-2 cells were grown on round coverslips in a 12-well culture plate and serum-starved for 2 days. After stabilizing in fresh serum-free medium for 1 h, the cells were incubated with 1 µg/ml LPA or 10 µM purified PA for 30 min and fixed with 3.7% (v/v) formaldehyde in Dulbecco's phosphate-buffered saline (DPBS) (1.2 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 0.9 mM CaCl₂, 2.7 mM KCl, 0.5 mM MgCl₂, and 138 mM NaCl, pH 7.5) for 30 min. Then, the cells were permeabilized with 0.2% (v/v) Triton X-100 in DPBS for 15 min and stained with 0.165 µM of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phallacidin for 30 min at room temperature. Stained cells were mounted on slide glasses with Gelvatol and observed with a laser scanning confocal microscope (Carl Zeiss LSM 410). Gelvatol was prepared by mixing 100 ml of 0.23% polyvinyl alcohol in DPBS with 50 ml of glycerol. Samples were excited by a 488 nm argon laser, and the images were filtered by a longpass 515 nm filter. Three-dimensional images were constructed from 5-10 serial images (each 1-µm thick) made by automatic optical sectioning.

Scrape Loading of C3 Transferase-- C3 transferase, prepared by the procedures of Leem et al. (23), was loaded into Rat-2 cells according to the modified procedures of Malcolm et al. (21). Briefly, Rat-2 cells, grown on a 100-mm culture dish, were scraped in 1 ml of DPBS containing 200 ng of C3 transferase and resuspended in culture medium. Then, cell suspensions were transferred into a six-well culture plate and cultured for 1 day. The cultures were serum-starved by sequential incubation with culture medium containing 0.5% (v/v) fetal bovine serum and serum-free medium each for 1 day and then used for measurements of [Ca²⁺]_i. The incorporation of C3 transferase was checked by immunostaining and co-incorporation of fluorescein isothiocyanate-conjugated dextrans.

Measurement of [Ca²⁺]_i-- [Ca²⁺]_i was measured by the use of a laser scanning confocal microscope (22). Cells, grown on coverslips and serum-starved for 2 days, were incubated with 4 µM fluo-3,AM in serum-free medium for 40 min and washed three times with serum-free medium. Each coverslip containing stained cells was mounted on a perfusion chamber (self-designed), subjected to a confocal laser scanning microscope (Carl Zeiss LSM 410), and then scanned every second with a 488 nm excitation argon laser and a 515 nm longpass emission filter. Agonists were added to the cells by using an automatic pumping system (self-designed). Sometimes, cells were pretreated with 500 units/ml A. niger catalase for 30 min or 20 mM NAC for 1 h. All images (about 200 images) from the scanning were processed to analyze changes of [Ca²⁺]_i in a single cell level. The results were expressed as the relative fluorescence intensity.

Measurement of Total Inositol Phosphates (IPs)-- Rat-2 cells were cultured in six-well plates and then labeled with 4 µCi/well myo-[³H]inositol for 2 day in inositol-free medium during serum starvation. Following incubation with 20 mM LiCl for 10 min, cells were stimulated with 10 µM purified PA or 1 µg/ml LPA for 15 min. 3 ml of ice-cold methanol was added for 30 min on ice and then water-soluble layer was separated by the procedures of Bligh and Dyer (24). The water-soluble upper layer was dried in a Speed Vac concentrator, re-dissolved in 1 ml of H₂O, and then applied to 1 ml of AG 1-X8 resin (200-400 mesh, formate form). After washing the column with 10 ml of 5 mM Na₂B₄O₇, 60 mM HCOONH₄, total IPs were eluted from the column with 2 ml of 0.1 mM HCOOH, 1.0 M HCOONH₄ (22) and then counted using a scintillation counter.

Translocation of RhoA-- Cells were incubated with 10 µM PA or 1 µg/ml LPA for 30 min, scraped in DPBS, and harvested by microcentrifugation. The cells were then resuspended in 0.2 ml of lysis buffer (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, pH 7.5) and disrupted by twenty passes through a 21.1-gauge needle on ice. The cytosolic fraction was separated from the particulate fraction by centrifugation at 10,000 × g for 1 h and precipitated with 5 volumes of acetone. SDS-polyacrylamide gel electrophoresis was performed using 12% acrylamide, and proteins were transferred onto polyvinylidine difluoride membranes using a Novex wet transfer unit. The membranes were blocked overnight with TBS (DPBS containing 0.01% Tween 20) with 5% (w/v) non-fat dried milk. The blots were incubated for 2 h with anti-RhoA antibody in TBS and further incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. Then, the blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

	RESULTS

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PA Increases [Ca²⁺]_i from PA-sensitive Stores-- In order to study the mechanism by which PA induces the increase in [Ca²⁺]_i, the level of [Ca²⁺]_i was measured after treating cells with purified PA in Rat-2 fibroblasts. As shown in Fig. 1A, PA caused a rapid, but transient, increase in [Ca²⁺]_i, consistent with previous reports (4, 17). The Ca²⁺ response was only slightly attenuated when external Ca²⁺ was eliminated by treating with EGTA, suggesting that PA increased [Ca²⁺]_i by triggering Ca²⁺ efflux from internal stores rather than stimulating Ca²⁺ influx from extracellular source. LPA also increased [Ca²⁺]_i, but EGTA inhibited sustained Ca²⁺ response to LPA, indicating that LPA elevated [Ca²⁺]_i by triggering both Ca²⁺ efflux from internal stores and the influx of extracellular Ca²⁺ (Fig. 1B). In contrast, EGTA completely blocked the increase of cytosolic Ca²⁺ produced by arachidonic acid (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Changes of [Ca2+]i by PA (A), LPA (B), or arachidonic acid (C). Serum-starved Rat-2 cells were labeled with 4 µM fluo-3,AM for 40 min and treated with 10 µM PA, 1 µg/ml LPA, or 100 µM arachidonic acid for the indicated times, in the absence (EGTA) or presence (EGTA+) of 4 mM EGTA. Then, [Ca2+]i was monitored using a laser scanning confocal microscope as explained under "Materials and Methods." Results are expressed as the relative fluorescence intensity (RFI). Each trace is a single cell representative from at least four separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   No production of inositol phosphates by PA. Rat-2 cells were cultured in six-well culture plates and labeled with 4 µCi/well myo-[3H]inositol for 2 days in serum-free medium without inositol. The cells were incubated with 20 mM LiCl for 10 min and then stimulated with 10 µM PA or 1 µg/ml LPA for 15 min. The amount of inositol phosphates was determined as described under "Materials and Methods." Results are the means ± S.D. from three separate experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Changes of [Ca2+]i by serial incubation with LPA and PA (A) or PA and LPA (B) in the absence of extracellular Ca2+. Serum-starved cells were labeled with 4 µM fluo-3,AM for 40 min and then incubated with 1 µg/ml LPA and 10 µM PA (A) or 10 µM PA and 1 µg/ml LPA (B) as shown, in the presence of 4 mM EGTA. [Ca2+]i was monitored as explained in the legend of Fig. 1. Each trace is a single cell representative from four separate experiments.

PA Increases [Ca²⁺]_i by RhoA-- Since it has been reported that PA stimulated stress fiber formation, which was inhibited by C3 transferase (8), we have investigated the possibility that RhoA was also involved in the PA-induced increase of [Ca²⁺]_i. First, we have tested possible activation of RhoA by PA by measuring the translocation of RhoA, since it is known that RhoA is redistributed from the cytosolic to the particulate fraction after being activated (21). As shown in Fig. 4, most of RhoA was localized in the cytosolic fraction in unstimulated cells, and a fraction of RhoA was translocated to the membrane fraction in response to PA. As expected from a previous report (21), LPA also activated the translocation of RhoA to the membrane fraction. Next, the role of RhoA in the PA-induced increase of [Ca²⁺]_i was investigated by scrape loading of C3 transferase into Rat-2 cells. It is known that C3 transferase inhibits the activity of RhoA by ADP-ribosylation at Asp⁴¹ (20). The effect of incorporated C3 transferase on RhoA was shown by the inhibition of stress fiber formation in response to PA (Fig. 5A). In the control cells, scraped without C3 transferase, PA stimulated stress fiber formation, which was completely inhibited in C3 transferase-loaded cells. C3 transferase also blocked the stress fiber formation induced by LPA (data not shown). The effect of C3 transferase on the elevation of [Ca²⁺]_i by PA was shown in Fig. 5B. C3 transferase inhibited the increase of [Ca²⁺]_i stimulated by PA, suggesting an essential role of RhoA in the PA-stimulated increase of [Ca²⁺]_i. However, the toxin had no significant effect on the elevation of [Ca²⁺]_i in response to LPA, possibly because LPA could release Ca²⁺ from IP₃-sensitive pool, even though PA-sensitive Ca²⁺ pool was blocked. Taken together, it was suggested that RhoA played an essential role in the PA-induced increase of [Ca²⁺]_i.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Translocation of RhoA by PA and LPA. Serum-starved cells were treated with 10 µM PA or 1 µg/ml LPA for 30 min and fractionated into the cytosolic and the membrane fraction. Proteins were extracted and separated on a 12% SDS-PAGE and then RhoA was detected by Western blotting as explained under "Materials and Methods."

View larger version (67K): [in this window] [in a new window]   Fig. 5.   Effects of C3 transferase on the stress fiber formation (A) and the level of [Ca2+]i (B). A, cells were scraped with buffer (panels a and b) or 200 ng of C3 transferase (panels c and d) and serum-starved for 2 days. The cells were then stimulated with control (panels a and c) or 10 µM PA (panels b and d) for 30 min, and F-actin was stained and observed using a confocal laser scanning microscope as described under "Materials and Methods." The bar is 20 µm. B, cells were loaded with C3 transferase and stimulated with 10 µM PA or 1 µg/ml LPA. [Ca2+]i was monitored using a laser scanning confocal microscope as described in the legend of Fig. 1. Results are expressed as percent of control response (unstimulated cells) ± S.D. from three separate determinations. Each determination is the mean of at least 10 cells.

The Role of H₂O₂ in the PA-induced Ca²⁺ Increase-- It has been reported that H₂O₂ stimulated the increase of [Ca²⁺]_i in various cells (29-31). Recently, we have observed that PA produced intracellular H₂O₂ by over 6-fold and the increase was blocked by C3 transferase in Rat-2 cells.² So we have investigated any possible role of H₂O₂ in the PA-induced rise of [Ca²⁺]_i. First, we measured the changes of [Ca²⁺]_i after treating the cells with exogenous H₂O₂ (Fig. 6A). H₂O₂ induced a transient elevation of [Ca²⁺]_i as did PA, consistent with previous reports (30, 31). The transient response of [Ca²⁺]_i to H₂O₂ was not largely affected by the pretreatment with EGTA, indicating that H₂O₂ increased [Ca²⁺]_i from the internal stores as did PA (Fig. 1). To confirm the role of H₂O₂ in the regulation of [Ca²⁺]_i, the cells were preincubated with H₂O₂ scavengers, catalase and NAC, and then the changes of [Ca²⁺]_i by PA were investigated (Fig. 6, B and C). Catalase completely blocked the increase of [Ca²⁺]_i stimulated by PA, but had no effect on the subsequent increase by LPA (Fig. 6B). NAC also blocked the Ca²⁺ response to PA, but not to LPA (Fig. 6C). These results suggested that intracellular H₂O₂ may play a role in the PA-induced increase of [Ca²⁺]_i.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Increase of [Ca2+]i by H2O2 (A) and effects of catalase (B) and NAC (C) on the elevation of [Ca2+]i. A, cells were treated with H2O2 for the indicated times in the absence (EGTA) or presence (EGTA+) of 4 mM EGTA, and [Ca2+]i was monitored as explained in the figure legend of Fig. 1 (B and C). Cells were incubated with 500 units/ml catalase for 30 min (B) or 20 mM NAC for 1 h (C) and then stimulated with 10 µ PA and 1 µg/ml LPA. [Ca2+]i was monitored as explained in the legend of Fig. 1. Each trace is a single cell representative from three separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Changes of [Ca2+]i by serial incubation with PA and H2O2 (A), H2O2 and PA (B), or H2O2 and LPA (C) in the absence of extracellular Ca2+. Serum-free cells were sequentially treated with 10 µ PA and 0.5 mM H2O2 (A), 0.5 mM H2O2 and 1 µg/ml LPA (B), or 0.5 mM H2O2 and 1 µg/ml LPA (C) in the presence of 4 mM EGTA. [Ca2+]i was monitored as described in the legend of Fig. 1. Each trace is a single cell representative from three separate experiments.

	DISCUSSION

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In the present report we have shown that PA increased [Ca²⁺]_i from PA-sensitive internal stores, and the transient Ca²⁺ response was dependent on RhoA and H₂O₂. PA induced a transient increase of [Ca²⁺]_i in the absence of extracellular Ca²⁺, whereas the sustained Ca²⁺ increase by LPA was dependent on the extracellular Ca²⁺. PA stimulated the translocation of RhoA to the membrane fraction, and C3 transferase inhibited the PA-induced elevation of [Ca²⁺]_i. H₂O₂ scavengers, catalase and NAC, also blocked the rise of [Ca²⁺]_i by PA. Furthermore, the depletion of PA-sensitive Ca²⁺ stores inhibited the Ca²⁺ response to H₂O₂ and vice versa.

Now, it is likely that PA stimulates Ca²⁺ release from PA-sensitive stores, which is not dependent on IP₃. In Jurkat T cells, the depletion of IP₃-sensitive Ca²⁺ stores by incubation with an anti-CD3 antibody OKT3 (an IP₃-generating drug), in the presence of EGTA, did not affect on the elevation of [Ca²⁺]_i by the subsequent incubation of PA (17). In the same cells, heparin inhibited the rise in [Ca²⁺]_i by IP₃, but did not by PA, suggesting that PA increased [Ca²⁺]_i from IP₃-insensitive stores. It has been also reported that sphingosine 1-phosphate could produce PA by activating phospholipase D and also increased [Ca²⁺]_i from internal stores by an IP₃-independent mechanism in Swiss 3T3 cells (25, 32). Consistent with the previous reports, our results showed that PA produced a transient increase of [Ca²⁺]_i from PA-sensitive stores without the production of IP₃ (Figs. 1 and 2).

To our understanding, this is the first report that the rise of [Ca²⁺]_i by PA was dependent on RhoA. PA activated the translocation of RhoA to the membrane fraction, and C3 transferase inhibited the transient Ca²⁺ response to PA (Fig. 4). It has been reported that C3 transferase induced in vivo ADP-ribosylation in a dose-dependent manner in Rat-2 cells (23). Previously, there have been reports indicating possible roles of RhoA in the regulation of [Ca²⁺]_i and smooth muscle contraction (33-35). In C3H 10 1/2 cells, microinjection of C3 transferase inhibited the increase of [Ca²⁺]_i in response to thrombin and platelet-derived growth factor (33). Ca²⁺ waves induced by IP₃ were retarded by C3 transferase in Xenopus oocytes (34). It has been also reported that RhoA increased Ca²⁺ sensitivity of arterial smooth muscle contraction (35). Thus, it is likely that RhoA plays an essential role in the regulation of [Ca²⁺]_i.

H₂O₂ is known to increase [Ca²⁺]_i in various cells, including smooth muscle cells, macrophages, and endothelial cells (29-31). In rat alveolar macrophages, H₂O₂ stimulated the release of Ca²⁺ from IP₃-independent stores, which was essential for respiratory burst (30). The role of H₂O₂ in the elevation of [Ca²⁺]_i was also shown in endothelial cells (31). Superoxide and H₂O₂ produced by the incubation with xanthine oxidase and xanthine at nontoxic doses induced a transient rise of [Ca²⁺]_i, but the Ca²⁺ response was mainly induced by H₂O₂ rather than superoxide in these cells (31). Our results also supported the previous reports. H₂O₂ caused a transient Ca²⁺ increase in the presence or absence of extracellular Ca²⁺. H₂O₂ scavengers, NAC and catalase, completely inhibited the rise of [Ca²⁺]_i by PA. Furthermore, we have observed that PA increased the amount of intracellular H₂O₂ and the increase was blocked by C3 transferase.² Considering an essential role of RhoA in the PA-stimulated increase of [Ca²⁺]_i, it was concluded that PA increased [Ca²⁺]_i by the activation of RhoA and the subsequent production of intracellular H₂O₂.

Interestingly, our results suggested that LPA triggered Ca²⁺ release from PA-sensitive stores. We have previously observed that LPA produced PA by stimulating phospholipase D in Rat-2 cells (23). The depletion of PA-sensitive Ca²⁺ stores did not block the subsequent Ca²⁺ increase by LPA, but preincubation with LPA blocked the Ca²⁺ response to PA (Fig. 3), indicating that LPA triggered Ca²⁺ efflux from PA-sensitive stores. C3 transferase, which inhibited the elevation of [Ca²⁺]_i by PA, had no inhibitory effect on the Ca²⁺ response to LPA (Fig. 5). In addition, H₂O₂ scavengers, catalase and NAC, inhibited the Ca²⁺ elevation by PA, but not by LPA (Fig. 6). PA activated the translocation of RhoA to the membrane fraction and the production of H₂O₂. The differential effects of C3 transferase and H₂O₂ scavengers on the Ca²⁺ response to PA and LPA were explained by the production of IPs by LPA (Fig. 2). Thus, it is likely that LPA increases [Ca²⁺]_i from both PA- and IP₃-sensitive stores.

What is the possible role of the intracellular H₂O₂ and Ca²⁺ increased by PA? One possibility is to play a role in the actin polymerization stimulated by PA. It has been reported that Ca²⁺ may be involved in the actin polymerization by regulating the interaction of actin with Ca²⁺-dependent actin-binding proteins such as gelsolin and villin (36). In Rat-2 cells, PA stimulated RhoA and the elevation of intracellular H₂O₂ and [Ca²⁺]_i. C3 transferase inhibited the production of H₂O₂² and stress fiber formation (Fig. 5) activated by PA. Catalase also inhibited PA-induced stress fiber formation (data not shown). H₂O₂ increased [Ca²⁺]_i (Figs. 6 and 7) and stimulated stress fiber formation as did PA (data not shown). So it was suggested that PA activated stress fiber formation by a sequential activation of RhoA and the increase of intracellular H₂O₂ and [Ca²⁺]_i. However, H₂O₂ alone produced fragmented F-actins in the Rat-2 cells loaded with C3 transferase,³ indicating that stress fiber formation may require additional kinases activated by RhoA, such as Rho kinase and protein kinase N (37-39). So, RhoA may regulate PA-induced stress fiber formation by activating its two downstream components, H₂O₂/Ca²⁺ and RhoA-activated kinases, even though it is necessary to elucidate their roles in the actin polymerization activated by PA.