Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF. Objectives: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis. Methods: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-b activation in vitro. Measurements and Main Results: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; a-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-b. TGF-b induced expression of LDH5 via hypoxia-inducible factor 1a (HIF1a). Importantly, overexpression of both HIF1a and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-b to induce differentiation. Furthermore, inhibition of both HIF1a and LDH5 inhibited TGF-b-induced myofibroblast differentiation. Conclusions: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pHdependent activation of TGF-b. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders.

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 AktS473. 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 J2 (15d-PGJ2), 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 A1 (PGA1), a structural analogue of 15d-PGJ2 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-PGJ2 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.

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-b is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) g is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARg ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-b-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of a-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARg ligand, 15-deoxy-D 12,14 -prostaglandin J 2 (15 d-PGJ 2 ), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARg-independent pathway. CDDO and 15 d-PGJ 2 contain an a/b unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A 1 and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ 2 lacking the electrophilic center is much less potent. CDDO does not alter TGF-b-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-b-responsive genes. Overall, these data indicate that certain PPARg ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-b-mediated profibrogenic activities through pathways independent of PPARg. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

The ability of the cell to sense environmental conditions and alter gene expression in response to them is critical to its survival. In Saccharomyces cerevisiae, the Tor1/2 serine/threonine kinases are global regulators situated at the top of a signal cascade reported to receive and transmit nutritional signals associated with the nitrogen supply of the cell. At the other end of that cascade is Gln3, one of two transcriptional activators responsible for most nitrogen catabolic gene expression. When nitrogen is in excess, Tor1/2 are active, and Gln3 is phosphorylated and localizes to the cytoplasm. If Tor1/2 are inhibited by rapamycin or mutation, Gln3 becomes dephosphorylated, accumulates in the nucleus, and mediates nitrogen catabolite repression (NCR)-sensitive transcription. The observations that Gln3 also accumulates in the nuclei of cells provided with poor nitrogen sources or during nitrogen starvation has led to the conclusion that Tor1/2 control intracellular Gln3 localization and NCR-sensitive transcription by regulating Gln3 phosphorylation/dephosphorylation. To test this model, we compared Gln3 phosphorylation states and intracellular localizations under a variety of physiological conditions known to elicit different levels of NCRsensitive transcription. Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support NCR-sensitive transcription. (ii) Gln3-Myc 13 is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization. (iii) Gln3-Myc 13 dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment. These data suggest that rapamycin treatment and growth with poor nitrogen sources bring about nuclear accumulation of Gln3 but likely do so by different mechanisms or by a common mechanism involving molecules other than Gln3 and/or other than the levels of Gln3-Myc 13 phosphorylation thus far detected by others and ourselves.

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Gln3p is one of two well characterized GATA family transcriptional activation factors whose function is regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus where they bind GATA sequences upstream of nitrogen catabolite repression (NCR)-sensitive genes and activate their transcription. Conversely, in excess nitrogen, these GATA sequences are unoccupied by Gln3p and Gat1p because these transcription activators are excluded from the nucleus. Ure2p binds to Gln3p and Gat1p and is required for NCR-sensitive transcription to be repressed and for nuclear exclusion of these transcription factors. Here we show the following. (i) Gln3p residues 344-365 are required for nuclear localization. (ii) Replacing Ser-344, Ser-347, and Ser-355 with alanines has minimal effects on GFP-Gln3p localization. However, replacing Gln3p Ser-344, Ser-347, and Ser-355 with aspartates results in significant loss of its ability to be concentrated in the nucleus. (iii) N and C termini of the Gln3p region required for it to complex with Ure2p and be excluded from the nucleus are between residues 1-103 and 301-365, respectively. (iv) N and C termini of the Ure2p region required for it to interact with Gln3p are situated between residues 101-151 and 330-346, respectively. (v) Loss of Ure2p residues participating in either dimer or prion formation diminishes its ability to carry out NCR-sensitive regulation of Gln3p activity.Saccharomyces cerevisiae is increasingly used as a model to identify the functions of mammalian proteins as well as how their production and activities are regulated and integrated. One of the gene families shared by S. cerevisiae and higher eukaryotes is the GATA family of DNA-binding proteins. In animal cells, GATA family proteins were originally shown to be responsible for regulating globin gene expression (1). However, they are now known to regulate a diverse set of developmental functions (2, 3). In yeast, the GATA family proteins Gln3p, Gat1p/Nil1p, Dal80p, Deh1p/Gzf3p have been studied as the main regulators of nitrogen catabolic gene expression (4-6), and recently their regulatory functions also been shown to be far more diverse (7,8). Gln3p and Gat1p are transcriptional activators, whereas Dal80p and Deh1p repress transcription by competing with these activators for binding to their target GATA sequences (4)(5)(6)(9)(10)(11) HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptGln3p and Gat1p function is also dramatically regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus, bind to the GATA sequences of their target promoters, and activate transcription (8,9,(12)(13)(14)(15). On the other hand, when nitrogen is in excess, Gln3p and Gat1p are excluded from the nucleus, and nitrogen catabolite repression (NCR) 1 -sensitive gene expression is repressed. Nuclear exclusion of Gln3p and Gat1p requires Ure2p, the first NCR regulator identified (16...

Corneal scarring, whether caused by trauma, laser refractive surgery, or infection, remains a significant problem for humans. Certain ligands for peroxisome proliferator-activated receptor gamma (PPARγ) have shown promise as antiscarring agents in a variety of body tissues. In the cornea, their relative effectiveness and mechanisms of action are still poorly understood. Here, we contrasted the antifibrotic effects of three different PPARγ ligands (15-deoxy-Δ12,14-prostaglandin J2, troglitazone, and rosiglitazone) in cat corneal fibroblasts. Western blot analyses revealed that all three compounds reduced transforming growth factor (TGF)-β1-driven myofibroblast differentiation and up-regulation of α-smooth muscle actin, type I collagen, and fibronectin expression. Because these effects were independent of PPARγ, we ascertained whether they occurred by altering phosphorylation of Smads 2/3, p38 mitogen-activated protein kinase, stress-activated protein kinase, protein kinase B, extracellular signal-regulated kinase, and/or myosin light chain 2. Only p38 mitogen-activated protein kinase phosphorylation was significantly inhibited by all three PPARγ ligands. Finally, we tested the antifibrotic potential of troglitazone in a cat model of photorefractive keratectomy-induced corneal injury. Topical application of troglitazone significantly reduced α-smooth muscle actin expression and haze in the stromal ablation zone. Thus, the PPARγ ligands tested here showed great promise as antifibrotics, both in vitro and in vivo. Our results also provided new evidence for the signaling pathways that may underlie these antifibrotic actions in corneal fibroblasts.

Gln3 and Gat1/Nil1 are GATA-family transcription factors responsible for transcription of nitrogen-catabolic genes in Saccharomyces cerevisiae. Intracellular Gln3 localization and Gln3-dependent transcription respond in parallel to the nutritional environment and inhibitors of Tor1/2 (rapamycin) and glutamine synthetase (L-methionine sulfoximine, MSX). However, detectable Gln3 phosphorylation, though influenced by nutrients and inhibitors, correlates neither with Gln3 localization nor nitrogen catabolite repression-sensitive transcription in a consistent way. To establish relationships between Gln3 and Gat1 regulation, we performed experiments parallel to those we previously reported for Gln3. Gat1 and Gln3 localization are similar during steady-state growth, being cytoplasmic and nuclear with good and poor nitrogen sources, respectively. Localization correlates with Gat1- and Gln3-mediated transcription. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions and rapamycin-treatment; (ii) their opposite responses to MSX-treatment, i.e. that cytoplasmic Gln3 becomes nuclear following MSX addition, whereas nuclear Gat1 becomes cytoplasmic; and (iii) their phosphorylation levels in the above situations. In instances where Gln3 phosphorylation can be straightforwardly demonstrated to change, Gat1 phosphorylation (in the same samples) appears invariant. The only exception was following carbon starvation, where Gat1, like Gln3, is hyperphosphorylated in a Snf1-dependent manner. However, neither carbon starvation nor MSX treatment elicits Snf1-independent Gat1 hyperphosphorylation, as observed for Gln3.