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Cell Biology International (2011) 35, 875–881 (Printed in Great Britain)
Lysophosphatidic acid (LPA) stimulates mouse mammary epithelial cell growth
In Suh Yuh1
Department of Animal Biotechnology, College of Animal Life Sciences, Kangwon National University, KNU Ave 1, Chunchon 200701, Korea

LPA (lysophosphatidic acid) is a bioactive phospholipid having diverse effects on various types of tissues. When NMuMG (normal murine mammary gland) cells were cultured in the presence of 0–10 μM LPA, cell numbers were increased by dose dependency for the 6-day culture periods (P<0.05). In DNA synthesis assay, 10 μM LPA induced 4.5-fold more DNA synthesis compared with control (P<0.05). In addition, the cultured cell density in the given area was increased by LPA treatment. MMP (matrix metalloproteinase) inhibitor GM6001 and EGFR [EGF (epidermal growth factor) receptor] tyrosine kinase inhibitor AG1478 [tyrphostin AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline] significantly decreased LPA-induced DNA synthesis and cell growth without cell death (P<0.05). To test the hypothesis that LPA-induced cell growth is mediated through LPA subtype receptors, LPA subtype receptor gene expressions were amplified by PCR. NMuMG cells expressed LPA1 and LPA2 receptor genes in the presence of 10% FBS (fetal bovine serum). LPA treatments increased ERK1/2 (extracellular-signal-regulated kinase) phosphorylation at 30 min and then dephosphorylated at 2 h after treatment. LPA treatment phosphorylated at tyrosine residues on a variety of Gi and PI3-dependent signal transducers in NMuMG cells. These results suggest that LPA subtype receptors play a role as the active transactivator of EGFR-associated kinases as well as direct growth regulator in mammary tissues.

Key words: LPA receptor, mammary epithelial cells, protein tyrosine phosphorylation, transactivation of EGF receptor tyrosine kinase

Abbreviations: EDG, endothelial differentiation gene, EGF, epidermal growth factor, EGFR, EGF receptor, ERK1/2, extracellular-signal-regulated kinase 1/2, FBS, fetal bovine serum, GAP, GTPase-activating protein, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GPCR, G-protein-coupled receptor, GSK3, glycogen synthase kinase 3, HB-EGF, heparin-binding EGF, HRP, horseradish peroxidase, LPA, lysophosphatidic acid, LP, lysophospholipid, MAPK, mitogen-activated protein kinase, MMP, matrix metalloproteinase, NMuMG, normal murine mammary gland, PI3K, phosphatidylinositol 3-kinase, PLC, phospholipase C, RT–PCR, reverse transcription–PCR, Syk, spleen tyrosine kinase, TGFα, transforming growth factor α, ZAP-70, ζ-chain (T-cell receptor)-associated protein kinase of 70 kDa


1. Introduction

LPA (lysophosphatidic acid) is the common name for monoacyl-sn-glycero-3-phosphate and one of LPs (lysophospholipids). The major LPs are LPAs, sphingosine 1-phosphate, lysophsophatidylcholine and sphingosylphosphorylcholine. The major pool of LPA is serum and body fluids such as saliva and other extracellular fluids. The physiological concentration of albumin-bound LPA is 1-5 μM range in human serum (Baker et al., 2001). The mechanisms of LPA synthesis vary and depend on the specificity of cell or tissue at least for the local production. LPAs are produced through various phospholipase pathways that involve monoacylglycerol kinase, phospholipase A1, phospholipase A2 and lyso-phospholipase D (identical with autotaxin) (Tokumura et al., 2002). Particularly, phospholipases A2 and D activate phosphatidic acids to synthesize bioactive LPA.

LPA has diverse effects on various types of tissues. It can affect a variety of cellular functions, which include cell proliferation, smooth muscle contraction, platelet aggregation, cell migration, protection of apoptosis, modulation of chemotaxis and morphogenesis (Ishii et al., 2004). It interacts with at least three to four distinct GPCRs (G-protein-coupled receptors), LPA1, LPA2, LPA3 and LPA4 (An et al., 1998; Ishii et al., 2004). LPA1 [EDG-2 (endothelial differentiation gene 2)] is a high-affinity receptor for LPA. The mammalian (human and mouse, and rat) lpa1 gene encodes 45 kDa proteins consisting of 364 amino acids with seven putative transmembrane domains. LPA1 and LPA2 couple with three types of G-proteins, Gi/o, Gq and G12/13 (Ishii et al., 2000). LPA2 (EDG-4) receptors are expressed in testis, kidney, lung, thymus, spleen, prostate, peripheral blood leucocytes and stomach of adult mice and in human (Ishii et al., 2000). LPA2 induces cellular responses, DNA synthesis, SRE (serum response element) activation, MARK [MAP (microtubule-associated protein)-regulating kinase/microtubule affinity-regulating kinase] activation, adenylate cyclase inhibition, PLC (phospholipase C)/Ca2+ activation, Rho activation and PI3K (phosphatidylinositol 3-kinase)/Akt (also known as protein kinase B) activation. LPA2 receptor-induced cellular responses are pretty similar to those of LPA1-induced responses. LPA3 (EDG-7) was isolated as an orphan GPCR gene by degenerative PCR-based cloning and homology searches (Im et al., 2000). LPA3 can mediate other LPA receptor-induced signalling such as PLC activation, Ca2+ mobilization, adenylate cyclase inhibition/activation, and MAPK (mitogen-activated protein kinase) activation (Im et al., 2000; Ishii et al., 2000).

It has been known that GPCR agonists induce EGFR [EGF (epidermal growth factor) receptor] transactivation in various cell types. Two possible mechanisms have been proposed, phosphorylation of the EGFR by the tyrosine kinase src activation (Daub et al., 1997; Luttrell et al., 1997; Luttrell and Luttrell, 2004) and MMP (matrix metalloproteinase)-mediated release of EGFR ligands (Prenzel et al., 1999; Pierce et al., 2001; McCole et al., 2002; Santiskulvong and Rozengurt, 2003). In the first case, some GPCRs and G-protein subunits directly regulate src activity. In the second case, EGFR ligands, such as HB-EGF (heparin-binding EGF) and TGFα (transforming growth factor α), are shed from the cell membrane by members of the ADAM (a disintegrin and metalloproteinase) family of MMPs. These shed EGFR ligands activate src and phosphorylate EGFR on tyrosine residues.

G-protein modulated the undifferentiated mammary epithelial cell growth in vitro (Shamay et al., 1990; Yuh and Sheffield, 1998). In spite of the some feasibilities of GPCR existence in mammary tissue from the previous studies, the possibility that the LPA receptors are presented in mammary tissue and these receptor-mediated pathways are involved in mammary development has received little attention. In addition, the GPCR activation which transactivates receptor tyrosine kinases through various growth factors on cell membranes depends upon the cell types. The possible roles of LPA and the interaction of LPA with other growth factor receptor pathways in mammary tissue are not clear. Thus the objective of this study is to determine whether LPA receptor stimulates growth of mammary epithelium and the growth stimulatory effect of LPA is modulated by transactivation of receptor tyrosine kinase or not.

2. Materials and methods

2.1. Cell culture

The NMuMG (normal murine mammary gland; Owens et al., 1974) cells were maintained at 37°C in an atmosphere of 95% air/5% CO2 and 100% humidity. Log-phase cells were routinely cultured in tissue culture flasks containing DMEM (Dulbecco's modified Eagle's medium; pH 7.4), supplemented with 10% FBS (fetal bovine serum), 50 units penicillin and 50 μg/ml streptomycin. For dose response of LPA for cell growth, cells were attached for 24 h in the presence of DMEM and 10% FBS and media was changed to serum-free DMEM for 24 h to enter G0/G1cells. The growth-arrested cells were treated with 0–100 μM of LPA for 6 days (media and treatments were refreshed every other day), trypsinized in trypsin-EDTA solution for 15 min and then counted cell numbers with a haemacytometer. To measure cell growth effect of GM6001 or AG1478 [tyrphostin AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline] on LPA treatment, cells were cultured with 0.2% FBS in DMEM as basal medium after 24 h cell attachment, the given concentration of GM6001 or AG1478 was added and then 10 μM LPA was added into half of GM6001 or AG1478 treatment groups at 30 min after GM6001 or AG1478 treatment. Each treatment was refreshed every other day thrice for 6-day incubation periods and then cell numbers were measured with haemacytometer.

2.2. Treatments

LPA (purchased from Sigma-Aldrich Co., U.S.A.), potent broad-spectrum inhibitor of MMPs [GM6001 (galardin); N-{(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl}-L-tryptophan methylamide, purchased from Calbiochem, U.S.A.], EGFR kinase inhibitor (AG1478, 4-(3-chloroanino)-6,7-dimethoxyquinazoline, purchased from Calbiochem). GM6001 and AG1478 were dissolved in DMSO to make 10 mM stocks of both reagents. The final concentration of solvent (DMSO) applied to no-addition control was diluted by 1/1000-fold and did not affect DNA synthesis and cell growth.

2.3. DNA synthesis

DNA synthesis was measured as incorporation of 3H-thymidine (70-86 Ci/mmol, Dupont, Willmington, DE, U.S.A.) into DNA by 1-h pulse of 1 μCi/ml beginning 18 h after treatment. The radioactivity incorporated into trichloroacetic acid-precipitable materials was measured as follows: cells were rinsed three times with TBS (Tris-buffered saline; 150 mM NaCl and 50 mM Tris, pH 7.5), three times with ice-cold 10% trichloroacetic acid and three times with ice-cold 100% ethanol. Cells were dried, dissolved in 0.5 M NaOH containing 0.1% Triton X-100, neutralized with HCl and then counted by liquid scintillation.

2.4. Total RNA preparation for quantitative RT–PCR (reverse transcription–PCR)

Total RNAs were isolated using QIA shredder, RNeasy kit (Qiagen, Hilden, Germany), and RNAlater RNA stablization reagents (Ambion Inc., Austin, TX, U.S.A.). On isolation, total RNA was frozen at −80°C until RT–PCR analysis.

2.5. RT–PCR and PCR products

Total RNAs were reverse transcribed in the presence of AMV-RT (avian myeloblastosis virus reverse transcriptase) and random hexamer primer (Promega, Madison, WI, U.S.A.). Reaction was performed at 37°C for 1.5 h and 95°C for 10 min. The primers used for the RT–PCR analysis have been reported previously (Moller et al., 2001). The sequences of the primer used were as follows: LPA1 receptor (349 bp product): sense, 5′-TCTTCTGGGCCATTTTCAAC-3′; antisense, 5′-TGCCTRAAGGTGGCGCTCAT-3′. LPA2 receptor (798 bp product): sense, 5′-CCTACCTCTTCCTCATGTTC-3′; antisense, 5′-TAAAGGGTGGAGTCCATCAG-3′. LPA3 receptor (382 bp product): sense, 5′-GGAATTGCCTCTGCAACATCT-3′; antisense, 5′-GAGTAGATGATGGGGTTCA-3′. GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 246 bp product): sense, 5′-GATGACATCAAGAAGGTGGTGAA-3′; antisense, 5′-GTCTTACTCCTTGGAGGCCATGT-3′. PCR was performed for 30 cycles with each cycle consisting of 30 s denaturation at 95°C, 30 s annealing at 57°C, 30 s extension at 72°C, and final 5 min extension at 72°C. PCR products were electrophoresed on a 5% polyacrylamide gel (Laemmli, 1970) and visualized by ethidium bromide staining.

2.6. Measurement of MAPK activity

NMuMG cells were cultured as described in the routine cell culture method to reach 80% confluent state. Prior to stimulation, cells were serum starved for 24 h and then stimulated according to the given incubation times. The reactions were stopped by placing culture dishes on the ice and by briefly washing cells with ice-cold PBS (pH 7.4). The cells were lysed in lysis buffer [15 mM NaCl/50 mM Tris/HCl, pH 7.8/Nonidet P40, 1% (w/v)/leupeptin (1 μM)/aprotinin (0.1 μM)]. The lysates were collected with rubber policemen and clarified by centrifugation (10 min, 13000 g, 4°C). The supernatant was resolved by SDS/12% PAGE. Phosphorylation of p44/42 MAPK [ERK1/2 (extracellular-signal-regulated kinase 1/2)] was detected by protein immunoblotting using MAPK rabbit polyclonal phosphor-antibody with anti-rabbit IgG HRP (horseradish peroxidase) conjugate as secondary antibody (Promega). The membranes were stripped by incubation in PBST (PBS-0.05% Tween-20, pH 7.4) on a shaker, and re-probed with rabbit anti-MAPK antibody (Sigma, St. Louis, MO, U.S.A.) with anti-rabbit IgG HRP conjugate as second antibody (Promega) to quantify the total MAPK loaded on to each lane. Quantification of phosphor ERK1/2 phosphorylation was performed after development of membranes with enhanced chemiluminescence (ECL system, Amersham Pharmacia Biotech) by scanning on image analysis system (Vilver Lourmat, France). The degree of ERK1/2 phosphorylation was normalized to total MAPK intensity within each treatment.

2.7. Protein tyrosine phosphorylation

The growth-arrested cells (3×106 cells/plate) were treated with 10 μM LPA for 0, 5 and 30 min. After incubation of 10 μM LPA for the given time periods, cell culture media were removed and cells were washed twice with ice-cold TBS (50 mM Tris, pH 7.5, 150 mM NaCl and 1.5 mM PMSF). For the whole cell extraction, Triton extraction solution (15 mM Tris, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.1 mM DTT (dithiothreitol), 0.5% Triton X-100, 10 μg/ml leupeptin and 0.5 mM PMSF) was added and cells were scraped by rubber policeman. Cell debris was pelleted by centrifugation at maximum speed in a desktop centrifuge at 4°C for 15 min. Supernatant was collected for AntibodyArray™ assay (Hypromatrix, Worcester, MA, U.S.A.). Trial AntibodyArray™ membranes were incubated with blocking solution (5% non-fat dried skimmed milk powder in TBST, 150 mM NaCl, 25 mM Tris and 0.05% Tween-20, pH 7.5) for 1 h at room temperature with slow shaking and then the membrane was incubated with mammary epithelial whole cell extracts for 2 h at room temperature with slow shaking. AntibodyArray™ membranes were washed thrice for 15 min each with TBST and then incubated with HRP-conjugated anti-phosphotyrosine antibody in TBST for 2 h at room temperature. AntibodyArray™ membranes were washed with TBST thrice for 15 min each and then peroxidase substrate with ECL system was applied and exposed to X-ray film as described in section 2.6. Experiments for protein tyrosine phosphorylation have two replications blocked by number of experiment repeats, and the magnitude of each protein phosphorylation was quantified by measuring the spot size on the computer image (Table 1).

Table 1 Protein tyrosine phosphorylation of NMuMG cells at 5 and 30 min after 10 μM LPA treatment

The screening of protein tyrosine phosphorylation was performed with the Trial AntibodyArray™ assay kit (Hypromatrix, Worcester, MA, U.S.A.). The detailed procedures are described in section 2. P, phosphorylated; –, dephosphorylated. References present the general knowledge of the known effectors.

Phosphorylated proteins 5 min 30 min Known effectors References
Erb2(Neu) P P EGF and EGF family (Fan et al., 2005; Shida et al., 2005)
pp120 P P Insulin (Najjar et al., 1995)
ZAP-70 kinase P TCR (T-cell receptor) engagement, Syk family (Au-Yeung et al., 2009)
c-fos P EGF, LPA, prolactin, oestrogen (Guzman et al., 2005)
IRAK P P Interleukin-1 (Cao et al., 1996)
Flt-3/2 P P Fms-like tyrosine kinase 3 (Flt3) ligand, VEGF (vascular endothelial growth factor) (Dehlin et al., 2008; Gille et al., 2001)
PI3 kinase, p85 P P EGF, LPA (Hutchinson et al., 2001; Xu et al., 2008)
Ras-GAP P EGF, LPA(Gi) (Radeff-Huang et al., 2004)
Syk P Non-receptor cytoplasmic tyrosine kinase in haemopoietic cell (Navara, 2004; Zhang et al., 2009)
c-Raf-1 P EGF (Schulze et al., 2004)
P38-MAPK P LPA (Saatian et al., 2006)
GSK3α P LPA (Fang et al., 2002)
c-Jun P LPA (Saatian et al., 2006)
Akt1/2 P EGF, LPA1, LPA2 (Chen et al., 2010)

2.8. Statistical analysis

Treatments were made in duplicate or triplicate, and were repeated on three or four separate occasions. Results were analysed by ANOVA using a randomized complete block design model. Overall means were compared by the pre-planned comparison (Snedecor and Cochran, 1980). Unless otherwise stated, significance was set at P<0.05.

3. Results

3.1. Cell growth by LPA addition

When NMuMG cells were cultured in the presence of 0–100 μM LPA, cell numbers were increased by dose dependency for 6-day culture periods. Addition of 1 or 10 μM of LPA induced 1.45- or 2.20-fold increase in cell number compared to the control (P<0.05, Figure 1). In the microscopic observation, addition of 10 μM LPA stimulated cell proliferation and increased cell density in the given culture area of the plate compared to control (Figure 2). Interestingly, 10 μM of LPA changed the cell organization and morphology compared with control. Addition of 100 μM LPA showed detachment of cells and cellular toxicity from microscopic observation.

3.2. PCR analysis of LPA subtype receptor gene expression

To examine whether mammary epithelial cells express LPA subtype receptors or do not in the growing cells, LPA subtype receptors were screened with PCR analysis. PCR products of the expressed LPA1 (348 bp) and LPA2 (798 bp) receptor genes were observed in the RT reaction lanes treated with avian myeloblastosis virus RT enzyme but were not in those without RT enzyme (Figure 3). These results support the hypothesis that LPA regulates mammary epithelial cell growth and extracellular organization through their receptors.

3.3. DNA synthesis and cell growth on LPA treatment by altering MMP or EGFR tyrosine kinase activity

To test whether LPA-induced mammary epithelial cell growth is partially altered by the change of MMP activity or EGFR tyrosine kinase activity or not, NMuMG cells were pre-incubated with broad-spectrum MMP inhibitor (GM6001) or LPA (Figure 4). GM6001 alone (0–10 μM) did not affect DNA synthesis. However, addition of 1 or 10 μM of GM6001 for 30 min before LPA treatment significantly decreased LPA-induced DNA synthesis. When AG1478 was added into the 10 μM LPA treatment group for 30 min before LPA stimulation, DNA synthesis of 10 μM LPA was significantly decreased by addition of 0.1, 1 or 10 μM of AG1478. In addition, 1 or 10 μM of AG1478 completely blocked 10 μM LPA-induced DNA synthesis of the growing cell. In Figure 5, to investigate if GM6001 or AG1478 has toxic effect on cell growth or not, these reagents were added to 10 μM LPA treatment group for 6 days and cell numbers were measured. Addition of 1 and 10 μM GM6001 or AG1478 showed cell growth rates of 86.6 and 80.1% or 74.4 [pooled SE (standard error) = 10.8%] and 72.7% (pooled SE = 12.6%) to no-addition control showing no statistical differences within each reagent treatment group (P>0.05). However, addition of 10 μM GM6001 or 1 and 10 μM AG1478 significantly decreased 10 μM LPA-induced cell growth without cell death by 74.1% or 57.4 and 57.4% respectively (P<0.05) suggesting the specificities of these inhibitors to their target enzymes.

3.4. Activation of MAPK activity by LPA

To test whether LPA stimulates cell growth through MAPK pathways or not, cells were treated with 10 μM LPA for the indicated times in Figure 6. LPA significantly increased p44/42 MAPK (ERK1/2) phosphorylation at 30 min after treatment and then dephosphorylated at 2 h after treatment. These results indicate that LPA-induced mammary epithelia growth includes MAPK pathways. In the previous experiment, EGF and ATP analogues (purinergic receptor agonists) significantly increased ERK1/ERK2 phosphorylations at 5 min after treatment (Yuh, 2004). In the present study, the peak time of MAPK activation by LPA was slower than that of other mitogens such as EGF, ATP or irreversible ATP analogues.

3.5. Protein tyrosine phosphorylation by LPA

It has been reported that LPA1, LPA2 and LPA3 similarly couple to three types of G-proteins, Gi/o, Gq and G12/13 (Heringdorf and Jakobs, 2007). In the present study, addition of 10 μM LPA into NMuMG cells phosphorylated on tyrosine residues in the known Gi protein signal transducers such as ERK, p38, Akt, c-Jun and c-fos. LPA 10 μM also stimulated phosphorylation at tyrosine residue in PI3K and GSK3 (glycogen synthase kinase 3), a multifunctional serine/threonine kinase. In addition, LPA treatment of NMuMG cells increased phosphorylation of tyrosine residues in Erb2 (Neu) protein that were activated by transphosphorylation of tyrosine residues in Erb2 family heterodimers formed on the stimulation of EGF family ligands (Fan et al., 2005; Shida et al., 2005). This result further supports the hypothesis that LPA partially and indirectly transactivates the signal through EGFR tyrosine kinase in mammary tissue.

4. Discussion

The expressed PCR products of LPA1 and LPA2 receptor genes in Figure 3 suggest that the mammary epithelial cell growth and extracellular organization by addition of LPA were regulated by these LPA receptors. In previous reports, LPA2 that activates G12/Rho pathway regulates cell cytoskeleton rearrangements and cell rounding (Radeff-Huang et al., 2004; Heringdorf and Jakobs, 2007). In the present study, 10 μM LPA treatment changed cell morphology or organization as well as significantly increased cell numbers. The change of cell morphology and reorganization by LPA treatment may suggest the possibility of involvement of LPA2 receptors in mammary epithelial cells. In the previous experiments, physiological concentration of EGF (1–100 nM) did not change NMuMG cell morphology and extracellular organization during cell growth (results not shown). Thus morphological change of cells by addition of LPA might be the LPA receptor-mediated main effect, if the biological effect on morphological changes is not linked to transactivation effect of EGFR tyrosine kinase.

It has been reported that GPCR agonists, including LPA, induced EGFR transactivation presumably by direct regulation of src activity by GPCRs (Luttrell and Luttrell, 2004) and/or by the family of MMP-mediated release of EGFR ligands such as HB-EGF and TGFα (McCole et al., 2002; Borrell-Pages et al., 2003) and platelet-derived growth factor receptor-beta (Wang et al., 2003) with different cell types and culture systems. In the present experiment, the blockade of MMP and EGFR tyrosine kinase activity by GM6001 and AG1478 significantly inhibited the LPA-induced DNA synthesis and cell proliferation suggesting the involvement of the MMP and EGFR tyrosine kinase regulation by LPA in mammary epithelial cells. The higher concentration of AG1478 (1-10 μM) completely inhibited LPA-induced DNA synthesis compared to control level. Otherwise, addition of GM6001 or AG1478 alone showed no differences in cell growth compared with the no-addition control group (P>0.05); however, 1 and 10 μM AG1478 decreased LPA-induced cell growth by 57.4% for a 6-day culture period (P<0.05) indicating the involvement of specific AG1478 inhibitory effect on EGFR tyrosine kinase activity induced indirectly by LPA. In previous experiments, 1/1000-fold dilution of stock DMSO equivalent to DMSO concentration in 10 μM AG1478 not only affected cell growth but also DNA synthesis rate in NMuMG cells. Thus the complete inhibition of AG1478 on LPA-induced DNA synthesis might result from some interference effect in 3H-thymidine uptake into cells or other unknown effects for 24 incubation period. The slower activation of MAPK by LPA than by other mitogens such as EGF and ATP analogues may raise the possibility of indirect pathway involvement of LPA in mammary epithelial cell growth.

In the present study, addition of 10 μM LPA in NMuMG cells phosphorylated tyrosine residues in ERK, p38, Akt, c-Jun and c-fos which were activated through LPA1 receptor-mediated Gi protein in various cell types (Saatian et al., 2006; Kim et al., 2008; Chen et al., 2010). LPA (10 μM) also stimulated PI3K and GSK3, which have been known to be regulated through PI3K activation pathway (Fang et al., 2002). It has been reported that activation of PI3K pathway resulted in the inhibition of pro-apototic signals and the stimulation of cell growth and survival in many different cell types (Hutchinson et al., 2001). Many of these phospho-proteins known as serine/threonine kinases are autophosphorylated or phosphorylate at serine/threonine residues in substrate proteins, however, these proteins are also dual-specificity kinases differentially regulated by tyrosine and serine/threonine phosphorylation. In the present study, LPA treatment increased phosphorylation of tyrosine residues in Erb2 (Neu) protein. The increase of EGFR tyrosine kinase activity including Erb2 heterodimer on transactivation of NMuMG cells by LPA treatment might result in phosphorylation of tyrosine kinase specific sites in cytosolic downstream targets such as Ras-GAP (GTPase-activating protein), c-Raf, MAPK, c-Jun and c-fos. Interestingly, LPA phosphorylated the cytokine-associated and -activated proteins such as Syk (spleen tyrosine kinase), IRAK (interleukin-1-receptor-associated kinase) and ZAP-70 [ζ-chain (T-cell receptor)-associated protein kinase of 70 kDa]. These proteins might be phosphorylated on transphosphorylation on tyrosine residues by cleavage of a variety of cytokines genes which were bound on membranes. It has been known that Syk and ZAP-70 are haemopoietic cell-specific signalling proteins and play important role in lymphocyte maturation and immune cell activation. Recently, many studies have proved these proteins are expressed not only in haemopoietic cells but also in non-haemopoietic cell types including tumour cells (Navara, 2004). In addition, Syk protein is associated with actin and tubulin to organize intracellular cytoskeleton (Navara, 2004) and with integrin presumably to control cell motility (Zhang et al., 2009).

Overall results indicate that LPA regulates mammary epithelial cell growth and morphology not only by a direct LPA receptor-mediated pathway but also an indirect cellular signal pathway such as the transactivation of EGFR tyrosine kinase in mammary tissue.


The author thanks Dr Sheffield and Dr Milo Wiltbank at the University of Wisconsin-Madison, WI, U.S.A. for the use of laboratory facilities and also thank K.E. Lee, H.M. Kim and E.J. Shim who are undergraduate senior students at Department of Animal Biotechnology in Kangwon National University, Chunchon, Korea for some technical assistances and lab works.


This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.


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Received 27 September 2010/27 January 2011; accepted 15 February 2011

Published as Cell Biology International Immediate Publication 15 February 2011, doi:10.1042/CBI20100706

© The Author(s) Journal compilation © 2011 Portland Press Limited

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)