. PPAR␥ agonists inhibit TGF-␤ induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis.

Diseases such as chronic obstructive pulmonary disease and lung cancer caused by cigarette smoke affect millions of people worldwide. The aryl hydrocarbon receptor (AhR) is a ligandactivated transcription factor that influences responses to certain environmental pollutants such as tobacco smoke. However, the physiological function(s) of the AhR is unknown. Herein we propose that the physiologic role of the AhR is to limit inflammation. We show that lung fibroblasts from AhR ؊/؊ mice produce a heightened inflammatory response to cigarette smoke, typified by increased levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs), when compared with wild type (AhR ؉/؉ ) fibroblasts. This response was dependent on AhR expression as transient transfection of an AhR expression plasmid into AhR ؊/؊ fibroblasts significantly attenuated the smoke-induced COX-2 and PG production, confirming the anti-inflammatory role of the AhR. The AhR can interact with NF-B. However, the heightened inflammatory response observed in AhR ؊/؊ fibroblasts was not the result of NF-B (p50/p65) activation. Instead it was coupled with a loss of the NF-B family member RelB in AhR ؊/؊ fibroblasts. Taken together, these studies provide compelling evidence that AhR expression limits proinflammatory COX-2 and PG production by maintaining RelB expression. The association between RelB and AhR may represent a new therapeutic and more selective target with which to combat inflammation-associated diseases.Lung inflammation and diseases such as chronic obstructive pulmonary disease and cancer caused by cigarette smoke affect millions of people. The link between chronic inflammation, typified by heightened expression of cyclooxygenase-2 (COX-2; 2 also known as prostaglandin endoperoxide H synthase (PGHS-2)), and these diseases is well established (1-3). COX enzymes catalyze the transformation of arachidonic acid into prostaglandin (PG) H 2 (4, 5), which serves as a substrate for various synthases that generate PGs and thromboxanes (6). There are two COX isoforms. In most tissues, COX-1 is expressed constitutively (7), whereas COX-2 is rapidly up-regulated by a variety of inflammatory stimuli (5) such as interleukin (IL)-1 (8) and cigarette smoke (9).Cigarette smoke is a complex mixture containing more than 4800 compounds. Cigarette smoke and some of its components, such as benzo[a]pyrene (B[a]P), induce COX-2 expression in lymphocytes, epithelial cells, and fibroblasts (9 -11). Fibroblasts are the main cell type in the lung interstitium, are involved in tissue repair and remodeling (12), provide structural support to the alveolar compartment, and are an important target of cigarette smoke (9, 13-18). Importantly fibroblasts are one of the major cell types that express COX-2 and synthesize PGs in humans (19,20).Regulation of COX-2 expression involves several transcriptional regulators (5), including the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-Arnt-Sim transcription factor ...

The transcription factor aryl hydrocarbon receptor (AhR) plays an important role in the response to environmental pollutants. However, its role in normal physiology is unclear. To investigate the role of AhR in acute lung inflammation, control and AhR knockout (KO) mice were exposed to inhaled cigarette smoke or bacterial endotoxin. Smoke-induced lung inflammation was twofold to threefold more severe in AhR KO mice than controls. Intriguingly, levels of tumor necrosis factor-alpha and interleukin-6 in the bronchoalveolar lavage of air-exposed KO mice were equal to the levels seen in smoke-exposed controls, suggesting that AhR-deficient mice are inflammation prone. AhR KO mice challenged with inhaled endotoxin, which does not contain AhR ligands, also developed greater lung neutrophilia than controls, and bronchoalveolar lavage cells from AhR KO mice produced elevated levels of tumor necrosis factor-alpha and interleukin-6 when treated with endotoxin in vitro. Nuclear factor-kappaB DNA-binding activity was elevated in smoke-exposed AhR KO mice compared with controls and was associated with a rapid loss of RelB only in the KO mice. We propose that AhR is a previously unrecognized regulator of inflammation that interacts with nuclear factor-kappaB so that in the absence of AhR RelB is prematurely degraded, resulting in heightened inflammatory responses to multiple proinflam-matory stimuli.

IntroductionCigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.MethodsPrimary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.ResultsRvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.ConclusionsRvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.

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.