Lysophosphatidic Acid Induces Early Growth Response-1 (Egr-1) Protein Expression via Protein Kinase Cδ-regulated Extracellular Signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK) Activation in Vascular Smooth Muscle Cells*

Background: The intracellular signaling pathway leading to lysophosphatidic acid (LPA)-induced Egr-1 expression has been largely unknown. Results: PKCδ-mediated activation of ERK and JNK is required for LPA-induced Egr-1 expression. Conclusion: This study provides the first evidence of PKCδ regulation of ERK and JNK activation in LPA signaling and the role of PKCδ/ERK/JNK in LPA-induced gene expression. Significance: Identified intracellular molecules may serve as therapeutic targets. Lysophosphatidic acid (LPA) modulates vascular cell function in vitro and in vivo via regulating the expression of specific genes. Previously, we reported that a transcriptional mechanism controls LPA-induced expression of Egr-1 in vascular smooth muscle cells. Egr-1 is a master transcription factor mediating the expression of various genes that have been implied to modulate a broad spectrum of vascular pathologies. In this study, we determined the essential intracellular signaling pathway leading to LPA-induced Egr-1 expression. Our data demonstrate that activation of ERK1/2 and JNK, but not p38 MAPK, is required for LPA-induced Egr-1 expression in smooth muscle cells. We provide the first evidence that MEK-mediated JNK activation leads to LPA-induced gene expression. JNK2 is required for Egr-1 induction. Examining the upstream kinases that mediate ERK and JNK activation, leading to Egr-1 expression, we found that LPA-induced activation of MAPKs and expression of Egr-1 are dependent on PKC activation. We observed that LPA rapidly activates PKCδ and PKCθ. Overexpression of dominant-negative PKCδ, but not dominant-negative PKCθ, diminished activation of ERK and JNK and blocked LPA-induced expression of Egr-1 mRNA and protein. We also evaluated LPA receptor involvement. Our data reveal an intracellular regulatory mechanism: LPA induction of Egr-1 expression is via LPA cognate receptor (LPA receptor 1)-dependent and PKCδ-mediated ERK and JNK activation. This study provides the first evidence that PKCδ mediates ERK and JNK activation in the LPA signaling pathway and that this pathway is required for LPA-induced gene regulation as evidenced by Egr-1 expression.

Lysophosphatidic acid (LPA) 2 is produced by activated platelets (1) and is formed in oxidized LDL (2). High concentrations of LPA have been found in human atherosclerotic lesions (2) and human serum (1,3). Accumulated evidence indicates that LPA influences vascular cell functions, including smooth muscle cell (SMC) proliferation, migration, and dedifferentiation, which are controlled by LPA-induced gene expression (for review, see Ref. 4).
Previously, we have shown that LPA markedly induces Egr-1 (early growth response gene-1) expression in vascular SMCs (5). Egr-1, a zinc finger transcription factor, activates a set of genes implicated in the pathogenesis of restenosis and atherosclerosis with subsequent thrombosis. The products of these genes include proinflammatory cytokines, chemokines, adhesion molecules, growth factors, coagulation factors, and matricellular modulators, such as tumor necrosis factor-␣, IL-2, monocyte chemotactic protein-1, intercellular adhesion molecule-1, CD44, platelet-derived growth factor A-and B-chains, fibroblast growth factor-2, transforming growth factor-␤1, tissue factor, plasminogen activator inhibitor-1, urokinase-type plasminogen activator, 5-lipoxygenase, thrombospondin, and metalloproteinases (6,7). Thus, Egr-1 plays a vital role in regulating the expression of a set of important genes, leading to vascular diseases. An important step toward understanding the mechanism of vascular diseases is understanding the intracel-* This work was supported, in whole or in part, by National Institutes of lular mechanism by which Egr-1 expression is regulated in vascular cells in response to lipid mediators that are accumulated in the vascular environment. In a previous study, we demonstrated that transcriptional regulation controls LPA-induced egr-1 gene expression and that both the cAMP response element and serum response element motifs of the egr-1 promoter are required for LPA-induced egr-1 promoter activation (5). In the present study, we sought to determine the intracellular signaling pathway leading to LPA-induced Egr-1 expression. Our results demonstrate the roles of LPA receptors and the specific PKC and MAPK in regulation of Egr-1 expression in vascular SMCs. We discovered several previously unrevealed intracellular molecular links in the LPA signaling pathway and their functions in LPA-induced gene expression.
Tissue Culture-SMCs were prepared from explants of excised aortas of rats as described previously (8). Cells were maintained in DMEM containing 10% fetal bovine serum. Cells were made quiescent by incubation in serum-free DMEM for 48 h. LPA was dissolved in PBS, and a final concentration of 25 M was used.
Northern Blot Analysis-Total cellular RNA was isolated using TRIzol reagent according to the manufacturer's instructions. Total RNA (6 -8 g) was subjected to denaturing electrophoresis on formaldehyde-agarose gels. RNA was blotted onto Nytran membranes (Schleicher & Schüll) and hybridized with radiolabeled cDNA probes as described previously (5).
Western Blot Analysis-Rat SMCs were rinsed with cold PBS and lysed in Western blot lysis buffer (50 mM Tris-HCl (pH 6.8), 8 M urea, 5% ␤-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).
Adenoviral Constructs and Adenoviral Infection of SMCs-Adenoviruses encoding PKC were constructed as previously described (9). Adenoviral wild-type and dominant-negative PKC␦ constructs were kindly provided by Dr. Motoi Ohba (Showa University, Tokyo, Japan). SMCs in DMEM containing 10% FBS were infected for 24 h with either wild-type or dominant-negative PKC isotypes.
Conventional RT-PCR Assay-Expression of LPA receptor mRNA was evaluated by RT-PCR. Total RNA was isolated from SMCs using an RNeasy mini-prep kit (Qiagen). The first strand of cDNA was reverse-transcribed using the ThermoScript RT-PCR system. The cDNA products were amplified using Gene-Amp PCR core reagents. The amplification conditions were as follows: 2 min at 95°C; 25-31 cycles for 45 s at 95°C, 45 s at 55°C, and 1 min at 72°C; followed by a final extension for 5 min at 72°C. The PCR products were analyzed by electrophoresis on a 1.0% agarose gel.
Real-time PCR Assay-The primers were designed using Primer Express 3.0 software (Applied Biosystems). Real-time PCR was performed on an ABI 7300 real-time PCR system (Applied Biosystems) under the following conditions: 95°C for 10 min and 40 cycles at 95°C for 30 s, 58°C for 1 min, and 72°C for 1 min.
siRNA Treatment-Cells were transfected with non-silencing, JNK1, or JNK2 siRNA (Qiagen) for 48 h using Lipofectamine RNAiMAX reagent (Invitrogen) following the instructions provided by the manufacturer. On day 3, cells were starved for 24 h prior to LPA treatment.
Statistical Analysis-The means Ϯ S.E. were calculated using Excel statistical Software, and statistical significance (p value) was determined by Student's two-tailed t test. A p value of Ͻ 0.05 was considered statistically significant.

Activation of ERK and JNK MAPKs Is Required for LPA-induced
Egr-1 Expression-We previously demonstrated that a transcriptional mechanism controls LPA-induced egr-1 gene expression (5). Our results further showed that nuclear serum response factor (SRF) and cAMP response element-binding protein (CREB) mediate LPA-induced egr-1 gene transcription in SMC nuclei (5). However, the upstream intracellular signal-ing pathway, which leads to LPA-induced Egr-1 expression, has been elusive. To first determine whether MAPKs are involved in LPA-induced Egr-1 expression, we measured LPA-induced MAPK activation. Rat SMCs were starved for 48 h prior to LPA treatment. At various time points of LPA treatment, SMCs were washed with PBS and lysed for the detection of intracellular phosphorylation of various MAPKs: ERK, JNK, and p38 MAPK. As shown in Fig. 1A, LPA markedly and transiently induced activation of ERK kinase (MEK), ERK, JNK, and p38 MAPK in SMCs. The activation peaks of these kinases in SMCs by LPA were at ϳ2-5 min.
To determine which of these kinases are involved in LPAinduced Egr-1 expression, we tested whether pretreatment with the ERK kinase (MEK) inhibitor U0126, p38 MAPK inhibitor SB203580, or JNK inhibitor SP600125 had any effect on LPA-induced Egr-1 expression. As shown in Fig. 1B, pretreatment of SMCs with 2.5 M U0126, but not with 2.5 M SB203580, completely blocked LPA-induced Egr-1 mRNA expression. SP600125 dose-dependently blocked LPA-induced Egr-1 mRNA (Fig. 1, C and D). These data show that activation of MEK/ERK and JNK is required, but p38 does not play a role in LPA-induced egr-1 gene expression in SMCs. We next evaluated the effects of these specific inhibitors on LPA-induced Egr-1 protein synthesis. Consistent with the data shown in Fig.  1 (B-D), pretreatment of cells with 2.5 and 5.0 M U0126 completely blocked LPA-induced Egr-1 protein expression; however, SB203580 at two effective doses (2.5 and 5.0 M) had no effect on LPA-induced Egr-1 protein synthesis (Fig. 1E). We also observed that SP600125 dose-dependently blocked Egr-1 protein expression (Fig. 1, F and G). These results indicate that Because LPA was dissolved in PBS, PBS was used as a system control. ␤-Actin was used as a loading control (Ctr). B, effect of U0126 or SB203580 on LPA-induced Egr-1 mRNA accumulation. SMCs were pretreated with U0126 (U0) or SB203580 (SB) at 2.5 M for 40 min, followed by LPA stimulation for 30 min. Egr-1 mRNA expression was detected by Northern blot analysis using 32 P-labeled rat Egr-1 cDNA fragments. Because U0126 or SB203580 was dissolved in dimethyl sulfoxide (DMSO), a sample treated with dimethyl sulfoxide alone served as a control. 28 S rRNA was used as a loading control. C, effect of SP600125 on LPA-induced Egr-1 mRNA expression. SMCs were pretreated with various doses of SP600125 for 40 min and then stimulated with LPA for 30 min. Egr-1 mRNA expression was determined by Northern blot analysis. Conditions were as described for B. D, the results of the Northern blot analysis were quantified by densitometry. Data are means Ϯ S.E. from three experiments (p Ͻ 0.01, each SP600125 ϩ LPA sample versus LPA-alone sample). E, effect of U0126 or SB203580 on LPA-induced Egr-1 protein expression. SMCs were pretreated with U0126 or SB203580 at various doses as indicated for 40 min, and cells were then stimulated with LPA for 1 h. Egr-1 protein synthesis was detected by Western blot analysis. F, effect of SP600125 on LPA-induced Egr-1 protein expression. SMCs were pretreated with SP600125 at various doses for 40 min, followed by LPA treatment for 1 h. Egr-1 protein expression was detected by Western blot analysis. G, the results of Western blot analysis were quantified by densitometry. Data are means Ϯ S.E. from three experiments (p Ͻ 0.01, each SP600125 ϩ LPA sample versus LPA-alone sample).
LPA activation of MEK/ERK and JNK is required for Egr-1 protein expression, whereas p38 MAPK does not have a role. Therefore, the data shown in Fig. 1 demonstrate that LPA-induced Egr-1 expression depends on the MEK/ERK and JNK cascades.
PKC Mediates MAPK Activation, Leading to LPA-induced Egr-1 Expression-To search for the upstream intracellular mediator that controls ERK and JNK MAPK activation, leading to LPA-induced Egr-1 expression, we examined the role of PKC in MAPK activation and Egr-1 synthesis. Pretreatment of SMCs with the general PKC inhibitor GF109203X dose-dependently blocked LPA-induced activation of MEK, ERK, and JNK (Fig. 2,  A and B), as well as LPA-induced expression of Egr-1 mRNA and protein (Fig. 2C). These results indicate that PKC-mediated activation of MEK/ERK and JNK leads to LPA induction of Egr-1 expression.
Specific PKC Isotypes Are Involved in LPA Induction of Egr-1 Expression in SMCs-To determine the specific PKC isotype function in LPA induction of Egr-1 expression, we evaluated activation of specific PKC isotypes in SMCs in response to LPA stimulation. As shown in Fig. 3A, PKC␦ and PKC were activated very rapidly and markedly by LPA, with phosphorylation peaks at ϳ30 s and 2 min. However, there was no significant phosphorylation detected for other isotypes of PKC in response to LPA (Fig. 3A), suggesting that novel PKC group members PKC␦ and PKC may be involved in LPA-induced Egr-1 expression.  We next tested the roles of PKC␦ and PKC in Egr-1 expression using the pharmacological compound rottlerin, which has been reported to inhibit the activity of PKC (10) and PKC␦ (11). As shown in Fig. 3B, pretreatment of SMCs with 20 M rottlerin completely blocked LPA-induced phosphorylation of PKC, PKC␦, ERK, and JNK, suggesting that activation of PKC and PKC␦ may be involved in ERK and JNK activation. Conversely, in a reciprocal experiment, we tested whether ERK activation has a feedback effect on activation of PKC and PKC␦ or has a role in activation of PKC and PKC␦. Our results show that 5 M U0126, a specific MEK inhibitor, completely blocked activation of ERK, a substrate of MEK, but had no effect on phosphorylation of PKC and PKC␦ (Fig. 3B), indicating that MEK and ERK are not the upstream regulators of either PKC or PKC␦ and do not have an effect on activation of PKC and PKC␦. Furthermore, we observed that rottlerin at a low dose of 5 M completely inhibited LPA-induced Egr-1 mRNA expression (Fig. 3C). Together, these data support the possibility that LPA-induced activity of PKC and PKC␦ may regulate activation of their downstream molecules ERK and JNK, leading to Egr-1 expression. (12)(13)(14)(15); however, the cross-talk between individual MAPK family members in LPA signaling is not well documented compared with that in growth factor signaling. Especially, MEK/ERK-regulated JNK activation in LPA signaling that leads to LPA induction of gene expression has not been revealed. We observed that U0126 at a low dose of 5 M completely blocked activation of ERK (a substrate of MEK) and JNK, indicating that MEK/ERK activation is required for LPA-induced JNK phosphorylation and activation (Fig. 3B). This result, together with the results shown in Fig. 1, indicates that MEK/ERK-regulated JNK activation leads to LPA-induced gene expression. To the best of our knowledge, these data document that the cross-talk between the MEK/ ERK pathway and the JNK pathway in LPA signaling contributes to LPA-induced gene expression as evidenced by Egr-1 expression.

MEK/ERK-regulated JNK Activation Leads to LPA-induced Gene Expression in Vascular SMCs-LPA activation of MAPK in various cell types has been reported
PKC␦, but Not PKC, Activates ERK and JNK, Leading to Egr-1 Expression-Because the pharmacological compound rottlerin has an inhibitory effect on PKC (10) and PKC␦ (11) and also considering the possible nonspecific effect of this pharmacological inhibitor, we further evaluated whether and which novel PKC mediates Egr-1 expression via activation of ERK and JNK by employing a dominant-negative approach. We determined the effect of overexpression of dominant-negative PKC␦ in adenovirus on activation of ERK and JNK in SMCs as well as on expression of Egr-1 in comparison with the effect of dominant-negative PKC. In these experiments, we included wildtype PKC␦ or PKC as a control. As shown in Fig. 4A, overexpression of dominant-negative PKC␦ in SMCs significantly blocked LPA-induced activation of MEK, ERK, and JNK. In contrast, overexpression of dominant-negative PKC had no effect on activation of MEK, ERK, and JNK induced by LPA (Fig.  4B). We also observed that neither wild-type PKC␦ nor wildtype PKC had a significant effect on MAPK activation (Fig. 4,  A and B). These results indicate that activated PKC␦, but not activated PKC, is required for LPA-induced MEK, ERK, and JNK activation. We next evaluated the effects of dominant-negative PKC␦ and dominant-negative PKC on Egr-1 expression. As shown in Fig. 4 (C and D), we found that overexpression of dominant-negative PKC␦, but not dominant-negative PKC, markedly blocked Egr-1 expression. Overexpression of wildtype PKC␦ and PKC had no significant effect on Egr-1 expression (Fig. 4, C and D). Therefore, the results shown in Fig. 4 support the conclusion that LPA signaling leads to intracellular activation of both PKC␦ and PKC, but only activated PKC␦, and not PKC, is required for MEK/ERK and JNK activation, which in turn, mediates Egr-1 expression.
JNK2 Is Required for LPA-induced Egr-1 Expression in SMCs-We found that the JNK inhibitor SP600125 dose-dependently blocked LPA-induced Egr-1 expression (Fig. 1, C, D, F, and G) and that ERK kinase (MEK) inhibitor U0126 completely blocked JNK activation (Fig. 3B) and Egr-1 expression (Fig. 1, B  and E). These results indicate that MEK-mediated JNK activation is required for LPA-induced Egr-1 expression. To confirm the JNK role in LPA-induced Egr-1 expression, we determined the isoforms of JNK expressed in rat SMCs and their functions in Egr-1 expression. As shown in Fig. 5A, JNK1 and JNK2, but not JNK3, were expressed in SMCs. We next tested whether knockdown of either JNK1 or JNK2 affects LPA-induced Egr-1 expression. Our results show that knockdown of JNK2, but not JNK1, blocked Egr-1 expression (Fig. 5B), indicating that JNK2 is required for the LPA signaling pathway leading to Egr-1 expression.
LPA 1 Is the Main Receptor Responsible for LPA-induced Egr-1 Expression-LPA is known to mediate gene expression via its cognate G-protein-coupled receptors. Thus far, at least five G-protein-coupled LPA receptors have been reported (16). LPA 1 , LPA 2 , and LPA 3 share very high homology (16). To determine which LPA receptors mediate the LPA signal leading to Egr-1 expression in SMCs, we performed RT-PCR analyses in various reaction cycles (25-31 cycles) to determine LPA receptor expression levels. As shown in Fig. 6A, we found that LPA 1 was expressed predominantly in rat SMCs. The relative abundance of the receptors was determined by real-time PCR analysis with the following ratio: LPA 1 Ͼ Ͼ LPA 2 Ͼ LPA 3 (Fig. 6B). To evaluate the role of the predominant form, LPA 1 , in LPAinduced kinase activation and Egr-1 expression, we pretreated SMCs with various doses of Ki16425, a potent inhibitor of LPA 1 and LPA 3 . As shown in Fig. 6C, pretreatment with Ki16425 dose-dependently blocked activation of PKC␦, MEK, ERK, and JNK and expression of Egr-1, indicating that LPA 1 is the major LPA receptor on the plasma membrane that mediates the LPA signal leading to the intracellular activation of PKC␦, which then activates the ERK and JNK pathways, leading to Egr-1 transcription in the nuclei of SMCs.

DISCUSSION
LPA, a bioactive phospholipid produced by activated platelets, has been found to accumulate at high concentrations in atherosclerotic lesions. LPA is formed during the oxidation of LDL (2). Evidence has shown that accumulation of this lipid mediator may serve as an important risk factor for development of atherosclerosis and thrombosis (4). LPA affects many func-tions of various vascular cells through mediating the expression of a variety of genes in these cells. In vascular SMCs, LPA induces expression of several genes, such as the coagulation initiator tissue factor (15), IL-6 (14), monocyte chemotactic protein-1 (17), and Egr-1 (5). Among these genes, Egr-1 is a nuclear transcription factor that reportedly mediates expression of monocyte chemotactic protein-1, IL-6, and tissue factor (18 -22), all of which are known to play important roles in the development of atherosclerosis and thrombosis. Therefore, understanding the intracellular signaling pathway leading to expression of the key regulatory transcription factor Egr-1 is essential to understanding how to prevent activation of the  Egr-1 pathway and how to minimize the effect of Egr-1 and its downstream target gene products and ultimately to reduce atherogenesis and thrombosis.
In this study, we focused on understanding the intracellular signaling cascade in LPA-stimulated Egr-1 expression in primary rat SMCs. Our data reveal an essential intracellular signaling cascade in the mediation of the LPA signal leading to Egr-1 up-regulation. We demonstrated that LPA via its cognate receptor LPA 1 activates PKC␦, which in turn mediates activation of MEK/ERK and JNK, leading to Egr-1 transcription and translation.
Previously, we established that the transcriptional regulation mechanism controls LPA-induced egr-1 gene expression (5). We showed that LPA regulates Egr-1 expression via transcription factors CREB and SRF, which bind to the cis-acting cAMP response element and serum response element in the egr-1 promoter region. These results established a novel role for CREB in mediating LPA-induced gene expression. Additionally, we also showed that LPA-induced protein-DNA binding activity is caused by LPA-induced rapid phosphorylation of CREB and SRF.
In this study, performed to determine the intracellular regulatory mechanism upstream of Egr-1 transcription, our data reveal an essential role for PKC␦ in LPA-induced Egr-1 expression. To our knowledge, although LPA signaling has been widely studied, the role of PKC␦ in LPA-induced gene expression has been largely unknown. We observed a rapid activation of both PKC␦ and PKC in vascular SMCs in response to LPA stimulation. Rottlerin, an inhibitor of both PKC␦ and PKC (10,11), blunted LPA induction of Egr-1, suggesting that PKC␦, PKC, or both may be involved in Egr-1 expression. However, our approach using overexpression of dominant-negative PKC␦ or PKC defined PKC␦, but not PKC, as the essential regulator mediating the LPA signal leading to Egr-1 expression.
Our data further demonstrate that an essential role for PKC␦ in LPA-induced Egr-1 expression is via the MEK/ERK-regulated JNK pathway. To date, LPA activation of PKC, MAPK, or PKC/MAPK in various cell types (12)(13)(14)23), LPA activation of PKC␦ (24,25), and PKC␦ regulation of IL-8 expression in bronchial epithelial cells (25,26), as well as ERK involvement in LPA-induced Egr-1 expression (27), have been reported. However, the regulatory relationship between PKC␦ and MAPK in the LPA signaling pathway has not been documented. Furthermore, the functional role of PKC␦-activated MAPK in LPA signaling has not been revealed. In this study, our data not only demonstrate that PKC␦ mediates activation of MEK/ERK and JNK, but also reveal the functional role of the PKC␦-mediated MEK/ERK and JNK cascade that up-regulates the egr-1 gene. Therefore, these data provide the first evidence of PKC␦ regulation of MAPK in the LPA signaling pathway and reveal for the first time that PKC␦-regulated MAPK (ERK and JNK) leads to LPA-induced gene expression in living cells, i.e. LPA induction of Egr-1 expression is mediated by the PKC␦-activated MAPK (ERK and JNK) pathway. In addition, our data demonstrate that the cross-talk between the MEK/ERK and JNK pathways contributes to LPA-induced gene expression. JNK2 is required for LPA-induced Egr-1 expression.
We previously showed that LPA induces Egr-1 transcription via transactivation of the CREB and SRF transcription factors and binding of these transcription factors to the cis-elements in the egr-1 promoter in the nuclei of vascular SMCs (5). Because CREB is one of the major downstream targets of ERK1/2 (28 -30) and activation of the SAPK/JNK pathway is required for activation of the chromosomal SRF-controlled reporter gene (31), it is likely that the individual ERK pathway or the MEK/ ERK pathway, in conjunction with the JNK pathway, cooperatively mediates Egr-1 expression via the CREB and SRF transcription factors in the nuclei of SMCs.
It is important to note that Egr-1, a zinc finger transcription factor, plays a key master regulatory role in multiple cardiovascular pathological processes, including atherosclerosis, cardiac hypertrophy, ischemia, and angiogenesis (6), and was recently described as a major link between infection and atherosclerosis (32,33). Our data clearly establish a signaling pathway involving LPA 1 and the PKC␦-activated MAPK (ERK and JNK) cascade in regulating LPA induction of Egr-1 synthesis. These results provide new insights into the cellular mechanisms by which LPA exerts its effects in SMCs. The identified intracellular molecules involved in the LPA signaling pathway that leads to the up-regulation of Egr-1 may serve as therapeutic targets in the management of vascular disease.