Phosphatidic Acid-induced Elevation of Intracellular Ca2+ Is Mediated by RhoA and H2O2 in Rat-2 Fibroblasts*

Zee-Won LeeDagger , Soo-Mi KweonDagger , Byung-Chul Kim§, Sun-Hee Leem, Incheol Shinparallel , Jae-Hong Kim§, and Kwon-Soo HaDagger **

From the Dagger  Biomolecule Research Group, Korea Basic Science Institute, Taejon 305-333, Korea, the § Laboratory of Molecular and Cellular Genetics, Institute of Environment and Life Science, Hallym University, Chun-Cheon, Kangwon-do 200-702, Korea, the  Department of Biology, Dong-A University, Pusan 604-714, Korea, and the parallel  Department of Biochemistry, College of Medicine, Hanyang University, Seoul 133-701, Korea

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have investigated possible roles of RhoA and H2O2 in the elevation of intracellular Ca2+ ([Ca2+]i) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca2+]i in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca2+]i, but the sustained Ca2+ response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca2+]i by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca2+ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca2+]i by PA. H2O2 also induced a transient increase of [Ca2+]i as did PA. H2O2 scavengers, catalase and N-acetyl-L-cysteine, completely blocked the rise of [Ca2+]i stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca2+ response to H2O2, and the incubation with H2O2 also blocked the PA-induced rise of [Ca2+]i. Thus, it was suggested that PA stimulated Ca2+ release from PA-sensitive, but not inositol 1,4,5-trisphosphate-sensitive, Ca2+ stores by the activation of RhoA and intracellular H2O2.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma 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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).


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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.

Since there have been inconsistent reports on the involvement of IP3 in PA-induced increase of [Ca2+]i (4, 17), the amount of IPs was measured after treating Rat-2 cells with purified PA in the presence of 20 mM LiCl (Fig. 2). The hydrolysis of phosphoinositides by phospholipase C is known to increase inositol phosphates in the presence of LiCl (16, 25). As expected from the previous reports (22, 26-28), incubation with LPA increased IPs by about 4-fold over the control level. However, PA did not cause any changes in the amount of IPs. So PA appears to induce the increase of [Ca2+]i by triggering Ca2+ efflux from IP3-insensitive internal stores.


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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.

Next, we have investigated whether LPA could trigger Ca2+ release from PA-sensitive stores, since it has been reported that LPA increased the level of PA by stimulating phospholipase D in Rat-2 cells (23). The preincubation with PA in the presence of EGTA completely blocked the elevation of [Ca2+]i induced by the subsequent addition of PA (data not shown), indicating that the incubation with PA was able to deplete PA-sensitive Ca2+ pool. LPA-sensitive Ca2+ pool was also depleted by the incubation with LPA (data not shown). So if LPA triggered Ca2+ efflux from PA-sensitive pool, the preincubation with LPA should block the elevation of [Ca2+]i induced by PA. As shown in Fig. 3A, the preincubation with LPA, in the presence of EGTA, completely blocked the increase of [Ca2+]i stimulated by PA. However, the depletion of PA-sensitive Ca2+ pool by the incubation with PA showed a small inhibitory effect on the subsequent Ca2+ response to LPA (Fig. 3B). Thus, these results strongly suggested that LPA increased [Ca2+]i from IP3-sensitive pools and also from PA-sensitive (IP3-independent) Ca2+ pool.


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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.


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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."


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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.


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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.

The role of H2O2 was further confirmed by serial incubations of Rat-2 cells with H2O2 and PA in the presence of EGTA (Fig. 7). The incubation with PA induced a transient increase of [Ca2+]i, but the subsequent incubation with H2O2 did not cause any changes in [Ca2+]i (Fig. 7A). Preincubation with H2O2 also blocked the Ca2+ response to PA (Fig. 7B), suggesting that PA and H2O2 caused Ca2+ release from the same stores. However, the depletion of H2O2- or PA-sensitive Ca2+ stores did not block the subsequent response of [Ca2+]i to LPA (Figs. 3B and 7C). Thus, it was concluded that H2O2 increased [Ca2+]i by triggering Ca2+ efflux from PA-sensitive, but not from IP3-sensitive stores.


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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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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Science and Technology (97-NN-04-A-01) and from the Korea Science and Engineering Foundation (96-0401-13-3).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.

** Senior Researcher of Korea Basic Science Institute. To whom all correspondence should be addressed. Tel.: 82-42-865-3422; Fax: 82-42-865-3419; E-mail: ksha{at}comp.kbsi.re.kr.

1 The abbreviations used are: PA, phosphatidic acid; [Ca2+]i, intracellular Ca2+; DPBS, Dulbecco's phosphate-buffered saline; IP3, inositol 1,4,5-trisphosphate; IPs, inositol phosphates; LPA, lysophosphatidic acid; NAC, N-acetyl-L-cysteine.

2 I. Shin, S.-M. Kweon, Z.-W. Lee, S. I. Kim, C. O. Joe, J.-H. Kim, Y. M. Park, and K.-S. Ha, manuscript in preparation.

3 Z.-W. Lee and K.-S. Ha, unpublished observations.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42[Medline] [Order article via Infotrieve]
  2. Exton, J. H. (1997) J. Biol. Chem. 272, 15579-15582[Free Full Text]
  3. Bocckino, S. B., Wilson, P. B., and Exton, J. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6210-6213[Abstract]
  4. Moolenaar, W. H., Kruijer, W., Yilly, B. C., Verlaan, I., BierMan, A. J., and de Laat, S. W. (1986) Nature 323, 171-173[Medline] [Order article via Infotrieve]
  5. Knauss, T. C., Jaffer, F. E., and Abboud, H. E. (1990) J. Biol. Chem. 265, 14457-14463[Abstract/Free Full Text]
  6. Ha, K.-S., and Exton, J. H. (1993) J. Cell Biol. 123, 1789-1796[Abstract]
  7. Ha, K.-S., Yeo, E.-J., and Exton, J. H. (1994) Biochem. J. 303, 55-59[Medline] [Order article via Infotrieve]
  8. Cross, M. J., Roberts, S., Ridley, A. J., Hodgkin, M. N., Stewart, A., Claesson-Welsh, L., and Wakelam, M. J. O. (1996) Curr. Biol. 6, 588-597[Medline] [Order article via Infotrieve]
  9. Exton, J. H. (1990) J. Biol. Chem. 265, 1-4[Abstract/Free Full Text]
  10. Ha, K.-S., and Exton, J. H. (1993) J. Biol. Chem. 268, 10534-10539[Abstract/Free Full Text]
  11. Jones, G. A., and Carpenter, G. (1993) J. Biol. Chem. 268, 20845-20850[Abstract/Free Full Text]
  12. Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L., and Bell, R. M. (1996) J. Biol. Chem. 271, 8472-8480[Abstract/Free Full Text]
  13. Bellavite, P., Corsa, F., Dusi, S., Grzeskowiak, M., Della-Bianca, V., and Rossi, F. (1988) J. Biol. Chem. 263, 8210-8214[Abstract/Free Full Text]
  14. Thelen, M., Dewald, B., and Baggiolini, M. (1993) Physiol. Rev. 73, 797-821[Free Full Text]
  15. Tokumura, A., Moriyama, T., Minamino, H., Hayakawa, T., and Tsukatani, H. (1997) Biochim. Biophys. Acta 1344, 87-102[Medline] [Order article via Infotrieve]
  16. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205[CrossRef][Medline] [Order article via Infotrieve]
  17. Sakano, S., Takemura, H., Yamada, K., Imoto, K., Kaneko, M., and Ohshika, H. (1996) J. Biol. Chem. 271, 11148-11155[Abstract/Free Full Text]
  18. Putney, J. W., Jr., Weiss, S. J., van de Walle, C. M., and Haddas, R. A. (1980) Nature 284, 345-347[Medline] [Order article via Infotrieve]
  19. Salmon, D. M., and Honeyman, T. W. (1980) Nature 284, 344-345[Medline] [Order article via Infotrieve]
  20. Takai, Y., Sasaki, T., Tanaka, K., and Nakanishi, H. (1995) Trends Biochem. Sci. 20, 227-231[CrossRef][Medline] [Order article via Infotrieve]
  21. Malcolm, K. C., Elliott, C. M., and Exton, J. H. (1996) J. Biol. Chem. 271, 13135-13139[Abstract/Free Full Text]
  22. Jung, H. I., Shin, I., Park, Y. M., Kang, K. W., and Ha, K.-S. (1997) Mol. Cells 7, 431-437[Medline] [Order article via Infotrieve]
  23. Leem, S.-H., Shin, I., Kweon, S.-M., Kim, S. I., Kim, J.-H., and Ha, K.-S. (1997) J. Biochem. Mol. Biol. 30, 337-341
  24. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  25. Mattie, M., Brooker, G., and Spiegel, S. (1994) J. Biol. Chem. 269, 3181-3188[Abstract/Free Full Text]
  26. van Corven, E. J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H. (1989) Cell 59, 45-54[Medline] [Order article via Infotrieve]
  27. Moolenaar, W. H., van der Bend, R. L., van Corven, E. J., Jalink, K., Eichholtz, T., and van Blitterswijk, W. J. (1992) Cold Spring Harbor Symp. Quant. Biol. 57, 163-167[Medline] [Order article via Infotrieve]
  28. van Corven, E. J., van Rijswijk, A., Jalink, K., van der Bend, R. L., van Blitterswijk, W. J., and Moolenaar, W. J. (1992) Biochem. J. 281, 163-169[Medline] [Order article via Infotrieve]
  29. Roveri, A., Coassin, M., Maiorino, M., van Amsterdam, F. T., Ratti, E., and Ursini, F. (1992) Arch. Biochem. Biophys. 297, 265-270[Medline] [Order article via Infotrieve]
  30. Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171[CrossRef][Medline] [Order article via Infotrieve]
  31. Volk, T., Hensel, M., and Kox, W. J. (1997) Mol. Cell. Biochem. 171, 11-21[CrossRef][Medline] [Order article via Infotrieve]
  32. Desai, N. N., Zhang, H., Olivera, A., Mattie, M. E., and Spiegel, S. (1992) J. Biol. Chem. 267, 23122-23128[Abstract/Free Full Text]
  33. Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513[Medline] [Order article via Infotrieve]
  34. Kato, N. (1996) Biochem. Biophys. Res. Commun. 226, 580-584[CrossRef][Medline] [Order article via Infotrieve]
  35. Hirata, K., Kikuchi, A., Sasaki, T., Kuroda, S., Kaibuchi, K., Matsuura, Y., Seki, H., Saida, K., and Takai, Y. (1992) J. Biol. Chem. 267, 8719-8722[Abstract/Free Full Text]
  36. Stossel, T. P. (1993) Science 260, 1086-1094[Medline] [Order article via Infotrieve]
  37. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibichi, K. (1996) Science 273, 245-248[Abstract]
  38. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648[Abstract]
  39. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650[Abstract]


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