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. 2005 Feb;32(2):135-41.
doi: 10.1165/rcmb.2004-0126OC. Epub 2004 Nov 11.

Prostaglandin E(2) inhibits fibroblast migration by E-prostanoid 2 receptor-mediated increase in PTEN activity

Eric S White  1 , Rachelle G Atrasz, Emily G Dickie, David M Aronoff, Vuk Stambolic, Tak W Mak, Bethany B Moore, Marc Peters-Golden
Affiliations

Prostaglandin E(2) inhibits fibroblast migration by E-prostanoid 2 receptor-mediated increase in PTEN activity

Eric S White et al. Am J Respir Cell Mol Biol. 2005 Feb.

Erratum in

  • Am J Respir Cell Mol Biol. 2008 Aug;39(2):252

Abstract

An increased migratory phenotype exists in lung fibroblasts derived from patients with fibroproliferative lung disease. Prostaglandin E(2) (PGE(2)) suppresses fibroblast migration, but the receptor(s) and mechanism(s) mediating this action are unknown. Our data confirm that treatment of human lung fibroblasts with PGE(2) inhibits migration. Similar effects of butaprost, an E-prostanoid (EP) 2 receptor-specific ligand, implicate the EP2 receptor in migration-inhibitory signaling. Further, migration in fibroblasts deficient for the EP2 receptor cannot be inhibited by PGE(2) or butaprost, confirming the central role of EP2 in mediating these effects. Our previous data suggested that phosphatase and tensin homolog on chromosome ten (PTEN), a phosphatase that opposes the actions of phosphatidylinositol-3-kinase (PI3K), may be important in regulating lung fibroblast motility. We now report that both PGE(2) and butaprost increase PTEN phosphatase activity, without a concomitant increase in PTEN protein levels. This contributes to EP2-mediated migration inhibition, because migration in PTEN-null fibroblasts is similarly unaffected by EP2 receptor signaling. Increased PTEN activity in response to EP2 stimulation is associated with decreased tyrosine phosphorylation on PTEN, a mechanism known to regulate enzyme activity. Collectively, these data describe the novel mechanistic finding that PGE(2), via the EP2 receptor, decreases tyrosine phosphorylation on PTEN, resulting in increased PTEN enzyme activity and inhibition of fibroblast migration.

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Conflict of interest statement

Conflict of Interest Statement : E.S.W. has no declared conflicts of interest; R.G.A. has no declared conflicts of interest; E.G.D. has no declared conflicts of interest; D.M.A. has no declared conflicts of interest; V.S. has no declared conflicts of interest; T.W.M. has no declared conflicts of interest; B.B.M. has no declared conflicts of interest; and M.P-G. has no declared conflicts of interest.

Figures

Figure 1
Figure 1
PGE2 inhibits bFGF-induced fibroblast migration. (A ) Migration of human lung fibroblasts in the presence or absence of bFGF, PGE2, or butaprost. Results are expressed as the mean number of migrated cells per hpf ± SEM. Experiments were performed in triplicate wells, and the experiment was repeated twice with representative results shown (*P < 0.001). (B ) The EP2 receptor is the predominant EP receptor expressed on IMR-90 cells. Bands corresponding to the EP1 and EP3 receptors were present but extremely faint. EP4 was undetectable. The results are representative of two separate experiments. (C ) Migration of wild-type (C57Bl/6) (white bars) or EP2-null (black bars) murine lung fibroblasts in the presence or absence of bFGF, PGE2, or butaprost. Results are expressed as the mean number of migrated cells per hpf ± SEM. Experiments were performed in triplicate wells, and the experiment was repeated twice with representative results shown (*P < 0.001).
Figure 2
Figure 2
PGE2 and butaprost treatment of lung fibroblasts increases PTEN activity. (A ) PTEN activity in IMR-90 cells increased 40% compared with control conditions by 6 h after PGE2 treatment and continuing through 18 h (*P < 0.001). PTEN activity returned to the control level by 24 h after treatment. Total PTEN levels were unchanged throughout the time course. (B ) PTEN activity in IMR-90 cells was increased 3.5-fold after butaprost treatment as compared with control conditions (*P < 0.001). PTEN activity returned to that of control by 24 h. Total PTEN levels were unchanged throughout the time course. (C ) Increased PTEN activity is dependent on EP2 signaling. PTEN activity increases in a time-dependent fashion after butaprost treatment in C57Bl/6 cells (white bars), but not EP2-null cells (black bars). (D ) Intracellular production of cyclic AMP (cAMP) in IMR-90 cells after butaprost (triangles) or PGE2 (squares) stimulation, reported in arbitrary units. The ratio of cAMP increase after butaprost as compared with PGE2 is reported below the graph. The results are representative of two independent experiments. (E ) Butaprost treatment resulted in a time-dependent diminution in S473 phospho-Akt. Control indicates IMR-90 cells in serum-free media alone. The data are representative of two sets of lysates prepared independently. Total Akt levels were unaffected. Numbers above lanes indicate relative density of phospho-Akt to Akt.
Figure 3
Figure 3
PTEN activity is required for EP2-mediated inhibition of fibro-blast migration. Migration in wild-type (C57Bl/6) (white bars) murine embryonic fibroblasts and pten-null (black bars) embryonic fibroblasts. Baseline migration of pten-null fibroblasts was significantly greater than control fibroblasts. In the presence of bFGF, both control and pten-null fibroblasts demonstrated increased migration. Whereas bFGF-induced migration of wild-type fibroblasts was significantly inhibited by either butaprost or PGE2 (*P < 0.001), migration in pten-null cells could not be inhibited by either agent.
Figure 4
Figure 4
Butaprost treatment of lung fibroblasts results in decreased PTEN tyrosine phosphorylation. (A ) IMR-90 lung fibroblasts treated with butaprost (500 nM) were lysed, separated by electrophoresis, and were immunoblotted with antibodies against PTEN phosphorylated on S380 or S380T382T383, or total PTEN. No change in phosphorylation of these residues was observed after butaprost treatment. (B ) IMR-90 lung fibro-blasts treated with butaprost (500 nM) were lysed and PTEN was immunoprecipitated. Immunoprecipitates were separated by electrophoresis and immunoblotted with antibody against phosphotyrosine. Numbers above lanes indicate relative density of phosphotyrosine to total PTEN. Tyrosine phosphorylation of PTEN decreased over time after butaprost treatment. To ensure equal protein loading, blots were stripped and reprobed for PTEN.
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