PPAR
RXR and its Brothers

Emerging PPARγ-Independent Role of PPARγ Ligands in Lung Diseases

INTRODUCTION
The Peroxisome Proliferator Activated Receptor gamma (PPAR gamma) is a member of the nuclear receptor superfamily that, upon binding the heterodimeric partner RXR (the 9-cis retinoic acid receptor), is able to modulate target gene transcription. PPAR binds to specific DNA sequences (PPAR Response Elements or PPRE) located on the promoters of target genes, and can be activated by poly-unsaturated fatty acids, prostaglandins (PGJ2) and by a class of oral anti-diabetic drugs, the thiazolidinediones (TZD). PPAR gamma is expressed in different kind of solid tumors and his activation by specific ligands results in growth inhibition of several epithelial cancer cell lines, such as pancreatic adenocarcinoma, colon cancer, hepatocellular carcinoma, adrenal and thyroid carcinoma and lung disease thus suggesting a possible role for TZD in cancer treatment.

FIGURA 1 : PPARγ ligands have multiple PPARγ-independent effects. In the classical PPARγ-dependent pathway, ligand-bound PPARγ forms a heterodimer with RXR and binds to PPARγ-response elements (PPREs) which leads to modulation of transcription. However, PPARγ ligands also exhibit direct effects that do not involve transcriptional activation by PPARγ/RXR. These direct effects may involve PPARγ protein interacting with PPARγ ligands in a “non-classical manner” (not involving RXR or PPRE) or may be completely independent of PPARγ (functioning even in the complete absence of PPARγ protein, i.e., direct effects). PPARγ-independent effects can alter multiple cellular programs including regulation of differentiation, inflammation, apoptosis and may be of significant therapeutic interest.
PPARγ ligands show great promise in moderating lung inflammation, as antiproliferative agents in combination to enhance standard chemotherapy in lung cancer and as treatments for pulmonary fibrosis, a progressive fatal disease with no effective therapy. Some of these effects occur when PPARγ is pharmaceutically antagonized or genetically PPARγ and are thus independent of classical PPARγ-dependent transcriptional control. Many PPARγ ligands demonstrate direct binding to transcription factors and other proteins, altering their function and contributing to PPARγ-independent inhibition of disease phenotypes. These PPARγ-independent mechanisms are of significant interest because they suggest new therapeutic uses for currently approved drugs and because they can be used as probes to identify novel proteins and pathways involved in the pathogenesis or treatment of disease, which can then be targeted for further investigation and drug development.

MECHANISM OF ACTION IN LUNG DISEASE
The molecular mechanism underling the anti-cancer effects mediated by PPARg activation and by PPARg ligands are still largely unknown. Moreover, a detailed understanding of the interactions between PPARg, its ligands and the other factors involved in the regulation of cell behaviour is missing. Some data suggest that PPARg activation promotes cell differentiation, inhibits cell cycle progression and induce apoptosis.
The mechanisms through which PPARg activation inhibits cancer cell growth in vitro and in vivo remain controversial. Suggested mechanisms include cell cycle arrest, induction of cell differentiation and apoptosis.
In Vitro Models of Pulmonary Fibrosis and PPAR Ligands There is a growing body of evidence that both natural and synthetic PPARγ agonists have powerful antifibrotic effects in vitro , and these results are beginning to be translated to preclinical animal models. Typical experiments examine differentiation of human lung fibroblasts to myofibroblasts in vitro and associated changes in expression of profibrotic cytokines and matrix proteins. A variety of nontransformed human lung fibroblast (HLF) cell lines are used including fetal, neonatal, adult nonfibrotic, and adult fibrotic (derived from patient biopsies).We and others reported that rosiglitazone and 15d-PGJ2 inhibited TGFβ-driven myofibroblast differentiation of primary HLFs .Expression of a dominant PPARγ was able to reverse the inhibitory effect of rosiglitazone more effectively than 15d-PGJ2, suggesting that rosiglitazone can act via both PPARγ-dependent and -independent mechanisms while 15d-PGJ2 acts predominantly via an independent mechanism . Since primary HLFs express abundant PPARγ and RXR proteins and are capable of PPARγ-dependent transcriptional regulation, this suggested that that the antifibrotic effects of the PPARγ agonists were mediated through both PPARγ-dependent and -independent mechanisms. We also investigated CDDO, a triterpenoid originally identified in herbal preparations with anti-inflammatory properties. We determined that CDDO has an EC50 for inhibition of myofibroblast differentiation that is 20-fold lower than 15d-PGJ2 and 400-fold lower than rosiglitazone, and it acts independently of PPARγ as confirmed by pharmacological and genetic approaches [78]. Recently, we reported that 15d-PGJ2 and CDDO inhibit TGFβ-induced phosphorylation of phosphotidyl-inositol 3-kinase-protein kinase B (PI3K-Akt) and focal adhesion kinase (FAK), but not TGFβ -induced p38-MAPK phosphorylation, and that the mechanism was independent of PPARγ [79]. We also noted that there is a strong correlation between the ability of a PPARγ ligand to inhibit Akt phosphorylation with its ability to suppress myofibroblast differentiation . We find that rosiglitazone is a weak inhibitor of TGFβ-induced Akt phosphorylation and may be therefore a poor choice as an antifibrotic treatment.

FIGURA 2: PPARγ ligands inhibit TGFβ-induced Akt phosphorylation and myofibroblast differentiation with varied potency. Primary human lung fibroblasts were treated with TGFβ (5 ng/mL), alone or in combination with three PPARγ ligands (CDDO (1 μM), 15d-PGJ2 (5 μM) and rosiglitazone (20 μM)). Protein lysates were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. The potency of a PPARγ ligand to inhibit Akt phosphorylation corresponds to its ability to inhibit myofibroblast differentiation. While CDDO inhibits both Akt phosphorylation and αSMA potently, rosiglitazone is a weak inhibitor of both.

PPARg agonists are able to induce arrest in G1 or G2/M phase by inducing cell cycle inhibitors (i.e. p21, p27, p18), down-regulating cyclines (cyclin D1) and genes downstream of p53 signalling, such as GADD135.
The G1 phase arrest often results in induction of apoptosis through up-regulation of pro-apoptotic proteins such as BAX and BAD by TZD administration.
In lung cancer the anti-proliferative mechanism of TZD action involves an increase in PTEN expression which in turn causes an inhibition of PI3K/AKT expression and AKT phosphorylation (14). This inhibitory effect on cell growth is enhanced by rapamycin, an inhibitor of mTOR. In pancreatic cancer cells lines PPARg activation up-regulates PTEN and inhibits PI3K activity.

CONCLUSION
Discovered serendipitously, the off-target, or PPARγ-independent effects of PPARγ ligands may prove as interesting and therapeutically useful as their PPARγ-dependent effects. PPARγ agonists have potent PPARγ-independent effects in vitro and in vivo, regulating proinflammatory responses and acting to promote apoptosis and inhibit differentiation, which may be beneficial in treating cancer and fibrosing diseases. Clinical trials are underway, investigating the currently approved TZDs in novel diseases, and investigating novel agonists such as CDDO and its derivatives.
The role of PPAR-γ as a potential therapeutic target for fibrotic lung diseases remains undefined but it’s important to use PPARγ ligands as probes to uncover novel disease regulatory pathways which can then be targeted by new, specific therapies. Cyclin D1, Keap-1, Akt, and FAK are examples of disease targets that have been identified through the PPARγ-independent effects of PPARγ ligands. It is now possible, through mass spectrometry and other techniques, to determine exactly how these compounds bind to their targets and alter their function. This should allow the development of new compounds that have specific targeting activity against their new targets, while eliminating or reducing their affinity for PPARγ and thus reducing or eliminating PPAR-dependent activity and its associated side effects. As these ligands enter clinical trials, there is an urgent need to understand their PPAR-dependent and -independent mechanisms of action for the future of targeted and personalized medicines.

Bibliografia

Emerging PPARγ-Independent Role of PPARγ Ligands in Lung Diseases

PPAR-γ agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis

Role of PPARgamma nuclear receptors in cancerogenesis.

Pleiotropic effects of PPAR gamma activators in neoplastic, inflammatory and fibrotic diseases.