Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Nov 17;1863(2):229–235. doi: 10.1016/j.bbamcr.2015.11.012

Cross-talk between lysophosphatidic acid receptor 1 and tropomyosin receptor kinase A promotes lung epithelial cell migration

Ling Nan 1,2, Jianxin Wei 2, Anastasia M Jacko 2, Miranda K Culley 2, Jing Zhao 2, Viswanathan Natarajan 3, Haichun Ma 1,†, Yutong Zhao 2,#,†
PMCID: PMC4767279  NIHMSID: NIHMS738969  PMID: 26597701

Abstract

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which plays a crucial role in regulation of cell proliferation, migration, and differentiation. LPA exerts its biological effects mainly through binding to cell-surface LPA receptors (LPA1–6), which belong to the G protein-coupled receptor (GPCR) family. Recent studies suggest that cross-talk between receptor tyrosine kinases (RTKs) and GPCRs modulates GPCRs-mediated signaling. Tropomyosin receptor kinase A (TrkA) is a RTK, which mediates nerve growth factor (NGF)-induced biological functions including cell migration in neuronal and non-neuronal cells. Here, we show LPA1 transactivation of TrkA in murine lung epithelial cells (MLE12). LPA induced tyrosine phosphorylation of TrkA in both time- and dose-dependent manners. Down-regulation of LPA1 by siRNA transfection attenuated LPA-induced phosphorylation of TrkA, suggesting a cross-talk between LPA1 and TrkA. To investigate the molecular regulation of the cross-talk, we focused on the interaction between LPA1 and TrkA. We found that LPA induced interaction between LPA1 and TrkA. The LPA1/TrkA complex was localized on the plasma membrane and in the cytoplasm. The C-terminus of LPA1 was identified as the binding site for TrkA. Inhibition of TrkA attenuated LPA-induced phosphorylation of TrkA and LPA1 internalization, as well as lung epithelial cell migration. These studies provide a molecular mechanism for the transactivation of TrkA by LPA, and suggest that the cross-talk between LPA1 and TrkA regulates LPA-induced receptor internalization and lung epithelial cell migration.

Keywords: LPA, GPCR, RTK, transactivation, receptor internalization, cell migration

Graphical abstract

graphic file with name nihms738969f8.jpg

1. Introduction

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, is detectable in various biological tissues and fluids, notably plasma [1] and bronchoalveolar lavage fluids (BAL) [2]. LPA is a crucial mediator of physiological processes including cell proliferation, survival, migration, differentiation, adhesion and morphology, as well as pathological processes in cancer, fibrosis, bone metabolism and reproduction [3, 4]. In the setting of wound repair, LPA is released from serum by a platelet-dependent pathway [1] at the site of acute injury [5, 6], and then LPA promotes cell migration [7] and thus enhances wound healing [8].

LPA exerts its biological effects by binding cognate, cell-surface LPA receptors (LPAR), which belong to G protein-coupled receptor (GPCR) family. Six LPA receptor genes, LPAR1-LPAR6 in humans and Lpar1-Lpar6 in mice and non-human species, have been identified to encode the receptors LPA1-LPA6 [9, 10] which have a wide tissue distribution. The isoforms of the transmembrane LPA receptors are coupled to different α-subunits of G-proteins, such as Gi (LPA1–3, 6), Gq (LPA1–5), and G12/13 (LPA1–2, 4–6), which are responsible for LPA-induced different signaling responses [11, 12].

Receptor tyrosine kinases (RTKs) are another major class of cell surface transmembrane proteins. Ligand canonically activates RTKs by binding to the extracellular domain to induce dimerization of the receptors and auto-phosphorylation of the tyrosine residues in the cytosolic domain. Recently, cross-talk between RTKs and GPCRs has been discovered [13–15]. For example, dopamine receptor D4 transactivates platelet-derived growth factor receptor β (PDGFRβ). Inhibition of PDGFRβ attenuates dopamine-induced activation of extracellular signal-regulated kinase (Erk1/2) [16], suggesting that PDGFRβ transactivation by dopamine receptor D4 is involved in dopamine-mediated signaling. We and others have previously reported that LPA induced epidermal growth factor receptor (EGFR) transactivation in human bronchial epithelial cells [17, 18]. The cross-talk between LPA1 and EGFR regulates LPA-induced activation of transcriptional factor CEBPβ and interleukin-8 (IL-8) production.

TrkA, a member of the RTK family, is a receptor for nerve growth factor (NGF). NGF activates TrkA signaling pathways, thus influencing neuronal cell proliferation, differentiation, and survival [19, 20]. Recent studies have suggested that NGF and its receptor can mediate inflammation, particularly in the lung [21]. Transactivation of TrkA by GPCRs has been reported. Adenosine treatment induced tyrosine phosphorylation of TrkA [22], indicating that adenosine receptor, a GPCR, cross-talks with TrkA. Moughal et al. reported that LPA1 contributes to NGF-induced phosphorylation of p42/44 MAPK in pheochromocytoma 12 (PC12) cells [23]. Still, little is known about TrkA receptor activation upon stimulation with LPA in lung epithelial cells. Here, we demonstrate LPA induces phosphorylation of TrkA receptor, LPA1 interaction with TrkA, and the cross-talk between LPA1 and TrkA contributes to LPA-induced lung epithelial cell migration.

2. Method and Material

2.1. Cell culture and reagents

The murine lung epithelial cell line (MLE12 cells) (American Type Culture Collection, Manassas, VA, USA) was cultured in HITES medium complemented with 10% fetal bovine serum. Cells were maintained in a 37°C incubator in the presence of 5% CO2. V5 antibody was purchased from Invitrogen (Carlsbad, CA). β-actin antibody, scrambled siRNA, LPA1 siRNA, control shRNA, TrkA shRNA, batimastat (BB94), and LPA were from Sigma Aldrich (St. Louis, MO). GM6001 was from Enzo Life Science (Farmingdale, NY). Antibodies to phospho (pY674/Y675)-TrkA and Myc tag were from Cell Signaling Technology (Danvers, MA). Antibody to TrkA was from EMD MilliPore (Billerica, MA). Antibodies to phospho (p)-Erk1/2, Erk1/2, and TrkA inhibitor (TrkAi) were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies and ECL kit for detection of proteins by Western blotting was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade.

2.2. Preparation of cell lysates and Western blotting

After the indicated treatments, cells were rinsed with ice-cold PBS and lysed in 120 µl of lysis buffer containing 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β-glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 µg/ml protease inhibitors, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. Cell lysates were sonicated on ice for 12 seconds and centrifuged at 1,000 g for 10 min at 4°C to remove the debris. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc) using BSA as standard. Equal amounts of cell lysates (20 µg) were subjected to 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and then membranes were blocked with 5% (wt/vol) BSA in 25 mM Tris·HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween 20 (TBST) for 30 min, and incubated with primary antibodies in 5% (wt/vol) BSA in TBST for 2 h. The membranes were washed at least three times with TBST at 10 min intervals and then incubated with mouse, rabbit or goat horseradish peroxidase-conjugated secondary antibody (1:2,000–5,000) for 1 h. They were then developed with the enhanced chemiluminescence detection system according to the manufacturer's instructions.

2.3. Immunofluorescence staining

MLE12 cells were grown on glass bottom dishes until 80–90% confluence. After treatment, cells were fixed with 3.7% formaldehyde for 20 min, blocked with 1% BSA in TBST for 30 min, then immunostained with TrkA, PY-TrkA, V5 tag, Myc tag, cortactin antibody, or phalloidin (F-actin) followed by three time washes with PBS, and incubated with the fluorescent probe-conjugated secondary antibodies. Images were captured by a Nikon ECLIPSE TE 300 inverted microscope.

2.4. Transfection of LPA1-Myc, TrkA-V5, TrkA shRNA, and LPA1 siRNA

MLE12 cells grown on 6-well plates or glass bottom dishes (60–70% confluence) were transfected with V5-tagged LPA1 (LPA1-V5), Myc-tagged-LPA1 (LPA1-Myc), V5-tagged-TrkA (TrkA-V5), TrkA short-hairpin RNA (TrkA shRNA) plasmids (4 µg), or LPA1 siRNA (125 nM) using Lonza electroporation transfection according to the manufacturer's protocol. LPA1-Myc or TrkA-V5 transfected cells were analyzed after 48 h. TrkA shRNA and LPA1 siRNA transfected cells were analyzed after 72 h.

2.5. Cell migration assay

MLE12 cells were cultured in 6-well plates until 100% confluence. Monolayers were scratched using a sterile 10 µl pipette tip. Cells were digitally photographed at 0 and 18 h after stimulation. The extent of cell migration was quantified using ImageJ software. The percentage of wound closure was calculated as follows: [(pre-migration area – migration area)/pre-migration area] × 100.

2.6. Statistical analysis

All results were subjected to statistical analysis using Microsoft Excel, and, wherever appropriate, the data were also analyzed by Student's t-test and expressed as means ± SD. Data were collected from at least three independent experiments, and p<0.05 was considered significant.

3. Results

3.1. LPA induces tyrosine phosphorylation of TrkA

We have previously shown that LPA transactivates EGFR in human lung epithelial cells [18]. To investigate whether LPA receptors cross-talk with TrkA, we examined the effect of LPA treatment on TrkA activation in murine lung epithelial cells (MLE12). MLE cells were treated with LPA (5 µM) for 1–120 min, and then the cell lysates were analyzed by Western blotting with antibodies to PY674/Y675-TrkA and total TrkA. LPA induced tyrosine phosphorylation of TrkA at 15 min, reaching a peak level at 30 min, without altering TrkA expression (Fig. 1A). Additionally, MLE12 cells were treated with various doses of LPA (0–10 µM) for 30 min. As shown in Fig. 1B, LPA induced phosphorylation of TrkA in a dose-dependent manner. To further confirm the phosphorylation of TrkA by LPA, MLE cells overexpressing V5 tagged TrkA (TrkA-V5) were treated with LPA (5 µM) for 30 min. Localization of TrkA-V5 and Y674/Y675 phosphorylated TrkA were examined by immunostaining. TrkA-V5 is predominately found on the plasma membrane and is also detectable in the cytoplasm (Fig. 2). Phosphorylated TrkA was not detectable in the untreated cells, but LPA increased phosphorylated TrkA on the plasma membrane and in the cytoplasm. Tyrosine 674/675 residues are in the SH1 domain, which regulates catalytic activity of the TrkA tyrosine kinase [24]. These data suggest that LPA induces activation of TrkA in murine lung epithelial cells.

Figure 1. LPA induces tyrosine phosphorylation of TrkA.

Figure 1

Open in a new tab

A. MLE cells were treated with LPA (5 µM) for 0–120 min. B. MLE12 cells were treated with various doses of LPA (0–10 µM) for 30 min. Cell lysates from both time- and dose-dependent challenges were analyzed using Western blotting with antibodies to PY674/Y675 (PY)-TrkA and total TrkA. Shown are representative images from three independent experiments.

Figure 2. Transactivated TrkA is mainly localized on the cell surface.

Figure 2

Open in a new tab

MLE cells overexpressing TrkA-V5 were treated with LPA (5 µM) for 30 min, and then cells were fixed. Localization of TrkA-V5 and phosphorylated TrkA were examined by immunostaining. Shown are representative images.

3.2. LPA induces phosphorylation of TrkA through LPA1 binding to TrkA

LPA1 is the major LPA receptor in MLE12 cells [25]. To determine whether LPA-induced transactivation of TrkA is mediated by LPA1, LPA1 was downregulated by LPA1 siRNA transfection prior to LPA treatment. LPA induced phosphorylation of TrkA and Erk1/2 in scrambled siRNA transfected cells, while LPA1 siRNA knockdown attenuated the effects of LPA and reduced LPA1 levels (~90%) (Fig. 3A), suggesting that LPA1 is critical for LPA-induced transactivation of TrkA. We have shown that LPA-induced transactivation of EGFR and activation of Erk1/2 requires activation of matrix metalloproteases (MMPs) [17]. To determine if transactivation of TrkA behaves via a similar mechanism, MLE cells were treated with MMPs inhibitor (GM6001) with and without LPA. GM6001 reduced LPA-induced phosphorylation of Erk1/2, which is consistent with our previous finding [17], while it had no effect on LPA-induced phosphorylation of TrkA (Fig. 3B). This data was confirmed by another broad-spectrum MMP inhibitor, BB94 (Fig. 3C). These results indicate LPA transactivation of TrkA, unlike the cross-talk between LPA1 and EGFR, is not dependent on MMPs activation.

Figure 3. LPA-induced phosphorylation of TrkA is mediated by LPA1, but not MMPs.

Figure 3

Open in a new tab

A. MLE12 cells were transfected with scrambled (scram) siRNA or LPA1 siRNA (125 nM, 72 h) prior to LPA (5 µM, 30 min) treatment. The cell lysates were analyzed by Western blotting with antibodies to PY-TrkA, total TrkA, P-Erk1/2, total Erk1/2, and LPA1. B. MLE cells were pretreated with a MMPs inhibitor GM6001 (20 µM) for 1 h, then cells were treated with LPA (5 µM) for 30 min. The cell lysates were analyzed by Western blotting with antibodies to PY-TrkA, total TrkA, P-Erk1/2 and total Erk1/2. C. MLE cells were pretreated with a MMPs inhibitor BB94 (20 µM) for 1 h, then cells were treated with LPA (5 µM) for 30 min. The cell lysates were analyzed by Western blotting with antibodies to PY-TrkA, total TrkA, P-Erk1/2 and total Erk1/2.

To investigate the mechanism by which LPA induces TrkA phosphorylation, we examined whether LPA1 associates with TrkA in response to LPA treatment. MLE cells overexpressing V5 tagged LPA1 (LPA1-V5) were treated with LPA (5 µM) for 0–30 min. The cell lysates were analyzed by immunoprecipitation with a V5 antibody followed by Western blotting with a TrkA antibody. LPA1-V5 binding to TrkA was observed after LPA treatment at 15 min (Fig. 4A). Furthermore, the interaction between endogenous LPA1 and TrkA were observed at 5 min after LPA treatment (Fig. 4B). The different binding times observed from Fig. 4A and Fig. 4B may be due to that the two forms of LPA1 (overexpressed or endogenous) exhibit the different TrkA binding rates. The data also suggests that the interaction between the receptors is transient. To gain additional evidence about whether LPA1 interacts with phosphorylated TrkA, MLE cells overexpressing both Myc tagged LPA1 (LPA-Myc) and TrkA-V5 were treated with LPA (5 µM) for 15 min. As shown in Fig. 4C, LPA induces co-localization of LPA1 and phosphorylated TrkA on the plasma membrane and in the cytoplasm, suggesting that LPA1 associates with transactivated TrkA in response to LPA treatment. Taken together, these results suggest that LPA induced TrkA phosphorylation, but the interaction between the receptors is transient. In response to LPA treatment, LPA1 binds and transactivates TrkA, and then disassociates with TrkA. LPA1 contains a long c-terminus (34 amino acids), which is associated with interaction with other adaptive proteins, such as GIPC (GAIP-interacting protein, C terminus) [26]. More specifically, we demonstrated an LPA1 truncation mutant (LPAC350-V5), which lacks 350–365 amino acids at the c-terminus, failed to associate with TrkA (Fig. 4A), suggesting that the fragment (350–365 amino acids) of LPA1 contains a TrkA docking site. These results illustrate that LPA induces transactivation of TrkA through increasing interaction with TrkA.

Figure 4. LPA induces interaction between LPA1 and TrkA.

Figure 4

Open in a new tab

A. MLE cells overexpressing with LPA1-V5 and LPA1 truncation mutant (LPAC350-V5), were treated with LPA (5 µM) for 0–30 min. The cell lysates were analyzed by immunoprecipitation with a V5 antibody, followed by Western blotting with a TrkA antibody. Input lysates were analyzed by Western blotting with antibodies to TrkA and V5. B. MLE cells were treated with LPA (5 µM) for 0–30 min. The cell lysates were analyzed by immunoprecipitation with a V5 antibody, followed by Western blotting with a TrkA antibody. Input lysates were analyzed by Western blotting with TrkA and β-actin antibodies. C. MLE cells were co-transfected with LPA1-Myc and TrkA-V5, and then cells were treated with LPA (5 µM) for 15 min. Cells were fixed, and localization of LPA1-Myc and phosphorylated TrkA were examined by immunostaining. PY-TrkA: green; LPA1-Myc: red; DAPI (stain for nuclei): blue. Arrows and orange indicate co-localization of PY-TrkA and LPA1-Myc. Shown are representative images.

3.3. Cross-talk between LPA1 and TrkA regulates LPA1 internalization

Plasma membrane receptor internalization plays a feedback role in limiting receptor activation and its downstream signaling. To investigate whether TrkA is involved in LPA-induced LPA1 internalization, MLE12 cells were co-transfected with LPA1-Myc and TrkA-V5 plasmids prior to LPA treatment. LPA induced LPA1-Myc internalization, while the effect was reduced in cells over-expressing TrkA-V5 (Fig. 5), suggesting that TrkA may promote LPA signaling by limiting LPA1 receptor internalization.

Figure 5. TrkA attenuates LPA1 internalization.

Figure 5

Open in a new tab

MLE cells overexpressing with LPA1-Myc and TrkA-V5 were treated with LPA (5 µM) for 30 min. Cells were fixed, and localization of LPA1-Myc and TrkA-V5 were examined by immunostaining. LPA1-Myc: red; TrkA-V5: green; DAPI (for nuclei): blue. LPA1-Myc/TrkA co-localization: orange. Arrows indicate localization on plasma membrane. Asterisks indicate nuclei. Dashed lines indicate plasma membrane. Shown are representative images.

3.4. TrkA contributes to LPA-induced lung epithelial cell migration

Cell migration is essential for normal embryonic development, immune system function, and angiogenesis, as well as lung epithelial wound repair after injury. LPA has been known as a stimulator for cell migration [27, 28]. To investigate the effect of TrkA on LPA-induced cell migration in lung epithelial cells, we examined whether inhibition or reduction of TrkA attenuates LPA signaling and cell migration. Pretreatment with a TrkA tyrosine kinase inhibitor, TrkAi (1–10 µM), attenuated the LPA-induced phosphorylation of TrkA in a dose-dependent manner (Fig. 6A). This further supports our conclusion that LPA induces activation of TrkA and suggests that TrkAi could be used to investigate the effect of TrkA on LPA-induced cell migration. MLE12 cells were scratched in cell culture dishes and cell migration distances were observed by measuring the area of wound closure. As shown in Fig. 6B and 6C, LPA treatment increased cell migration, which is consistent with previous reports [29, 30], while inhibition of TrkA by TrkAi significantly diminished LPA-induced cell migration. Further, cells were transfected with control shRNA or TrkA shRNA prior to LPA treatment. TrkA shRNA reduced TrkA protein levels around 60%, and significantly attenuated LPA-induced cell migration (Fig. 6D). Cortactin, a cytoskeletal-associated protein, has been known to regulate cell migration [31]. LPA increased cortactin co-localization with F-actin at the leading edge of migrating cells, while the effect was attenuated by TrkAi (Fig. 7). Taken together, these data suggest the cross-talk between LPA1 and TrkA plays a critical role in LPA-induced cell migration in lung epithelial cells.

Figure 6. TrkA contributes to LPA-induced lung epithelial cell migration.

Figure 6

Open in a new tab

A. MLE cells were pretreated with a TrkA tyrosine kinase inhibitor, TrkAi (1–10 µM), for 1 h, and then cells were treated with LPA (5 µM) for 3 h. The cell lysates were analyzed by Western blotting with antibodies to PY-TrkA and total TrkA. Shown are representative images from three independent experiments. B. MLE12 cells were cultured in 6-well plates until 100% confluence. Monolayers were scratched using a sterile 10 µl pipette tip. Cells were treated with TrkAi (5 µM, 1 h) prior to LPA treatment (5 µM, 18 h). Cells were digitally photographed at 0 and 18 h after LPA stimulation. C. The extent of cell migration was quantified using ImageJ software. The percentage of wound closure was calculated as follows: [(pre-migration area – migration area)/pre-migration area] × 100. D. Cells were transfected with control shRNA or TrkA shRNA for 3 days, and then cells were scratched using a sterile 10 µl pipette tip. After LPA (5 µM) treatment, cell migration were examined as described above. Cell lysates were analyzed by TrkA and β-actin immunoblotting.

Figure 7. TrkA regulates LPA-induced cortactin accumulation at leading edge of migrating cells.

Figure 7

Open in a new tab

MLE cells were pretreated with TrkAi (5 µM, 1 h) prior to LPA (5 µM, 30 min) treatment. Cells were fixed, and localization of cortactin and F-actin were examined by Immunostaining. Cortactin: green; F-actin: red. Arrows represent leading edge. Shown are representative images.

4. Discussion

In the present study, we demonstrate that LPA induces Y674/Y675 phosphorylation of TrkA through LPA1 in MLE cells. The LPA1 receptor, specifically the c-terminus, is critical for its interaction with TrkA. The receptors cross-talk attenuates LPA1 internalization. Lastly, transactivation of TrkA increases cell migration in lung epithelial tissues exposed to injury.

Studies have illustrated complex, nonlinear signaling cascades generated from the cross-talk between GPCRs and RTKs that coordinates the plethora of extracellular stimuli under several physiological and pathological conditions [13, 32–34]. Transactivation of RTKs by GPCR agonists in particular has been demonstrated in recent years, specifically transactivation of EGFR by ligands such as LPA, thrombin, sphingosine 1-phosphate, endothelin-1, prostaglandin F2α, and parathyroid hormone [35–38]. LPA is of particular interest in the lungs because, during lung injury, LPA concentrations increase in the airspaces [39]. Additionally, LPA binding to LPA1 has been linked to idiopathic pulmonary fibrosis, an example of an aberrant wound healing response, further implicating the role of LPA/LPA1 in the injury response in the lungs [39]. TrkA, our RTK of interest, has been studied in cases of pulmonary physiology and pathology when stimulated with NGF, but not yet with LPA [21, 40]. Our studies first show that LPA increases Y674/Y675 phosphorylation of TrkA in both time and dose-dependent manners. The effect of LPA on TrkA transactivation is localized primarily on the plasma membrane, indicating a novel ligand-receptor association in lung epithelial cells. Moughal et al. had shown that LPA induced Y490 phosphorylated TrkA into the nuclei of PC12 cells [23]. The contradictory results may be due to cell type specificity and different phospho sites of TrkA observed between studies. Tyrosine 490 is localized in juxtamembrane domain of TrkA, its phosphorylation triggers Shc binding to TrkA, but it is not involved in TrkA catalytic activity. Tyrosine residues Y674/Y675 are localized in catalytic domain of TrkA, the phosphorylation of Y674/Y675 regulates TrkA catalytic activity. It is possible that different phosphorylation sites trigger different behaviors of TrkA, including localization.

Silencing of LPA1 by LPA1 siRNA attenuated phosphorylation of TrkA and Erk1/2 in the presence of LPA, confirming our hypothesis of a GPCR-RTK cross-talk. Erk1/2 are protein serine/threonine kinases apart of the Ras-Raf-MEK-Erk signal transduction cascade which ultimately regulates processes like cell adhesion, cell cycle progression, cell migration, cell survival, differentiation, proliferation, and transcription [41]. The increase of phosphorylation of Erk1/2 shown in the presence of LPA is consistent with previous studies that have shown transactivation of RTKs, particularly EGFR, mediate Erk1/2 activation by LPA. Although this study supports transactivation of TrkA by LPA/LPA1 may be linked to Erk1/2, the exact mechanism of Erk1/2 activation remains undefined. A previous study suggests Erk1/2 acting as a critical component in the signaling cascade linking LPA receptor activation to CREB phosphorylation [42], expanding our understanding of potential downstream transcription factor targets.

The intricate mechanism of GPCR transactivation of RTKs can be categorized as either the ligand-dependent triple-membrane-passing-signal (TMPS) or ligand-independent. The former depends on activation of membrane-bound matrix metalloproteases (MMPs) such as the A Disintegrin and Metalloprotease (ADAM) family members, while the latter depends on activation of secondary messengers such as Ca2+ ions, protein kinase C (PKC), the non-receptor protein tyrosine kinases Src and Pyk, and β-arrestin which, in turn, induce tyrosine phosphorylation and subsequent activation of RTKs [14]. Treatment of MLE cells with broad-spectrum MMPs inhibitors (GM6001 or BB94) in the presence of LPA resulted in no attenuation of phosphorylated TrkA, suggesting the mechanism of transactivation is ligand shedding-independent. Our study reveals a new model: GPCR ligand induces transient GPCR/RTK interation. The transactivation of GPCRs by RTK agonists has been shown to require GPCR-RTK complex formation with intracellular scaffolding proteins and sometimes phosphorylation of transactivated GPCRs [43]. Characterization of the exact interaction of LPA1 and TrkA, and potentially other proteins will require further experimentation.

The cross-talk between LPA1 and TrkA regulates LPA1 internalization but the effects of this receptor-regulated process remain largely uncharacterized. The Src family of non-receptor tyrosine kinases composes a group of signal transducers that are activated by extracellular stimuli and regulate a variety of cellular functions including proliferation, survival, and migration that could be contributing to LPA1 internalization. Agonist-induced activation of several GPCRs, including LPA receptors, have been shown to increase the activity of Src-family tyrosine kinases, and Src has been shown to be a critical regulator of GPCR activity, affecting receptor internalization, desensitization, and coupling of ERK1/2 to RTK [42]. The GPCR-RTK complex we have proposed could include a Src-family non-receptor tyrosine kinase that mediates LPA1 internalization and even ERK1/2 signaling.

Ligand-receptor binding in the LPA/LPA1 signaling pathway promotes migration and enhances wound healing in vivo [7, 8]. In current study, we investigated the effect of TrkA on LPA-induced cell migration in lung epithelial cells and found that TrkA tyrosine kinase inhibitor or reduction of TrkA level significantly attenuated the LPA-induced cell migration. The effect of LPA-induced transactivation of TrkA promotes cell migration and thus not only has implications in wound healing after lung injury but also potentially in aberrant wound healing (i.e. idiopathic pulmonary fibrosis) and inflammation as well as lung tumor growth and metastasis. LPA is generated from lysoposphatidylcholine by action of lysophospholipase D (autotaxin). We have shown that autotaxin induced lung epithelial cell migration through both LPA/LPA1 and autotaxin/LPA1 pathways [44]. The effect of autotaxin on cell migration may be through LPA production and cross-talk between LPA1 and TrkA. An understanding of LPA1-TrkA cross-talk could provide a new, drugable target for lung injury reparation and lung tumor cell migration.

5. Conclusions

In conclusion, we provide evidence that LPA-induced transactivation of TrkA is LPA1 mediated and there is an important binding site at its c-terminus. Furthermore, cross-talk between LPA1 and TrkA regulates LPA1 internalization and LPA-induced cell migration in the setting of wound healing. This work further contributes to a growing understanding of the complex GPCRRTK cross-talk that informs cell signaling, specifically for the first time in pulmonary epithelial cells and in the context of wound healing.

Research Highlights.

  • LPA induces tyrosine phosphorylation of TrkA.

  • LPA induces interaction between LPA1 and TrkA.

  • TrkA reduces LPA1 internalization.

  • The cross-talk between LPA1 and TrkA regulates cell migration.

Acknowledgements

This study was supported by the US National Institutes of Health (R01 HL112791, PO1 HL114453-01A1 to Y.Z., R01GM115389 to J.Z.), American Heart Association 12SDG9050005 (J.Z.), and American Lung Association Biomedical Research Grant RG350146 (J.Z.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

All authors declare no conflict of interest.

References

  • 1.Aoki J, Taira A, Takanezawa Y, Kishi Y, Hama K, Kishimoto T, Mizuno K, Saku K, Taguchi R, Arai H. Serum lysophosphatidic acid is produced through diverse phospholipase pathways. The Journal of biological chemistry. 2002;277:48737–48744. doi: 10.1074/jbc.M206812200. [DOI] [PubMed] [Google Scholar]
  • 2.Georas SN, Berdyshev E, Hubbard W, Gorshkova IA, Usatyuk PV, Saatian B, Myers AC, Williams MA, Xiao HQ, Liu M, Natarajan V. Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology. 2007;37:311–322. doi: 10.1111/j.1365-2222.2006.02626.x. [DOI] [PubMed] [Google Scholar]
  • 3.Tigyi G. Aiming drug discovery at lysophosphatidic acid targets. British journal of pharmacology. 2010;161:241–270. doi: 10.1111/j.1476-5381.2010.00815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, Mutoh T, Lin ME, Teo ST, Park KE, Mosley AN, Chun J. LPA receptors: subtypes and biological actions. Annual review of pharmacology and toxicology. 2010;50:157–186. doi: 10.1146/annurev.pharmtox.010909.105753. [DOI] [PubMed] [Google Scholar]
  • 5.Eichholtz T, Jalink K, Fahrenfort I, Moolenaar WH. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. The Biochemical journal. 1993;291(Pt 3):677–680. doi: 10.1042/bj2910677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Goetzl EJ, An S. Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 1998;12:1589–1598. [PubMed] [Google Scholar]
  • 7.Sauer B, Vogler R, Zimmermann K, Fujii M, Anzano MB, Schafer-Korting M, Roberts AB, Kleuser B. Lysophosphatidic acid interacts with transforming growth factor-beta signaling to mediate keratinocyte growth arrest and chemotaxis. The Journal of investigative dermatology. 2004;123:840–849. doi: 10.1111/j.0022-202X.2004.23458.x. [DOI] [PubMed] [Google Scholar]
  • 8.Demoyer JS, Skalak TC, Durieux ME. Lysophosphatidic acid enhances healing of acute cutaneous wounds in the mouse. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2000;8:530–537. doi: 10.1046/j.1524-475x.2000.00530.x. [DOI] [PubMed] [Google Scholar]
  • 9.Jesionowska A, Cecerska E, Dolegowska B. Methods for quantifying lysophosphatidic acid in body fluids: a review. Analytical biochemistry. 2014;453:38–43. doi: 10.1016/j.ab.2014.02.021. [DOI] [PubMed] [Google Scholar]
  • 10.Yung YC, Stoddard NC, Chun J. LPA receptor signaling: pharmacology, physiology, and pathophysiology. Journal of lipid research. 2014;55:1192–1214. doi: 10.1194/jlr.R046458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Yanagida K, Ishii S. Non-Edg family LPA receptors: the cutting edge of LPA research. Journal of biochemistry. 2011;150:223–232. doi: 10.1093/jb/mvr087. [DOI] [PubMed] [Google Scholar]
  • 12.Ishii I, Fukushima N, Ye X, Chun J. Lysophospholipid receptors: signaling and biology. Annual review of biochemistry. 2004;73:321–354. doi: 10.1146/annurev.biochem.73.011303.073731. [DOI] [PubMed] [Google Scholar]
  • 13.El Zein N, D'Hondt S, Sariban E. Crosstalks between the receptors tyrosine kinase EGFR and TrkA and the GPCR, FPR, in human monocytes are essential for receptors-mediated cell activation. Cellular signalling. 2010;22:1437–1447. doi: 10.1016/j.cellsig.2010.05.012. [DOI] [PubMed] [Google Scholar]
  • 14.Cattaneo F, Guerra G, Parisi M, De Marinis M, Tafuri D, Cinelli M, Ammendola R. Cell-surface receptors transactivation mediated by g protein-coupled receptors. International journal of molecular sciences. 2014;15:19700–19728. doi: 10.3390/ijms151119700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhao Y, Zhao J, Mialki RK, Wei J, Spannhake EW, Salgia R, Natarajan V. Lipopolysaccharide-induced phosphorylation of c-Met tyrosine residue 1003 regulates c-Met intracellular trafficking and lung epithelial barrier function. American journal of physiology. Lung cellular and molecular physiology. 2013;305:L56–L63. doi: 10.1152/ajplung.00417.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Gill RS, Hsiung MS, Sum CS, Lavine N, Clark SD, Van Tol HH. The dopamine D4 receptor activates intracellular platelet-derived growth factor receptor beta to stimulate ERK1/2. Cellular signalling. 2010;22:285–290. doi: 10.1016/j.cellsig.2009.09.031. [DOI] [PubMed] [Google Scholar]
  • 17.Zhao Y, He D, Saatian B, Watkins T, Spannhake EW, Pyne NJ, Natarajan V. Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase Cdelta, Lyn kinase, and matrix metalloproteinases. The Journal of biological chemistry. 2006;281:19501–19511. doi: 10.1074/jbc.M511224200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.He D, Natarajan V, Stern R, Gorshkova IA, Solway J, Spannhake EW, Zhao Y. Lysophosphatidic acid-induced transactivation of epidermal growth factor receptor regulates cyclo-oxygenase-2 expression and prostaglandin E(2) release via C/EBPbeta in human bronchial epithelial cells. The Biochemical journal. 2008;412:153–162. doi: 10.1042/BJ20071649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bartkowska K, Paquin A, Gauthier AS, Kaplan DR, Miller FD. Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development. Development. 2007;134:4369–4380. doi: 10.1242/dev.008227. [DOI] [PubMed] [Google Scholar]
  • 20.Zhang Y, Moheban DB, Conway BR, Bhattacharyya A, Segal RA. Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate NGF-induced differentiation. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2000;20:5671–5678. doi: 10.1523/JNEUROSCI.20-15-05671.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Freund-Michel V, Frossard N. The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacology & therapeutics. 2008;117:52–76. doi: 10.1016/j.pharmthera.2007.07.003. [DOI] [PubMed] [Google Scholar]
  • 22.Lee FS, Rajagopal R, Chao MV. Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors. Cytokine & growth factor reviews. 2002;13:11–17. doi: 10.1016/s1359-6101(01)00024-7. [DOI] [PubMed] [Google Scholar]
  • 23.Moughal NA, Waters C, Sambi B, Pyne S, Pyne NJ. Nerve growth factor signaling involves interaction between the Trk A receptor and lysophosphatidate receptor 1 systems: nuclear translocation of the lysophosphatidate receptor 1 and Trk A receptors in pheochromocytoma 12 cells. Cellular signalling. 2004;16:127–136. doi: 10.1016/j.cellsig.2003.08.004. [DOI] [PubMed] [Google Scholar]
  • 24.Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994;372:746–754. doi: 10.1038/372746a0. [DOI] [PubMed] [Google Scholar]
  • 25.Zhao J, He D, Su Y, Berdyshev E, Chun J, Natarajan V, Zhao Y. Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs. American journal of physiology. Lung cellular and molecular physiology. 2011;301:L547–L556. doi: 10.1152/ajplung.00058.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Varsano T, Taupin V, Guo L, Baterina OY, Jr, Farquhar MG. The PDZ protein GIPC regulates trafficking of the LPA1 receptor from APPL signaling endosomes and attenuates the cell's response to LPA. PloS one. 2012;7:e49227. doi: 10.1371/journal.pone.0049227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kang S, Han J, Song SY, Kim WS, Shin S, Kim JH, Ahn H, Jeong JH, Hwang SJ, Sung JH. Lysophosphatidic acid increases the proliferation and migration of adiposederived stem cells via the generation of reactive oxygen species. Molecular medicine reports. 2015 doi: 10.3892/mmr.2015.4023. [DOI] [PubMed] [Google Scholar]
  • 28.Shida D, Kitayama J, Yamaguchi H, Okaji Y, Tsuno NH, Watanabe T, Takuwa Y, Nagawa H. Lysophosphatidic acid (LPA) enhances the metastatic potential of human colon carcinoma DLD1 cells through LPA1. Cancer research. 2003;63:1706–1711. [PubMed] [Google Scholar]
  • 29.Kim J, Keys JR, Eckhart AD. Vascular smooth muscle migration and proliferation in response to lysophosphatidic acid (LPA) is mediated by LPA receptors coupling to Gq. Cellular signalling. 2006;18:1695–1701. doi: 10.1016/j.cellsig.2006.01.009. [DOI] [PubMed] [Google Scholar]
  • 30.Willier S, Butt E, Grunewald TG. Lysophosphatidic acid (LPA) signalling in cell migration and cancer invasion: a focussed review and analysis of LPA receptor gene expression on the basis of more than 1700 cancer microarrays. Biology of the cell / under the auspices of the European Cell Biology Organization. 2013;105:317–333. doi: 10.1111/boc.201300011. [DOI] [PubMed] [Google Scholar]
  • 31.MacGrath SM, Koleske AJ. Cortactin in cell migration and cancer at a glance. Journal of cell science. 2012;125:1621–1626. doi: 10.1242/jcs.093781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wang C, Wu LL, Liu J, Zhang ZG, Fan D, Li L. Crosstalk between angiotensin II and platelet derived growth factor-BB mediated signal pathways in cardiomyocytes. Chinese medical journal. 2008;121:236–240. [PubMed] [Google Scholar]
  • 33.Wuhanqimuge A, Itakura Y, Matsuki M, Tanaka M. Arioka, Lysophosphatidylcholine enhances NGF-induced MAPK and Akt signals through the extracellular domain of TrkA in PC12 cells. FEBS open bio. 2013;3:243–251. doi: 10.1016/j.fob.2013.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Domeniconi M, Chao MV. Transactivation of Trk receptors in spinal motor neurons. Histology and histopathology. 2010;25:1207–1213. doi: 10.14670/HH-25.1207. [DOI] [PubMed] [Google Scholar]
  • 35.Shah BH, Catt KJ. Matrix metalloproteinase-dependent EGF receptor activation in hypertension and left ventricular hypertrophy. Trends in endocrinology and metabolism: TEM. 2004;15:241–243. doi: 10.1016/j.tem.2004.06.011. [DOI] [PubMed] [Google Scholar]
  • 36.Luttrell LM. Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors. Canadian journal of physiology and pharmacology. 2002;80:375–382. doi: 10.1139/y02-045. [DOI] [PubMed] [Google Scholar]
  • 37.Eguchi S, Frank GD, Mifune M, Inagami T. Metalloprotease-dependent ErbB ligand shedding in mediating EGFR transactivation and vascular remodelling. Biochemical Society transactions. 2003;31:1198–1202. doi: 10.1042/bst0311198. [DOI] [PubMed] [Google Scholar]
  • 38.Waters CM, Connell MC, Pyne S, Pyne NJ. c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells. Cellular signalling. 2005;17:263–277. doi: 10.1016/j.cellsig.2004.07.011. [DOI] [PubMed] [Google Scholar]
  • 39.Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, Polosukhin V, Wain J, Karimi-Shah BA, Kim ND, Hart WK, Pardo A, Blackwell TS, Xu Y, Chun J, Luster AD. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nature medicine. 2008;14:45–54. doi: 10.1038/nm1685. [DOI] [PubMed] [Google Scholar]
  • 40.Freund-Michel V, Frossard N. Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells. Biochimica et biophysica acta. 2008;1783:1964–1971. doi: 10.1016/j.bbamcr.2008.05.014. [DOI] [PubMed] [Google Scholar]
  • 41.Roskoski R., Jr ERK1/2 MAP kinases: structure, function, and regulation. Pharmacological research : the official journal of the Italian Pharmacological Society. 2012;66:105–143. doi: 10.1016/j.phrs.2012.04.005. [DOI] [PubMed] [Google Scholar]
  • 42.Olianas MC, Dedoni S, Onali P. Antidepressants activate the lysophosphatidic acid receptor LPA(1) to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts. Biochemical pharmacology. 2015;95:311–323. doi: 10.1016/j.bcp.2015.04.002. [DOI] [PubMed] [Google Scholar]
  • 43.Natarajan K, Berk BC. Crosstalk coregulation mechanisms of G protein-coupled receptors and receptor tyrosine kinases. Methods in molecular biology. 2006;332:51–77. doi: 10.1385/1-59745-048-0:51. [DOI] [PubMed] [Google Scholar]
  • 44.Zhao J, He D, Berdyshey E, Zhong M, Salgia R, Morris AJ, Smyth SS, Natarajan V, Zhao Y. Autotaxin induces lung epithelial cell migration through lysoPLD activity-dependent and -independent pathways. Biochemical Journal. 2001;439:45–55. doi: 10.1042/BJ20110274. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES