PPARγ ligands have both anti-inflammatory and antiscarring effects. In primary human lung fibroblasts, rosiglitazone and 15 d-PGJ2
inhibit expression of α-SMA and collagen (24
). Others have demonstrated in animal models of lung fibrosis, as well as in other organs, that PPARγ ligands have antifibrotic effects (20
). Here, we report that CDDO is a novel antifibrotic agent that potently inhibits TGF-β–stimulated differentiation of human lung fibroblasts to myofibroblasts via a largely PPARγ-independent pathway, with an inhibitory concentration causing a 50% drop (40 nM) that is 20-fold lower than 15 d-PGJ2
and 400-fold lower than rosiglitazone.
Because of the exciting potential of PPARγ ligands as antifibrotic agents, it is important to understand their mechanism of action. The anti-inflammatory and antifibrotic effects of the both synthetic and natural PPARγ ligands have been reported to involve both PPARγ-dependent and -independent mechanisms (22
). The PPARγ-dependent mechanism requires ligand binding to PPARγ, heterodimerization with RXR and its ligand, nuclear translocation, binding to the PPRE, and induction of gene transcription, whereas the PPARγ-independent mechanism or mechanisms have not yet been fully determined.
To investigate whether the antifibrotic effects of CDDO and 15 d-PGJ2
are PPARγ dependent or independent, we used a small molecule inhibitor of PPARγ and overexpression of a dominant-negative PPARγ construct. GW9662, an irreversible PPARγ antagonist, did not restore myofibroblast differentiation in fibroblasts treated with TGF-β and CDDO or 15 d-PGJ2
(). Similarly, when lung fibroblasts were transfected with LV-PPARγ-DN, CDDO and 15 d-PGJ2
were still able to significantly inhibit TGF-β–driven myofibroblast differentiation (). Interestingly, when PPARγ activity was blocked with either GW9662 or the DN construct, treatment with TGF-β resulted in significantly greater induction of α-SMA than in cells with intact PPARγ signaling cascades treated with TGF-β. This suggests that, in the absence of potent exogenous agonists, PPARγ opposes TGF-β–stimulated myofibroblast differentiation via a PPARγ-dependent mechanism involving endogenous or media-derived PPARγ ligands, such as eicosanoids and other fatty acids (21
). Blocking PPARγ function removes this opposing regulatory pathway, increasing the profibrotic effects of TGF-β. Note that, although rosiglitazone is a strong PPARγ-dependent promoter of adipogenesis, it exhibits little PPARγ-independent activity, and is a relatively poor inhibitor of myofibroblast differentiation compared with other agents (24
). We hypothesize that myofibroblast differentiation is regulated by both PPARγ-dependent and PPARγ-independent pathways, but that the PPARγ-independent mechanisms are much stronger, and compounds that access the independent pathways are more effective at inhibiting TGF-β–stimulated myofibroblast differentiation at lower concentrations. Therefore, compounds that access PPARγ-independent pathways are likely to have greater clinical potential as antifibrotic agents.
Inspection of the structures of the PPARγ ligands suggests that the strong PPARγ-independent effects of 15 d-PGJ2
and CDDO might be mediated by their electrophilic α/β-unsaturated ketone. These compounds are susceptible to Michael addition reactions by nucleophiles of free sulfhydryls, such as occur in glutathione and accessible cysteine residues in cellular proteins (Figure E3B) (30
), and it has been reported that 15 d-PGJ2
modifies the activity of the estrogen receptor by covalently binding to its DNA binding domain (48
). We tested this hypothesis using CAY10410, an analog of 15 d-PGJ2
that lacks the α/β-unsaturated ketone and is unable to undergo this reaction (Figure E3A). CAY10410 was a much less effective inhibitor of α-SMA expression than 15 d-PGJ2
at the same concentration (). We further demonstrated the importance of the electrophilic carbon by using two other strong electrophilic compounds (DSPS and PGA1
), which potently decreased TGF-β–induced expression of α-SMA (). These new findings support the concept that the electrophilic carbon has a central role in mediating inhibition of TGF-β–stimulated myofibroblast differentiation by a PPARγ-independent pathway.
PPARγ-independent effects of PPARγ ligands have been reported in several papers; however, no common pathway applicable to all cell types has been identified. For example, 15 d-PGJ2
decreases Smad2/3 phosphorylation in human hepatic stellate cells (49
), whereas it inhibits nuclear localization without affecting phosphorylation in human glomerular mesangial cells (50
). Our data demonstrate that, in primary human lung fibroblasts, neither 15 d-PGJ2
nor CDDO decrease TGF-β–induced phosphorylation of Smad2/3, or alter Smad localization. It has been reported that Smad3, but not Smad2, is required for the fibrotic response (51
); however, the antibodies used is this study detect both Smad2 and Smad3. Therefore, a role for Smad3 alone is not completely ruled out.
Alternately, pioglitazone inhibits profibrotic changes in mouse glomerular mesangial cells by suppressing AP-1 activity (41
). However, neither 15 d-PGJ2
nor CDDO inhibits AP-1 activation in human lung fibroblasts (). In fact, both ligands alone activated an AP-1 reporter, and cotreatment with TGF-β and either ligand resulted in more AP-1 activity than TGF-β alone (). We have found that 15 d-PGJ2
and CDDO induce heme oxygenase-1 (HO-1), an enzyme involved in protecting cells from oxidative stress (H. E. Ferguson and colleagues, unpublished data), and it has been reported that HO-1 induces AP-1 activity (53
). We hypothesize that, because 15 d-PGJ2
and CDDO are strong electrophiles and can react with and consume glutathione (Figure E3), they induce oxidative stress and HO-1 in lung fibroblasts, leading to enhanced AP-1 activity.
We report here the novel finding that CDDO inhibits acetylation of CBP/p300. CBP/p300 is a critical transcriptional cofactor, the activity of which is regulated in part by acetylation (54
). CBP/p300 contains a HAT domain that can acetylate the core histones, H3 and H4, which relaxes the chromatin, making it accessible to other transcription factors. The HAT domain of CBP/p300 can acetylate other transcription factors as well, and it has been shown that acetylation of Smad3 is required for the expression of TGF-β–responsive genes, including α-SMA (16
). Upon binding of TGF-β to its receptor, Smad3 is phosphoryated, recruits Smad4, and the Smad3/4 complex is translocated to the nucleus, where it binds to the promoters of TGF-β–responsive genes (Figure E4). Smad3 recruits binding of the CBP/p300 cofactor, which becomes acetylated itself by an unknown mechanism. The HAT domain of CBP/p300 acetylates Smad3, which is required for transcriptional activation, and the core histones, H3 and H4, which converts the chromatin from a closed, transcriptionally inactive conformation to an open, transcriptionally active conformation. Acetylation of CBP/p300 results in enhanced HAT activity (54
). We have shown that CDDO dramatically inhibits acetylation of CBP/p300 (). Therefore, we hypothesize that CDDO inhibits myofibroblast differentiation by inhibiting acetylation of CBP/p300, which, in turn, inhibits acetylation of Smad3 and histones, thereby blocking transcription from TGF-β–responsive genes.
Overall, we have demonstrated for the first time that PPARγ ligands, such as CDDO and other small molecules with electrophilic carbons, are potent inhibitors of TGF-β–induced myofibroblast differentiation and extracellular matrix production in human lung fibroblasts that act via a PPARγ-independent pathway. This pathway may involve a novel mechanism of action for CDDO, inhibiting α-SMA expression by dysregulating acetylation of CBP/p300. CDDO is an orally active agent, with a long half-life, and is in clinical trials for the treatment of several forms of cancer, including leukemia, solid tumors, and myelomas. The translational potential of this compound is thus high. Our results also suggest that developing other electrophilic compounds that target PPARγ-independent pathways may be of great clinical utility for scarring diseases of the lungs and other tissues.