Geniece M. Lehmann

Electrophilic Peroxisome Proliferator–Activated Receptor-γ Ligands Have Potent Antifibrotic Effects in Human Lung Fibroblasts

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta 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) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-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 PPARgamma ligand, 15-deoxy-Delta(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 PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta 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-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. 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.

p53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p21CIP1/WAF1

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53(-) cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53(-) cells consistent with MAAA/mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21CIP1/WAF1 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53(+) cells but persisted in p53(-) cells. Arsenite induced p53-S15-P and p21CIP1/WAF1 only in p53(+) cells. P21CIP1/WAF1-siRNA-treated p53(+) cells were similar to p53(-) cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45α and 14-3-3σ are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21CIP1/WAF1 induction.

Ocular fibroblast diversity: implications for inflammation and ocular wound healing.

PURPOSE Various ocular and orbital tissues differ in their manifestations of inflammation, although the reasons for this are unclear. Such differences may be due to behaviors exhibited by resident cell types, including fibroblasts. Fibroblasts mediate immune function and produce inflammatory mediators. Chronic stimulation of ocular fibroblasts can lead to prolonged inflammation and, in turn, to impaired vision and blindness. Interleukin (IL)-1β, which is produced by various cells during inflammation, is a potent activator of fibroblasts and inducer of the expression of inflammatory mediators. The hypothesis for this study was that that human fibroblasts derived from distinct ocular tissues differ in their responses to IL-1β and that variations in the IL-1 signaling pathway account for these differences. METHODS Human fibroblasts were isolated from the lacrimal gland, cornea, and Tenons capsule and treated with IL-1β in vitro. Cytokine and prostaglandin (PG)E(2) production were measured by ELISA and EIA. Cyclooxygenase (Cox)-2 expression was detected by Western blot. Components of the IL-1 signaling pathway were detected by flow cytometry, ELISA, Western blot, and immunofluorescence. RESULTS Cytokine and PGE(2) production and Cox-2 expression were greatest in corneal fibroblasts. VEGF production was greatest in Tenons capsule fibroblasts. Variations in IL-1 receptor and receptor antagonist expression, IκBα degradation and p65 nuclear translocation, however, did not account for these differences, but overexpression of the NF-κB member RelB dampened Cox-2 expression in all three fibroblast types. CONCLUSIONS The results highlight the inherent differences between ocular fibroblast strains and provide crucial insight into novel, tissue-specific treatments for ocular inflammation and disease, such as RelB overexpression.

Regulation of Lymphocyte Function by PPARγ: Relevance to Thyroid Eye Disease-Related Inflammation

Thyroid eye disease (TED) is an autoimmune condition in which intense inflammation leads to orbital tissue remodeling, including the accumulation of extracellular macromolecules and fat. Disease progression depends upon interactions between lymphocytes and orbital fibroblasts. These cells engage in a cycle of reciprocal activation which produces the tissue characteristics of TED. Peroxisome proliferator-activated receptor-γ (PPARγ) may play divergent roles in this process, both attenuating and promoting disease progression. PPARγ has anti-inflammatory activity, suggesting that it could interrupt intercellular communication. However, PPARγ activation is also critical to adipogenesis, making it a potential culprit in the pathological fat accumulation associated with TED. This review explores the role of PPARγ in TED, as it pertains to crosstalk between lymphocytes and fibroblasts and the development of therapeutics targeting cell-cell interactions mediated through this signaling pathway.

Role of Peroxisome Proliferator-Activated Receptor Gamma and Its Ligands in the Treatment of Hematological Malignancies

Peroxisome proliferator-activated receptor gamma (PPARγ) is a multifunctional transcription factor with important regulatory roles in inflammation, cellular growth, differentiation, and apoptosis. PPARγ is expressed in a variety of immune cells as well as in numerous leukemias and lymphomas. Here, we review recent studies that provide new insights into the mechanisms by which PPARγ ligands influence hematological malignant cell growth, differentiation, and survival. Understanding the diverse properties of PPARγ ligands is crucial for the development of new therapeutic approaches for hematological malignancies.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARβ/δ and PPARγ) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.