Lysophosphatidylcholine activates mesangial cell PKC and MAP kinase by PLCgamma -1 and tyrosine kinase-Ras pathways

Babu V. Bassa1,2, Daeyoung D. Roh1,2, Nosratola D. Vaziri2, Michael A. Kirschenbaumdagger ,1,2, and Vaijinath S. Kamanna1,2

1 Nephrology Section, Department of Veterans Affairs Medical Center, Long Beach 90822; and 2 Division of Nephrology and Hypertension, Department of Medicine, University of California, Irvine, California 92697


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although lysophosphatidylcholine (LPC)-mediated cellular responses are attributed to the activation of protein kinase C (PKC), relatively little is known about the upstream signaling mechanisms that regulate the activation of PKC and downstream mitogen-activated protein (MAP) kinase. LPC activated p42 MAP kinase and PKC in mesangial cells. LPC-mediated MAP kinase activation was inhibited (but not completely) by PKC inhibition, suggesting additional signaling events. LPC stimulated protein tyrosine kinase (PTK) activity and induced Ras-GTP binding. LPC-induced MAP kinase activity was blocked by the PTK inhibitor genistein. Because LPC increased PTK activity, we examined the involvement of phospholipase Cgamma -1 (PLCgamma -1) as a key participant in LPC-induced PKC activation. LPC stimulated the phosphorylation of PLCgamma -1. PTK inhibitors suppressed LPC-induced PKC activity, whereas the same had no effect on phorbol 12-myristate 13-acetate-mediated PKC activity. Other lysophospholipids [e.g., lysophosphatidylinositol and lysophosphatidic acid (LPA)] also induced MAP kinase activity, and only LPA-induced MAP kinase activation was sensitive to pertussis toxin. These results indicate that LPC-mediated PKC activation may be regulated by PTK-dependent activation of PLCgamma -1, and both PKC and PTK-Ras pathways are involved in LPC-mediated downstream MAP kinase activation.

atherogenic lipoproteins; oxidatively modified lysophosphatidylcholine; intracellular signaling; atherosclerosis; glomerulosclerosis; protein kinase C; mitogen-activated protein kinase; phospholipase Cgamma -1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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A GROWING BODY OF EVIDENCE suggests that lysophosphatidylcholine (LPC), a principal component of oxidatively (ox) modified forms of low-density lipoproteins (LDL), can modulate vascular cell activation processes, culminating in the gene expression of various cytokines, growth factors, and other proteins associated with vascular diseases (19, 20, 28, 43, 44). Similarly, we have shown that the incubation of glomerular endothelial cells with LPC induced intercellular adhesion molecule 1 (ICAM-1) expression and increased monocyte adhesion to glomerular endothelial cells (9). LPC associated with ox-LDL or exogenous LPC has been shown to induce selective impairment of receptor-mediated endothelium-dependent arterial relaxation (17). The in vivo generation of LPC involved in pathobiological cellular events is not completely understood; however, the hydrolysis of sn-2 fatty acid of phosphatidylcholine by plasma lecithin-cholesterol acyltransferase can produce LPC (14). Furthermore, phospholipase A2 associated with apolipoprotein B of LDL has been implicated in the generation of LPC during oxidative modification of LDL (31). After its formation, LPC, a relatively polar molecule, gets associated with serum albumin or LDL and is carried to various cellular sites to participate in cellular, metabolic, and pathophysiological pathways. Increased concentrations of plasma LPC (including in LDL fractions) have been observed in atherosclerosis and nephrosis in humans and experimental animals (14, 34). Higher circulating levels of LPC have also been reported during ischemia in both animals and humans (1, 26).

Recently, we have reported that the stimulation of mesangial cells with LDL, ox-LDL (with higher potency), and LPC (major active component of ox-LDL) increased the activation of p42 mitogen-activated protein (MAP) kinase (2). MAP kinases are a family of serine threonine kinases that are proposed to converge diverse signal transduction events associated with various cellular responses, including mitogenesis and differentiation (33). Atherogenic lipoproteins (e.g., LDL, ox-LDL, or its major components) can activate cellular protein kinase C (PKC; see Refs. 18 and 29), and PKC in turn can activate MAP kinases through the activation of Raf-1 and a MAP kinase kinase pathway (12, 40, 22). The activation of membrane or cytoplasmic protein tyrosine kinase (PTK) pathways may regulate both PKC and downstream MAP kinase activation through phospholipase Cgamma -1 (PLCgamma -1) and Ras-mediated pathways (25, 27). However, the involvement of any of the above-noted multiple signaling events in atherogenic lipoprotein- and LPC-mediated responses are not known so far.

Based on the current understanding, the activation of PKC has been the primary mechanism implicated in several actions of LPC (18, 29, 36). However, the molecular basis for the activation of PKC, especially the identification of the upstream signaling mechanism(s) of the phosphoinositide turnover and the generation of diacylglycerol (DAG), which is absolutely required for the activation of PKC, is poorly understood so far. Furthermore, the involvement of LPC-induced PKC and/or other signaling events in the regulation of MAP kinase is not clearly understood. With the use of stably transformed murine glomerular mesangial cells (SV40 transformed) and primary cultures of rat glomerular mesangial cells (in selective MAP kinase experiments), this study was designed to identify the upstream signaling mechanisms of LPC-mediated PKC activation and the involvement of PKC and/or PTK- and Ras-mediated pathways in downstream MAP kinase activation by LPC. We report here that LPC, through the stimulation of phosphorylation of PLCgamma -1 and activation of Ras, important members of the mitogenic signaling cascade, regulates PKC activation and downstream MAP kinase signaling. The activation of membrane or cytoplasmic tyrosine kinases may be an early principal event in LPC-mediated signaling processes.


    MATERIALS AND METHODS
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Materials. Antibodies for PKC (alpha , beta , and gamma  reacting), PKC-zeta , p42 MAP kinase, PLCgamma -1, Shc, pp60src, and phosphotyrosines were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ras antibody (clone Y 13-259) was obtained from Calbiochem (San Diego, CA). [32P]ATP was obtained either from Amersham (Arlington Heights, IL) or from DuPont New England Nuclear Research Products (Boston, MA). Pertussis toxin (PTX), genistein, tyrphostin B46, and bisindolylmaleimide GF109203-X (GFX) were obtained from Calbiochem (La Jolla CA). Protein A-Sepharose, myelin basic protein (MBP), phorbol 12-myristate 13-acetate (PMA), LPC (palmitoyl and oleolyl), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), FBS, and other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Cell culture and treatment. The murine mesangial cells (stably transformed with SV40) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were routinely grown in DMEM supplemented with 5% FBS. Primary cultures of rat glomerular mesangial cells were isolated by a previously described procedure (30), and cells for MAP kinase assays were used within 4-10 passages. Cell cultures were prepared by seeding 5 × 105 cells/well in 30-mm six-well cluster dishes. When cell monolayers were ~80% confluent, the medium was replaced by serum-free DMEM. After 24 h, the medium was replaced by fresh serum-free DMEM and LPC, or other agonists and protein kinase inhibitors were added in appropriate quantities. LPC and other lysophospholipid stock solutions were prepared in absolute ethanol (10-20 mM). A working solution was prepared by diluting the stock solution (in serum-free DMEM) and then adding aliquots of this solution to the cultures at selected concentrations. A stock solution of PMA (10 mg/ml) was prepared in ethanol, and working solutions were prepared by diluting the stock appropriately in serum-free DMEM. GFX, herbimycin, and genistein were dissolved in DMSO. Appropriate quantities of DMSO (5-10 µl) were added to the control dishes. After being incubated for selected time intervals, cells were washed with 1 ml of cold Hanks' balanced salt solution and were processed with an appropriate cell lysis buffer for the determination of PKC, PLCgamma -1, MAP kinase, tyrosine kinase, or Ras activities.

Assay of p42 MAP kinase. Mesangial cells were stimulated with LPC and other agonists at various concentrations for different time periods. After the stimulation, cells were scraped in 0.5 ml of MAP kinase lysis buffer [50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM vanadate, 40 mM paranitrophenyl phosphate, 1 µM pepstatin, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 1% Nonidet P-40 (NP-40)]. MAP kinase in cell lysate (20-30 µg) was immunoprecipitated using goat polyclonal anti-p42 MAP kinase antibody (0.4 µg) and protein A-Sepharose (50%). After washing, the kinase activity of the immune complex was assayed with MBP as a substrate in a reaction mixture containing 7.5 mM HEPES, pH 7.5, 10 mM magnesium acetate, 50 µM ATP, and 4 µCi [32P]ATP in a total volume of 40 µl (35). The reaction was performed at 30°C for 20 min, and the reaction was terminated by adding 20 µl of 3× Laemmli sample buffer. Samples were heated at 80°C for 5 min and resolved by SDS-PAGE (10%). The gel was stained with Coomassie blue, dried, and exposed to X-ray film for 1-4 h. The MBP bands were then cut out, and the radioactivity was measured by liquid scintillation spectrometry. MAP kinase activity was expressed as picomoles phosphate incorporated in MBP per milligram cell lysate protein.

Assay of PKC. Cell cultures were prepared as described above. After incubation with LPC at appropriate concentrations and time intervals, the cells were washed and disrupted in 20 mM Tris · HCl, 0.5 mM EGTA, 0.25 mM sucrose, 50 µM digitonin, and 50 µg/ml leupeptin at pH 7.5. After centrifugation at 13,000 g, the membrane fraction was solubilized in a buffer containing 1.0% Triton X-100, 20 mM Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 5 µg/ml leupeptin. The PKC protein from this membrane solution was precipitated with anti-PKC antibody that had been conjugated to protein A-Sepharose (30 µl of 50% protein A-Sepharose, 0.5 µg anti-PKC antibody) by mixing for 2 h at 4°C. The immune complex was resuspended in 15 µl of micellar mixture prepared as described earlier (11), and 25 µl of the assay buffer (10 mM Tris · HCl, pH 7.5, 5 mM MgCl2, 50 mM CaCl2, 5 µM ATP, 0.6 mg/ml histone III, and 2.5 µCi [gamma -32P]ATP) were then added. The kinase reaction was carried out at 30°C for 10 min, and the reaction was terminated by the addition of 25 µl of Laemmli sample buffer. After being heated at 80°C for 5 min, the samples were resolved on SDS-PAGE (10% gel). The gels were dried and exposed to X-ray films for autoradiography. The degree of histone phosphorylation, as a measure of PKC activity, was determined by densitometric scanning of autoradiograms.

PKC activation was also assessed by determining the membrane association of PKC isoforms (e.g., alpha /beta , gamma , and zeta ) by Western blot analysis in control and LPC-treated cells. Mesangial cells (serum starved for 24 h) were stimulated with LPC (10 and 25 µM) for 5-15 min. Cells were scraped in PBS and sonicated for 15 s. The membrane fraction was isolated by centrifugation at 100,000 g for 30 min. The membrane pellet was solubilized in a lysis buffer (25 mM HEPES, pH 7.8, 10 mM EGTA, 10 µg/ml leupeptin, 1 mM PMSF, 50 mM NaF, 0.5 M NaCl, 0.2 mM Na3VO4, 1.5% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS), samples were resolved on SDS-PAGE (12% gel), and proteins were transferred to the nitrocellulose membrane. The membranes were probed with antibodies specific for PKC isoforms.

Ras activation analysis. Ras activation analysis was done by the method of Downward et al. (4) and modified as described below. Cells were cultured in six-well plates as described earlier. After 24 h of serum starvation, cells were incubated for 3 h in phosphate-free DMEM containing 300 µCi of [32P]orthophosphate. Cells were then stimulated with LPC (10 µM) for various time periods. The cells were then washed with Tris-buffered saline and scraped in 0.5 ml of Ras lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 9 µg/ml PMSF, 1% Triton X-100, 1 mg/ml BSA, and 10 mM benzamidine). The cell lysate was first mixed with an equal volume of a solution of 1 M NaCl, 0.1% SDS, and 1% sodium deoxycholate and was then treated with 100 µl of BSA-coated charcoal (4). After a brief mixing and centrifugation, the supernatant was immunoprecipitated with anti-Ras antibody (0.5 µg, conjugated to protein A-Sepharose) by mixing for 40 min at 4°C. The beads were washed four times with Ras lysis buffer, and the guanine nucleotides were then eluted with potassium phosphate buffer (1 M, pH 3.4) by heating at 80°C for 5 min. GTP and GDP were resolved by TLC on polyethyleneimine cellulose plates and were detected by autoradiography.

Phosphorylation of PLCgamma -1 assay. After 24 h of serum starvation, cells were stimulated with LPC (10-25 µM) for 5-20 min. Cells were washed and lysed with a buffer containing 10 mM Tris · HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and 1% Triton X-100. After a brief centrifugation, the cell lysate was immunoprecipitated with anti-PLCgamma -1 conjugated with protein A-Sepharose. The immune complex was mixed with Laemmli sample buffer, heated at 80°C for 5 min. The sample was resolved on SDS-PAGE (12.5% gel), transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine, and phosphorylated PLCgamma -1 was detected by enhanced chemiluminescent (ECL) reagents.

Assay of membrane tyrosine kinase activity. Tyrosine kinase activity in mesangial cell membranes was measured by an in vitro phosphorylation technique as described in instructions for a commercially available assay kit (Life Technologies, GIBCO-BRL, Grand Island, NY). Mesangial cell membranes were prepared by centrifugation at 21,000 rpm for 20 min. Membrane preparations were incubated on ice with 10 µM LPC for 30 min. Tyrosine kinase activity of the activated membranes was determined in the presence or in the absence of genistein (100 µM) using a synthetic peptide [12-amino acid sequence surrounding tyrosine phosphorylation site in pp60src, specific for epidermal growth factor (EGF) receptor] as a substrate and [gamma -32P]ATP. The tyrosine kinase activity was expressed as counts per minute (cpm), phosphate incorporation, per microgram of membrane protein.

For cellular protein tyrosine phosphorylation studies, quiescent mesangial cells were treated with LPC (25 µM) for 5-10 min, and the cells were lysed. The cell lysate was immunoprecipitated with anti-phosphotyrosine antibodies. The immunoprecipitates were incubated with [gamma -32P]ATP in a kinase assay buffer (50 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.01% NP-40, and 75 mM NaCl) for 20 min at 30°C. The samples were resolved on 7.5% SDS-PAGE, and phosphorylated proteins were detected by autoradiography. In additional studies, the phosphorylation of Shc and the activity of pp60src were measured. For Shc phosphorylation studies, LPC-treated cells were lysed and immunoprecipitated with anti-Shc antibodies. The immunoprecipitates were resolved on SDS-PAGE, transferred to the membranes, and immunoblotted with anti-phosphotyrosine antibodies, and the phosphorylation was detected by the ECL method. For Src activity measurement, LPC-treated cells were lysed and immunoprecipitated with anti-Src antibodies, and the Src activity was determined by using enolase as a substrate (8).

Statistical analysis. Results are presented as representative studies or by displaying mean values ± SE for three to four separate experiments, each assayed in duplicate or triplicate. A Student's t-test was used to compare the means, and a P value of <0.05 was considered significant.


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The incubation of mesangial cells with LPC (10-25 µM) for 15 min to 3 h, as used in this study, did not alter the viability, cell number, or cellular protein content as assessed by the trypan blue exclusion criterion and by measuring cell number and protein content, respectively. The cell numbers in control and LPC-treated cells were as follows (1 × 106/dish, average of 2 determinations): 15 min incubation, control = 1.98, 10 µM LPC = 2.30, 25 µM LPC = 2.25; 3 h incubation, control = 1.76, 10 µM LPC = 1.63, 25 µM LPC = 1.61. The viability of cells in control and LPC-treated cells for 15 min to 3 h was similar (95-98% viable, as assessed by trypan blue exclusion method).

Activation of PKC by LPC and inhibition by tyrosine kinase inhibitors. One of the aims of the present studies is to understand the mechanism by which LPC activates PKC. For the assay of PKC, membrane-bound PKC was purified by immnoprecipitation with anti-PKC (reacting with alpha , beta , and gamma  isoforms), and the kinase assay was performed using histone III as a substrate. We found this method of assaying PKC to be more reproducible, as we encountered high background radioactivity in the phosphocellulose disc method used for the separation of the substrate. The activity of PKC in the membrane fraction, obtained from LPC-treated cells, increased by ~2.5- to 3.5-fold during 5-10 min of stimulation (Fig. 1A). Preincubation of cells with the PTK inhibitor herbimycin completely suppressed the activation of PKC by LPC. The quantitative densitometric analysis (as arbitrary units, average from 2 experiments) of the autoradiograms for PKC activity showed the following data: control = 1.02; 10 µM LPC, 5 min = 2.39; 25 µM LPC, 5 min = 3.09; 10 µM LPC, 10 min = 3.32; 25 µM LPC, 10 min = 3.42. The corresponding quantitative data for LPC-induced PKC activity in the presence of herbimycin was as follows: control = 1.02; 10 µM LPC + herbimycin, 5 min = 0.85; 25 µM LPC + herbimycin, 5 min = 1.01; 10 µM LPC + herbimycin, 10 min = 1.13; 25 µM LPC + herbimycin, 10 min = 1.47. PMA markedly activated PKC activity, and this activation was not inhibited by herbimycin (quantitative arbitrary units: control = 1.02; PMA = 6.4; PMA + herbimycin = 6.2). Similarly, another tyrosine kinase inhibitor, genistein (which is structurally different from herbimycin), also suppressed the activation of PKC by LPC (quantitative data: control = 1.77; 10 µM LPC, 10 min = 2.95; 10 µM LPC + genistein, 10 min = 1.81; Fig. 1B). Additionally, pretreatment of cells with genistein did not significantly alter PMA-induced PKC activity (Fig. 1B).



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Fig. 1.   Effect of lysophosphatidylcholine (LPC) on protein kinase C (PKC) activity: role of herbimycin and genistein (Gen) on the activation of PKC by LPC. A: mesangial cells were stimulated with LPC (10 or 25 µM) for 10 min. In additional experiments, cells were preincubated with herbimycin (5 µM) or vehicle (DMSO) for 1 h before stimulation of cells with LPC (10 or 25 µM) or PMA (100 nM) for 10 min. Cells were processed for the determination of PKC activity as described in MATERIALS AND METHODS. C, control; Inc. time, incubation time; PMA, phorbol 12-myristate 13-acetate. B: mesangial cells (serum starved for 24) were incubated in the absence or presence of genistein (100 µM) for 90 min. Cells were then treated with 10 or 25 µM LPC or PMA (100 nM) for 10 min, and cells were then processed for the determination of PKC activity as described in MATERIALS AND METHODS.

In other studies, Western blot analysis of membrane association of PKC isoforms indicated that LPC induced the membrane association of alpha /beta and gamma  isoforms of PKC (by 2- to 3-fold compared with controls; Fig. 2A), but LPC had no effect on the membrane association of PKC-zeta (Fig. 2B). The PKC antibody used in Fig. 2A recognizes all three isoforms of PKC (alpha , beta , and gamma ). Because the molecular masses for alpha  and beta  PKC isoforms are very similar (~82 kDa), only a single band was noted for both alpha  and beta  isoforms, whereas the gamma  isoform was detected as a separate band of molecular mass of ~78 kDa.



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Fig. 2.   Effect of LPC on membrane association of PKC-alpha /beta and -gamma isoforms (A) and PKC-zeta (B). Mesangial cells (serum starved for 24 h) were stimulated with LPC (10 and 25 µM) for 5-15 min. Cellular membrane fractions (100,000 g) were resolved on SDS-PAGE, and proteins were transferred to the nitrocellulose membrane. Membranes were probed with antibodies specific for either alpha /beta /gamma or zeta  isoforms of PKC. Membrane association of alpha /beta , gamma , or zeta  PKC isoforms was detected by the enhanced chemiluminescent (ECL) method. PKC antibody used in A recognizes all 3 isoforms of PKC (alpha , beta , and gamma ; PKC-MC5, cat no. sc-80; Santa Cruz Biotechnology). Because the molecular masses for alpha  and beta  PKC isoforms are very similar (~82 kDa), only single band was noted for both alpha  and beta  isoforms, whereas the gamma  isoform was detected as a separate band of molecular mass ~78 kDa.

Activation of MAP kinase by LPC: Its partial dependency on the activity of PKC. Activated PKC can stimulate the activity of MAP kinases in the cell. In the present studies, we examined the effect of LPC on the p42 MAP kinase isoform because preliminary studies indicated that the p42 isoform is more predominant in the cell type used. LPC activated MAP kinase in a time- and dose-dependent manner (Fig. 3). Significant activation of MAP kinase occurred as early as 5 min with both 5 and 10 µM doses. With the use of 5 and 10 µM doses of LPC, peak activities were observed at 15 min, and the activity returned to basal levels between 30 min and 3 h. [Control values were pooled as they did not vary significantly at different time points (Fig. 3).] On the other hand, with a 25 µM dose, maximum activation of MAP kinase (~3-fold) occurred at 10 min, and the activation persisted for a longer period. At 3 h of stimulation with 25 µM of LPC, the MAP kinase activity was about twofold higher than that of controls (Fig. 3). LPC did not affect MAP kinase protein content as measured by Western blot analysis. The quantitative arbitrary data (measured by densitometric scanning of Western blots) for MAP kinase content were as follows: control = 65.8; 25 µM LPC, 10 min = 65.2; 25 µM LPC, 3 h = 62.2 (data are average of 2 experiments). In contrast, phosphatidylcholine (parent molecule for the formation of LPC) did not induce MAP kinase activity (data not shown). Previous studies reported by this and other laboratories have shown similar morphological and functional characteristics between stably transformed murine mesangial cells and the primary cultures of mesangial cells (24, 30, 32). In the present study, using primary cultures of rat glomerular cells, we have found that LPC at 10 and 25 µM stimulated the MAP kinase activity by 125 and 160%, respectively.


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Fig. 3.   Effect of LPC on the activity of p42 mitogen-activated protein kinase (MAPK): time and dose response. Mesangial cells (serum starved for 24 h) at ~80% confluence in 30-mm dishes were incubated with various concentrations of LPC for selected time intervals as indicated. Cells were then washed with cold Hanks' balanced salt solution and were extracted with MAPK assay buffer. The p42 MAPK activity in the extracts was determined as described in MATERIALS AND METHODS.

To test the PKC dependency of LPC-mediated MAP kinase activation, in the first method, the cells were treated for 24 h under serum-free conditions with 10 nM PMA to deplete the cellular PKC. PMA (10 nM) stimulated MAP kinase activity by about fourfold in 15 min (Table 1). However, when the cells were preincubated for 24 h with 10 nM PMA to deplete PKC, further addition of PMA did not activate MAP kinase, confirming the degree of PKC depletion (Table 1). On the other hand, depletion of cellular PKC did not completely suppress the activation of MAP kinase by LPC. In PKC-depleted cells, LPC at the doses of 10 and 25 µM induced MAP kinase activity by 38 and 89%, respectively (Table 1). Under the same experimental conditions, in control cells (without PKC depletion), LPC at 10 and 25 µM doses activated MAP kinase by 135 and 138%, respectively (Table 1). In the second method, the PKC inhibitor GFX was used to inhibit PKC activity (37). Cells were incubated with GFX (5 µM) for 30 min before the addition of 10 or 25 µM LPC, and the activity of MAP kinase was determined at 15 min of incubation. As shown in Table 2, GFX suppressed MAP kinase activity stimulated by LPC, at 10 and 25 µM, only by 31 and 63%, respectively.

                              
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Table 1.   Effect of depletion of cellular PKC (by incubation with PMA, 24 h) on the activation of p42 MAP kinase by LPC


                              
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Table 2.   Effect of GFX on stimulation of p42 MAP kinase by LPC

Effect of PTX on the activation of MAP kinase by LPC and other lysophospholipids. We also studied the effect of PTX on the activation of MAP kinase by LPC and other lysophospholipids to understand the role played by the Gi proteins. PTX covalently modifies and inactivates Gi proteins (7). In addition to LPC, LPI and LPA (but not LPE) activated MAP kinase (Table 3). In these studies, cells were incubated with PTX (500 ng/ml) for 6 h before stimulation with LPC or other lysophospholipids (10 µM, 15 min). Thrombin, a known agonist for G protein-mediated activation of MAP kinase, was also included as a positive control. Preincubation of cells with PTX did not affect the activation of MAP kinase by either LPC or LPI (Table 3). The induction of MAP kinase activation by LPA and thrombin was significantly suppressed by PTX (Table 3).

                              
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Table 3.   Effect of LPC, LPI, LPE, LPA, and TBN on MAP kinase activity in the absence or presence of PTX

Effect of genistein on the activation of MAP kinase by LPC: Involvement of PTK. Because Gi protein-mediated pathways did not appear to play a role in LPC stimulation of MAP kinase in mesangial cells and because PKC inhibition did not completely block LPC-induced MAP kinase, we next proceeded to examine if the inhibition of tyrosine kinases would modulate the LPC-induced MAP kinase activity. Cells were incubated with 50-100 µM genistein for 150 min before the addition of LPC (10 or 25 µM), and the MAP kinase activity was determined after 10 or 15 min of stimulation. Genistein nearly completely blocked LPC-induced MAP kinase activity, and genistein had no significant effect on basal MAP kinase activity (Fig. 4). The MAP kinase activities (pmol/mg protein), from three experiments, in LPC-treated cells (15 min) in the absence or presence of genistein (100 µM) were as follows: control = 17.6 ± 2.5; 10 µM LPC = 37.8 ± 0.6; 10 µM LPC + genistein = 20.9 ± 6.1; 25 µM LPC = 47.8 ± 5.8; and 25 µM LPC + genistein = 21.8 ± 5.1. Similar inhibition of MAP kinase activity by genistein was also observed in cells stimulated with LPC (10 and 25 µM) for 10 min (data not shown). Parallel experiments using 50 µM of genistein also showed marked inhibition of LPC-induced MAP kinase (data not shown). Incubation of cells with genistein at similar concentrations and incubation time did not alter either the morphology or viability of cells (data not shown). Similarly, tyrphostin B46 (50-100 µM, another specific PTK inhibitor) inhibited LPC-induced mesangial cell MAP kinase activity (data not shown).


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Fig. 4.   Effect of genistein on the activation of p42 MAPK by LPC in mesangial cells. Cells at nearly 80% confluence in 30-mm dishes were serum starved for 24 h and were then preincubated in the absence or presence of 100 µM genistein (stock dissolved in DMSO) for 3 h. Cells were then stimulated with LPC (10 or 25 µM) for 10 or 15 min. Extraction of the cells and assay of p42 MAPK activity were done as described in MATERIALS AND METHODS. MBP, myelin basic protein.

Effect of LPC on PTK activity. Because LPC-mediated activation of MAP kinase was found to be dependent on PKC and PTK, additional experiments were performed to examine the direct effect of LPC on mesangial cell PTK activity. In these experiments, plasma membranes (isolated from control cells) were incubated with 10 µM LPC on ice, and the membrane tyrosine kinase activity was subsequently determined in these activated membranes. LPC at 10 µM concentrations significantly increased membrane PTK activity by 20.3% (PTK activity, as cpm/µg membrane protein, in control = 26,586 ± 590 and in 10 µM LPC = 32,183 ± 869, data are means ± SE of 3 separate determinations, statistically significant at P < 0.05, control vs. LPC treatment). Furthermore, addition of genistein completely inhibited the stimulation of PTK activity by LPC (data not shown). Additionally, the effect of LPC on the tyrosine phosphorylation of cellular proteins (membrane/cytoplasmic) was assessed. The results indicated that LPC induced a modest increase (40-60% compared with control) in the phosphorylation of mainly two proteins in the molecular mass range of ~80 and 140 kDa (Fig. 5). Additional studies indicated that LPC had no effect on the activation of Shc and Src. The quantitative arbitrary data (as measured by densitometric scanning of Western blots) for the activation of Shc were as follows: control = 38.3; 25 µM LPC, 5 min = 40.2; 25 µM LPC, 30 min = 39.6 (data are average of 2 experiments). Similarly, the quantitative arbitrary data for the activation of Src were as follows: control = 51.0; 25 µM LPC, 5 min = 49.9; 25 µM LPC, 30 min = 44.5 (data are average of 2 experiments).


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Fig. 5.   Effect of LPC on cellular protein tyrosine phosphorylation profile. Quiescent mesangial cells were treated with LPC (25 µM) for 5-10 min, and the cells were lysed. Cell lysate was immunoprecipitated with anti-phosphotyrosine antibodies. Immunoprecipitates were incubated with [gamma -32P]ATP in a kinase assay buffer for 20 min at 30°C. Samples were resolved on 7.5% SDS-PAGE, and phosphorylated proteins were detected by autoradiography.

Effect of LPC on PLC-gamma 1 phosphorylation. In the cell, DAG is one of the principal activators of PKC. Phospholipases are the enzymes that produce DAG by the hydrolysis of phospholipids during cell signaling. Among various phospholipases, only PLCgamma -1 is known to be activated by membrane tyrosine kinases and to be involved in the phosphoinositide turnover and the generation of DAG. The observation that the activation of PKC and MAP kinase by LPC was dependent on tyrosine kinases prompted us to explore the possibility that the activation of PLC-gamma 1 was involved in LPC-mediated effects. Cells were incubated with 10 µM LPC for 5-20 min and were processed for the assessment of PLCgamma -1 phosphorylation. As shown in Fig. 6, the stimulation of mesangial cells with LPC markedly, but transiently (within 5-10 min), stimulated the phosphorylation of PLCgamma -1. The phosphorylation of PLCgamma -1 induced by LPC returned to basal levels in 20 min of stimulation (Fig. 6). The quantitative densitometric data for the phosphorylation of PLCgamma -1 were as follows: control = 0.46; 10 µM LPC, 5 min = 1.52; 10 µM LPC, 10 min = 1.24; 10 µM LPC, 20 min = 0.20. 


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Fig. 6.   Effect of LPC on phospholipase Cgamma -1 (PLCgamma -1) phosphorylation. Mesangial cells (serum starved for 24 h) were stimulated with LPC (10 µM) for 5-20 min, and the cells were lysed. An aliquot of cell lysate was immunoprecipitated with anti-PLCgamma -1 conjugated with protein A-Sepharose. Immune complex was mixed with Laemmli sample buffer, heated at 80°C for 5 min. Sample was resolved on SDS-PAGE (12.5% gel), transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine, and phosphorylated PLCgamma -1 was detected by ECL reagents.

Activation of Ras by LPC. In the present studies, we also investigated the effect of LPC on Ras because Ras is an important member of the tyrosine kinase signaling cascade, and it lies outside the cross talk between the PKC and the growth factor signaling cascade. Serum-starved cells were first incubated with [32P]orthophosphate for 3 h. LPC (10 µM) was then added, and the cells were processed after 2, 5, 10, or 60 min of incubation. FBS (5%) was used as a positive control. As shown in Fig. 7, LPC markedly increased GTP binding to Ras within 2 min, and the effect lasted for nearly 1 h. The ratios of GTP to GDP associated with Ras were as follows: control = 0.032; FBS = 0.09; 10 µM LPC, 2 min = 0.124; 10 µM LPC, 5 min = 0.10; 10 µM LPC, 10 min = 0.08; 10 µM LPC, 1 h = 0.05. 


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Fig. 7.   Effect of LPC on the activation of Ras. Mesangial cells were grown in 30-mm dishes to 80% confluence. After 24 h of serum starvation, cells were labeled with [32P]orthophosphate (50 µCi · 2 ml-1 · dish-1) for 3 h. After this labeling period, cells were stimulated either with FBS (5%) for 2 min or with 10 µM LPC for 2 min to 2 h. Cellular Ras was then immunoprecipitated, and the guanine nucleotides associated with Ras were analyzed by TLC as described in MATERIALS AND METHODS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mesangial cells, specialized vascular pericytes within the glomerulus, perform a fundamental role in the homeostasis of glomerular function and are influenced by various circulating chemical modulators (3). Recently, we have reported that the activation of mesangial cells with circulating LDL and oxidatively modified LDL (with greater potency) stimulated the expression of various cytokines and growth factors involved in monocyte infiltration and proliferation of intrinsic resident cells (10, 15, 16, 30). Because increased levels of LPC is one of the major characteristics of ox-LDL, the atherogenic potential of ox-LDL to modulate cellular responses has been attributed to the presence of LPC (2). Presently, PKC activation is believed to be the principal mediator in various cellular responses stimulated by LPC (18, 29, 36). However, PKC activation alone may not explain all of the diverse actions of LPC, because, for example, the stimulation of endothelial cell ICAM-1 by LPC is insensitive to PKC inhibition (21). In many cell types, active forms of PKC are capable of activating MAP kinases by a pathway that involves Raf (12, 22, 40). As reported by us in our recent preliminary communication (2), LPC indeed markedly activated MAP kinases in murine mesangial cells. LPC was also shown to activate c-Jun NH2-terminal kinase (23). Our studies were originally aimed at identifying the principal early signaling events stimulated by LPC in mesangial cells. To understand the upstream intracellular signaling events stimulated by LPC, we assessed the activities of either MAP kinase or both PKC and MAP kinase under various experimental conditions. LPC activated both PKC and MAP kinase. Interestingly, the activation of both PKC and MAP kinase by LPC was inhibited by the tyrosine kinase inhibitors. The effect of these inhibitors (herbimycin and genistein) on PKC is noteworthy, because the activation of PKC is not mediated through its tyrosine phosphorylated state, and it is less likely that these inhibitors directly acted on PKC. It is possible that the upstream membrane/cytoplasmic tyrosine kinases may be involved in LPC-induced PKC activation. In this regard, LPC induced modestly the tyrosine phosphorylation of cellular proteins of the molecular mass range of 80 and 140 kDa. Based on the molecular mass range, we reasoned that these proteins may represent intracellular signaling molecules, including PLCgamma -1, pp60src, Shc, etc. However, we were unable to detect measurable activation of Shc or Src by LPC.

There are two classical pathways by which various agonists stimulate distinct forms of phospholipases to generate DAG, which is absolutely required for the activation of PKC. The first one is dependent on serpentine receptor- and Gi protein-mediated activation of PLC-beta (5). The inability of PTX to inhibit LPC-induced MAP kinase in our experiments apparently excludes this pathway as a participant in the activation of cells by LPC. Unlike LPC, LPA-mediated MAP kinase activation was suppressed by PTX in mesangial cells. LPA has been shown to stimulate MAP kinase through PTX-sensitive G protein-coupled pathways in other cell types (13). The second route is dependent on PTKs. For example, the activation of PKC by platelet-derived growth factor in many tissues involves phosphorylation and activation of PLCgamma -1 (42). The induction of DAG synthesis by EGF in A431 cells (38) or in Swiss 3T3 cells (41) and by LPI in thyroid cells (6) is also dependent on the phosphorylation and stimulation of PLCgamma -1 by receptor tyrosine kinases. The generation of DAG and the activation of PKC have been shown to be insensitive to PTX in all of these instances. Based on these earlier reports and our observations on the effects of PTK inhibitors on PKC activity, we conducted additional experiments to find out if LPC activated membrane tyrosine kinases and PLCgamma -1. LPC activated total membrane tyrosine kinase activity as assayed using a synthetic peptide substrate. Furthermore, LPC markedly, but transiently, activated PLCgamma -1, and the time course of activation was consistent with the premise that PLCgamma -1 was involved in LPC-induced PKC and MAP kinase activation.

Our data indicate that both PKC-dependent and PKC-independent mechanisms contributed to the activation of MAP kinase by LPC. First, MAP kinase activity, especially in the early phases of activation, showed clear dependency on the availability of PKC, but the depletion or inhibition of PKC did not completely suppress the maximum activation of MAP kinase at the later period of 15 min. We also found that LPC did not activate PKC-zeta , suggesting that PMA-insensitive isoforms of PKC are not involved in LPC-mediated MAP kinase activation. Second, tyrosine kinase inhibitors nearly completely inhibited the activation of MAP kinase by LPC. The concentrations of PTK inhibitors used in these experiments were not known to inhibit the MAP kinase-phosphorylating dual specific kinase, namely MAP kinase kinase (40). Based on the conclusion that both PKC-dependent and PKC-independent pathways contributed to the activation of MAP kinase, we examined the effect of LPC on Ras. Ras is a G protein and is a key member of the growth factor signaling cascade that is upstream of the cross talk between PKC and the growth factor signaling cascades. LPC markedly stimulated Ras-GTP binding. It is also interesting because, in addition to activating the downstream members of the growth factor signaling cascade, Ras is also known to directly modulate gene expression (39).

As summarized in the proposed model (Fig. 8), our results suggest that the activation of a membrane tyrosine kinase is the key early event in LPC-induced cellular signaling responses. Activation of Ras and PLCgamma -1 by LPC and the inhibition of the activation of PKC and MAP kinase by tyrosine kinase inhibitors support this view. However, additional yet unidentified signaling molecule(s) may be involved in LPC-mediated cellular responses (Fig. 8). Because PKC, MAP kinases, and Ras are involved in modulating nuclear events associated with gene expression, our data may provide a more logical support for the earlier findings that LPC stimulates diverse responses in various cell types. Furthermore, because glomerular mesangial cells and vascular smooth muscle cells share many morphological and functional characteristics, the results presented in this study may shed light on the roles played by LPC and LDLs in the pathobiology of renovascular and vascular diseases, such as glomerulosclerosis and atherosclerosis.


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Fig. 8.   Proposed model for the involvement of tyrosine kinase pathway in LPC-mediated activation of PKC and mitogen-activated protein (MAP) kinase. Data suggest that the activation of membrane/cytoplasmic tyrosine kinase(s) plays a key role in LPC-induced mitogenic signaling responses. The specific upstream signaling events that modulate LPC-mediated activation of PLCgamma -1 and Ras, associated with PKC and MAPK activation, are yet to be identified. DAG, diacylglycerol; MEK, MAP kinase kinase.


    ACKNOWLEDGEMENTS

We thank Dr. Rama Pai for technical assistance in tyrosine kinase assay.


    FOOTNOTES

dagger Deceased 21 June 1997.

This work was supported by a Merit Review from the Department of Veterans Affairs.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: V. S. Kamanna, Nephrology Research Laboratories (151), Dept. of Veterans Affairs Medical Center, 5901 East Seventh St., Long Beach, CA 90822 (E-mail: kamanna.vaijinath_s.{at}long_beach.va.gov).

Received 7 August 1998; accepted in final form 7 May 1999.


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Am J Physiol Renal Physiol 277(3):F328-F337
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