Idiopathic pulmonary fibrosis (IPF) can lead to respiratory failure and death due to deteriorating respiratory function 
. There are currently few if any effective therapies. Therefore, novel antifibrotic drugs are urgently needed for the treatment of IPF and other scarring diseases.
PPAR-γ ligands are emerging as exciting potential therapeutics for inflammatory and fibrotic and other diseases 
. PPAR-γ activation induces adipogenesis and differentiation, and represses inflammation 
. PPAR-γ ligands including rosiglitazone and other members of TZD family are used for the treatment of type II diabetes 
. Clinical trials for CDDO are in progress, and it has been found to be orally active for the treatment of solid tumors and lymphoma 
. However, the mechanisms of anti-fibrotic actions of PPAR-γ ligands remain poorly understood. Therefore, we investigated the underlying molecular pathways targeted by distinct PPAR-γ ligands to explore the potential of PPAR-γ agonists as anti-fibrotic therapies.
Our previous work identified CDDO, a novel PPAR-γ ligand, as a potent anti-fibrotic agent of TGFβ-driven pro-fibrotic activity in vitro 
. TGFβ induces lung fibrosis in vivo 
and also stimulates phosphorylation of Akt in animal models 
and other human organs 
. Studies in fetal lung fibroblasts demonstrate the role of TGFβ-induced Akt pathway in myofibroblast differentiation 
. Here, we report that in both normal and IPF primary human lung fibroblasts, PPAR-γ ligands potently block myofibroblast differentiation via a PPAR-γ-independent mechanism by targeting the TGFβ-induced PI3K-Akt pathway involving FAK.
We investigated the role of PI3K-Akt pathway in TGFβ-stimulated myofibroblast differentiation using LY294004, a specific PI3K activity inhibitor, and by using a kinase-dead (KD-Akt) construct of Akt. Both LY294002 and the Akt mutant strongly blocked TGFβ-stimulated myofibroblast differentiation, confirming the central role of PI3K-Akt pathway in TGFβ-mediated myofibroblast differentiation in adult human normal and “diseased” IPF lung fibroblasts ( and ). Although, CDDO 
and 15d-PGJ2 
have been reported to reduce Akt phosphorylation in some studies, their mechanism of reduction of TGFβ-induced myofibroblast differentiation through Akt pathway is not yet reported. Here, we show that the suppression of TGFβ-induced phosphorylation of AktS473
is the central mechanism of action of CDDO and 15d-PGJ2
that leads to their anti-fibrotic activity. CDDO suppresses TGFβ-induced phospho-Akt more potently than 15d-PGJ2
, which correlates very well with the abilities of CDDO and 15d-PGJ2
to reduce TGFβ-induced myofibroblast differentiation (). Compared to CDDO and 15d-PGJ2
, rosiglitazone was relatively poorly effective at inhibiting TGFβ-induced Akt phosphorylation (data not shown). Interestingly, Kilter et al. reported that rosiglitazone facilitates rephosphorylation of Akt in rat myocardiocytes 
. We and others have reported that rosiglitazone has some anti-fibrotic effects in vitro 
, but is much less potent than either CDDO or 15d-PGJ2
. If rosiglitazone indeed facilitates re-phosphorylation of Akt, then it would result in a pro-fibrotic response that undercuts its anti-fibrotic effects, suggesting that rosiglitazone is not an optimal choice for treating fibrotic lung diseases. We did not investigate rosiglitazone further in this study.
CDDO and 15d-PGJ2
have electrophilic properties that rosiglitazone does not, and we have previously identified the electrophilic centers as important in the antifibrotic activity of these compounds. Building on these observations, here we demonstrate that the PPAR-γ-independent effects of CDDO and 15d-PGJ2
on Akt phosphorylation are dependent on the electrophilic properties (). These observations offer an additional possibility that CDDO and 15d-PGJ2
could directly bind to the active site of a signaling molecule involved in the Akt pathway. One study has shown that biotinylated CDDO is capable of binding and thus inactivating the active site of PTEN 
, and 15d-PGJ2
is capable of inhibiting the activities of proteins through direct covalent modification 
. Further studies involving biochemical approaches should help us understand the exact nature of action of compounds involving electrophilic carbon. To generate newer anti-fibrotic therapeutics, further investigation of the electrophilic properties of PPAR-γ ligands and similar compounds is necessary.
Because CDDO and 15d-PGJ2
block Akt phosphorylation in the absence of new transcription (), this suggests that CDDO and 15d-PGJ2
act to directly inhibit a kinase or activate a phosphatase that acts on Akt. We examined the role of three important upstream regulators of Akt phosphorylation: p38-MAPK, PTEN and FAK (). We chose to examine p38-MAPK because it is previously known to be a part of the PI3K/Akt pathway 
while other MAP kinases are involved in separate 
or even antagonistic pathways 
. TGFβ activates a number of signaling pathways including Smads, Akt and the MAPK-ERK, and while our data does not completely rule out that PPAR-γ ligands may act via other pathways, the ability of LY294002 and KD-Akt to almost completely block myofibroblast differentiation shows that the PI3K/Akt pathway is the most important pathway. Since TGFβ stimulates phosphorylation of p38-MAPK to activate myofibroblast differentiation by up-regulating Akt phosphorylation we examined involvement of p38-MAPK in primary HLF.
A proposed model showing the mechanism of action of electrophilic PPAR-γ ligands on TGFβ-induced myofibroblast differentiation.
In agreement with a previous report 
, we determined that p38-MAPK is phosphorylated following TGFβ treatment of HLF (), and MAPK inhibitor SB203580 reduced phosphorylation of Akt (data not shown). However, neither CDDO nor 15d-PGJ2
altered TGFβ-induced p38-MAPK phosphorylation, indicating that MAPK is likely not involved in inhibition of Akt by CDDO and 15d-PGJ2
. We were also able to exclude PTEN as a major mediator of the effects of PPAR-γ ligands in HLFs. PTEN can inhibit the Akt pathway by dephosphorylating Akt, and PTEN phosphatase is itself activated by dephosphorylation at Thr308
. Thus, if CDDO or 15d-PGJ2
blocked Akt phosphorylation via PTEN, we would expect increased PTEN phosphatase activity associated with increase in its dephosphorylation at T308. In fact, CDDO or 15d-PGJ2
do not change PTEN phosphorylation (). Interestingly, in human retinal epithelial cells, biotinylated CDDO (CDDO-Bt) binds to Cys124 within the active site of PTEN and inhibits the lipid phosphatase activity of PTEN in vitro 
. If PTEN activity inhibition was an important mechanism in our system, we would expect levels of phospho-Akt to increase in presence of CDDO; instead we observed the opposite.
Multiple reports show that TGFβ stimulates autophosphorylation-dependent activation of focal adhesion kinase (FAK) 
. For example in CCL20 lung fibroblasts, Xia et al demonstrated that β1-integrin signaling upregulates FAK phosphorylation and its physical interaction with PI3K-p85 resulting in phosphorylation of Akt 
. Although FAK is a widely accepted upstream regulator of Akt phosphorylation, it has been reported that FAK does not act upstream of Akt during TGFβ signaling in IMR90 human fetal lung fibroblasts 
. However, it is widely accepted that integrins are able to activate TGFβ 
and FAK 
, both of which are involved in myofibroblast differentiation. Our results demonstrate that PPAR-γ ligands are able to inhibit phosphorylation of FAK and limit the TGFβ-mediated fibrotic response in adult primary HLFs (). We confirmed both in our normal () and IPF () primary human cell strains that blocking FAK activity inhibited not only phosphorylation of Akt, but also expression of αSMA, confirming that FAK indeed regulates myofibroblast differentiation under normal and diseased conditions through activation of Akt pathway. To establish the therapeutic potential of PPAR-γ ligands we treated fibroblasts obtained from IPF patients, in parallel, with PPAR-γ ligands, two PI3K inhibitors and a FAK inhibitor and confirmed that CDDO and 15d-PGJ2
potently block myofibroblast differentiation of not only normal HLF but also diseased IPF fibroblasts () via PI3K-Akt and FAK pathways. Although by using a complimentary genetic approach we confirmed that overexpression of FAK is capable of upregulating Akt phosphorylation and myofibroblast differentiation (), at this point, we cannot rule out an additional mechanism of action of PPAR-γ ligands that would result in reduction of Akt phosphorylation ().
Current models of pulmonary fibrosis suggest that TGFβ, mechanical stress, or adhesion and integrin mediated activation of myofibroblast differentiation all contribute to upregulation of a fibrotic response. One very critical, central and shared event in all of these pathways involves upregulation of FAK activity, defined by its phosphorylation at Tyr397. It is conceivable that once TGFβ activates myofibroblast differentiation, the increased deposition of extracellular matrix proteins would cause additional mechanical stress on the cell surface leading to sustained and continual activation of FAK. Since FAK itself upregulates myofibroblast differentiation, once TGFβ initiates this process, sustained activation of FAK would be able to perpetuate the fibrotic response even in the absence of active TGFβ. Our work is the first report in any biological system demonstrating that PPAR-γ ligands reduce FAK activity by reducing FAK phosphorylation at Tyr397. Since FAK plays a cardinal role in myofibroblast differentiation, drugs that target the catalytic activity of FAK could be very valuable in the treatment of pulmonary fibrosis.
This study highlights a very important mechanism of action of CDDO and 15d-PGJ2
that involves down-regulation of PI3K-Akt pathway in both normal and IPF fibroblasts. Knowing that Akt is a central regulator of multiple cellular pathways including cell proliferation, cell cycle progression, inflammation and apoptosis 
, interfering with the Akt pathway can have multiple cellular and organ-wide effects. Although, we have noted sustained basal activity of Akt in untreated cells, the nature of Akt activation is largely inducible and dependent on upstream signaling molecules. Therefore, the use of Akt-inhibition as a potential therapy for pulmonary fibrosis is a very novel and exciting concept.
Overall, we propose that certain PPAR-γ ligands have tremendous translational potential as therapeutics for pulmonary fibrosis by not only inhibiting Akt but also FAK activation. Future in vivo studies involving PPAR-γ ligands will be pivotal in exploring the promising potential of PPAR-γ ligands as therapeutics for pulmonary fibrosis as well as other scarring diseases.