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. 2011 Jan 6;6(1):e15909.
doi: 10.1371/journal.pone.0015909.

PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis

Ajit A Kulkarni  1 , Thomas H Thatcher, Keith C Olsen, Sanjay B Maggirwar, Richard P Phipps, Patricia J Sime
Affiliations

PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis

Ajit A Kulkarni et al. PLoS One. .

Abstract

Transforming growth factor beta (TGFβ) induced differentiation of human lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription factors, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands inhibit TGFβ-mediated differentiation of human lung fibroblasts to myofibroblasts via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway leading to phosphorylation of Akt(S473). Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15d-prostaglandin J(2) (15d-PGJ(2)), inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF) fibroblasts, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A(1) (PGA(1)), a structural analogue of 15d-PGJ(2) with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of 15d-PGJ(2) and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of anti-fibrotic therapeutics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Inhibition of PI3K-Akt pathway by LY294002 inhibits myofibroblast differentiation.
Primary HLFs were treated with the PI3K inhibitor LY294002 (50µM) followed by TGFβ (5ng/ml) for 48 hours and A, immunoblots were performed to detect expression of the indicated proteins, and B, immunofluorescence for αSMA (green) was performed to assess the effects of PI3K inhibition on TGFβ-induced myofibroblast differentiation. DAPI (blue) was used to visualize nuclei. C, HLF cells were transfected with an empty vector or a dominant negative kinase-dead (KD) Akt construct, treated with TGFβ, and assayed for myofibroblast differentiation by Western blot. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. These data indicate that a functional PI3K-Akt pathway is essential for the TGFβ-induced myofibroblast differentiation in primary human lung fibroblast.
Figure 2
Figure 2. PPAR-γ ligands inhibit TGFβ-induced phosphorylation of Akt and myofibroblast differentiation in a dose-dependent manner.
A, Primary HLFs were grown until 70–80% confluent, serum starved for 24 hours and treated with the indicated concentrations of PPAR-γ ligands for 48 hours. Total cell lysates were prepared, and subjected to SDS-PAGE followed by immunoblotting. The blot was probed with antibodies against phospho-AktS473, stripped and probed to detect total Akt, αSMA and loading control GAPDH. The relative changes in the ratio of phospho-AktS473/total Akt (R.P.) and relative changes in the expression of αSMA/GAPDH (R.E.) are as indicated in the figure. The experiment was performed in triplicate and a representative blot is shown here. B, LDH release does not increase in response to 15d-PGJ2 or CDDO. Primary human lung fibroblasts were treated with either 5 µM 15d-PGJ2 or 1µM CDDO for 72 hours and LDH release was measured (nmol/min/mL). C, Primary human lung fibroblasts were transfected with a PPRE luciferase reporter and a CMV β-galactosidase construct. Cells were treated with either 5µM 15d-PGJ2 or 1 µM CDDO for 48 hrs and luciferase activity was measured. Background was subtracted and data normalized to β-galactosidase transfection efficiency and reported as fold induction of luciferase units over the untreated samples. These data represent three independent experiments (mean ± S.E. shown, **p≤0.01, *** p≤0.001, compared to untreated).
Figure 3
Figure 3. PPAR-γ ligands inhibit TGFβ-induced Akt phosphorylation and myofibroblast differentiation in a PPAR-γ-independent but electrophilic carbon-dependent manner.
HLF cells were treated with indicated compounds and/or TGFβ (5ng/ml) for 48 hours. Immunoblots were performed to assess the expression of indicated proteins. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. A, the ability of PPAR-γ ligands (CDDO (1µM) and 15d-PGJ2 (5µM)) to reduce p-Akt was not altered upon GW9662-mediated inhibition of PPAR-γ. GW9662 (5µM) inhibits PPAR-γ activity by a covalent bond formation with PPAR-γ protein . R.P. indicates relative changes in Akt phosphorylation compared to control sample, and R.E., relative changes in expression compared to control sample. B, PPAR-γ ligands contain electrophilic carbons. Here, positions of the electrophilic carbons in the structures of the compounds are marked. CAY10410 and PGA1 are structural analogues of 15-d-PGJ2. PGA1 has an electrophilic center but CAY10410 does not. DSPS, like CDDO, has two electrophilic centers. Cells were pre-treated with CAY10410 (5µM), PGA1 (10µM) and DSPS (10µM) for 30 minutes C, only compounds with an electrophilic carbon are able to reduce Akt phosphorylation, indicating that presence of an electrophilic carbon is essential for the observed reduction in the phosphorylation of Akt. All the experiments were performed in triplicate and representative images are shown here.
Figure 4
Figure 4. Actinomycin D partially inhibits TGFβ-induced phosphorylation of Akt but not PPAR-γ ligand-mediated inhibition of Akt phosphorylation.
Primary HLFs were pre-treated with transcription inhibitor ActD (1µg/ml), followed by PPAR-γ ligands (CDDO (1µM) and 15d-PGJ2 (5µM)) and TGFβ (5ng/ml). A, Immunoblots were performed to detect levels of indicated proteins upon inhibition of transcription. The experiment was performed with triplicate samples. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. B, The triplicate samples were measured by densitometry and the ratio of phospho to total Akt was determined and normalized to untreated and bar graphs were plotted. The significance was calculated by one way ANOVA. For samples without ActD treatment (open bars), * indicates difference (P≤0.05) over untreated and ** indicates difference (P≤0.05) over TGFβ-treated samples. For ActD-treated samples (black bars), # indicates difference (P≤0.05) over untreated and ## indicates difference (P≤0.05) over TGFβ-treated samples.
Figure 5
Figure 5. PPAR-γ ligands inhibit TGFβ-induced phosphorylation of Akt and FAK but not MAPK-p38 and PTEN.
Primary HLFs were pretreated with PPAR-γ ligands; CDDO (1µM) and 15d-PGJ2 (5µM) followed by TGFβ (5ng/ml). Cells were harvested and lysates analyzed by immunoblots at the indicated time. The ratio of phospho-protein to total protein was measured by densitometric analysis and normalized to untreated cells (untreated = 1.0). TGFβ-induced phosphorylation of A, AktS473 and D, FAKY397 was inhibited significantly by the PPAR-γ ligands but the phosphorylation of B, PTENT308 and C, p38-MAPK T180/Y182 was not affected. The statistical significance over TGFβ-treatment alone was calculated either by one way ANOVA on triplicate samples (A, B and C) or using unpaired t-test on duplicate samples (D) and is indicated as * where P≤0.05.
Figure 6
Figure 6. Pharmacological inhibition of FAK activity inhibits the PI3K-Akt pathway and myofibroblast differentiation.
A, Primary HLFs were treated in presence or absence of TGFβ and 10µM specific Src-FAK kinase inhibitor AG1879 (PP2) or its analogue, PP3, that does not inhibit FAK activity and immunoblots were performed to analyze expression of the indicated proteins. The FAK inhibitor AG1879, but not its analogue PP3, inhibited TGFβ-induced phosphorylation of FAKY397 and AktS473 and reduced myofibroblast differentiation as determined by expression of αSMA and calponin. B, HLF cells were transfected with the empty vector (V) or FAK overexpressing construct (HA-FAK) and assayed for myofibroblast differentiation by Western blot. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, irrelevant lanes excluded and representative lanes from a single experiment are shown here. These data indicate that FAK overexpression induces myofibroblast differentiation of primary human lung fibroblasts.
Figure 7
Figure 7. PPAR-γ ligands block myofibroblast differentiation of primary human IPF fibroblasts.
Primary IPF fibroblasts were treated with either A, two PI3K inhibitors LY294002 (50µM) or wortmannin (100nM) or B, a Src-FAK inhibitor AG1879 (10 and 20µM) or C, CDDO (1µM) or 15d-PGJ2 (5µM) followed by TGFβ (5ng/ml) for 48 hours. Protein lysates were prepared and immunoblots were performed to detect expression levels of the indicated proteins. The experiment was performed in triplicate on three independent IPF fibroblast strains. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from one representative set of data are shown here. These data confirm that CDDO and 15d-PGJ2 inhibit both, FAK and PI3K-Akt pathways to inhibit TGFβ-induced myofibroblast differentiation of primary IPF fibroblasts.
Figure 8
Figure 8. A proposed model showing the mechanism of action of electrophilic PPAR-γ ligands on TGFβ-induced myofibroblast differentiation.
TGFβ induces myofibroblast differentiation by activating SMAD, FAK and PI3K-Akt pathways. However, PPAR-γ ligands inhibit the TGFβ-induced PI3K-Akt pathway, partly by targeting FAK induced activation of Akt.
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