FKHR is a member of the FOXO subfamily of Forkhead transcription factors, which are important targets for insulin and growth factor signaling. FKHR contains three predicted protein kinase B phosphorylation sites (Thr-24, Ser-256, and Ser-319) that are conserved in other FOXO proteins. We have reported that phosphorylation of Ser-256 is critical for the ability of insulin and insulin-like growth factors to suppress transactivation by FKHR (Guo, S., Rena, G., Cichy, S., He, X., Cohen, P., and Unterman, T. (1999) J. Biol. Chem. 274, 17184 -17192) and for its exclusion from the nucleus (Rena, G., Prescott, A. R., Guo, S., Cohen, P., and Unterman, T. G. Recent studies have revealed that FOXO Forkhead transcription factors are important targets for mediating effects of insulin and growth factors on gene expression downstream from phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB; also known as Akt) (1-13). Early findings in this laboratory revealed that Forkhead transcription factors interact with insulin response sequences (IRSs) in the insulin-like growth factor-binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) genes (14, 15) and that signaling by PI3K and PKB mediate the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (16). Subsequent studies in Caenorhabditis elegans provided genetic evidence that DAF-16, a member of the FOXO subfamily of Forkhead transcription factors, is a major target for signaling by the insulin/IGF receptor-PI3K-PKB pathway in the nematode (17, 18). DAF-16 and its mammalian homologues, including FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) interact directly with IRSs from the IGFBP-1 promoter in a sequence-specific fashion in in vitro assays and in cells (1,5,9,19,20). In liver-derived cells, FOXO proteins stimulate the activity of promoters for IGFBP-1 (1), glucose-6 phosphatase (21,22), and PEPCK (23,24). In other cell types, FOXO proteins also stimulate the expression of proteins that inhibit cell cycle progression, including p27 Kip (25), Rb2 (26), and GADD45 (27,28), and proteins that promote cells death, including Bim (29) and Fas ligand (5). Thus, the ability to suppress transactivation by FOXO Forkhead proteins is important for insulin to regulate hepatic production of IGFBP-1 and glucose and for effects of growth factors on cell proliferation and survival.Several critical features distinguish FOXO proteins from other Forkhead family members and make them uniquely suited as mediators of insulin and growth factor action. X-ray crystallographic studies with the DBD of HNF-3␥ indicated that the DNA binding motif of Forkhead proteins, named the Forkhead box, or FOX box, contains three ␣-helices, a wing-like

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

Metabolic reprogramming greatly contributes to the regulation of macrophage activation. However, the mechanism of lipid accumulation and the corresponding function in tumor-associated macrophages (TAMs) remain unclear. With primary investigation in colon cancer and confirmation in other cancer models, here we determine that deficiency of monoacylglycerol lipase (MGLL) results in lipid overload in TAMs. Functionally, macrophage MGLL inhibits CB2 cannabinoid receptor-dependent tumor progression in inoculated and genetic cancer models. Mechanistically, MGLL deficiency promotes CB2/TLR4-dependent macrophage activation, which further suppresses the function of tumor-associated CD8+ T cells. Treatment with CB2 antagonists delays tumor progression in inoculated and genetic cancer models. Finally, we verify that expression of macrophage MGLL is decreased in cancer tissues and positively correlated with the survival of cancer patients. Taken together, our findings identify MGLL as a switch for CB2/TLR4-dependent macrophage activation and provide potential targets for cancer therapy.

SUMMARY Overnutrition activates proinflammatory program in macrophages to induce insulin resistance (IR), but its molecular mechanisms remain incompletely understood. Here we show that saturated fatty acid and lipopolysaccharide, two factors implicated in high-fat diet (HFD)-induced IR, suppress macrophage CGI-58 expression. Macrophage-specific CGI-58 knockout (MaKO) in mice aggravates HFD-induced glucose intolerance and IR, which is associated with augmented systemic/tissue inflammation and proinflammatory activation of adipose tissue macrophages. CGI-58-deficient macrophages exhibit mitochondrial dysfunction due to defective peroxisome proliferator-activated receptor (PPAR)-γ signaling. Consequently they overproduce reactive oxygen species (ROS) to potentiate secretion of proinflammatory cytokines by activating NLRP3 inflammasome. Anti-ROS treatment or NLRP3 silencing prevents CGI-58-deficient macrophages from over-secreting proinflammatory cytokines and from inducing proinflammatory signaling and IR in the co-cultured fat slices. Anti-ROS treatment also prevents exacerbation of inflammation and IR in HFD-fed MaKO mice. Our data thus establish CGI-58 as a suppressor of overnutrition-induced NLRP3 inflammasome activation in macrophages.

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver and liver injury. The current study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild type (WT), CYP2E1-knockin (KI) and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolylhydroxlase 2 which promotes HIF-1α degradation were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were co-localized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells which express CYP2E1 with ethanol plus arachidonic (AA) acid or ethanol plus buthionine sulfoximine (BSO) which depletes GSH caused loss of cell viability to greater extent than in HepG2 C34 cells which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis and liver injury.