Lysophosphatidylcholine Activates a Novel PKD2-Mediated Signaling Pathway That Controls Monocyte Migration
Objective— Monocyte activation and migration are crucial events in the development of atherosclerosis and other inflammatory diseases. This study examined the role of protein kinase D (PKD) in monocyte migration.
Method and Results— PKD2 is the predominant isoform of PKD expressed in monocytic THP-1 cells and primary human monocytes. Lysophosphatidylcholine (lysoPC), a prominent component of oxidized low-density lipoprotein, induces rapid and marked PKD activation in these cells. Using multiple approaches, including dominant-negative mutants and small interfering RNA knock-down, we found that lysoPC-induced PKD2 activation was required for the activation of both ERK and p38 MAPK. p38 MAPK mediation of lysoPC-induced monocytic cell migration was reported previously; our results reveal that the lysoPC-induced PKD2-p38 pathway controls monocyte migration.
Conclusions— This study provides the first evidence that (1) lysoPC activates PKD, (2) PKD2 has a novel role in p38 activation, and (3) the PKD2-activated p38 pathway is responsible for lysoPC-induced migration of THP-1 cells and human monocytes. Thus, PKD is a novel and functional intracellular regulator in both lysoPC signaling and monocyte migration. These results suggest a new role for PKD2 in the development of atherosclerosis and other inflammatory diseases.
Monocytes play an important role in many pathophysiological conditions, particularly when the progression of a disease stems from inflammation, as is the case in atherosclerosis. Oxidized low-density lipoprotein (oxLDL), as well as many growth factors, cytokines, adhesion molecules, and chemokines have been implicated in monocyte activation, migration, adhesion, and differentiation. It is believed that oxLDL, which has been found in atherosclerotic lesions, mediates the development of atherosclerosis.1,2
Accumulating evidence suggests that many of the cellular effects of oxLDL during early atherosclerosis development are caused by lysophosphatidylcholine (lysoPC), an important bioactive lipid component of oxLDL (for review see3). These effects include regulation of a broad range of cellular processes such as monocyte chemotaxis,4 monocyte growth factor production,5 and gene transcription.6 LysoPC is thought to be responsible for oxLDL-mediated migration of circulating monocytes, both directly4,7 and indirectly by stimulation of the release of monocyte chemoattractant protein-1 (MCP-1) from smooth muscle cells and endothelial cells.6,8 A previous study showed that lysoPC-induced THP-1 monocytic cell migration involves activation of p38 mitogen-activated protein kinase (MAPK).7 However, how p38 MAPK is activated by lysoPC remains elusive.
In an effort to better understand lysoPC-induced signaling cascades and cellular responses to lysoPC, we identified protein kinase D (PKD) as a new component in both the lysoPC-signaling pathway and monocyte activation. PKD is a relatively new serine/threonine protein kinase family consisting of 3 isoforms: PKD1, PKD2, and PKD3 (for review see9,10). Because PKD2 has only recently been identified, many of its biological functions remain to be explored. Our data demonstrated that lysoPC activates PKD2 in monocytes and the activated PKD2 mediates lysoPC-induced monocyte migration. These results reveal a novel cellular function of lysoPC-activated PKD2 in the regulation of monocyte migration.
Materials and Methods
LysoPC (1-Palmitoyl-2-hydroxy-sn-Glycero-3-Phosphocholine) was from Avanti Polar Lipids. Antibodies against PKD2 and phospho-PKD2 (Ser876 of human PKD2) were from Upstate Cell Signaling Solutions. Antibodies against PKD1, phospho-PKD-loop (phospho-Ser738/742 of human PKD1, phospho-Ser706/710 of human PKD2, and phospho-Ser731/735 of human PKD3), phospho-PKD1 C termini (phospho-Ser910 of human PKD1), ERK, phospho-ERK, p38, and phospho-p38 were from Cell Signaling Technology. An antibody against PKD3 was from Bethyl Laboratories. PKCδ antibody was from BD Biosciences. G2A antibody was from MBL International Corporation. Ficoll-Paque premium density gradient medium was from GE Healthcare. Alpha-naphthyl acetate esterase detection kit was from Sigma-Aldrich.
Isolation of Human Peripheral Blood Monocytes and Cell Culture
Human monocytes were isolated from heparinized whole blood by sequential centrifugation over a Ficoll-Paque premium density gradient medium and adherence to serum-coated cell culture flasks as described previously.11 Nonadherent cells were then removed. The adherent cells were detached with 5 mmol/L EDTA/PBS. Purity of yielded monocytes was more than 95% as determined by an α-naphthyl acetate esterase detection kit.11 The isolated human monocytes were then cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 2 mmol/L glutamine for 48 hours before addition of stimuli. THP-1 cells, a human leukemic monocytoid cell line, were obtained from American Type Culture Collection and were cultured under the same condition as described for monocytes.
Construction of PKD2 and Dominant-Negative PKD2
Full-length human PKD2 (HPKD2) was cloned into the pcDNA3.1A vector (Invitrogen). The construct of pcDNA3.1A-HPKD2-D695A encoding dominant-negative PKD2 was prepared by mutating an aspartic acid into an alanine at position 695 in human PKD2.12 Detailed information is given in the supplemental materials (available online at http://atvb.ahajournals.org).
DNA and siRNA Transfection
Cells were transfected with wild-type PKD2 or dominant-negative PKD2 construct with the use of Lipofectamine 2000 from Invitrogen. Transfection of siRNA was performed according to the manufacturer’s instructions (Qiagen). The specific PKD2 siRNA was designed and supplied by Qiagen; the control (nonsilencing) siRNA and PKCδ siRNA were also from Qiagen. G2A siRNA was from Invitrogen.
Western Blot Analysis
Cells were lysed and subjected to Western blot analysis as described previously.13
Cell Migration Assay
The cell migration was performed using a 24-well transwell (6.5-mm diameter, 5-μm pore size; Corning Costar). Briefly, cultured cells were harvested and resuspended in the medium (RPMI 1640 plus 1 mg/mL BSA) at a cell density of 5×106 cells/mL. LysoPC was premixed with the medium in the lower chamber of a transwell and incubated at 37°C for 20 minutes. Then 100 μL of cells were added to the top chamber of a transwell and incubated for 3 hours at 37°C in 5% CO2. Cells passing through the membrane were collected from the lower chamber and counted with a FACS Vantage SE flow cytometry system (BD Biosciences Immunocytometry Systems) and with a hemocytometer.
The means±SEs were calculated using Excel statistical software, and the statistical significance (probability value) was determined by the 2-tailed Student t test. A value of P<0.05 was considered statistically significant.
PKD2 Is the Predominant PKD Isoform Expressed in THP-1 Cells and in Primary Human Monocytes
To investigate the biological function of the relatively new protein kinase PKD, we first examined expression of the 3 isoforms of the PKD family. Cell lysates of THP-1 cells as well as cell lysates of HEK 293 and HeLa cells, which were used as controls, were examined by Western analysis using PKD1-, PKD2-, and PKD3-specific antibodies. As shown in Figure 1A, all 3 isoforms, PKD1, PKD2, and PKD3, were detected in HEK 293 cells. In HeLa cells, PKD2 and PKD3, but not PKD1, were detected. Interestingly, in monocytic THP-1 cells, only PKD2 was detected. Thus, PKD2 is the predominant isoform of PKD expressed in THP1 cells. We also examined the expression pattern of PKD isoforms in freshly isolated primary human monocytes. As shown in Figure 1B, PKD2 is the major isoform detected in human monocytes. Therefore PKD2 is the predominant isoform of PKD expressed in both monocytic THP1 cells and primary human monocytes.
LysoPC Induces Rapid and Marked Activation of PKD
To determine whether lysoPC activates PKD in monocytic cells, we examined PKD phosphorylation in THP-1 cells upon stimulation with lysoPC. Using a specific antibody (Cell Signaling Technology) that recognizes phosphorylated serine residues at the activation loop common to all 3 isoforms of PKD (for human PKD1, at Ser738 and Ser742; for human PKD2, at Ser706 and Ser710; and for human PKD3, at Ser731 and Ser735), we found that lysoPC (15 μmol/L) rapidly and markedly induced PKD phosphorylation in THP-1 cells with a peak (over 8-fold) at 2 minutes (Figure 2A and 2B). Also, lysoPC induced PKD activation in a dose-dependent manner (Figure 2C and 2D), and maximum phosphorylation occurred in the lysoPC concentration range of 15 to 20 μmol/L.
As shown in Figure 2A through 2D, when the blot was probed with antibodies specific for phosphorylated PKD1 (S910) and PKD2 (S876), phosphorylation of PKD2 was detected following the same time- and concentration-dependent pattern of the phosphorylation of the common activation loop of all 3 PKD isoforms. In contrast, phosphorylation of PKD1 (S910) was not detected. These results demonstrate that PKD2 is activated upon stimulation by lysoPC in THP-1 cells.
LysoPC also markedly induced phosphorylation of PKD2 in freshly isolated human monocytes, and the level of phosphorylation of PKD2 was similar as that of phosphorylation of p38 MAPK (Figure 2E).
PKD2 Is a Key Upstream Regulator of ERK and p38 MAPK in Response to LysoPC
LysoPC-induced ERK and p38 MAPK activation in THP-1 monocytic cells has been reported previously.7 However, the identity of the upstream signaling molecules that mediate ERK and p38 activation, in response to lysoPC, is still unknown. We explored the possible role of PKD2 in ERK and p38 activation by examining whether a dominant-negative PKD2 affects lysoPC-induced ERK and p38 activation. THP-1 cells were transfected with an empty vector or plasmids expressing either wild-type PKD2 or dominant-negative PKD2 mutant. As shown in Figure 3A, wild-type PKD2 and dominant-negative mutant PKD2 were expressed at equal levels in cells transfected with recombinant plasmids. It was found that upon stimulation with lysoPC, phosphorylation of PKD2, PKD activation loop, ERK, and p38 was detected in cells and that overexpression of wild-type PKD2 had no significant effect on the degree of lysoPC-induced phosphorylation of PKD2, PKD activation loop, ERK, and p38. However, in cells overexpressing dominant-negative PKD2, the degree of the phosphorylation of PKD2, PKD activation loop, ERK, and p38 was markedly reduced (PKD2 phosphorylation reduced by 85.7%, PKD activation loop phosphorylation by 83.0%, ERK phosphorylation by 66.2%, and p38 phosphorylation by 71.0%; Figure 3A through 3C). These results indicate that dominant-negative PKD2 inhibits lysoPC-induced ERK and p38 activation and strongly suggest that PKD2 is an upstream mediator of lysoPC-induced activation of both ERK and p38.
To further substantiate the finding that PKD2 functions as an upstream mediator in lysoPC-activated ERK and p38 pathways, we examined the effect of siRNA depletion of endogenous PKD2 on the phosphorylation of ERK and p38 in THP1 cells. As shown in Figure 3D through 3F, transfection of PKD2 siRNA reduced PKD2 protein expression by 76.1% and blocked PKD2 phosphorylation by 78.3%, PKD phosphorylation (activation loop) by 80.9%, ERK phosphorylation by 75.7%, and p38 phosphorylation by 82.2% in comparison with cells transfected with control siRNA (nonsilencing siRNA). In contrast, the PKCδ siRNA (an independent kinase control) had no effect on lysoPC-induced phosphorylation of PKD2 and PKD (activation loop) or phosphorylation of ERK and p38. The effect of PKD2 siRNA on lysoPC-induced phosphorylation of PKD2 and p38 in monocytes was also examined. As shown in Figure 3G, depletion of PKD2 by PKD2 siRNA blocked lysoPC-induced phosphorylation of PKD2 and p38. Taken together, the results obtained from the experiments using dominant-negative PKD2 construct (Figure 3A through 3C) and the siRNA depletion of PKD2 (Figure 3D through 3G) indicate that PKD2 is a new intermediate molecule in lysoPC-triggered signaling and mediates the activation of both ERK and p38 MAPK in monocytes.
PKD2 Mediates LysoPC-Induced Monocyte Migration
It has been reported that lysoPC possesses a potent chemotactic attraction for monocytes,4 and p38 mediates lysoPC-induced monocytic cell migration.7 Based on our observation that PKD2 functions as a novel mediator of lysoPC-induced activation of both ERK and p38 (Figure 3), it became interesting to determine whether PKD2 mediates lysoPC-induced monocyte migration. We first examined the effect of overexpression of dominant-negative PKD2 on lysoPC-induced THP-1 cell migration. The effect of transfection of wild-type PKD2 or the dominant-negative PKD2 on phosphorylation of ERK and p38 is shown in Figure 3A. We observed that overexpression of the wild-type PKD2 had no significant effect on lysoPC-induced THP-1 cell migration; in contrast, overexpression of dominant-negative PKD2 reduced lysoPC-induced cell migration by 67.6% (Figure 4A). These results strongly suggest that PKD2 mediates lysoPC-induced THP-1 cell migration.
We confirmed this finding by using another independent approach: siRNA downregulation of endogenous PKD2 expression. Transfection of PKD2-specific siRNA into THP1 cells efficiently blocked endogenous PKD2 protein expression, PKD2 phosphorylation, and MAPK phosphorylation, as shown in Figure 3D. We observed that transfection of the PKD2 siRNA reduced lysoPC-induced THP1 cell migration by 73.5%. In contrast, the nonsilencing siRNA (control) did not have a significant effect on lysoPC-induced cell migration (Figure 4B). Using primary human monocytes, we observed that PKD2 siRNA significantly reduced lysoPC-induced cell migration (by 78.6%, Figure 4C). Taken together, these data clearly indicate that PKD2 functionally mediates lysoPC-induced monocyte migration.
LysoPC Activation of PKD2 Is Mediated by Pertussis Toxin–Sensitive G Proteins and G2A Protein
A pertussis toxin (PTX)-sensitive pathway has been reported to mediate lysoPC-induced MAPK phosphorylation and arachidonic acid release in monocytic cells7,14; however, a PTX-insensitive pathway has also been reported to mediate lysoPC-induced calcium mobilization in monocytic cells.15 To our knowledge, whether G proteins contribute to the newly identified PKD2 activation in intact cells has not been reported. To determine whether and which groups of G proteins are responsible for lysoPC-induced PKD2 activation, we pretreated THP-1 cells with PTX, an established and specific Gi/o protein inhibitor, and found that PTX concentration-dependently blocked lysoPC-induced PKD2 activity (Figure 5A and 5B). Our data also showed that PTX blocked p38 activation (Figure 5C). These results indicate that the Gi/o group of G proteins mediates lysoPC-induced activation of PKD2 and p38 in monocytic cells and suggest that a membrane receptor mediated pathway regulates lysoPC-induced PKD2 activation. A recent report demonstrated that receptor G2A mediates lysoPC-induced chemotaxis to monocytic THP1 cells.16 To determine whether G2A mediates lysoPC-induced PKD2 activation in monocytic cells, we investigated the effect of depletion of G2A expression on lysoPC-induced PKD2 activation using the G2A-specific siRNA. Our results showed that depletion of G2A blocked lysoPC-induced PKD2 activation (Figure 5D), indicating that G2A functionally mediates the lysoPC signal leading to PKD2 activation.
Taken together, these results support the conclusion that lysoPC, via a novel PKD2-mediated p38 MAPK signaling pathway, mediates monocyte migration, an important biological function in the early development of atherosclerosis.
OxLDL is believed to be an important factor in the development of atherosclerosis. LysoPC is a prominent bioactive component of oxLDL and has been shown to induce monocyte migration,4 an early event in the development of atherosclerosis. Understanding how lysoPC affects monocyte function may be important in understanding the role of oxLDL in the development of atherosclerosis. This study demonstrates for the first time that lysoPC activates PKD in living cells. Our data further indicate that the specific PKD isoform expressed and activated in monocytic THP-1 cells and primary human monocytes is PKD2, which mediates phosphorylation of ERK and p38, providing a regulatory linkage between PKD2 and p38 MAPK. Our results reveal that PKD2 is a novel intermediate molecule in both lysoPC signaling and monocyte activation. Importantly, these results reveal that lysoPC induces monocyte migration via a novel signaling pathway specifically mediated by PKD2 and p38.
Our data provide the first evidence that Gi/o protein mediates PKD2 activation in living cells. The concentration-dependent inhibition of PKD2 activation by the Gi/o inhibitor PTX indicates that a specific G protein–coupled receptor is involved in mediating lysoPC signaling in monocytic cells. Ovarian cancer G protein–coupled receptor 1 (OGR1), G2A, GPR4, and T cell death-associated gene 8 (TDAG8) have been reported to bind various lipids.17–20 These G protein–coupled receptors share high amino acid sequence homologies. Although the functions of G2A and GPR4 are still under debate, a recent article reported that G2A is the crucial receptor mediating lysoPC-induced chemotaxis to THP-1 cells.16 Our results suggest that G2A mediates lysoPC-induced PKD2 activation.
PKDs are a new family of serine and threonine protein kinases. Of the 3 members of the PKD family (PKD1, PKD2 and PKD3)9,10 only the intracellular signaling pathway of PKD1 has been well studied. It has been shown that PKD1 mediates oxidative stress–induced NF-κB activation21 and BMP-2–induced JNK and p38 MAPK activation.22 However, PKD2 is a recently cloned new member of the PKD family,23 and much less is understood about its role in intracellular signaling pathways. Recently, it was reported that overexpression of PKD2 potentiates MEK/ERK/RSK signaling,24 suggesting that endogenous PKD2 may mediate extracellular stimulus-triggered MAPK activation. In the present study, our data show that PKD2 is the predominant isoform expressed in THP-1 cells and primary human monocytes and that transfection of PKD2 dominant-negative constructs markedly blocked lysoPC-induced phosphorylation of ERK and p38, indicating that endogenous PKD2 is an upstream regulator of MAPK activation. Knockdown of the endogenous PKD2 expression with the specific PKD2 siRNA also markedly inhibited lysoPC-induced phosphorylation of both ERK and p38, further confirming that lysoPC-induced endogenous PKD2 activity mediates the activation of both ERK and p38 in monocytes. Thus, the present study not only provides evidence that lysoPC induces PKD2 activation, but also provides evidence that PKD2 activation is essential for lysoPC-induced MAPK activation. To our knowledge, our results reveal for the first time that endogenous PKD2 is an upstream mediator of both ERK and p38 MAPK activation in living cells, in response to an extracellular stimulus.
PKC-dependent and independent activation of PKD has been reported (see reviews9,10). Our previous studies have shown that PKCδ mediates both thrombin- and angiotensin II–induced PKD activation in smooth muscle cells.25,26 PKCδ activation of PKD has also been found in other cell types in response to various stimuli.21,27–29 In the present study, the activation of PKCδ, PKCε, PKCθ, PKCα, and PKCζ was not detected in response to lysoPC stimulation in monocytic cells (data not shown), suggesting that none of these PKCs contributes to lysoPC-induced PKD activation in these cells. Furthermore, our data demonstrating that transfection of PKCδ siRNA has no effect on PKD2 activation support our conclusion that PKCδ has no role in lysoPC-induced PKD2 activation in monocytic cells.
Only in the last few years has the activation of this new kinase PKD been reported in vascular cells, including endothelial cells30 and smooth muscle cells,25,26 and in platelets.31 Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the regulation of myocardial contraction, hypertrophy, and remodeling. However, the understanding of PKD function in the constituent cells of the heart and its role in circulation is still in its infancy (see review in32). The present study provided new evidence of PKD activation in monocytes and explored the biological function of lysoPC-induced PKD activation. Our results identified that PKD2 is the upstream mediator of p38 and that the PKD2-p38 pathway modulates lysoPC-induced monocyte migration. These results provide evidence of the novel role of a PKD2-p38 pathway in cell migration. Therefore, our results suggest a possible new role of PKD2 in the development of atherosclerosis and possibly other inflammatory diseases.
The authors thank Dr Heather Williamson for her help in the experiments using human blood samples, and Dr Donald McGavin and Misty Bailey for critical reading of the manuscript.
Sources of Funding
This work was supported by National Institutes of Health grants HL074341 (to M.-Z.C.), AG026640 (to X.X.), and HL29582 (to G.M.C.). M.-Z.C. received a research award from the Philip Morris Foundation.
M.T. and F.H. contributed equally to this study.
Received July 24, 2008; revision accepted May 26, 2009.
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