- Email: [email protected]
Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells
Biochemical and Biophysical Research Communications 378 (2009) 264–268
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells Song Li, Junjie Zhang * The Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, No. 19 Xinjiekouwai Avenue, Beijing 100875, China
a r t i c l e
i n f o
Article history: Received 17 October 2008 Available online 21 November 2008
Keywords: Autotaxin LPS THP-1 cells PKR JNK p38 MAPK LPA
a b s t r a c t Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the b isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment. Ó 2008 Elsevier Inc. All rights reserved.
Autotaxin (ATX), also known as ENPP2 (ectonucleotide pyrophosphatase phosphodiesterase-2), has the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) [1]. Ectopic expression of ATX in Ras-transformed NIH3T3 cells stimulates their tumorigenesis and metastatic potential [2]. ATX is overexpressed in some malignant tumor tissues, including non-small cell lung cancer [3], breast cancer [4], and thyroid cancer [5]. Therefore, ATX is regarded as an attractive target in cancer therapy, and several ATX inhibitors have been developed recently [6,7]. ATX is also required for normal development since ATX deficiency in mouse leads to embryonic lethality. ATX-null mutants show impaired vessel formation, massive neural tube defects and asymmetric headfold, indicating a critical role for ATX in vascular and neuronal development [8,9]. Recently, it has been reported that ATX is highly expressed in the high endothelial venules (HEVs) of lymphoid organs to facilitate the entry of lymphocytes from the blood into secondary lymphoid organs, suggesting a profound function of ATX in the immune system [10]. However, how ATX expression is regulated in different tissues or cell lineages remains elusive [11]. LPA activates multiple signal transduction events via specific G-protein-coupled receptors [12], and has been implicated in
* Corresponding author. Fax: +86 10 58807721. E-mail address: [email protected] (J. Zhang). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.047
tumor progression and metastasis, inflammation, vascular disease, and neural development [13]. As an important lysophospholipid mediator in inflammation and immunity, LPA can modulate immune response by attracting and activating T-cells, B-cells and macrophages directly or influencing their interaction with other cell types [14,15]. So far, the mechanism by which LPA levels are regulated during inflammation and immune responses is essentially unknown. In the present study, we investigated the ATX-LPA axis in monocytic THP-1 cells. LPS was chosen as the stimulus, since it is a component of the gram-negative bacterial cell wall, one of the most potent activators of monocytes and macrophages. Stimulation of monocytes by LPS results in generation of a number of inflammatory mediators, growth factors and adhesion molecules [16]. We found that the expression of ATX was induced in the LPS-stimulated human monocytic THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and that ATX could enhance the migrations of monocytic THP-1 cells and Jurkat T cells by upregulating LPA levels. Materials and methods Reagents and antibodies. LPS (from Escherichia coli serotype 055:B5) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma–Aldrich. Extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 MAPK inhibitor SB202190, and JNK inhibitor SP600125 were obtained from Calbiochem (La Jolla, CA). PKR inhibitor 2-aminopurine (2-AP) was obtained from Sigma–Al-
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
drich. The primary antibodies against the phosphorylated forms of JNK(Thr183/Tyr185), p38 MAPK(Thr180/Tyr182), and ERK(Thr202/ Tyr204), as well as the primary antibodies against total ERK and total p38 MAPK were purchased from Cell Signaling Technology (Beverly, MA). The primary antibodies against total JNK and total PKR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibody against phosphorylated PKR (Thr 451) was from Upstate Biotechnology (Lake Placid, NY). Construction of plasmids. The DNA fragment encoding 58–157 amino acid residues of human ATX was amplified by PCR using ATX isoform b cDNA as template. The obtained DNA fragment was cloned into the pET21cc vector (Novagen), creating plasmid pET21c-ATX(58–157). The full length ATX isoform b cDNA was cloned into the pcDNA4/TO vector (Invitrogen), creating the plasmid pcDNA4-ATX. Cell culture. The THP-1 cells were cultured in RPMI-1640 medium supplemented with fetal bovine serum (10%), L-glutamine (2 mM), streptomycin (100 lg/ml) and penicillin (100 U/ml) at 37 °C in a humidified atmosphere containing 5% CO2. For experiments to detect the secreted ATX, the THP-1 cells were cultured in a conditioned serum-free medium with 250 lg/ml fatty-acid free BSA as described previously [17]. Preparation of anti-ATX antibody. Escherichia coli BL21 cells harboring pET-21c-ATX(58–157) were cultured at 37 °C in the presence of 1 mM IPTG to induce the expression of Autotaxin(58– 157)-His6 fusion protein, which was purified by Ni–NTA resins (Novagen) and then used to immunize rabbit to prepare anti-Autotaxin polyclonal antibody. Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted from THP-1 cells with Trizol (Invitrogen) according to the protocol supplied by manufacturer. Reverse transcription was performed with Reverse Transcription System (Promega). cDNA encoding ATX and GAPDH were amplified using specific primers: ATX-a forward (50 -CTCACCCTGCCAGAT CATGAG-30 ), ATX-a reverse (50 -CTCAGTTCTATCACATGTGACATC30 ), ATX-c forward (50 -GGATTGAAGCCAGCTCCTAAT-30 ), ATX-c reverse (50 -GCAACTGGTCAGATGGTCAGG-30 ), GAPDH forward (50 -TT AGC ACCCCTGGCCAAGG-30 ), and GAPDH reverse (50 -CTTACTCCTT GGAGGCCATG-30 ). The isoform of ATX expressed in THP-1 cells was characterized by RT-PCR as described previously [18] with two sets of primers for ATX-a (Set I) and ATX-c (Set II), respectively. ATX mRNA expression in THP-1 cells after LPS stimulation was detected by RT-PCR using the two primers for ATX-c (Set II). Detection of lysoPLD activity. The conditioned serum-free medium from THP-1 cells with or without exposure to LPS (2 lg/ml) was concentrated (40-fold) using Amicon Ultra 30,000 (Millipore). The lysoPLD activity in the concentrated conditioned medium was analyzed using fluorogenic substrate FS-3 as described previously [19]. Briefly, the assays were performed by mixing 50 ll concentrated medium with 10 lM FS-3 at 37 °C for 4 h. LysoPLD activity was measured by detecting the fluorescence increase with 494 and 520 nm as the excitation and emission wavelengths, respectively. Cell migration assay. The migrations of THP-1 and Jurkat cells were determined using transwell assays. THP-1 or Jurkat cells (1 105) were added to the upper chambers of Transwells (pore size, 5 lm; Costar 3421) and allowed to migrate for 3 h at 37 °C in the conditioned serum-free medium from the THP-1 cells transfected with pcDNA4-ATX plasmid or pcDNA4/TO vector. LPA (1 lM) or LPC (10 lM) was added to the upper chamber to analyze the cell chemokinetic response. For the pertussis toxin (PTX) inhibition experiments, the cells were pretreated with PTX (250 ng/ml; Fluka) for 2 h at 37 °C. Migrated cells were collected from the lower chamber and quantified with Beckman Z1 cell counter.
265
Results and discussion LPS induces ATX expression in monocytic THP-1 cells LPA is a lysophospholipid mediator in the blood, with immunomodulatory functions to affect cells and organs of the immune system. The mechanisms by which LPA levels are regulated in inflammation and immune responses remain elusive. Recently, ATX was found to have the lysoPLD activity to generate LPA from LPC in the blood [20]. In this study, we examined ATX expression in human monocytic THP-1 cells in the presence of LPS, a wellknown initiator of the inflammatory response. LPS (2 lg/ml) induced an increase in ATX mRNA expression in THP-1 cells in a time-dependent manner (Fig. 1A). Although both LPS and PMA (phorbol 12-myristate 13-acetate) can induce differentiation of THP-1 cells [21,22], LPS, but not PMA, upregulated ATX expression (Fig. 1A and B), suggesting that ATX induction is not directly related to differentiation. ATX expression was dose-dependently induced by various amounts of LPS (0.02–5 lg/ml) at both mRNA and protein levels (Fig. 1C), which resulted in an increase of the secreted ATX in culture medium as detected by western blot analysis (Fig. 1C). These results indicate that ATX induction is one of the targets of LPS in the monocytic THP-1 cells. LPS-induced ATX in THP-1 is characterized as isoform b with a LysoPLD activity The human ATX gene is organized in 27 exons, and three alternative splicing isoforms of ATX, named a, b, and c, have been reported [18]. Compared to the isoform b, the isoform a contains a 52 residue-insertion (exon 12) in the catalytic domain, and the isoform c contains a 25-residue insertion (exon 21) close to the nuclease-like domain (Fig. 1D). The isoform of LPS-induced ATX in THP-1 cells was characterized by RT-PCR with two sets of primers for ATX-a (Set I) and ATX-c (Set II) respectively. Based on the sizes of two fragments (237 and 405 bp, respectively) resulted from the amplifications (Fig. 1E, lanes 1 and 2), the LPS-induced ATX in THP-1 cells was identified as the b isoform. To determine whether the ATX protein secreted from THP-1 cells was functionally active, the ATX/lysoPLD activity in THP-1 culture medium was measured with FS-3 as substrate. Hydrolysis of the substrate by ATX/lysoPLD results in a measurable increase in fluorescence. The lysoPLD activity in THP-1 cell culture medium with LPS stimulation was significantly higher than the basal level activity in non-stimulated cell culture medium (Fig. 1F), indicating that LPS was able to increase lysoPLD activity in cell culture medium by inducing ATX expression. Activation of JNK and p38 MAPK is required for LPS-induced ATX expression To explore the signaling mechanisms of LPS-induced ATX expression, we initially focused on mitogen-activated protein kinase (MAPK) signaling pathways, since MAPKs are involved in LPS-mediated induction of gene expression [23]. It was found that LPS induced ERK, JNK, and p38 MAPK activation in THP-1 cells as detected by Western blot analysis with corresponding phosphospecific antibodies, while PMA induced ERK activation and a weak JNK activation, but not p38 MAPK activation (Fig. 2A), suggesting that the difference in MAPKs activation may be the reason for the different effects of LPS and PMA on ATX expression in THP-1 cells. When JNK or p38 MAPK signaling pathway was inhibited by its specific kinase inhibitor (SP600125 for JNK- and SB202190 for p38-mediated signaling; Fig. 2B), LPS-induced ATX expression in THP-1 cells was suppressed at both the mRNA and protein levels
266
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
Fig. 1. LPS induces ATX expression in monocytic THP-1 cells. (A,B) THP-1 cells were stimulated with LPS (2 lg/ml) (A) or PMA (100 nM) (B) for the indicated times. LPSinduced ATX mRNA expression in THP-1 cells was detected by RT-PCR. (C) After stimulation with various amounts of LPS (0.02–5 lg/ml) for 24 h, ATX mRNA levels in THP-1 cells were detected by RT-PCR, and the secreted ATX protein levels in culture medium were detected by Western blot analyses. (D) The alternative splicing forms of ATX. The predicted fragments from RT-PCR with Set I primers are 393, 237, and 237 bp for ATX-a, ATX-b, and ATX-c, respectively. The predicted fragments from RT-PCR with Set II primers are 405, 405, and 480 bp for ATX-a, ATX-b, and ATX-c, respectively. (E) Total RNA was extracted from THP-1 cells after stimulation with LPS (2 lg/ml) for 24 h. The designed Set I primers (lane 1) and Set II primers (lane 2) were used to discriminate the splicing form of ATX mRNA by RT-PCR as described previously [18]. (F) After stimulation with LPS (2 lg/ml) for 24 h, the lysoPLD activity in concentrated (40-fold) conditioned serum-free culture medium of THP-1 cells was analyzed using FS-3 as substrate. The conditioned serum-free medium without cell and the conditioned serum-free culture medium of unstimulated THP-1 cells were concentrated (40-fold) and used as controls. Data represent the mean and SD of triplicate determinations. Statistical analysis was performed using Student’s t test. Significance was assumed at a p value of less than 0.01 (*p < 0.01).
(Fig. 2C). SP600125 and SB202190 efficiently suppressed ATX expression at 10 and 5 lM, respectively (Fig. 2C, lanes 2 and 3). However, the MEK specific inhibitor PD98059, although it effectively blocked ERK activation (Fig. 2B), could not inhibit LPS-induced ATX expression even at a concentration as high as 50 lM (Fig. 2C, lane 1), indicating that the activation of JNK and p38 MAPK, but not ERK, is required for LPS-induced ATX expression. PKR functions as an upstream regulator of LPS-induced ATX expression The double-stranded RNA-activated serine/threonine kinase R (PKR) is regarded as an upstream molecule implicated in TLR signal transduction in response to bacterial LPS [24]. LPS could induce PKR phosphorylation in THP-1 cells, but PMA could not (Fig. 3A). The PKR activation in LPS-stimulated THP-1 cells was inhibited by PKR inhibitor 2-aminopurine (2-AP) (Fig. 3B). The pharmacologic inhibition of PKR activity by 2-AP significantly attenuated the LPS-induced activation of JNK and p38 MAPK (Fig. 3B), leading to the inhibition of LPS-induced ATX expression (Fig. 3C). 2-AP inhibited the LPS-induced ATX expression in a dose-dependent manner (Fig. 3C), and suppressed ATX expression to the basal level at 5 mM (Fig. 3C, lane 6). These data suggest that PKR activation is required for the LPS-induced ATX expression, and that PKR may function as an upstream regulator of LPS-induced ATX expression in THP-1 cells via affecting the JNK and p38 MAPK pathways.
ATX enhances migrations of THP-1and Jurkat cells via a Gi-signaling pathway To investigate the biological effects of ATX upregulation in THP-1 cells, ATX was ectopically expressed in THP-1 cells using the plasmid pcDNA4-ATX, with the empty vector pcDNA4/TO as control. Compared with the conditioned serum-free medium from the vectortransfected cells, the conditioned serum-free medium obtained from pcDNA4-ATX-transfected cells contained high-level secreted ATX (Fig. 4A). The ATX-rich culture medium had no significant effects on the migrations of THP-1 and Jurkat cells by itself, but it could significantly promote their migrations when LPC was supplied exogenously (Fig. 4B and C), suggesting that the ATX-induced cell migration is dependent on the presence of LPC. However, in the presence of LPA in the top chamber of the Transwells, migrations of THP-1 and Jurkat cells were enhanced significantly regardless ATX levels in the medium. In addition, both the ATX/LPC- and LPA-induced cell migrations were inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated signaling (Fig. 4B and C). These data support the notion that the ATX-induced cell migration is dependent on the LPA production from LPC and the LPA receptor-Gi signaling. Interestingly, migrations of THP-1 and Jurkat cells were not induced when LPA was added to the lower chambers of the Transwells, suggesting that LPA has a chemokinetic, but not chemotactic effect on THP-1 and Jurkat cells (data not shown).
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
267
Fig. 2. Activation of JNK and p38 MAPK is required for LPS-induced ATX expression in THP-1 cells. (A) The effects of PMA and LPS on MAPKs activation in THP-1 cells. Cells were treated with PMA (100 nM) or LPS (2 lg/ml) for the indicated times, and then cell lysates were subjected to SDS–PAGE and immunoblotting to detect the phosphorylation of ERK, JNK, or p38 MAPK. (B) Effects of specific inhibitors on LPS-induced MAPKs activation in THP-1 cells. Cells were pretreated with PD98059 (20 lM), SP600125 (10 lM), or SB202190 (5 lM), for 30 min prior to stimulation with LPS (2 lg/ml; 30 min). Cell lysates were subjected to SDS–PAGE and immunoblotting to detect the phosphorylation of ERK, JNK, or p38 MAPK. (C) Effects of MAPKs inhibitors on LPS-induced ATX expression in THP-1 cells. THP-1 cells were treated with PD98059 (50 lM), SP600125 (10 lM), SB202190 (5 lM) or DMSO (0.1%; a solvent control) for 30 min prior to LPS (2 lg/ml) treatment. After LPS stimulation for 24 h, ATX mRNA expression was measured by RT-PCR, and the ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. Data shown are representatives of three independent experiments.
Fig. 3. Activation of PKR is required for LPS-induced ATX expression in THP-1 cells. (A) The effects of PMA and LPS on PKR activation in THP-1 cells. Cells were treated with PMA (100 nM) or LPS (2 lg/ml) for the indicated times, and then cell lysates were subjected to- SDS–PAGE and immunoblotting to detect the phosphorylation of PKR. (B) 2-AP inhibited PKR, JNK and p38 MAPK activation in LPS-stimulated THP-1 cells. THP-1 cells were treated with agonists as indicated. Lane 1, untreated; lane 2, treated with 2-AP (5 mM) for 1 hr; lane 3, treated with LPS (2 lg/ml) for 30 min; lane 4, pretreated with 2-AP (5 mM) for 30 min before the 30 min stimulation with LPS (2 lg/ml). Cell lysates were subjected to SDS–PAGE and immunoblotting. Phosphorylated PKR, JNK and p38 MAPK were detected using phospho-specific antibodies. (C) 2-AP inhibited LPS-induced ATX mRNA and protein expression in a dose-dependent manner. THP-1 cells were pretreated with various amounts of 2-AP as indicated for 30 min before exposure to LPS (2 lg/ml). After LPS stimulation for 24 h, ATX mRNA expression was measured by RT-PCR, and the ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. Data shown are representatives of three independent experiments.
ATX has a lysoPLD activity to generate bioactive lysophospholipid LPA, which is involved in cell proliferation, survival, migration, and invasion, as well as many additional effects by
activating its specific receptors. In the present study, we demonstrated that ATX expression was induced in human monocytic THP-1 cells by LPS, and that the activation of PKR, JNK, and p38
268
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
A
References
Control ATX
B Cell number
25000 20000 Control
15000
ATX
10000 5000 0 Vehicle
C
LPC LPC+PTX LPA LPA+PTX
Cell number
25000 20000 Control
15000
ATX
10000 5000 0 Vehicle
LPC LPC+PTX LPA LPA+PTX
Fig. 4. Effects of ATX on the migrations of THP-1 and Jurkat cells. (A) THP-1 cells were transfected with the ATX-expressing plasmid pcDNA4-ATX or the vector pcDNA4/TO (Control). The conditioned serum-free medium was collected at 36 h post-transfection. ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. (B,C) Transwell migration assays of THP-1 (B) and Jurkat (C) cells. THP-1 or Jurkat cells (1 105) were added in the upper chambers of Transwells, incubated in the ATX-rich conditioned serum-free medium or in the control medium, and allowed to migrate for 3 h at 37 °C. To analyze the chemokinetic responses of the cells, LPC (10 lM) or LPA (1 lM) was added in the upper chambers. For the pertussis toxin (PTX) inhibition experiments, the THP-1 cells were pretreated with PTX (250 ng/ml) for 2 h at 37 °C. Migrated cells were collected from the lower chamber and quantified with Beckman Z1 cell counter. Data represent the mean and SD of triplicate determinations. Statistical analysis was performed using Student’s t test. Significance was assumed at a p value of less than 0.01 (*p < 0.01).
MAPK is required for the ATX induction. Further studies are needed to determine which downstream transcription factors of the JNK and p38 MAPK pathways are involved in ATX expression regulation in THP-1 cells. ATX enhanced the migrations of monocytic THP-1 cells and Jurkat T cells likely through producing LPA from LPC and then activating the LPA receptor-Gi mediated signaling pathways. Therefore, we hypothesize that bacterial infection may increase the motility of immune cells via upregulating ATX expression and increasing LPA levels in the microenvironment. Together with the published LPA effects on gene expression, cell migration, and apoptosis in B-cells, NK cells, and macrophages [25–27], our data support the importance of the ATX-LPA axis in inflammation and immune processes, and provide intriguing and important bases for further elucidation of the mechanisms by which LPA levels are regulated to stimulate cellular functions in inflammation responses. Acknowledgments This work was supported by grants from the National High Technology Research and Development Program (No. 2006AA02Z4A6), Science and Technology Program of Beijing Municipality (No. Z0006329000191), the National Nature Science Foundation of China (Nos. 30570409 and 30770030), the Beijing NOVA Program (No. 2005B47), and the Program for New Century Excellent Talents in University (No. NCET-05-0145). We thank Dr. Yan Xu (Indiana University, Indianapolis) for her advices and critical reading of this manuscript.
[1] M. Umezu-Goto, Y. Kishi, A. Taira, K. Hama, N. Dohmae, K. Takio, T. Yamori, G.B. Mills, K. Inoue, J. Aoki, H. Arai, Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production, J. Cell. Biol. 158 (2002) 227–233. [2] S.W. Nam, T. Clair, C.K. Campo, H.Y. Lee, L.A. Liotta, M.L. Stracke, Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cell, Oncogene 19 (2000) 241–247. [3] Y. Yang, L. Mou, N. Liu, M.S. Tsao, Autotaxin expression in non-small-cell lung cancer, Am. J. Respir. Cell. Mol. Biol. 21 (1999) 216–222. [4] S.Y. Yang, J. Lee, C.G. Park, S. Kim, S. Hong, H.C. Chung, S.K. Min, J.W. Han, H.W. Lee, H.Y. Lee, Expression of autotaxin (NPP-2) is closely linked to invasiveness of breast cancer cells, Clin Exp Metastas 19 (2002) 603–638. [5] A. Kehlen, N. Englert, A. Seifert, T. Klonisch, H. Dralle, J. Langner, C. Hoang-Vu, Expression, regulation and function of autotaxin in thyroid carcinomas, Int. J. Cancer 109 (2004) 833–838. [6] A.L. Parrill, U. Echols, T. Nguyen, T.C. Pham, A. Hoeglund, D.L. Baker, Virtual screening approaches for the identification of non-lipid autotaxin inhibitors, Bioorg. Med. Chem. 16 (2008) 1784–1795. [7] L.A. van Meeteren, V. Brinkmann, J.S. Saulnier-Blache, K.R. Lynch, W.H. Moolenaar, Anticancer activity of FTY720: phosphorylated FTY720 inhibits autotaxin, a metastasis-enhancing and angiogenic lysophospholipase D, Cancer Lett. 266 (2008) 203–208. [8] M. Tanaka, S. Okudaira, Y. Kishi, R. Ohkawa, S. Iseki, M. Ota, S. Noji, Y. Yatomi, J. Aoki, H. Arai, Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281 (2006) 25822–25830. [9] L.A. van Meeteren, P. Ruurs, C. Stortelers, P. Bouwman, M.A. van Rooijen, J.P. Pradere, T.R. Pettit, M.J. Wakelam, J.S. Saulnier-Blache, C.L. Mummery, W.H. Moolenaar, J. Jonkers, Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26 (2006) 5015–5022. [10] H. Kanda, R. Newton, R. Klein, Y. Morita, M.D. Gunn, S.D. Rosen, Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs, Nat. Immunol. 9 (2008) 415– 423. [11] L.A. van Meeteren, W.H. Moolenaar, Regulation and biological activities of the autotaxin-LPA axis, Prog. Lipid Res. 46 (2007) 145–160. [12] I. Ishii, N. Fukushima, X. Ye, J. Chun, Lysophospholipid receptors: signaling and biology, Annu. Rev. Biochem. 73 (2004) 321–354. [13] G.B. Mills, W.H. Moolenaar, The emerging role of lysophosphatidic acid in cancer, Nat. Rev. Cancer 3 (2003) 582–591. [14] M.H. Graler, E.J. Goetzl, Lysophospholipids and their G protein-coupled receptors in inflammation and immunity, Biochim. Biophys. Acta 1582 (2002) 168–174. [15] D.A. Lin, J.A. Boyce, Lysophospholipids as mediators of immunity, Adv. Immunol. 89 (2006) 141–167. [16] M. Guha, N. Mackman, LPS induction of gene expression in human monocytes, Cell. Signal. 13 (2001) 85–94. [17] M. Chen, K.L. O’Connor, Integrin alpha6beta4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells, Oncogene 24 (2005) 5125–5130. [18] A. Giganti, M. Rodriguez, B. Fould, N. Moulharat, F. Coge, P. Chomarat, J.P. Galizzi, P. Valet, J.S. Saulnier-Blache, J.A. Boutin, G. Ferry, A sdfsdaf, J. Biol. Chem. 283 (2008) 7776–7789. [19] A.J. Morris, S.S. Smyth, Measurement of autotaxin/lysophospholipase D activity, Methods Enzymol. 434 (2007) 89–104. [20] K. Nakamura, R. Ohkawa, S. Okubo, M. Tozuka, M. Okada, S. Aoki, J. Aoki, H. Arai, H. Ikeda, Y. Yatomi, Measurement of lysophospholipase D/autotaxin activity in human serum samples, Clin. Biochem. 40 (2007) 274–277. [21] E. Huberman, C.A. Jones, Control of cell differentiation and cell transformation in vitro by phorbol 12-myristate 13-acetate and 1 alpha,25-dihydroxyvitamin D3, Carcinog. Compr. Surv. 10 (1985) 263–273. [22] C.W. Chow, S. Grinstein, O.D. Rotstein, Signaling events in monocytes and macrophages, New Horiz. 3 (1995) 342–3451. [23] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499–511. [24] B.R. Williams, Signal integration via PKR, Sci. STKE 2001 (2001) RE2. [25] X. Hu, F.J. Mendoza, J. Sun, V. Banerji, J.B. Johnston, S.B. Gibson, Lysophosphatidic acid (LPA) induces the expression of VEGF leading to protection against apoptosis in B-cell derived malignancies, Cell Signal. 20 (2008) 1198–1208. [26] Y. Jin, E. Knudsen, L. Wang, A.A. Maghazachi, Lysophosphatidic acid induces human natural killer cell chemotaxis and intracellular calcium mobilization, Eur. J. Immunol. 33 (2003) 2083–2089. [27] C.L. Chang, M.E. Lin, H.Y. Hsu, C.L. Yao, S.M. Hwang, C.Y. Pan, C.Y. Hsu, H. Lee, Lysophosphatidic acid-induced interleukin-1 beta expression is mediated through Gi/Rho and the generation of reactive oxygen species in macrophages, J. Biomed. Sci. 15 (2008) 357–363.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Lipopolysaccharide induces autotaxin expression in human monocytic THP-1 cells Song Li, Junjie Zhang * The Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, No. 19 Xinjiekouwai Avenue, Beijing 100875, China
a r t i c l e
i n f o
Article history: Received 17 October 2008 Available online 21 November 2008
Keywords: Autotaxin LPS THP-1 cells PKR JNK p38 MAPK LPA
a b s t r a c t Autotaxin (ATX) is a secreted enzyme with lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a bioactive phospholipid involved in numerous biological activities, including cell proliferation, differentiation, and migration. In the present study, we found that bacterial lipopolysaccharide (LPS), a well-known initiator of the inflammatory response, induced ATX expression in monocytic THP-1 cells. The activation of PKR, JNK, and p38 MAPK was required for the ATX induction. The LPS-induced ATX in THP-1 cells was characterized as the b isoform. In the presence of LPC, ATX could promote the migrations of THP-1 and Jurkat cells, which was inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated LPA receptor signaling. In summary, LPS induces ATX expression in THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and the ATX induction is likely to enhance immune cell migration in proinflammatory response by regulating LPA levels in the microenvironment. Ó 2008 Elsevier Inc. All rights reserved.
Autotaxin (ATX), also known as ENPP2 (ectonucleotide pyrophosphatase phosphodiesterase-2), has the lysophospholipase D (lysoPLD) activity to convert lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) [1]. Ectopic expression of ATX in Ras-transformed NIH3T3 cells stimulates their tumorigenesis and metastatic potential [2]. ATX is overexpressed in some malignant tumor tissues, including non-small cell lung cancer [3], breast cancer [4], and thyroid cancer [5]. Therefore, ATX is regarded as an attractive target in cancer therapy, and several ATX inhibitors have been developed recently [6,7]. ATX is also required for normal development since ATX deficiency in mouse leads to embryonic lethality. ATX-null mutants show impaired vessel formation, massive neural tube defects and asymmetric headfold, indicating a critical role for ATX in vascular and neuronal development [8,9]. Recently, it has been reported that ATX is highly expressed in the high endothelial venules (HEVs) of lymphoid organs to facilitate the entry of lymphocytes from the blood into secondary lymphoid organs, suggesting a profound function of ATX in the immune system [10]. However, how ATX expression is regulated in different tissues or cell lineages remains elusive [11]. LPA activates multiple signal transduction events via specific G-protein-coupled receptors [12], and has been implicated in
* Corresponding author. Fax: +86 10 58807721. E-mail address: [email protected] (J. Zhang). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.047
tumor progression and metastasis, inflammation, vascular disease, and neural development [13]. As an important lysophospholipid mediator in inflammation and immunity, LPA can modulate immune response by attracting and activating T-cells, B-cells and macrophages directly or influencing their interaction with other cell types [14,15]. So far, the mechanism by which LPA levels are regulated during inflammation and immune responses is essentially unknown. In the present study, we investigated the ATX-LPA axis in monocytic THP-1 cells. LPS was chosen as the stimulus, since it is a component of the gram-negative bacterial cell wall, one of the most potent activators of monocytes and macrophages. Stimulation of monocytes by LPS results in generation of a number of inflammatory mediators, growth factors and adhesion molecules [16]. We found that the expression of ATX was induced in the LPS-stimulated human monocytic THP-1 cells via a PKR, JNK and p38 MAPK-mediated mechanism, and that ATX could enhance the migrations of monocytic THP-1 cells and Jurkat T cells by upregulating LPA levels. Materials and methods Reagents and antibodies. LPS (from Escherichia coli serotype 055:B5) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma–Aldrich. Extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 MAPK inhibitor SB202190, and JNK inhibitor SP600125 were obtained from Calbiochem (La Jolla, CA). PKR inhibitor 2-aminopurine (2-AP) was obtained from Sigma–Al-
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
drich. The primary antibodies against the phosphorylated forms of JNK(Thr183/Tyr185), p38 MAPK(Thr180/Tyr182), and ERK(Thr202/ Tyr204), as well as the primary antibodies against total ERK and total p38 MAPK were purchased from Cell Signaling Technology (Beverly, MA). The primary antibodies against total JNK and total PKR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibody against phosphorylated PKR (Thr 451) was from Upstate Biotechnology (Lake Placid, NY). Construction of plasmids. The DNA fragment encoding 58–157 amino acid residues of human ATX was amplified by PCR using ATX isoform b cDNA as template. The obtained DNA fragment was cloned into the pET21cc vector (Novagen), creating plasmid pET21c-ATX(58–157). The full length ATX isoform b cDNA was cloned into the pcDNA4/TO vector (Invitrogen), creating the plasmid pcDNA4-ATX. Cell culture. The THP-1 cells were cultured in RPMI-1640 medium supplemented with fetal bovine serum (10%), L-glutamine (2 mM), streptomycin (100 lg/ml) and penicillin (100 U/ml) at 37 °C in a humidified atmosphere containing 5% CO2. For experiments to detect the secreted ATX, the THP-1 cells were cultured in a conditioned serum-free medium with 250 lg/ml fatty-acid free BSA as described previously [17]. Preparation of anti-ATX antibody. Escherichia coli BL21 cells harboring pET-21c-ATX(58–157) were cultured at 37 °C in the presence of 1 mM IPTG to induce the expression of Autotaxin(58– 157)-His6 fusion protein, which was purified by Ni–NTA resins (Novagen) and then used to immunize rabbit to prepare anti-Autotaxin polyclonal antibody. Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted from THP-1 cells with Trizol (Invitrogen) according to the protocol supplied by manufacturer. Reverse transcription was performed with Reverse Transcription System (Promega). cDNA encoding ATX and GAPDH were amplified using specific primers: ATX-a forward (50 -CTCACCCTGCCAGAT CATGAG-30 ), ATX-a reverse (50 -CTCAGTTCTATCACATGTGACATC30 ), ATX-c forward (50 -GGATTGAAGCCAGCTCCTAAT-30 ), ATX-c reverse (50 -GCAACTGGTCAGATGGTCAGG-30 ), GAPDH forward (50 -TT AGC ACCCCTGGCCAAGG-30 ), and GAPDH reverse (50 -CTTACTCCTT GGAGGCCATG-30 ). The isoform of ATX expressed in THP-1 cells was characterized by RT-PCR as described previously [18] with two sets of primers for ATX-a (Set I) and ATX-c (Set II), respectively. ATX mRNA expression in THP-1 cells after LPS stimulation was detected by RT-PCR using the two primers for ATX-c (Set II). Detection of lysoPLD activity. The conditioned serum-free medium from THP-1 cells with or without exposure to LPS (2 lg/ml) was concentrated (40-fold) using Amicon Ultra 30,000 (Millipore). The lysoPLD activity in the concentrated conditioned medium was analyzed using fluorogenic substrate FS-3 as described previously [19]. Briefly, the assays were performed by mixing 50 ll concentrated medium with 10 lM FS-3 at 37 °C for 4 h. LysoPLD activity was measured by detecting the fluorescence increase with 494 and 520 nm as the excitation and emission wavelengths, respectively. Cell migration assay. The migrations of THP-1 and Jurkat cells were determined using transwell assays. THP-1 or Jurkat cells (1 105) were added to the upper chambers of Transwells (pore size, 5 lm; Costar 3421) and allowed to migrate for 3 h at 37 °C in the conditioned serum-free medium from the THP-1 cells transfected with pcDNA4-ATX plasmid or pcDNA4/TO vector. LPA (1 lM) or LPC (10 lM) was added to the upper chamber to analyze the cell chemokinetic response. For the pertussis toxin (PTX) inhibition experiments, the cells were pretreated with PTX (250 ng/ml; Fluka) for 2 h at 37 °C. Migrated cells were collected from the lower chamber and quantified with Beckman Z1 cell counter.
265
Results and discussion LPS induces ATX expression in monocytic THP-1 cells LPA is a lysophospholipid mediator in the blood, with immunomodulatory functions to affect cells and organs of the immune system. The mechanisms by which LPA levels are regulated in inflammation and immune responses remain elusive. Recently, ATX was found to have the lysoPLD activity to generate LPA from LPC in the blood [20]. In this study, we examined ATX expression in human monocytic THP-1 cells in the presence of LPS, a wellknown initiator of the inflammatory response. LPS (2 lg/ml) induced an increase in ATX mRNA expression in THP-1 cells in a time-dependent manner (Fig. 1A). Although both LPS and PMA (phorbol 12-myristate 13-acetate) can induce differentiation of THP-1 cells [21,22], LPS, but not PMA, upregulated ATX expression (Fig. 1A and B), suggesting that ATX induction is not directly related to differentiation. ATX expression was dose-dependently induced by various amounts of LPS (0.02–5 lg/ml) at both mRNA and protein levels (Fig. 1C), which resulted in an increase of the secreted ATX in culture medium as detected by western blot analysis (Fig. 1C). These results indicate that ATX induction is one of the targets of LPS in the monocytic THP-1 cells. LPS-induced ATX in THP-1 is characterized as isoform b with a LysoPLD activity The human ATX gene is organized in 27 exons, and three alternative splicing isoforms of ATX, named a, b, and c, have been reported [18]. Compared to the isoform b, the isoform a contains a 52 residue-insertion (exon 12) in the catalytic domain, and the isoform c contains a 25-residue insertion (exon 21) close to the nuclease-like domain (Fig. 1D). The isoform of LPS-induced ATX in THP-1 cells was characterized by RT-PCR with two sets of primers for ATX-a (Set I) and ATX-c (Set II) respectively. Based on the sizes of two fragments (237 and 405 bp, respectively) resulted from the amplifications (Fig. 1E, lanes 1 and 2), the LPS-induced ATX in THP-1 cells was identified as the b isoform. To determine whether the ATX protein secreted from THP-1 cells was functionally active, the ATX/lysoPLD activity in THP-1 culture medium was measured with FS-3 as substrate. Hydrolysis of the substrate by ATX/lysoPLD results in a measurable increase in fluorescence. The lysoPLD activity in THP-1 cell culture medium with LPS stimulation was significantly higher than the basal level activity in non-stimulated cell culture medium (Fig. 1F), indicating that LPS was able to increase lysoPLD activity in cell culture medium by inducing ATX expression. Activation of JNK and p38 MAPK is required for LPS-induced ATX expression To explore the signaling mechanisms of LPS-induced ATX expression, we initially focused on mitogen-activated protein kinase (MAPK) signaling pathways, since MAPKs are involved in LPS-mediated induction of gene expression [23]. It was found that LPS induced ERK, JNK, and p38 MAPK activation in THP-1 cells as detected by Western blot analysis with corresponding phosphospecific antibodies, while PMA induced ERK activation and a weak JNK activation, but not p38 MAPK activation (Fig. 2A), suggesting that the difference in MAPKs activation may be the reason for the different effects of LPS and PMA on ATX expression in THP-1 cells. When JNK or p38 MAPK signaling pathway was inhibited by its specific kinase inhibitor (SP600125 for JNK- and SB202190 for p38-mediated signaling; Fig. 2B), LPS-induced ATX expression in THP-1 cells was suppressed at both the mRNA and protein levels
266
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
Fig. 1. LPS induces ATX expression in monocytic THP-1 cells. (A,B) THP-1 cells were stimulated with LPS (2 lg/ml) (A) or PMA (100 nM) (B) for the indicated times. LPSinduced ATX mRNA expression in THP-1 cells was detected by RT-PCR. (C) After stimulation with various amounts of LPS (0.02–5 lg/ml) for 24 h, ATX mRNA levels in THP-1 cells were detected by RT-PCR, and the secreted ATX protein levels in culture medium were detected by Western blot analyses. (D) The alternative splicing forms of ATX. The predicted fragments from RT-PCR with Set I primers are 393, 237, and 237 bp for ATX-a, ATX-b, and ATX-c, respectively. The predicted fragments from RT-PCR with Set II primers are 405, 405, and 480 bp for ATX-a, ATX-b, and ATX-c, respectively. (E) Total RNA was extracted from THP-1 cells after stimulation with LPS (2 lg/ml) for 24 h. The designed Set I primers (lane 1) and Set II primers (lane 2) were used to discriminate the splicing form of ATX mRNA by RT-PCR as described previously [18]. (F) After stimulation with LPS (2 lg/ml) for 24 h, the lysoPLD activity in concentrated (40-fold) conditioned serum-free culture medium of THP-1 cells was analyzed using FS-3 as substrate. The conditioned serum-free medium without cell and the conditioned serum-free culture medium of unstimulated THP-1 cells were concentrated (40-fold) and used as controls. Data represent the mean and SD of triplicate determinations. Statistical analysis was performed using Student’s t test. Significance was assumed at a p value of less than 0.01 (*p < 0.01).
(Fig. 2C). SP600125 and SB202190 efficiently suppressed ATX expression at 10 and 5 lM, respectively (Fig. 2C, lanes 2 and 3). However, the MEK specific inhibitor PD98059, although it effectively blocked ERK activation (Fig. 2B), could not inhibit LPS-induced ATX expression even at a concentration as high as 50 lM (Fig. 2C, lane 1), indicating that the activation of JNK and p38 MAPK, but not ERK, is required for LPS-induced ATX expression. PKR functions as an upstream regulator of LPS-induced ATX expression The double-stranded RNA-activated serine/threonine kinase R (PKR) is regarded as an upstream molecule implicated in TLR signal transduction in response to bacterial LPS [24]. LPS could induce PKR phosphorylation in THP-1 cells, but PMA could not (Fig. 3A). The PKR activation in LPS-stimulated THP-1 cells was inhibited by PKR inhibitor 2-aminopurine (2-AP) (Fig. 3B). The pharmacologic inhibition of PKR activity by 2-AP significantly attenuated the LPS-induced activation of JNK and p38 MAPK (Fig. 3B), leading to the inhibition of LPS-induced ATX expression (Fig. 3C). 2-AP inhibited the LPS-induced ATX expression in a dose-dependent manner (Fig. 3C), and suppressed ATX expression to the basal level at 5 mM (Fig. 3C, lane 6). These data suggest that PKR activation is required for the LPS-induced ATX expression, and that PKR may function as an upstream regulator of LPS-induced ATX expression in THP-1 cells via affecting the JNK and p38 MAPK pathways.
ATX enhances migrations of THP-1and Jurkat cells via a Gi-signaling pathway To investigate the biological effects of ATX upregulation in THP-1 cells, ATX was ectopically expressed in THP-1 cells using the plasmid pcDNA4-ATX, with the empty vector pcDNA4/TO as control. Compared with the conditioned serum-free medium from the vectortransfected cells, the conditioned serum-free medium obtained from pcDNA4-ATX-transfected cells contained high-level secreted ATX (Fig. 4A). The ATX-rich culture medium had no significant effects on the migrations of THP-1 and Jurkat cells by itself, but it could significantly promote their migrations when LPC was supplied exogenously (Fig. 4B and C), suggesting that the ATX-induced cell migration is dependent on the presence of LPC. However, in the presence of LPA in the top chamber of the Transwells, migrations of THP-1 and Jurkat cells were enhanced significantly regardless ATX levels in the medium. In addition, both the ATX/LPC- and LPA-induced cell migrations were inhibited by pertussis toxin (PTX), an inhibitor of Gi-mediated signaling (Fig. 4B and C). These data support the notion that the ATX-induced cell migration is dependent on the LPA production from LPC and the LPA receptor-Gi signaling. Interestingly, migrations of THP-1 and Jurkat cells were not induced when LPA was added to the lower chambers of the Transwells, suggesting that LPA has a chemokinetic, but not chemotactic effect on THP-1 and Jurkat cells (data not shown).
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
267
Fig. 2. Activation of JNK and p38 MAPK is required for LPS-induced ATX expression in THP-1 cells. (A) The effects of PMA and LPS on MAPKs activation in THP-1 cells. Cells were treated with PMA (100 nM) or LPS (2 lg/ml) for the indicated times, and then cell lysates were subjected to SDS–PAGE and immunoblotting to detect the phosphorylation of ERK, JNK, or p38 MAPK. (B) Effects of specific inhibitors on LPS-induced MAPKs activation in THP-1 cells. Cells were pretreated with PD98059 (20 lM), SP600125 (10 lM), or SB202190 (5 lM), for 30 min prior to stimulation with LPS (2 lg/ml; 30 min). Cell lysates were subjected to SDS–PAGE and immunoblotting to detect the phosphorylation of ERK, JNK, or p38 MAPK. (C) Effects of MAPKs inhibitors on LPS-induced ATX expression in THP-1 cells. THP-1 cells were treated with PD98059 (50 lM), SP600125 (10 lM), SB202190 (5 lM) or DMSO (0.1%; a solvent control) for 30 min prior to LPS (2 lg/ml) treatment. After LPS stimulation for 24 h, ATX mRNA expression was measured by RT-PCR, and the ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. Data shown are representatives of three independent experiments.
Fig. 3. Activation of PKR is required for LPS-induced ATX expression in THP-1 cells. (A) The effects of PMA and LPS on PKR activation in THP-1 cells. Cells were treated with PMA (100 nM) or LPS (2 lg/ml) for the indicated times, and then cell lysates were subjected to- SDS–PAGE and immunoblotting to detect the phosphorylation of PKR. (B) 2-AP inhibited PKR, JNK and p38 MAPK activation in LPS-stimulated THP-1 cells. THP-1 cells were treated with agonists as indicated. Lane 1, untreated; lane 2, treated with 2-AP (5 mM) for 1 hr; lane 3, treated with LPS (2 lg/ml) for 30 min; lane 4, pretreated with 2-AP (5 mM) for 30 min before the 30 min stimulation with LPS (2 lg/ml). Cell lysates were subjected to SDS–PAGE and immunoblotting. Phosphorylated PKR, JNK and p38 MAPK were detected using phospho-specific antibodies. (C) 2-AP inhibited LPS-induced ATX mRNA and protein expression in a dose-dependent manner. THP-1 cells were pretreated with various amounts of 2-AP as indicated for 30 min before exposure to LPS (2 lg/ml). After LPS stimulation for 24 h, ATX mRNA expression was measured by RT-PCR, and the ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. Data shown are representatives of three independent experiments.
ATX has a lysoPLD activity to generate bioactive lysophospholipid LPA, which is involved in cell proliferation, survival, migration, and invasion, as well as many additional effects by
activating its specific receptors. In the present study, we demonstrated that ATX expression was induced in human monocytic THP-1 cells by LPS, and that the activation of PKR, JNK, and p38
268
S. Li, J. Zhang / Biochemical and Biophysical Research Communications 378 (2009) 264–268
A
References
Control ATX
B Cell number
25000 20000 Control
15000
ATX
10000 5000 0 Vehicle
C
LPC LPC+PTX LPA LPA+PTX
Cell number
25000 20000 Control
15000
ATX
10000 5000 0 Vehicle
LPC LPC+PTX LPA LPA+PTX
Fig. 4. Effects of ATX on the migrations of THP-1 and Jurkat cells. (A) THP-1 cells were transfected with the ATX-expressing plasmid pcDNA4-ATX or the vector pcDNA4/TO (Control). The conditioned serum-free medium was collected at 36 h post-transfection. ATX protein secreted from THP-1 cells into the culture medium was detected by Western blot analyses. (B,C) Transwell migration assays of THP-1 (B) and Jurkat (C) cells. THP-1 or Jurkat cells (1 105) were added in the upper chambers of Transwells, incubated in the ATX-rich conditioned serum-free medium or in the control medium, and allowed to migrate for 3 h at 37 °C. To analyze the chemokinetic responses of the cells, LPC (10 lM) or LPA (1 lM) was added in the upper chambers. For the pertussis toxin (PTX) inhibition experiments, the THP-1 cells were pretreated with PTX (250 ng/ml) for 2 h at 37 °C. Migrated cells were collected from the lower chamber and quantified with Beckman Z1 cell counter. Data represent the mean and SD of triplicate determinations. Statistical analysis was performed using Student’s t test. Significance was assumed at a p value of less than 0.01 (*p < 0.01).
MAPK is required for the ATX induction. Further studies are needed to determine which downstream transcription factors of the JNK and p38 MAPK pathways are involved in ATX expression regulation in THP-1 cells. ATX enhanced the migrations of monocytic THP-1 cells and Jurkat T cells likely through producing LPA from LPC and then activating the LPA receptor-Gi mediated signaling pathways. Therefore, we hypothesize that bacterial infection may increase the motility of immune cells via upregulating ATX expression and increasing LPA levels in the microenvironment. Together with the published LPA effects on gene expression, cell migration, and apoptosis in B-cells, NK cells, and macrophages [25–27], our data support the importance of the ATX-LPA axis in inflammation and immune processes, and provide intriguing and important bases for further elucidation of the mechanisms by which LPA levels are regulated to stimulate cellular functions in inflammation responses. Acknowledgments This work was supported by grants from the National High Technology Research and Development Program (No. 2006AA02Z4A6), Science and Technology Program of Beijing Municipality (No. Z0006329000191), the National Nature Science Foundation of China (Nos. 30570409 and 30770030), the Beijing NOVA Program (No. 2005B47), and the Program for New Century Excellent Talents in University (No. NCET-05-0145). We thank Dr. Yan Xu (Indiana University, Indianapolis) for her advices and critical reading of this manuscript.
[1] M. Umezu-Goto, Y. Kishi, A. Taira, K. Hama, N. Dohmae, K. Takio, T. Yamori, G.B. Mills, K. Inoue, J. Aoki, H. Arai, Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production, J. Cell. Biol. 158 (2002) 227–233. [2] S.W. Nam, T. Clair, C.K. Campo, H.Y. Lee, L.A. Liotta, M.L. Stracke, Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cell, Oncogene 19 (2000) 241–247. [3] Y. Yang, L. Mou, N. Liu, M.S. Tsao, Autotaxin expression in non-small-cell lung cancer, Am. J. Respir. Cell. Mol. Biol. 21 (1999) 216–222. [4] S.Y. Yang, J. Lee, C.G. Park, S. Kim, S. Hong, H.C. Chung, S.K. Min, J.W. Han, H.W. Lee, H.Y. Lee, Expression of autotaxin (NPP-2) is closely linked to invasiveness of breast cancer cells, Clin Exp Metastas 19 (2002) 603–638. [5] A. Kehlen, N. Englert, A. Seifert, T. Klonisch, H. Dralle, J. Langner, C. Hoang-Vu, Expression, regulation and function of autotaxin in thyroid carcinomas, Int. J. Cancer 109 (2004) 833–838. [6] A.L. Parrill, U. Echols, T. Nguyen, T.C. Pham, A. Hoeglund, D.L. Baker, Virtual screening approaches for the identification of non-lipid autotaxin inhibitors, Bioorg. Med. Chem. 16 (2008) 1784–1795. [7] L.A. van Meeteren, V. Brinkmann, J.S. Saulnier-Blache, K.R. Lynch, W.H. Moolenaar, Anticancer activity of FTY720: phosphorylated FTY720 inhibits autotaxin, a metastasis-enhancing and angiogenic lysophospholipase D, Cancer Lett. 266 (2008) 203–208. [8] M. Tanaka, S. Okudaira, Y. Kishi, R. Ohkawa, S. Iseki, M. Ota, S. Noji, Y. Yatomi, J. Aoki, H. Arai, Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281 (2006) 25822–25830. [9] L.A. van Meeteren, P. Ruurs, C. Stortelers, P. Bouwman, M.A. van Rooijen, J.P. Pradere, T.R. Pettit, M.J. Wakelam, J.S. Saulnier-Blache, C.L. Mummery, W.H. Moolenaar, J. Jonkers, Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26 (2006) 5015–5022. [10] H. Kanda, R. Newton, R. Klein, Y. Morita, M.D. Gunn, S.D. Rosen, Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs, Nat. Immunol. 9 (2008) 415– 423. [11] L.A. van Meeteren, W.H. Moolenaar, Regulation and biological activities of the autotaxin-LPA axis, Prog. Lipid Res. 46 (2007) 145–160. [12] I. Ishii, N. Fukushima, X. Ye, J. Chun, Lysophospholipid receptors: signaling and biology, Annu. Rev. Biochem. 73 (2004) 321–354. [13] G.B. Mills, W.H. Moolenaar, The emerging role of lysophosphatidic acid in cancer, Nat. Rev. Cancer 3 (2003) 582–591. [14] M.H. Graler, E.J. Goetzl, Lysophospholipids and their G protein-coupled receptors in inflammation and immunity, Biochim. Biophys. Acta 1582 (2002) 168–174. [15] D.A. Lin, J.A. Boyce, Lysophospholipids as mediators of immunity, Adv. Immunol. 89 (2006) 141–167. [16] M. Guha, N. Mackman, LPS induction of gene expression in human monocytes, Cell. Signal. 13 (2001) 85–94. [17] M. Chen, K.L. O’Connor, Integrin alpha6beta4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells, Oncogene 24 (2005) 5125–5130. [18] A. Giganti, M. Rodriguez, B. Fould, N. Moulharat, F. Coge, P. Chomarat, J.P. Galizzi, P. Valet, J.S. Saulnier-Blache, J.A. Boutin, G. Ferry, A sdfsdaf, J. Biol. Chem. 283 (2008) 7776–7789. [19] A.J. Morris, S.S. Smyth, Measurement of autotaxin/lysophospholipase D activity, Methods Enzymol. 434 (2007) 89–104. [20] K. Nakamura, R. Ohkawa, S. Okubo, M. Tozuka, M. Okada, S. Aoki, J. Aoki, H. Arai, H. Ikeda, Y. Yatomi, Measurement of lysophospholipase D/autotaxin activity in human serum samples, Clin. Biochem. 40 (2007) 274–277. [21] E. Huberman, C.A. Jones, Control of cell differentiation and cell transformation in vitro by phorbol 12-myristate 13-acetate and 1 alpha,25-dihydroxyvitamin D3, Carcinog. Compr. Surv. 10 (1985) 263–273. [22] C.W. Chow, S. Grinstein, O.D. Rotstein, Signaling events in monocytes and macrophages, New Horiz. 3 (1995) 342–3451. [23] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499–511. [24] B.R. Williams, Signal integration via PKR, Sci. STKE 2001 (2001) RE2. [25] X. Hu, F.J. Mendoza, J. Sun, V. Banerji, J.B. Johnston, S.B. Gibson, Lysophosphatidic acid (LPA) induces the expression of VEGF leading to protection against apoptosis in B-cell derived malignancies, Cell Signal. 20 (2008) 1198–1208. [26] Y. Jin, E. Knudsen, L. Wang, A.A. Maghazachi, Lysophosphatidic acid induces human natural killer cell chemotaxis and intracellular calcium mobilization, Eur. J. Immunol. 33 (2003) 2083–2089. [27] C.L. Chang, M.E. Lin, H.Y. Hsu, C.L. Yao, S.M. Hwang, C.Y. Pan, C.Y. Hsu, H. Lee, Lysophosphatidic acid-induced interleukin-1 beta expression is mediated through Gi/Rho and the generation of reactive oxygen species in macrophages, J. Biomed. Sci. 15 (2008) 357–363.