Adaptive thermogenesis in brown adipose tissue involves activation of pannexin-1 channels.

OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of ß-adrenergic receptor (ßAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by ß3AR stimulation via a novel mechanism that involves Gßγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of ß3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective ß3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gßγ-dependent Panx1 channel activation is involved in ß3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of ß3AR activation may have therapeutic implications.

Liver-specific loss of Perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1ß, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy.

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation.

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase ß-dependent (IKKß-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.

Mixed lineage kinase 3 modulates ß-catenin signaling in cancer cells.

Expression of ß-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3ß (GSK3ß)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/ß-catenin signaling or mutations of members of the degradation complex) can result in ß-catenin stabilization and activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control ß-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between ß-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce ß-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with ß-catenin and phosphorylate it in vitro. Overexpression of GSK3ß-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3ß pathway. Surprisingly, despite stabilizing ß-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A ß-catenin. These resulted in reduced expression of ß-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease ß-catenin/TCF interaction but promoted interaction between ß-catenin and KLF4, a known repressor of ß-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and ß-catenin resulted in significant G(2)/M arrest. These studies provide a novel insight toward the regulation of ß-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of ß-catenin signaling.

Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells.

BACKGROUND: The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal (GI) cells. The studies described here were undertaken to elucidate in detail the signaling pathways mediating the migratory responses of amidated gastrin (G17) and to understand the involvement of the serine/threonine kinase Glycogen Synthase Kinase-3 beta (GSK3beta) in this. RESULTS: Our results indicate that incubation of gastric cancer cells overexpressing CCK2 receptor (AGSE cells) with G17 results in a dose and time dependent increase of GSK3betaSer9 phosphorylation, indicative of an inhibition of the kinase. Pretreatment with a pharmacological inhibitor of PI3Kinase pathway (Wortmannin) was unable to antagonize G17-induced GSK3betaSer9 phosphorylation, suggesting that this might involve PI3Kinase-independent pathways. Treatment with G17 was also associated with increased Snail expression, and beta-catenin nuclear translocation, both of which are GSK3beta downstream targets. Pretreatment with a pharmacological inhibitor of GSK3beta (AR-A014418) augmented Snail expression and beta-catenin nuclear translocation in the absence of G17, whereas overexpression of a phosphorylation deficient mutant of GSK3beta (S9A) abrogated Snail promoter induction. These suggested that G17 modulates Snail and beta-catenin pathways via inhibiting GSK3beta. In addition, overexpression of GSK3beta wild type (WT) or S9A mutant inhibited G17-induced migration and MMP7 promoter induction. G17 studies designed following small interference RNA (siRNA)-mediated knockdown of Snail and beta-catenin expression indicated a significant reduction of G-17-induced migration and MMP7 promoter induction following combined knockdown of both proteins. CONCLUSION: Our studies indicate that inhibition of GSK3beta is necessary to activate G17-induced migratory pathways in gastric cancer cells. Inhibition of GSK3beta leads to an induction of Snail expression and beta-catenin nuclear translocation, both of which participate to promote G17-induced migration.

Mixed lineage kinase-3/JNK1 axis promotes migration of human gastric cancer cells following gastrin stimulation.

Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

Caspase-mediated cleavage of beta-catenin precedes drug-induced apoptosis in resistant cancer cells.

A delicate balance between cell death and survival pathways maintains normal physiology, which is altered in many cancers, shifting the balance toward increased survival. Several studies have established a close connection between the Wnt/beta-catenin pathway and tumorigenesis, aberrant activation of which might contribute toward increased cancer cell growth and survival. Extensive research is underway to identify therapeutic agents that can induce apoptosis specifically in cancer cells with minimal collateral damage to normal cells. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis specifically in tumor cells, many cancer cells develop resistance, which can be overcome by combinatorial treatment with other agents: for example, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands. To identify the molecular target mediating combinatorial drug-induced apoptosis, we focused on beta-catenin, a protein implicated in oncogenesis. Our results show that co-treatment of TRAIL-resistant cancer cells with TRAIL and the PPARgamma ligand troglitazone leads to a reduction of beta-catenin expression, coinciding with maximal apoptosis. Modulation of beta-catenin levels via ectopic overexpression or small interference RNA-mediated gene silencing modulates drug-induced apoptosis, indicating involvement of beta-catenin in regulating this pathway. More in-depth studies indicated a post-translational mechanism, independent of glycogen synthase kinase-3beta activity regulating beta-catenin expression following combinatorial drug treatment. Furthermore, TRAIL- and troglitazone-induced apoptosis was preceded by a cleavage of beta-catenin, which was complete in a fully apoptotic population, and was mediated by caspases-3 and -8. These results demonstrate beta-catenin as a promising new target of drug-induced apoptosis, which can be targeted to sensitize apoptosis-resistant cancer cells.