Loss of miR-24-3p promotes epithelial cell apoptosis and impairs the recovery from intestinal inflammation.

While apoptosis plays a significant role in intestinal homeostasis, it can also be pathogenic if overactive during recovery from inflammation. We recently reported that microRNA-24-3p (miR-24-3p) is elevated in the colonic epithelium of ulcerative colitis patients during active inflammation, and that it reduced apoptosis in vitro. However, its function during intestinal restitution following inflammation had not been examined. In this study, we tested the influence of miR-24-3p on mucosal repair by studying recovery from colitis in both novel miR-24-3p knockout and miR-24-3p-inhibited mice. We observed that knockout mice and mice treated with a miR-24-3p inhibitor had significantly worsened recovery based on weight loss, colon length, and double-blinded histological scoring. In vivo and in vitro analysis of miR-24-3p inhibition in colonic epithelial cells revealed that inhibition promotes apoptosis and increases levels of the pro-apoptotic protein BIM. Further experiments determined that silencing of BIM reversed the pro-apoptotic effects of miR-24-3p inhibition. Taken together, these data suggest that miR-24-3p restrains intestinal epithelial cell apoptosis by targeting BIM, and its loss of function is detrimental to epithelial restitution following intestinal inflammation.

Cathelicidin Suppresses Colon Cancer Metastasis via a P2RX7-Dependent Mechanism.

The antimicrobial peptide cathelicidin inhibits development of colitis-associated colon cancer. However, the role of cathelicidin in colon cancer metastasis remains unknown. We hypothesized that cathelicidin is effective in inhibiting colon cancer metastasis. Human colon cancer HT-29 cells were injected intravenously into nude mice. Control HA-tagged adeno-associated virus (HA-AAV) or cathelicidin-overexpressing AAV (CAMP-HA-AAV) were injected intravenously into nude mice on the same day. Four weeks later, the nude mice were assessed for lung and liver metastases. Human colon cancer SW620 cells were used to study the effect of cathelicidin on cell migration and cytoskeleton. Incubation of SW620 cells with cathelicidin dose-dependently reduced cell migration, disrupted cytoskeletal structure, and reduced ßIII-tubulin (TUBB3) mRNA expression. The addition of the P2RX7 antagonist KN62, but not the FPRL1 antagonist WRW4, prevented the LL-37-mediated inhibition of cell migration and TUBB3 mRNA expression. The CAMP-HA-AAV-overexpressing group showed significantly reduced human CK20 protein (by 60%) and TUBB3 mRNA expression (by 40%) in the lungs and liver of the HT-29-loaded nude mice, compared to the HA-AAV control group. Intraperitoneal injection of KN62 reversed the CAMP-HA-AAV-mediated inhibition of human CK20 and TUBB3 expression in the lungs and liver of HT-29-loaded nude mice. In conclusion, cathelicidin inhibits colon cancer metastasis via a P2RX7-dependent pathway.

Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition.

BACKGROUND AND PURPOSE: Lipocalin-2 is a pro-inflammatory adipokine up-regulated in obese human subjects and animal models. Its circulating levels are positively correlated with the unfavourable lipid profiles, elevated blood pressure and insulin resistance index. Augmented lipocalin-2 has been found in patients with cardiovascular abnormalities.The present study was designed to investigate the role of lipocalin-2 in regulating endothelial function and vascular reactivity. EXPERIMENTAL APPROACH: Wild-type and lipocalin-2 knockout (Lcn2-KO) mice were fed with either a standard chow or a high-fat diet. Blood pressures and endothelium-dependent relaxations/contractions were monitored at 2 week intervals. RESULTS: Systolic blood pressure was elevated by high-fat diet in wild-type mice but not in Lcn2-KO mice. Endothelial dysfunction, reflected by the impaired endothelium-dependent relaxations to insulin and augmented endothelium-dependent contractions to ACh, was induced by high-fat diet in wild-type mice. In contrast, Lcn2-KO mice were largely protected from the deterioration of endothelial function caused by dietary challenges. The eNOS dimer/monomer ratio, NO bioavailability, basal and insulin-stimulated PKB/eNOS phosphorylation responses were higher in aortae of Lcn2-KO mice. Administration of lipocalin-2 attenuated endothelium-dependent relaxations to insulin and promoted endothelium-dependent contractions to ACh. It induced eNOS uncoupling and elevated COX expression in the arteries. Treatment with sulphaphenazole, a selective inhibitor of cytochrome P450 2C9, improved endothelial function in wild-type mice and blocked the effects of lipocalin-2 on both endothelium-dependent relaxations to insulin and endothelium-dependent contractions to ACh, as well as eNOS uncoupling. CONCLUSIONS: Lipocalin-2, by modulating cytochrome P450 2C9 activity, is critically involved in diet-induced endothelial dysfunction.

Lipocalin-2 deficiency attenuates insulin resistance associated with aging and obesity.

OBJECTIVE: The proinflammatory cytokines/adipokines produced from adipose tissue act in an autocrine and/or endocrine manner to perpetuate local inflammation and to induce peripheral insulin resistance. The present study investigates whether lipocalin-2 deficiency or replenishment with this adipokine has any impact on systemic insulin sensitivity and the underlying mechanisms. METHODS AND RESULTS: Under conditions of aging or dietary-/genetic-induced obesity, lipocalin-2 knockout (Lcn2-KO) mice show significantly decreased fasting glucose and insulin levels and improved insulin sensitivity compared with their wild-type littermates. Despite enlarged fat mass, inflammation and the accumulation of lipid peroxidation products are significantly attenuated in the adipose tissues of Lcn2-KO mice. Adipose fatty acid composition of these mice varies significantly from that in wild-type animals. The amounts of arachidonic acid (C20:4 n6) are elevated by aging and obesity and are paradoxically further increased in adipose tissue, but not skeletal muscle and liver of Lcn2-KO mice. On the other hand, the expression and activity of 12-lipoxygenase, an enzyme responsible for metabolizing arachidonic acid, and the production of tumor necrosis factor-alpha (TNF-alpha), a critical insulin resistance-inducing factor, are largely inhibited by lipocalin-2 deficiency. Lipocalin-2 stimulates the expression and activity of 12-lipoxygenase and TNF-alpha production in fat tissues. Cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC), an arachidonate lipoxygenase inhibitor, prevents TNF-alpha expression induced by lipocalin-2. Moreover, treatment with TNF-alpha neutralization antibody or CDC significantly attenuated the differences of insulin sensitivity between wild-type and Lcn2-KO mice. CONCLUSIONS: Lipocalin-2 deficiency protects mice from developing aging- and obesity-induced insulin resistance largely by modulating 12-lipoxygenase and TNF-alpha levels in adipose tissue.

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti-aging and metabolic effects of sirtuins.

Sirtuins are a family of NAD(+)-dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti-aging effects of human homologues of sirtuins, SIRT1-7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti-aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI-TOF/TOF-MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co-immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti-aging and metabolic regulatory activities of sirtuins.