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First published online 28 March 2006
doi: 10.1242/jcs.02858

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Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling

Ophthalmology and Visual Sciences Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA

View larger version (18K): [in a new window]   Fig. 1. Wounding induces activation of PLD. Corneal epithelial cells were seeded in the presence of agarose droplets (Block et al., 2004) and labeled with [3H]myristic acid. The agarose was removed and 1-butanol added for 5-minute intervals, and the amounts of radioactivity in phosphatidylbutanol (PtdBu) quantified (for details, see Materials and Methods). (A) Autoradiogram of thin-layer plates. (B) Time course of activation. The values in this and the following figures are means ± s.d. of triplicate determinations, except where noted. All experiments were performed at least three times with consistent results. (C) Effect of tyrphostin AG 1478 on PLD activation. Corneal epithelial cells were wounded and activities measured at 0-5 minutes with 10 µM UO126 or 10 µM tyrphostin AG 1478, as indicated. Control blots showed that the phosphorylation of the EGFR was completely blocked (data not shown, but see Fig. 7C,D).

View larger version (106K): [in a new window]   Fig. 2. C8-PA promotes a motile phenotype. (A) Corneal epithelial cells were seeded in the presence of agarose droplets and photographed using phase contrast optics. `0' is before removal of agarose droplets. `Untreated', `+EGF' and `+C8-PA' are 1 day after removal of the agarose droplets with no treatment, or in the presence of 100 ng/ml EGF or 250 µM C8-PA, as indicated. Bars, 50 µm. (B) Induction of vinculin by wounding. Corneal epithelial cells were seeded as in A. The agarose droplets were removed, and the cells were harvested at the indicated times. Extracts were then subjected to SDS-PAGE and blotted with an anti-vinculin antibody, and the same samples blotted with an anti-ß-actin antibody as a loading control. (C) MDCK cells were treated with 10 ng/ml HGF or 250 µM C8-PA overnight, as indicated. `Reverted' refers to cells that had been treated with C8-PA overnight and transferred to fresh medium without C8-PA for 8 hours.

View larger version (88K): [in a new window]   Fig. 3. Analysis of phenotype induced by overexpression of PLD2, and localization of endogenous PLD2. (A) MDCK cells were infected with the adenovirus coding for wild-type or an enzymatically inactive mutant of PLD2 and photographed using phase contrast optics. The vector also codes for the green fluorescent protein, and the infected cells are shown. The graph shows PLD activities in infected cultures. Bars, 20 µm. (B) Cells were infected overnight with adenovirus coding for the indicated construct, or treated with C8-PA for 5 hours and stained with Alexa Fluor® 546 phalloidin or immunostained for E-cadherin. As in A, virtually all cells in the fields were infected. (C) Localization of PLD2. Corneal epithelial cells infected with adenovirus coding for PLD2(wt) or uninfected cells were stained with an anti-PLD2 antibody. Unspecific staining of uninfected cells was determined by pre-incubating the antiserum with the immunizing peptide (`blocked'). Uninfected cells 8 hours after induction of wounding were also stained (right panel). Arrows indicate staining of lamellipodia. The lamellipodial staining was blocked by preincubation of the antibody with the peptide (data not shown). A confocal section close to the tissue culture plastic is depicted, so the nuclei are not visible.

View larger version (14K): [in a new window]   Fig. 4. C8-PA enhances wound healing in MDCK cells. (A) Dose response. Wounds were induced as described previously (Block et al., 2004), and healing allowed to proceed for 14 hours in the presence of the indicated concentrations of C8-PA. (B) Healing in the presence of 375 µM C8-PA, 10 µM UO126 or 10 µM tyrphostin AG 1478, as indicated.

View larger version (26K): [in a new window]   Fig. 5. C8-PA does not induce phosphorylation of MARCKS. MDCK cells were stimulated with 100 nM PMA, 500 µM C8-DAG, or 500 µM C8-PA for 5 or 25 minutes, and extracts were immunoblotted with an anti-phospho-MARCKS antibody. Lower panel is a parallel blot probed with an anti-MARCKS antibody.

View larger version (15K): [in a new window]   Fig. 6. Inhibition of PLD blocks cell motility in corneal epithelial cells. (A) Inhibition of healing in wounds of confluent layers of cells. Healing was allowed to proceed in the presence of EGF (200 ng/ml), and alcohols [1-butanol (1-BuOH) and tert-BuOH] (0.45%). (B) Inhibition of EGF-induced chemokinesis. Cells were seeded in Transwell® migration chambers in the presence of the indicated compounds in both the top and bottom chambers. Alcohols were used at 0.40%, and C8-PA at 125 µM. (C) Cells were pre-treated with the indicated concentrations of 1-butanol for 8 hours. The 1-butanol was washed out, and the cells were allowed to migrate for another 8 hours in the presence of EGF.

View larger version (60K): [in a new window]   Fig. 7. C8-PA-induced scattering requires ERK1/2 and EGFR activities. (A) MDCK cells were incubated overnight with 250 µM C8-PA and 50 µM LY294002, 10 µM UO126, or 10 µM tyrphostin AG 1478 as indicated, and photographed using phase contrast optics. Bar, 50 µm. (B) Time-course for ERK1/2 activation. MDCK cells were treated with 375 µM C8-PA or 10 ng/ml HGF for the indicated periods of time and extracts were prepared and immunoblotted with an antibody against the activated forms of ERK1/2. (C) Activation of the EGFR by C8-PA in MDCK cells. Cells were pre-incubated with 10 µM UO126, 10 µM tyrphostin AG 1478, 50 µM GM6001, and then treated with C8-PA for 15 minutes or 10 ng/ml EGF for 10 minutes. Equal loads in the lanes were verified by staining the blots with Ponceau S (see Materials and Methods). (D) Activation of the EGFR by C8-PA in human corneal epithelial cells. Conditions were as in C. Preincubation with 50 µg/ml CRM 197 or 20 µg/ml antibody was for 6 hours.

View larger version (16K): [in a new window]   Fig. 8. Addition of exogenous PA activates cellular PLD. (A) Time course. 375 µM C8-PA was added to MDCK cells and the cellular PLD activities assayed as described in Materials and Methods at the indicated times after addition. The values are means of duplicate determinations and the error bars are the ranges of the values. (B) Effects of various agents on PLD activation by C8-PA. Cells were stimulated with 375 µM C8-PA after 4 hours pre-incubation with 50 µM brefeldin A, or 15 minutes preincubation with 10 µM UO126 or 10 µM tyrphostin AG 1478. PMA was used at 0.1 µM in the presence or absence of 10 µM C8-PA. LysoC6-PA was used at 10 µM and 1-oleoyl-glycero-3-phosphate (lyso phosphatidic acid; LPA) complexed with lipid-free bovine serum albumin was used at 2 µM. This experiment was performed in the absence of serum. (C) Lack of increase of PtdIns(4,5)P2 levels by C8-PA was assayed at the indicated times after addition of 500 µM C8-PA to MDCK cells. `5'kin' cells were infected with adenovirus coding for phosphatidylinositol 4-phosphate 5'-kinase as a positive control.

View larger version (16K): [in a new window]   Fig. 9. Down-regulation of response to C8-PA does not down-regulate response to C8-DAG. MDCK cells were untreated or pre-treated with 500 µM C8-PA overnight. The cells were then untreated or stimulated with 375 µM C8-PA or C8-DAG, as indicated, for 15 minutes and PLD activities were measured.

View larger version (12K): [in a new window]   Fig. 10. Model of PLD signaling in cell motility in corneal epithelial cells. Wounding stimulates PLD and/or EGFR through unknown mechanisms. PA synthesis is amplified through a positive feed-back loop. Increased PA leads to activation of the EGFR, and PLD can conversely be activated by EGFR signaling. Both EGFR and PA signaling are necessary for epithelial cell motility.

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