Reverse crosstalk of TGFβ and PPARβ/δ signaling identified by transcriptional profiling

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and transforming growth factor-β (TGFβ) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFβ, which is counteracted by PPARβ/δ activation. This is mediated, at least in part, by the TGFβ-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARβ/δ ligands, indicating that TGFβ and PPARβ/δ signals are integrated by chromatin-associated complexes. A second class represents TGFβ-induced genes that are downregulated by PPARβ/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARβ/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARβ/δ is able to influence the expression of distinct sets of both TGFβ-repressed and TGFβ-activated genes in both directions.     

VLDL hydrolysis by LPL activates PPAR-α through generation of unbound fatty acids

Recent evidence suggests that lipoproteins serve as circulating reservoirs of peroxisomal proliferator activated receptor (PPAR) ligands that are accessible through lipolysis. The present study was conducted to determine the biochemical basis of PPAR-α activation by lipolysis products and their contribution to PPAR-α function in vivo. PPAR-α activation was measured in bovine aortic endothelial cells following treatment with human plasma, VLDL lipolysis products, or oleic acid. While plasma failed to activate PPAR-α, oleic acid performed similarly to VLDL lipolysis products. Therefore, fatty acids are likely to be the PPAR-α ligands generated by VLDL lipolysis. Indeed, unbound fatty acid concentration determined PPAR-α activation regardless of fatty acid source, with PPAR-α activation occurring only at unbound fatty acid concentrations that are unachievable under physiological conditions without lipase action. In mice, a synthetic lipase inhibitor (poloxamer-407) attenuated fasting-induced changes in expression of PPAR-α target genes. Apolipoprotein CIII (apoCIII), an endogenous inhibitor of lipoprotein and hepatic lipase, regulated access to the lipoprotein pool of PPAR-α ligands, because addition of exogenous apoCIII inhibited, and removal of endogenous apoCIII potentiated, lipolytic PPAR-α activation. These data suggest that the PPAR-α response is generated by unbound fatty acids released locally by lipase activity and not by circulating plasma fatty acids.     

PPARδ activation attenuates hepatic steatosis in Ldlr?/? mice by enhanced fat oxidation,reduced lipogenesis,and improved insulin sensitivity

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr^−/− mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1^−/− hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1^−/− hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1^−/− hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.     

Adipose triglyceride lipase is a TG hydrolase of the small intestine and regulates intestinal PPARα signaling

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [³H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [³H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.     

GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation

Background

After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl₄) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl₄ alone or CCl₄ co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ.

Results

We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl₄/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway.

Conclusions

This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.     

Improving Pseudomonas alcaligenes lipase’s diastereopreference in hydrolysis of diastereomeric mixture of menthyl propionate by site-directed mutagenesis

The resolutions of racemic diastereomeric mixtures of menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because of the inherently low diastereopreference of PaL, covalent docking and molecular dynamic (MD) simulations were used to investigate possible avenues of improvement. Rational site-directed mutagenesis of PaL revealed residues V180 and A272 to be the hotspots for diastereopreference. The double V180L/A272F mutant exhibited the highest degree of diastereopreference, as the diastereomeric ratio of (2S, 5R) L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) and (2R, 5R) D-isoneomenthol (dr2) (diastereomeric ratios dr1 and dr2 increased to 4.65 and 2.13 times that of wild-type PaL). MD simulation analysis indicated that these mutations decrease the flexibility of the surrounding protein regions. The combination of increased steric exclusion and decreased flexibility results in less favorable binding of the non-target substrates, (2R, 5S) L-neomenthyl propionate and (2R, 5R) D-isoneomenthyl propionate, to the V180L/A272F mutant. These results confirmed and further improved our previously proposed model of the diastereomer recognition mechanism based on the combined effect of steric exclusion and regional flexibility.     

Skin-targeted inhibition of PPAR β/δ by selective antagonists to treat PPAR β/δ-mediated psoriasis-like skin disease in vivo

We have previously shown that peroxisome proliferator activating receptor ?/δ (PPAR β/δ is overexpressed in psoriasis. PPAR β/δ is not present in adult epidermis of mice. Targeted expression of PPAR β/δ and activation by a selective synthetic agonist is sufficient to induce an inflammatory skin disease resembling psoriasis. Several signalling pathways dysregulated in psoriasis are replicated in this model, suggesting that PPAR β/δ activation contributes to psoriasis pathogenesis. Thus, inhibition of PPAR β/δ might harbour therapeutical potential. Since PPAR β/δ has pleiotropic functions in metabolism, skin-targeted inhibition offer the potential of reducing systemic adverse effects. Here, we report that three selective PPAR β/δ antagonists, GSK0660, compound 3 h, and GSK3787 can be formulated for topical application to the skin and that their skin concentration can be accurately quantified using ultra-high performance liquid chromatography (UPLC)/mass spectrometry. These antagonists show efficacy in our transgenic mouse model in reducing psoriasis-like changes triggered by activation of PPAR β/δ. PPAR β/δ antagonists GSK0660 and compound 3 do not exhibit systemic drug accumulation after prolonged application to the skin, nor do they induce inflammatory or irritant changes. Significantly, the irreversible PPAR β/δ antagonist (GSK3787) retains efficacy when applied topically only three times per week which could be of practical clinical usefulness. Our data suggest that topical inhibition of PPAR β/δ to treat psoriasis may warrant further exploration.     

Hyaluronan regulates PPARγ and inflammatory responses in IL-1β-stimulated human chondrosarcoma cells, a model for osteoarthritis

The carbohydrate polymer, hyaluronan, is a major component of the extracellular matrix in animal tissues. Exogenous hyaluronan has been used to treat osteoarthritis (OA), a degenerative joint disease involving inflammatory changes. The underlying mechanisms of hyaluronan in OA are not fully understood. Pro-inflammatory interleukin (IL)-1β downregulates peroxisome proliferator-activated receptor gamma (PPARγ), and increases expression of matrix metalloproteinases (MMPs) which are responsible for the degeneration of articular cartilage. The effects of low- and high-molecular-weight hyaluronan (oligo-HA and HMW-HA) on the inflammatory genes were determined in human SW-1353 chondrosarcoma cells. HMW-HA antagonized the effects of IL-1β by increasing PPARγ and decreasing cyclooxygenase (COX)-2, MMP-1, and MMP-13 levels. It promoted Akt, but suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) signaling, indicating anti-inflammatory effects. In contrast, the cells had overall opposite responses to oligo-HA. In conclusion, HMW-HA and oligo-HA exerted differential inflammatory responses via PPARγ in IL-1β-treated chondrosarcoma cells.