Lysophosphatidic acid sensitizes mechanical stress-induced Ca2+ response via activation of phospholipase C and tyrosine kinase in cultured smooth muscle cells

Life

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LYSOPHOSPHATIDIC ACID SENSITIZES MECHANICAL STRESS-INDUCED Ca*+ RESPONSE VIA ACTIVATION OF PHOSPHOLIPASE C AND TYROSINE KINASE IN CULTURED SMOOTH MUSCLE CELLS Hisayuki Ohata*, Hiromi Aizawa, and Kazutaka Momose Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, l-S-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan (Receivedin final form January

13,1997)

We previously reported that lysophosphatidic acid (LPA) sensitized mechanical stress-induced intracellular free Ca2+ concentration response (Biochem. Biophys. Res. Commun. 208, 19-25, 1995). In the present study, the signal transduction pathway of the sensitizing effect of LPA was investigated in cultured longitudinal muscle cells from guinea pig ileum. Suramin, a putative LPA receptor antagonist, did not affect the response in the presence of 30 nM LPA, suggesting that the response is induced via activation of suramin-insensitive LPA receptor. Neither pertussis toxin nor wortmannin inhibited the LPA-sensitized response, indicating that Gi/c- and phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathways are not involved in the sensitizing effect. C3 ADP ribosyltransferase had no effect on the response, whereas formation of actin-stress fiber in the presence of LPA was completely inhibited, suggesting rho-related cytoskeletal change is not involved in the response. In contrast, a phospholipase C (PLC) inhibitor, U73122, completely inhibited the response, but broad spectrum kinase inhibitors, staurosporine and H7, had no effect on the response. In addition, tyrosine kinase inhibitor, genistein, but not tyrphostin partially inhibited the response. These results suggest that LPA sensitizes the mechanical stress-induced response via activation of PLC, but not protein kinase C. Additionally, tyrphostin-insensitive tyrosine kinase, which is related to other pathway than Gu,- and rho-mediated pathways, may be involved in the response. KeyWordc lysophosphatidic acid,intracellularcalchunsignal& mechanotransdudon, phosphollpaseC,tyrhe kinase,cuhure4Ismooth musclecells Many cell types can expose to several types of mechanical stress from extracellular environment and adjacent cells in multicellular animals. Mechanotransduction systems are considered to play an important role in conversion of externally applied mechanical stress to signals that regulate cellular function and metabolism not only in sensory organs but also in nonsensory tissues such as osteoblastic cells (1). vascular endothelial cells (2), mammary epithelial cells (3), lung epithelial cells (4), vascular smooth muscle (5,6), gingival fibroblasts (7), cardiac muscle (8) and skeletal muscle (9). In most of these cell types, a transient increase in intracellular free Ca2+concentration ([Caz+]i) has been observed when cells expose to mechanical stress, and it is considered to be related to Ca2+ influx through mechanosensitive ion channels (10). However, little information is available about the signal transduction pathway that mediates conversion of mechanical stress to the specific cellular response including [Caz+]imobilization. *Correspondence to: Dr. Hisayuki Ohata, Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Phone +81-33784-8212. FAX +81-3-3784-3232.

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We have previously reported that lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, sensitizes response in [Caz+]i to mechanical stress in cultured longitudinal muscle cells from guinea pig ileum (I 1) and cultured human lung epithelial cells (12). LPA is rapidly produced and released by thrombin-stimulated platelets (13) and perhaps other cell types into the extracellular environment, and is present in mammalian serum in active form at micromolar concentrations (13, 14). In addition, recent study indicates that nonpancreatic phospholipase A2 secreted during inflammation could be actively involved in the generation of novel lipid mediator LPA (15). Accordingly, our previous findings indicated a possibility that LPA might play an significant role in mechanotransduction systems as an endogenous modulator in situ. However, the signal transduction pathways that mediate the sensitizing effect of LPA is unknown. Moreover, it is possible that a portion of the signal transduction pathways activated by LPA may overlap with the pathways that mediate the mechanotransduction systems. It is also well established that LPA evokes multiple biological responses by activating a specific G protein coupled receptors, which are linked with stimulation of phospholipase C (PLC) and inhibition of adenylate cyclase (16). In addition, recent studies indicate that LPA receptor also couples to novel routes, notably activation of the small GTP-binding protein rho to trigger actinbased cytoskeletal events (17). Since it has recently been suggested that membrane-anchored filamentous actin may regulate Ca 2+ flux in cells responding to mechanical stretch (7), LPA may sensitize the mechanotransduction systems via rho-mediated cytoskeletal changes. The clarification of the signaling pathways that mediate the sensitizing effect of LPA on the response to mechanical stress is important to know physiological role in LPA, and must also contribute to elucidation of the molecular mechanisms of the mechanotransduction systems. In the present study, to clarify the signal transduction pathways that mediate the sensitizing effect of LPA on mechanical stress-induced response, we examined the effect of several specific inhibitors of the signaling pathways related to LPA-induced cellular response on the mechanical stress-induced [Ca2+]i mobilization sensitized by LPA in cultured longitudinal muscle cells from guinea pig ileum. The results show that LPA sensitizes the mechanical stress-induced response via activation of PLC and tyrosine kinase, but not through pertussis toxin-sensitive and rho-mediated pathways. Materials

and Methods

Materials: Fluo-3 acetoxymzthyl ester (fluo-3/AM) was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Lysophosphatidic acid (LPA: from egg yolk lecithin) were purchased from Serdary Research Laboratories (Ontario, Canada). Pertussis toxin, staurosporine, tyrphostin 25 and wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Thapsigargin, C3 ADP ribosyltransferase from Clostridium boturinum (C3), genistein, fluorescein phalloidin and l -(6(( 17 a -3-methoxyestra- 1,3,5( 1O)-trien- 17-yl)amino)hexyl)- 1H-pyrrole-2,5-dione (U73 122) were obtained from Wako Pure Chemicals (Osaka, Japan). Suramin was purchased from Biomol Research Laboratories (PA, USA). I-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) was obtained from Seikagaku Co. (Tokyo, Japan). Cremophor EL was purchased from Nacalai Tesque Inc. (Kyoto, Japan). All other chemicals were commercial products of the highest available grade of purity. LPA was dissolved in water at 10 pM. Staurosporine, tyrphostin 25, thapsigargin, genistein, U73 122 and wortmannin were dissolved in dimethylsulfoxide as the final concentration of dimethylsulfoxide in experimental medium was less than 0.2% to avoid its effect on the mechanical stress-induced response. Other inhibitors were dissolved in water. Cell culture: The cultured longitudinal muscle cells were obtained from guinea pig ileum as described previously (18). The longitudinal muscle was carefully stripped off and placed in Tyrode-Hepes solution containing (n&I) NaCl, 137; KCI, 2.7; CaCl2, 1.8; MgC12, i -0; glucose, 5.6; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), 8.4; pH 7.4. The longitudinal muscle segments were cut into small pieces. Small explants of longitudinal muscle were cultured in minimum essential medium (MEM, Gibco) containing 20% fetal calf serum on 25-mm-diameter glass coverslips inside 35-mm Petri dishes and maintained at 37oC under humidified conditions of 95% air - 5% CO2. After 24 hr, the culture medium was replaced with MEM containing 10% fetal calf serum, and the medium was changed every 3 days until the cells were grown to complete confluency. The confluent cells of secondary culture were used for the experiments.

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Measurement of lCa2+]i: To determine the [Ca*+]i, the cultured cells on a coverslip were washed several times with Tyrode-Hepes solution, and were incubated with the same solution containing 5 pM fluo-UAM and 0.03% cremophor EL at room temperature for 1 hour. After loading, the coverslips were rinsed several times with Tyrode-Hepes solution. After washing, the coverslip was mounted in an experimental chamber. Fluorescence images of fluo-3 were collected under the various conditions using a Bio-Rad MRCSOO confocal laser scanning attachment mounted on a Nikon Diaphot inverted microscopy as described previously (11). Excitation wavelength of 488 nm was provided by an Argon laser and was attenuated with 1% neutral density filter to minimize photobleaching and photodamage. Green fluorescence of fluo-3 was collected using a 5 IO-nm long pass dichroic reflector and a 515-nm long pass emission filter. The temperature was kept at 32oC, since intracellular fluorescence of fluo-3 decreased rapidly at 37oC, probably by dye leakage. The mean intensity of fluo-3 fluorescence for each individual cell was obtained by averaging the intensity of each pixel within a rectangular zone centered over the cell. ADDlication : Mechanical stress was applied to the cells as described previously (I I). Bath solution was perpendicularly spritzed onto the fluo-3-loaded cells from pipette at an appropriate constant flow rate (1.8 ml/min) for 3 seconds. Tip of the pipette was settled at 3 mm right over the interest cells and the inside diameter of the tip was 1 mm. This mechanical stress did not affect acquisition of the images of fluo-3 fluorescence and induced the reproducible response, though it may act as mixed mechanical stress of both flow and pressure stress. Staining actin filaments: Cells on coverslip were fixed in 4% paraformaldehyde for IO min and permeabilized with 0.1% Triton X-100 for 10 min. Coverslip was rinsed in PBS, and permeabilized cells were incubated with fluorescein-phalloidin (150 units/ ml) for 30 min. After rinsing in PBS, cells were viewed on a Bio-Rad MRC-500 confocal laser scanning attachment mounted on a Nikon Diaphot inverted microscopy using 60 x 1.4 oil immersion objective. Sm Data were expressed as the mean + S.E. of more than three experiments. were analyzed for statistical significance by an one-way analysis of variance (ANOVA).

Data

Results Bindine site of LPA for the sensitizine effect of LPA: Mechanical stress by spritzing bath solution at the flow rate of 1.8 ml/mm for 3 seconds onto the cultured longitudinal muscle cells caused a [Ca2+]i transient in the presence of 30 nM LPA, but not in the absence of LPA as shown in (Fig. 1A). This phenomenon clearly shows that LPA sensitizes the mechanical stress-induced [Caz+]i transients. To test whether the response depends on binding of LPA to the membrane receptor, we examined the effect of suramin, the only known LPA antagonist (19), on the sensitizing effect. However, the pretreatment with 300 pM suramin did not affect the response as shown in Fig. 1. On the other hand, the LPA-induced sensitization was immediately abolished by washout of LPA. : To clarify whether activation of Gu, mediates the LPAInvolvement of Gi/,_mediated sensitized response to mechanical stress, effect of pretreatment of pertussis toxin (1 pg/ml) for 2 hours on the response was examined. Pertussis toxin did not significantly inhibit the sensitizing effect of LPA (data not shown). Role of DhoSpholiDase C and nrotein kinase C: To elucidate whether activation of PLC by LPA is required for the LPA-sensitized response to mechanical stress, we examined the effect of a putative PLC inhibitor, U73122 (20), on the LPA-sensitized response to mechanical stress. As shown in Fig. 2, addition of 3 uM U73122 completely inhibited the LPA-sensitized response. Also 1 pM U73 122 significantly decreased the percentage of cells responded to the mechanical stress to less than 20% of the control response as shown in Fig. 2B. In this connection, we have confirmed that 3 pM U73122 does not affect the transient increase in [Caz+]i induced by 1 uM thapsigargin, an inhibitor of the endoplasmic reticulum Ca z+-ATPase pump (21) (data not shown).

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Fig. 1. Effect of suramin on changes in [Caz+]i to mechanical stress in cultured smooth muscle cells in the presence of LPA. A: Open and closed circles represent the time courses of changes in [Caz+]i to the mechanical stress in two different cells in the same microscopic field. As shown at the top of the panel, 30 nM LPA and 300 pM suramin were added to the cells. Allows show applications of the mechanical stress as described in Materials and Methods. B: Percentages of cells responded to the mechanical stress in 30 cells in the presence or absence of 30 nM LPA or 300 pM suramin as shown at the bottom of the panel were represented as the mean f SE. of 3 experiments, respectively. Cell responded to the mechanical stress was defined as the cell where intensitv of fluo-3 fluorescence increases to more than 30% of the baseline level within 30 seknds after the mechanical stress.

0.0

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Effect of U73 122 on changes in [Caz+]i to mechanical stress in the presence of LPA. A: Open and closed circles represent the time courses of changes in [Ca*+]i to the mechanical stress in two different cells in the same microscopic field. As shown at the top of the panel, 30 nM LPA and 3 pM U73122 were added to the cells. Allows show applications of the mechanical stress. B: Percentages of cells responded to the mechanical stress in 30 cells treated with 0, 1 or 3 pM U73 122 in the presence or absence of 30 nM LPA as shown at the bottom of the panel were represented as the mean rf:S.E. of 3-4 experiments, respectively. Significant differences, *p&01.

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To test involvement of activation of PKC in the LPA-sensitized response to mechanical stress, we then examined that the effect of broad spectrum kinase inhibitors, staurosporine (22) and H7 (23), on the response. Neither the pretreatment of 100 nM staurosporine for 10 min nor the pretreatment of 30 @4 H7 for 5 min inhibited the response as shown in Fig. 3. 80 60

40

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Fig. 3. Effects of staurosporine and H7 on changes in [Ca*+]i to mechanical stress in the presence of LPA. Percentages of cells responded to the mechanical stress in 30 cells pretreated with or without 100 nM staurosporine (SP) for 10 min (A) or 30 pM H7 for 5 min (B) in the presence or absence of 30 nM LPA as shown at the bottom of the panel were represented as the mean a SE. of 4 and 3 experiments, respectively.

Fig. 4. Effect of C3 on changes in [Caz+]i to mechanical stress and on distribution of actin filaments in the presence of LPA. A: Percentages of cells responded to the mechanical stress in 30 cells pretreated with 5 pg/ml C3 for 72 hours in the presence (LPA) or absence (Cant) of 30 nM LPA were represented as the mean f S.E. of 4 experiments. B, C: Actin filaments were stained with fluorescein phalloidin as described in Materials and Methods, after confirming the [Ca2+]i response to the mechanical stress in the presence of 30 nM LPA in cells pretreated with (C) or without (B) 5 pg/ml C3 for 72 hours. Scale bar represents 50 pm.

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Jnvolvement of rho-mediated cvtoskeletal changes: To examine whether rho-related cytoskeletal changes induced by LPA are required for the LPA-sensitized response to mechanical stress, we examined the effect of C3 ADP ribosyltransferase, which inactivates rho protein (24), on the response. The pretreatment of C3 (5 clg/ml) for 72 hours had no effect on the response as shown in Fig. 4A, whereas formation of a&in-stress fiber in the presence of LPA was clearly inhibited by the pretreatment (Fig. 4C) as compared with that in nontreated cells (Fig. 4B). PI3-kinase-mediated uath av: To determine whether activation of PI3-kinase mediates the LPAsensitized response to mechanical stress, effect of wortmannin, a potent inhibitor of PI3-kinase (25) on the response was examined. Pretreatment of 100 nM wortmannin had no effect on the sensitizing effect of LPA (data not shown).

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Fig. 5. Effects of genistein (A) and tyrphostin (B) on changes in [Ca*+]i to mechanical stress in the presence of LPA. Percentages of cells responded to the mechanical stress in 30 cells pretreated with or without 100 PM genistein (GS) for 5 min (A) or 100 uM tyrphostin (TP) for 20 min (B) in the presence or absence of 30 nM LPA as shown at the bottom of the panel were represented as the mean Z!Z SE. of 8 and 4 experiments, respectively. Significant differences, *p
Discussion We have previously reported that LPA sensitizes mechanical stress-induced response in [Ca2+]1 depending on Caz+ influx through Gds+-sensitive channels, probably stretch-activated ion channels (11, 12), following Ca*+ release from ryanodine-sensitive intracellular stores (32). However, the signal transduction pathway involved in the sensitization is unknown. Since it is possible that a portion of the signal transduction pathway activated by LPA may overlap with that in the mechanotransduction systems, we tried to clarify the pathway using several inactivators of the signaling pathways activated by LPA in the present study.

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Most of cellular responses caused by LPA were inhibited by suramin, the only known LPA antagonist (19X but high concentration of suramin did not have any effect on the LPA-sensitized response to the mechanical stress (Fig. 1). The result shows that the response is not caused by binding of LPA to suramin-sensitive receptor. On the other hand, the results that the sensitizing effect was caused by LPA of nanomolar concentrations, and was immediately abolished by washout of LPA suggest that the effect is induced by binding of LPA to a specific binding site on plasma membrane, probably suramin-insensitive type of receptor. Expression of a suramin-insensitive type of LPA receptor has been recently shown in epithelial cell like avian salt gland cells (33). The results in the present study also suggest that the cultured smooth muscle cells may express the same suramin-insensitive type of LPA receptor as that is expressed in avian salt gland cells. Several responses to LPA including inhibition of adenylate cyclase, activation of MAP kinase and stimulation of DNA synthesis are mediated by a pertussis toxin-sensitive Gi/o (34). In the yeast Succharomyces cerevisiae, a member of the MAP kinase group has been implicated in osmosensing, one of the mechanotransduction systems (35). Accordingly, there is a possibility that LPA-induced activation of MAP kinase cascade through Gil0 is involved in the sensitizing effect. To clarify the possibility, we examined the effect of pertussis toxin on the LPA-sensitized response to the mechanical stress. However, pertussis toxin did not have significant effect on the sensitization, suggesting that the activation of Gu,-coupled pathways including MAP kinase cascade by LPA is not involved in the sensitizing effect. Involvement of PLC activation in stretch-induced myogenic activation in cerebral arteries has been reported (5,6). Since it is well established that LPA activates phosphoinositide-specific PLC with PKC activation in various cell types (16), it is possible that activation of PLC and/or PKC by LPA is involved in the LPA-sensitized response to the mechanical stress. A putative PLC inhibitor, U73 122 (20) completely inhibited the sensitizing effect (Fig. 2). The concentration of the inhibitor used to block the effect could be sufficiently low to lead the specific action, because it did not affect the [Ca’+]i transient induced by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ATPase pump (21). These results indicate that signaling pathway through the activation of PLC is required for the LPA-sensitized response to the mechanical stress. However it is unlikely that the LPA-induced sensitization is caused by only activation of PLC, because it is not sensitized by Ca2+mobilizing agonists such as histamine and carbachol, which activate PLC leading inositol 1,4,5trisphosphate (IPs)-induced Ca2+ release from intracellular stores, as shown in the previous study (11). In addition, since staurosporine (22) and H7 (23) have no apparent effect of the LPA-induced sensitization (Fig. 3), PKC is unlikely to play a role in the response. These results suggest that other factor changed by activation of PLC than IPs-induced Ca2+ release and activation of PKC is involved in the sensitization. Furthermore, the results also suggest that neither cyclic AMPdependent protein kinase (PKA) nor cyclic GMP-dependent protein kinase (PKG) is involved in the sensitization, because H7 inhibits with a similar potency PKC, PKA, and PKG (23). Recent studies indicated that LPA receptor also coupled to rho to trigger actin-based cytoskeletal events (17). In addition, it might be a PLC-induced decrease in phosphoinositide levels somehow leads to rho activation (16). On the other hand, it has recently been suggested that membraneanchored filamentous actin may regulate Ca2+ flux in human fibroblasts responding to mechanical stretch (7). Therefore, it was possible that LPA might sensitize the mechanotransduction systems via rho-related cytoskeletal changes. However, contrary to the expectation, the LPA-induced sensitization was not affected by the inactivation of rho using C3 (24), whereas formation of actinstress fiber in the presence of LPA was clearly inhibited by the pretreatment of C3 (Fig. 4). The result shows that the pathway mediated by the activation of rho is not required for the LPAsensitized response to the mechanical stress. Additionally, the result also suggests that the response to the mechanical stress in the presence of LPA is not involved in changes in actin filaments. It has been shown that activation of PI3-kinase is required for cytoskeletal reorganization such as membrane ruffling (36, 37) and platelet aggregation (38). LPA-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) has been found in Swiss 3T3 cells (26), in megakaryoblastic cells (39) and in human platelets (38). To determine whether activation of PI3kinase mediates the LPA-sensitized response to the mechanical stress, effect of wortmannin, a potent inhibitor of PI3-kinase (25), on the response was examined. However, wortmannin had no

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effect on the sensitizing effect of LPA, suggesting that the activation pathway is not involved in the sensitizing effect of LPA.

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of PI3-kinase-mediated

It has recently been reported that LPA-induced protein tyrosine phosphorylations are associated with PLC activation (40). Therefore, it was possible that activation of tyrosine kinase related with PLC activation might be involved in the LPA-induced sensitization. According to the expectation, a tyrosine kinase inhibitor, genistein (29) significantly attenuated the sensitizing effect (Fig. 5A), suggesting that the activation of tyrosine kinase by LPA is also involved in the LPA-induced sensitization. Furthermore, it was considered that the tyrosine kinase required for the sensitization is not involved in Gu,- and rho-mediated pathways, because neither pertussis toxin nor C3 inhibited the LPA-induced sensitization. The supposition was also supported by the result that tyrphostin, which inhibits a tyrosine kinase in upstream of rho (31), did not affect the sensitization (Fig. 5B). These results suggest that the activation of tyrosine kinase is important factor for the sensitizing effect, but further studies are required to clarify whether the tyrosine kinase required for the sensitization is located in downstream of PLC activation or in parallel pathway in addition to identify the target protein of the tyrosine kinase. It has recently been reported that platelet endothelial cell adhesion molecule 1 is rapidly tyrosine-phosphorylated in vascular endothelial cells exposed to flow, hyper- and hypo-osmotic shocks (41). It is possible that LPA-induced tyrosine phosphorylation may have positive effect on the mechanical stress-induced tyrosine phosphorylation as shown in endothelial cells. To clarify the possibility, we are planning to assay for LPAinduced tyrosine phosphorylation. In conclusion, the results presented here suggest that the LPA-induced activations of PLC and tyrphostin-insensitive tyrosine kinase, but not PKC-, Giio- PI3-kinaseand rho-mediated pathways, contribute to the regulation of mechanotransduction systems in smooth muscle cells, although further studies are required to elucidate how the activations of PLC and tyrosine kinase affect the mechanotransduction systems.

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