Lysophosphatidic acid (LPA) is a potent bioactive lysophospholipid. Accumulated evidence supports a role for LPA in inflammation. To profile LPA-induced cytokine production in vascular smooth muscle cells (SMCs), we used a cytokine antibody array system and found that LPA prominently induces the secretion of IL-6 and monocyte chemoattractant protein (MCP)-1 from human aortic SMCs (HASMCs). The mechanism by which LPA induces MCP-1 expression in SMCs has been previously reported. However, LPA induction of IL-6 secretion from vascular SMCs and its regulatory mechanism are unknown. The present study reveals that LPA induces the expression of IL-6 mRNA and protein in HASMCs as well as the secretion of IL-6 protein in a time-dependent manner. Our results demonstrate that LPA-specific receptor 1 (LPA1) mediates LPA-induced IL-6 secretion and that LPA induction of IL-6 is independent of the EGF receptor pathway. Our data further show that PKC-mediated p38 MAPK is responsible for the IL-6 secretion. Finally, small interfering RNA depletion experiments revealed that p38α is specifically responsible for the LPA-induced IL-6 secretion. The present study profiles the regulatory relationship between LPA and multiple cytokines in vascular SMCs for the first time, provides the first evidence that LPA upregulates IL-6 in vascular SMCs, and reveals the regulatory mechanism of LPA-induced IL-6 production in HASMCs. In light of the emerging roles of LPA and IL-6 in vascular inflammation, the understanding of the regulatory mechanism may contribute to the treatment and prevention of cardiovascular disorders.
- bioactive lipids
- vascular smooth muscle cells
- lysophosphatidic acid
- protein kinase C
- LPA-specific receptor 1
lysophosphatidic acid (LPA) is produced by activated platelets (17) and formed in oxidized LDL (46). High concentrations of LPA have been found in human atherosclerotic lesions (46) and human serum (up to ∼6 μM) (3, 17). Accumulated evidence indicates that LPA influences vascular cell functions, including smooth muscle cell (SMC) proliferation, migration, and dedifferentiation (for a review, see Ref. 47). LPA induction of the expression of cytokines, such as monocyte chemoattractant protein (MCP)-1, IL-8, and IL-1β in vascular endothelial cells (33, 34), MCP-1 in vascular SMCs (29), and IL-1β in macrophages (7), has recently been reported. Inflammatory cytokines play an important role in the development of atherosclerosis and its complications, specifically thrombosis (11, 50). Understanding the mechanism by which LPA affects cytokine release from vascular SMCs may contribute to understanding the mechanism of vascular inflammation and atherosclerosis.
To profile the cytokine expression regulated by LPA in vascular SMCs, we applied an array-based technology-cytokine antibody assay, which detects the expression of 60 cytokines in an array format. The results reveal that LPA prominently upregulates the release of IL-6 and MCP-1 from vascular SMCs. LPA induction of IL-6 in vascular cells has not been previously reported. IL-6, a circulating cytokine, induces SMC proliferation (26) and exacerbates early atherosclerosis in mice (25). IL-6 also initiates an acute phase response by altering liver protein synthesis and increasing the production of fibrinogen, plasminogen activator inhibitor (PAI)-1, and the inflammatory biomarker C-reactive protein. Therefore, the production of IL-6 by vascular SMCs in peripheral atheroma generates a systemic prothrombotic response by increasing levels of circulating PAI-1 and fibrinogen (for a review, see Ref. 41).
LPA exerts its effects via its cognate G protein-coupled receptors (GPCRs) (9, 52). LPA-specific receptors 1 (LPA1), 2 (LPA2), and 3 (LPA3) belong to the same endothelial differentiation gene (Edg) family; these receptors have been well characterized (1, 4, 24, 27, 35) and share very high homology. Reportedly, LPA also binds to peroxisome proliferator-activated receptor (PPAR)-γ (37). Using the recently developed LPA receptor antagonists and the specific PPAR-γ inhibitor, we determined the specific involvement of LPA receptors in the present study.
The LPA-triggered signaling pathway leads to LPA-induced gene expression and cellular functions (38). We have previously reported that LPA-induced expression of blood coagulation initiator tissue factor in rat vascular SMCs requires the activation of Gi protein and the ERK1/2 pathway (13) and that LPA-induced early growth response factor (Egr)-1 expression in vascular SMCs depends on the phosphorylation of both cAMP response element-binding factor (CREB) and serum response factor (SRF) (12). Our previous results also identified that LPA1, MAPK, p42, and p38α mediate LPA-induced PC3 cell migration (21). In the present study, we determined how LPA regulates IL-6 release from human aortic SMCs (HASMCs), and the results revealed, for the first time, the mechanism by which LPA induces IL-6 release from vascular SMCs.
MATERIALS AND METHODS
LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was purchased from Avanti Polar Lipids (Alabaster, AL); the human cytokine antibody array C series 1000 kit was from Raybiotech (Norcross, GA); and nonsilencing control small interfering (si)RNA and p38α MAPK siRNA were from Millipore (Billerica, MA). TRIzol reagent, siRNA transfection reagent lipofectamine RNAi MAX, and the ThermoScript RT-PCR system were from Invitrogen (Carlsbad, CA). The RNeasy kit was from Qiagen (Valencia, CA). GeneAmp PCR core reagents were from Applied Biosystems (Foster City, CA); [32P]dCTP was from MP Biochemicals (Solon, OH); and the DNA labeling kit was from GE Healthcare (Piscataway, NJ). Pertussis toxin (PTX), SB-203580, U-0126, SP-600125, GF-109203X, and ki16425 were from BioMol (Plymouth Meeting, PA). The LPA3 receptor antagonists (compound 19b and phosphorothioate diester compound 7) were kind gifts from Dr. Duane D. Miller (Department of Pharmaceutical Sciences, The University of Tennessee) and Dr. Glenn D. Prestwich and Dr. Yong Xu (Department of Medicinal Chemistry, The University of Utah), respectively (16, 43). The antibody against human IL-6 was from R&D Systems (Minneapolis, MN). Antibodies against human ERK, p38 MAPK, JNK, phospho-ERK, phospho-p38, phospho-JNK, p38α MAPK, and p38γ MAPK were from Cell Signaling Technology (Beverly, MA). The antibody against p38β MAPK (E-20) was from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody against p38δ MAPK was from Upstate (Lake Placid, NY). Amicon ultracentrifugal filter devices were from Millipore.
HASMCs supplied by Cascade Biologics (Portland, OR) were cultured in medium 231 with special SMGS supplements (Cascade Biologics). Cells were starved for 24 h in serum-free medium before stimulation with LPA.
Concentration of secreted proteins in the culture media and cytokine antibody array.
At various time points after LPA stimulation as indicated in the text, conditioned media were collected and concentrated 20-fold using Amicon Ultra-4 at 4°C. Concentrated proteins in the media were used for the cytokine antibody array test according to the manufacturer's instructions (RayBiotech) and for Western blot analysis.
The expression of mRNA was evaluated by RT-PCR. Total RNA was isolated from HASMCs using an RNeasy mini-prep kit (Qiagen). The first strand of cDNA was reverse transcribed using the ThermoScipt RT-PCR system (Invitrogen). The cDNA products were amplified using GeneAmp PCR core reagents (Applied Biosystems). The amplification conditions were as follows: 5 min at 95°C; 27, 31, or 35 cycles of 30 s at 95°C; 30 s at 56°C; and 1 min at 72°C; followed by a final extension for 10 min at 72°C. The primers used were as follows: LPA1, forward 5′-ATGGCTGCCATCTCTACTTCCATCCC-3′ and reverse 5′-CTAAACCACAGAGTGGTCATTGCTGTG-3′; LPA2, forward 5′-ATGGTCATCATGGGCCAGTGCTACTAC-3′ and reverse 5′-TCAGTCCTGTTGGTTGGGTTGAGCC-3′; and LPA3, forward 5′-CGATGACTGGACAGGAAC-3′ and reverse 5′-GCCTGCAGTTCAGGCCGTCG-3′. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel.
Northern blot analysis.
Total RNA of the cells was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and subjected to electrophoresis in formaldehyde-agarose gels. RNA was transferred onto nylon membranes (Amersham Biosciences) and hybridized with radiolabeled cDNA probes. A 618-bp fragment of human IL-6 cDNA was amplified by PCR and purified to be used as a probe to detect human IL-6 mRNA. The primers used were as follows: forward 5′-CTACATTTGCCGAAGAGCCCTC-3′ and reverse 5′-ATGAACTCCTTCTCCACAAGCGCC-3′.
Western blot analysis.
HASMCs were rinsed with cold PBS and lysed in Western blot lysis buffer [50 mM Tris·HCl (pH 6.8), 8 M urea, 5% β-mercaptoethanol, 2% SDS, and protease inhibitors] with sonication for 20 s on ice. Cellular proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore). Membranes were then probed with the specific antibodies, and the specific protein bands were visualized by ECL-Plus (GE Healthcare).
Nonsilencing siRNA (20 nM) and p38α MAPK siRNA (20 nM) were transfected into HASMCs using the RNAi MAX transfection reagent according to the manufacturer's instructions (Invitrogen). Nonsilencing siRNA was used as a negative control. Forty-eight hours after transfection, cells were starved for 24 h followed by treatment either with or without LPA.
All data are representative of a minimum of three experiments. Results are expressed as means ± S.E. Comparisons between multiple groups were performed using one-way ANOVA with Dunnett's posthoc t-tests. A single comparison analysis was made using two-tailed unpaired Student t-tests. P values of <0.05 for ANOVA or t-tests were considered to be statistically significant.
Cytokine profiling data reveals that LPA markedly induces IL-6 and MCP-1 secretion from HASMCs.
Accumulated evidence suggests that LPA promotes inflammation (20, 54). To establish the regulatory relationship between LPA and multiple cytokines in vascular SMCs, we determined the cytokine secretion profile using HASMCs stimulated by LPA in a cytokine antibody array system that contains 60 different anti-cytokine antibodies (RayBiotech). Cytokine antibodies on the array membrane are shown in Fig. 1A, top. Serum-starved HASMCs were treated or untreated with LPA for 16 h, and conditioned media were collected and applied to the array assay. In our previous publications, we have examined concentration dependency of LPA in the expression of Egr-1 and tissue factor in vascular SMCs (12, 13), which showed that LPA at 10 μM highly induces the expression of these genes. Also, 10 μM LPA is in the range of pathological concentrations found in atherosclerotic lesions in vivo (46). Therefore, 10 μM LPA was used in the present study. Our results showed that LPA markedly induced the secretion of IL-6 and MCP-1 in the conditioned medium of HASMCs (Fig. 1A, bottom). LPA induction of MCP-1 in SMCs has been previously reported (29); however, LPA induction of IL-6 in the vascular wall system has not been documented. Our cytokine array finding provides the first evidence that LPA induces IL-6 secretion from vascular SMCs.
LPA induction of the secretion of IL-6 was confirmed by Western blot analysis.
To substantiate findings in the LPA-stimulated cytokine secretion array, we measured IL-6 protein secretion in the conditioned medium using an established method: Western blot analysis. Conditioned media were collected at various time points and concentrated using Amicon ultra-4. The concentrated media samples were then analyzed by Western blot analysis. As shown in Fig. 1B, LPA induced IL-6 secretion from HASMCs in a time-dependent manner, with a peak induction (9.5-fold) at 15 h. In contrast, LPA had no effect on IL-6 secretion from human aortic endothelial cells (data not shown), indicating that LPA regulation of IL-6 release is cell type specific.
LPA-induced IL-6 secretion in conditioned media is due to LPA induction of the expression of IL-6 mRNA and IL-6 protein in HASMCs.
To assess the possibility that LPA-induced secretion of IL-6 in the conditioned medium is due to LPA-increased levels of IL-6 mRNA and proteins in HASMCs, we determined the expression of IL-6 mRNA and protein in cultured HASMCs. Cells were serum starved for 24 h and then treated with 10 μM LPA for various time periods. Total RNA and protein were extracted from cells, and the expression of IL-6 mRNA and protein were detected by Northern blot analysis and Western blot analysis, respectively. As shown in Fig. 2A, LPA markedly induced IL-6 mRNA expression, which peaked at 9 h and declined at 15 h. LPA strikingly induced IL-6 protein expression (Fig. 2B). IL-6 protein expression was detectable at 3 h, and the peak time point was ∼15 h (Fig. 2B). These results indicate that LPA-induced secretion of IL-6 is due to LPA-increased IL-6 mRNA expression and intracellular protein production.
LPA receptor LPA1 mediates LPA-induced IL-6 secretion, but PPAR-γ and EGF receptor pathways are not involved.
To determine the pathway through which LPA exerts its functions, we first evaluated the involvement of LPA receptors. The expression of LPA receptors in HASMCs was determined by RT-PCR. Total RNA was extracted from the cultured cells and applied to RT-PCR using specific primers for different cycles from 27 to 35, as described in materials and methods. We found that LPA1 was predominantly expressed in HASMCs, and LPA2 was also expressed in HASMCs, whereas the level of LPA3 was extremely low (Fig. 3A). The expression levels of LPA receptors in HASMCs were shown to be the following: LPA1 >> LPA2 > LPA3. To determine the role of LPA (Fig. 3E) receptors in LPA-induced IL-6 expression, we applied the LPA1/LPA3-selective antagonist ki16425 to treat HASMCs for 40 min. Cells were then stimulated with LPA for 15 h, and IL-6 protein secretion in the conditioned medium was detected by Western blot analysis. We observed that 0.5 or 5 μM ki16425 almost completely blocked LPA-induced IL-6 secretion (Fig. 3B), suggesting that either LPA1 or LPA3 may mediate the LPA-induced IL-6 release. To further assess the effect of LPA1 and LPA3 in IL-6 expression, we pretreated cells with the LPA3 antagonists phosphorothioate diester compound 7 (43) and compound 19b (16) and found that neither compound 7 nor compound 19b had any effect on LPA-induced IL-6 expression (Fig. 3, C and D); in comparison, 5 μM ki16425 completely blocked the IL-6 release, indicating that LPA3, which is expressed at very low levels, has no significant role in LPA-induced IL-6 expression. Therefore, LPA1 is responsible for LPA-induced IL-6 expression in HASMCs.
In addition to LPA-specific plasma membrane receptors, nuclear receptor PPAR-γ has also been reported to serve as an intracellular receptor and to transduce the LPA signal to downstream molecules (37). To examine whether PPAR-γ has a role in LPA-induced IL-6 release from HASMCs, we determined the effect of the PPAR-γ inhibitor GW-9662 on LPA-induced IL-6 release. We observed that pretreatment of HASMCs with various concentrations of GW-9662 (0.1–10 μM) for 40 min had no effect on the secretion of IL-6 induced by LPA. The dose of 1 μM has been reported to efficiently block PPAR-γ activation in vascular SMCs (19). These results indicate that the PPAR-γ pathway has no role in the LPA induction of IL-6 release (Fig. 3E).
Previously, it has been reported that LPA, via the activation of the tyrosine kinase EGF receptor (EGFR), mediates the LPA signal in several cell types, including Rat-1 fibroblast (14), prostate cancer PC3 cells (31), and human bronchial epithelial cells (53). However, an LPA-induced signaling pathway completely independent of EGFR activation in rat pheochromocytoma PC12 cells has also been reported (15); whether LPA induction of any gene expression in vascular SMCs depends on the activation of EGFR is currently unknown. To examine whether LPA activates EGFR, and whether LPA-induced IL-6 expression is dependent on EGFR activation in HASMCs, we first examined the effect of the selective EGFR inhibitor AG-1478 on LPA-induced IL-6 secretion. As shown in Fig. 3F, pretreatment of cells with 10 μM AG-1478, a dose that has been used to completely block EGFR activation (23), had no effect on LPA-induced IL-6 secretion, indicating that EGFR is not involved in the LPA-triggered signaling pathway leading to IL-6 release. We further investigated whether LPA stimulates EGFR activation in HASMCs compared with EGF stimulation. As shown in Fig. 3G, EGFR was expressed in HASMCs. EGF (50 ng/ml) markedly induced EGFR activation in HASMCs; however, LPA had no detectable effect on EGFR activation over a wide range of time periods. Taken together, LPA-induced IL-6 release from HASMCs is mediated by LPA1 and is independent of PPAR-γ and EGFR pathways.
PTX-sensitive G protein and PKC mediate LPA-induced IL-6 secretion.
The results shown in Fig. 3 demonstrated that GPCR LPA1 mediates LPA-induced IL-6 secretion. We next determined which G protein is involved in IL-6 secretion. Pretreatment with PTX, a Gi/o protein inhibitor, completely blocked LPA-induced IL-6 secretion, indicating that Gi/o protein is responsible for the LPA induction of IL-6 secretion (Fig. 4A). To determine whether PKC is involved in LPA-induced IL-6 secretion, we evaluated the effect of the pan PKC inhibitor GF-109203X on IL-6 release. As shown in Fig. 4B, pretreatment of cells with GF-109203X for 40 min completely inhibited IL-6 secretion induced by LPA, indicating that PKC activation is required for LPA-induced IL-6 secretion from HASMCs.
LPA activates p38 MAPK, ERK, and JNK in HASMCs, but only p38 MAPK activation is required for LPA-induced IL-6 secretion.
To further determine the intracellular signaling pathways involved in LPA-induced IL-6 secretion, we assessed the effect of LPA on the activation of MAPK pathways in HASMCs. Starved cells were treated with LPA for various time periods; we found that LPA activated the three MAPKs in HASMCs: ERK, p38, and JNK. The maximum effect occurred at ∼2–15 min and declined to basal levels after 30 min of treatment (Fig. 5A), suggesting that the activation of MAPKs may be involved in LPA-induced IL-6 secretion. To determine whether and which specific MAPK is functionally involved in the LPA induction of IL-6 secretion, we treated HASMCs with subtype-specific MAPK inhibitors and examined their effects on LPA-induced IL-6 secretion. As shown in Fig. 5B, p38 MAPK inhibitor SB-203580 dose dependently blocked IL-6 secretion; in contrast, neither the JNK inhibitor SP-600125 nor the ERK kinase inhibitor U-0126 had an effect on LPA-induced IL-6 secretion (Fig. 5, C and D). These data demonstrated that LPA-activated p38 MAPK, but not ERK or JNK, mediates IL-6 secretion from HASMCs.
LPA1, Gi/o protein, and PKC mediate LPA-induced p38 MAPK activation in HASMCs.
The above results showed that LPA1, Gi/o proteins, PKC, and p38 MAPK mediate LPA-induced IL-6 secretion. To evaluate whether the LPA signal via LPA1, Gi/o proteins, and PKC leads to p38 MAPK activation, we first examined the effect of the LPA1/LPA3 antagonist ki16425 and LPA3 antagonist compound 7 on LPA-induced p38 activation. As shown in Fig. 6A, pretreatment with ki16425 but not compound 7 completely blocked the LPA induction of p38 phosphorylation, indicating that LPA1 mediates p38 activation. We next observed that treatment with the PKC inhibitor GF-109203X completely blocked p38 phosphorylation induced by LPA (Fig. 6A), indicating that p38 activation is dependent on PKC activation. Finally, we assessed the effect of PTX on LPA-induced p38 activation. As shown in Fig. 6B, pretreatment with 100 ng/ml PTX completely blocked p38 activation, indicating that Gi/o proteins mediate p38 activation.
p38α (but not other isoforms of p38) mediates LPA-induced IL-6 secretion.
The p38 MAPK family includes four isoforms: p38α, p38β, p38γ, and p38δ. To further identify the specific isoforms of p38 that are responsible for LPA-induced IL-6 secretion, we first examined the expression of p38 MAPK isoforms in HASMCs. Western blot analysis results showed that p38α and p38γ, but not p38β and p38δ, are expressed in cultured HASMCs (Fig. 7A). Considering our result that SB-203580 (5 μM) nearly completely blocked LPA-induced IL-6 secretion (Fig. 5B) and the fact that SB-203580 is a specific inhibitor for p38α and p38β (39), we assumed that p38α may be responsible for IL-6 secretion. To test this hypothesis, we examined whether the specific p38α siRNA affected IL-6 expression induced by LPA. As shown in Fig. 7B, transfection of p38α siRNA specifically blocked 85% of p38α expression with no detectable effect on p38γ expression; interestingly, p38α siRNA blocked 93% of IL-6 secretion. These data strongly support the conclusion that p38α mediates LPA-induced IL-6 secretion from HASMCs.
The present study provides the first profile of LPA-induced cytokine expression in vascular SMCs. Our data demonstrate that LPA induces prominent secretion of MCP-1 and IL-6 from SMCs. We further show that the LPA-induced production of IL-6 mRNA and IL-6 protein in SMCs precedes the resultant secretion of IL-6 protein from SMCs. The results from the present study reveal that LPA-induced IL-6 expression is mediated by its cognate receptor LPA1 via a Gi/o protein-mediated, PKC-dependent p38α activation pathway.
LPA is produced in cells and in body fluids, including the blood. In serum or plasma, LPA is predominantly produced from lysophospholipids by a plasma enzyme called autotaxin. LPA is also produced from phosphatidic acid by its deacylation, catalyzed by phospholipase A-type enzymes (2). Accumulated evidence has shown that 1) LPA is produced by platelets (32, 36); 2) LPA is formed during mild oxidation of LDL (46); 3) fairly high concentrations of LPA (0.6–0.7 μM) are present in circulating blood and the LPA concentrations in serum prepared from platelet-rich plasma are 5- to 10-fold higher than in platelet-poor plasma (3); 4) LPA accumulates in atherosclerotic lesions (46); and 5) the lysophospholipase D/autotaxin responsible for the synthesis of LPA in the blood can bind to the surface of lymphocytes and activated platelets through interactions with integrin receptors (28, 42), suggesting localized LPA production at cell surfaces. These lines of evidence strongly suggest that LPA is important in cardiovascular regulation. The present study reveals the relationship between LPA and the production of the inflammatory cytokine IL-6 in vascular SMCs.
IL-6 levels appear to be predictive of future coronary artery disease (22) and are elevated in patients with unstable angina compared with those with stable angina (6). Patients with persistently elevated IL-6 levels and unstable angina demonstrate a worse in-hospital outcome (5). IL-6 produced in SMCs may travel to the liver, where it elicits the acute-phase response, resulting in the release of C-reactive protein, fibrinogen, and PAI-1 (41). Our results showing that LPA markedly induces IL-6 secretion from vascular SMCs suggest a possible role for LPA via IL-6 in the regulation of vascular disease. The fact that LPA does not induce IL-6 release from vascular endothelial cells suggests possible distinct regulatory mechanisms in these specific cell types in the vascular wall in response to LPA.
Regarding the mechanism of LPA induction of IL-6, we demonstrated that LPA cognate receptor LPA1 is highly expressed in SMCs and that LPA via LPA1 mediates IL-6 secretion in vascular SMCs. However, in ovarian cancer cells and granulose-lutein cells, it has been reported that LPA induces IL-6 expression via LPA2 (8, 18). These results suggest specific roles of LPA receptors in mediating LPA function in specific cell types. LPA1 may be a useful therapeutic target moderating LPA-induced vascular inflammatory disorders.
The induction of IL-6 mRNA expression and IL-6 protein secretion by growth factors, cytokines, and oxysterol 7-ketocholesterol has been previously reported. The regulation patterns, i.e., the immediate-early regulation and delayed regulation in response to these stimuli, have been demonstrated in the literature. For instance, ANG II rapidly induces IL-6 mRNA expression in both vascular SMCs and cardiac fibroblasts, with the peak induction at ∼30 min to 1 h (44, 45); however, thrombin stimulates a quick induction of IL-6 mRNA expression in vascular SMCs, with the peak induction at ∼30 min to 1 h, whereas in lung fibroblasts and blood monocytes, thrombin induces delayed IL-6 mRNA accumulation, with an induction peak at ∼6–12 h (30, 48). A recent study (49) showed that oxysterol 7-ketocholesterol induces IL-6 mRNA expression in vascular SMCs in a delayed pattern as well, peaking at 24–48 h (49). The regulation mechanisms in response to various stimuli in these different cell types are also different. ANG II-induced IL-6 expression depends on the activation of NF-κB and CREB in vascular SMCs (44); however, the ANG II induction of IL-6 expression is independent of the NF-κB pathway in cardiac fibroblasts (45). Moreover, EGFR-dependent ERK and p38 pathways are required for the thrombin induction of IL-6 expression (51); however, our results indicate that EGFR-independent p38 activation mediates LPA-induced IL-6 expression in vascular SMCs. The delayed induction of IL-6 mRNA expression in vascular SMCs suggests another intermediate factor regulatory mechanism, which warrants further investigation. Although LPA induces the rapid expression of transcription factor Egr-1 in vascular SMCs (12), Egr-1 is likely to be excluded as having a role in LPA-induced IL-6 expression because treatment with a p38 MAPK inhibitor had no effect on LPA-induced Egr-1 expression (data not shown).
The present study reveals that the activation of p38 MAPK, but not that of ERK or JNK MAPK, contributes to the LPA induction of IL-6 secretion in vascular SMCs. In accord with our present findings, in granulose-lutein cells, p38 MAPK was also found to be responsible for LPA-induced IL-6 expression (8). In OVCAR-3 ovarian cancer cells, p38 MAPK was found to be mainly responsible for LPA-induced IL-6 expression, and JNK and ERK are probably also involved (18). However, in another ovarian cancer cell type, SK-OV3, apparently phosphatidylinositol 3-kinase (PI3K) but not p38 mediates LPA-induced IL-6 expression (10). In addition to p38 MAPK and PI3K, ERK was also reported to mediate LPA-induced IL-6 secretion in dendritic cells (40). These results indicate that specific kinases downstream of LPA cognate receptors are responsible for LPA-induced IL-6 secretion. We further identified that p38α is responsible for LPA-stimulated IL-6 secretion in SMCs, providing the first evidence of the role of p38α in mediating the LPA induction of cytokine expression.
The upstream kinases responsible for MAPK activation leading to the LPA induction of IL-6 secretion have not been previously evaluated. Our data have shown that PKC mediates p38 activation leading to IL-6 secretion in SMCs in response to LPA stimulation. It has been shown that LPA, via the activation of the EGF plasma membrane receptor, transduces the LPA signal to activate downstream genes in several types of cells (14). However, in human SMCs, AG-1478, a tyrosine kinase inhibitor of EGFR, has no effect on LPA-induced IL-6 secretion (Fig. 3F), despite the presence of EGFRs in HASMCs (Fig. 3G); in addition, we did not detect LPA induction of EGFR phosphorylation in HASMCs (Fig. 3G). Together, these results indicate that EGFRs do not contribute to LPA-induced IL-6 secretion from HASMCs.
In summary, we have provided evidence that in SMCs, LPA1 mediates p38 MAPK activation via a Gi/o protein and PKC-dependent pathway, which is independent of EGFR activation, leading to IL-6 secretion induced by LPA. In light of the emerging roles of LPA and IL-6 in vascular inflammation, our present study in revealing the molecular mechanisms of LPA induction of IL-6 secretion in vascular cells provides valuable information that may contribute to the treatment and prevention of cardiovascular disorders.
This work was supported by National Institutes of Health Grants HL-074341 (to M.-Z. Cui) and AG-026640 (to X. Xu).
No conflicts of interest are declared by the author(s).
The authors thank Dr. Glenn D. Prestwich and Dr. Yong Xu (Department of Medicinal Chemistry, The University of Utah) for phosphorothioate diester compound 7, Dr. Duane D. Miller (Department of Pharmaceutical Sciences, The University of Tennessee) for compound 19b, and Dr. Donald McGavin and Misty Bailey for critical reading of the manuscript.
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