TNF-α Antagonizes the Effect of Leptin on Insulin Secretion through FOXO1-Dependent Transcriptional Suppression of LepRb in INS-1 Cells.

Proinflammatory cytokines play a causal role in the development of hyperinsulinemia and T2MD. FOXO1, a transcription factor which is known to enhance proinflammation, was recently shown to be involved in obesity-induced ß cell dysfunction. However, molecular mechanisms for the association remained elusive. In this study, we first found that both leptin (10 nM) and TNF-α (20 ng/ml) significantly inhibited glucose-stimulated insulin secretion (GSIS) of INS-1E cells. When in combination, the GSIS function of INS-1E cells was significantly increased compared with that of the leptin alone treatment, indicating that TNF-α attenuated the inhibiting effect of leptin on GSIS of INS-1E cells. Similarly, we found that TNF-α has the same inhibitory effect on leptin in regulating insulin synthesis and secretion, and the survival and apoptosis of insulin cells. Further studies showed that TNF-α blocks leptin pathway by reducing the expression of leptin receptor (LepRb, also called OBRb) and inhibiting the activation of STAT3, a key molecule involved in the leptin signaling pathway in INS-1E cells. Besides, the downregulated expression of phosphorylated FOXO1 was found to be involved in the possible mechanism of TNF-α. Overexpression of constitutively active FOXO1 markedly aggravated the LepRb reduction by TNF-α treatment of INS-1E cells, and the endogenous FOXO1 knockdown abolished the effect of TNF-α on INS-1E cells. Furthermore, we have proved that FOXO1 could directly bind to the promoter of LepRb as a negative transcription regulator. Taken together, the results of this study reveal that TNF-α-induced LepRb downregulated in pancreatic ß cells and demonstrate that transcriptional reduction of FOXO1 might be the primary mechanism underlying TNF-α promoting INS-1E leptin resistance and ß cell dysfunction. Conclusions. Our current studies based on INS-1E cells in vitro indicate that the inflammatory factor TNF-α plays an important role in the development of INS-1E leptin resistance and glucose metabolism disorders, probably through FOXO1-induced transcription reduction of LepRb promoter in pancreatic ß cells, and FOXO1 may be a novel target for treating ß cell dysfunction in obesity-induced hyperinsulinemia and T2DM.

Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression.

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.

Metformin increases hepatic leptin receptor and decreases steatosis in mice.

In addition to the ascertained efficacy as antidiabetic drug, metformin is increasingly being used as weight-loss agent in obesity, and as insulin sensitizer in nonalcoholic fatty liver disease (NAFLD). However, the mechanisms underlying these effects are still incompletely understood. Emerging evidence suggest metformin as leptin sensitizer to mediate the weight-loss effect in the brain. In this study, we investigated effects of metformin on expression of leptin receptors in liver and kidney in mice. C57BL/6 mice were fed with chow diet (CD) or high-fat diet (HF) for 5months. Afterward, mice were treated with metformin (50mg/kg or 200mg/kg) for 15days. Metabolic parameters and hepatic gene expression were analyzed at the end of the treatment. We also tested the effects of metformin on plasma-soluble leptin receptor (sOB-R) levels in newly diagnosed type 2 diabetes mellitus (T2DM) patients, and assessed its effect on hepatosteatosis in mice. Results showed that metformin upregulates the expression of leptin receptors (OB-Ra, -Rb, -Rc, and -Rd) in liver but not kidney. The stimulation effect is dose-dependent in both chow and HF mice. Upregulation of OB-Rb, long signaling isoform, needs a relatively higher dose of metformin. This effect was paralleled by increased sOBR levels in mice and T2DM patients, and decreased hepatic triglyceride (TG) content and lipogenic gene expression, including sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FAS) and acetyl-CoA carboxylase-1 (ACC-1). Taken together, these data identify hepatic leptin receptor as target gene being upregulated by metformin which may enhance leptin sensitivity in liver to alleviate steatosis.

Macrophage TCF-4 co-activates p65 to potentiate chronic inflammation and insulin resistance in mice.

Transcription factor 4 (TCF-4) was recently identified as a candidate gene for the cause of type 2 diabetes, although the mechanisms have not been fully elucidated. In the present study, we demonstrated that the TCF-4 transgene in macrophages aggravated high-fat diet (HFD)-induced insulin resistance and chronic inflammation, characterized by the elevation of proinflammatory cytokines in the blood, liver and white adipose tissue, as well as a proinflammatory profile of immune cells in visceral fats in mice. Mechanistically, TCF-4 functioned as a co-activator of p65 to amplify the saturated free fatty acid (FFA)-stimulated promoter activity, mRNA transcription and secretion of proinflammatory cytokines in primary macrophages. Blockage of p65 with a specific interfering RNA or inhibitor could prevent TCF-4-enhanced expression of proinflammatory cytokines in FFA/lipopolysaccharide-treated primary macrophages. The p65 inhibitor could abolish macrophage TCF-4 transgene-aggravated systemic inflammation, glucose intolerance and insulin resistance in HFD-treated mice. In addition, we demonstrated that the mRNA expression of TCF-4 in the peripheral blood monocytes from humans was positively correlated to the levels of interleukin (IL)-1ß, tumour necrosis factor α, IL-6 and fasting plasma glucose. In summary, we identified TCF-4 as a co-activator of p65 in the potentiation of proinflammatory cytokine production in macrophages and aggravation of HFD-induced chronic inflammation and insulin resistance in mice.

Lost expression of ADAMTS5 protein associates with progression and poor prognosis of hepatocellular carcinoma.

Altered expression of ADAMTS5 is associated with human carcinogenesis and tumor progression. However, the role of ADAMTS5 in hepatocellular carcinoma (HCC) is unclear. This study analyzed ADAMTS5 expression in HCC tissues and tested for association with clinicopathological and survival data from HCC patients and then explored the role of ADAMTS5 in HCC cells in vitro. Paraffin blocks from 48 HCC patients were used to detect ADAMTS5 and vascular endothelial growth factor (VEGF) expression and microvessel density (MVD). A normal liver cell line and HCC cell lines were used to detect ADAMTS5 expression and for ADAMTS5 manipulation. ADAMTS5 cDNA was stably transfected into HCC cells and ADAMTS5 expression assessed by Western blot analysis. Tumor cell-conditioned growth medium was used to assess human umbilical vein endothelial cell migration and Matrigel tube formation. Xenograft assay was performed to determine the role of ADAMTS5 in vivo. The data showed that the expression of ADAMTS5 was reduced in HCC, which was inversely associated with VEGF expression, MVD, and tumor size and associated with poor overall survival of HCC patients. Lentivirus-mediated ADAMTS5 expression significantly inhibited tumor angiogenesis by downregulating in vitro expression of VEGF and inhibiting migration and tube formations, and also inhibited tumor growth and VEGF expression and reduced MVD in vivo in a mouse xenograft model. Taken together, these results suggest that ADAMTS5 plays a role in suppression of HCC progression, which could be further studied as a promising novel therapeutic target and a potential prognostic marker in HCC.

Macrophage CGI-58 deficiency promotes IL-1ß transcription by activating the SOCS3-FOXO1 pathway.

Over-nutrition induces low-grade inflammation that dampens insulin sensitivity, but the underlying molecular mediators are not fully understood. Comparative gene identification-58 (CGI-58) is an intracellular lipolytic activator. In the present study, we show that in mouse visceral fat-derived macrophages or human peripheral blood monocytes, CGI-58 negatively and interleukin (IL)-1ß positively correlate with obesity. Saturated non-esterified fatty acid (NEFA) suppresses CGI-58 expression in macrophages and this suppression activates FOXO1 (forkhead box-containing protein O subfamily-1) through inhibition of FOXO1 phosphorylation. Activated FOXO1 binds to an insulin-responsive element in IL-1ß promoter region to potentiate IL-1ß transcription. Gain- and loss-of-function studies demonstrate that NEFA-induced CGI-58 suppression activates FOXO1 to augment IL-1ß transcription by dampening insulin signalling through induction of SOCS3 (suppressor of cytokine signalling 3) expression. CGI-58 deficiency-induced SOCS3 expression is NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome-dependent. Our data thus identified a vicious cycle (IL-1ß-SOCS3-FOXO1-IL-1ß) that amplifies IL-1ß secretion and is initiated by CGI-58 deficiency-induced activation of the NLRP3 inflammasome in macrophages. We further show that blocking this cycle with a FOXO1 inhibitor, an antioxidant that inhibits FOXO1 or IL-1 receptor antagonist alleviates chronic inflammation and insulin resistance in high-fat diet (HFD)-fed mice. Collectively, our data suggest that obesity-associated factors such as NEFA and lipopolysaccharide (LPS) probably adopt this vicious cycle to promote inflammation and insulin resistance.

Association Between a Variant in ADAMTS5 and the Susceptibility to Hepatocellular Carcinoma in a Chinese Han Population.

A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is considered to be an important anti-angiogenic protein, in which the first TSR domain is crucial for its anti-angiogenic activity. Previous study showed that ADAMTS5 plays a role in suppression of hepatocellular carcinoma (HCC) progression through its anti-angiogenic activity. The rs2380585 G>A single-nucleotide polymorphism (SNP) is a missense mutation, located in the ADAMTS5 first TSR domain coding sequence (CDS). In this study, we investigated the impacts of ADAMTS5 rs2380585 polymorphism on the risk and progress of hepatocellular carcinoma. A total of 220 HCC patients and 220 controls in a Chinese Han population were enrolled and genotyped. The associations between SNPs and HCC incidence and progression were analyzed with logistic regression model. We found that individuals with the ADAMTS5 rs2380585 A allele was significantly associated with decreased HCC risk (OR = 0.348, 95 % CI 0.236-0.512; p = 0.000). Individuals having the ADAMTS5 rs2380585 polymorphic genotype (GA+AA) had an OR of 0.348 (95 % CI 0.201-0.600; p = 0.000) for developing HCC, compared with individuals having the ADAMTS5 rs2380585 ancestral genotype. However, stratified analyses did not find any evident gene-covariates interaction. The SNP of rs2380585 was irrelevant to the frequencies of clinicopathological characteristics. Our results for the first time indicate that ADAMTS5 rs2380585 polymorphism contributes to HCC susceptibility.

HBx inhibits CYP2E1 gene expression via downregulating HNF4α in human hepatoma cells.

CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on -318 to -294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1 expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis.

Macrophage CGI-58 deficiency activates ROS-inflammasome pathway to promote insulin resistance in mice.

Overnutrition activates a 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 oversecreting proinflammatory cytokines and from inducing proinflammatory signaling and IR in the cocultured 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.

Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo.

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. This 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-knock-in (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 prolyl hydroxylase 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 colocalized 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 acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a 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 in 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.

TNF-α up-regulates protein level and cell surface expression of the leptin receptor by stimulating its export via a PKC-dependent mechanism.

Increasing evidence suggests that inflammation/cytokines may modulate hypothalamic responses to leptin, which is a key regulator of energy homeostasis and inflammatory/stress responses. We investigated a possible role of TNF-α, a key early mediator of inflammation, in regulating the expression and trafficking of the long-isoform leptin receptor (LEPRb), the primary mediator of leptin signaling, in cultured cells. We found that TNF-α in a wide range of concentrations up-regulated LEPRb protein level and soluble LEPR (sLEPR) release via ectodomain shedding of LEPRb in multiple cell types, including neuronal cells. TNF-α also acutely increased LEPRb cell surface expression and leptin-induced STAT3 phosphorylation. In contrast, TNF-α had no significant effects on the protein level or cell surface expression of several other transmembrane proteins, including the transferrin receptor and cadherin. The stimulatory effects of TNF-α on LEPRb cell surface expression and sLEPR release were not dependent on de novo protein synthesis or functional lysosomes but were blocked by brefeldin A, suggesting that an intact Golgi or continuous endoplasmic reticulum to Golgi transport of newly synthesized proteins is required for these effects. However, TNF-α did not increase the half-life of cell surface LEPRb. Protein kinase C (PKC) inhibitor GF109203X abrogated the effects of TNF-α, whereas the pan-PKC activator phorbol 12-myristate 13-acetate mimicked the TNF-α effects. Taken together, our results suggest that TNF-α, via activation of PKC, regulates anterograde trafficking and/or degradation of LEPRb in the biosynthetic pathway, leading to concomitant increases in LEPRb protein level, cell surface expression, and sLEPR production. The finding that LEPRb cell surface expression and sLEPR production, key modulators of leptin sensitivity and bioavailability, are direct targets of TNF-α signaling could have a potentially important implication in the regulation of leptin signaling activity in different pathophysiological conditions as diverse as obesity and sepsis.

FOXO1 involvement in insulin resistance-related pro-inflammatory cytokine production in hepatocytes.

OBJECTIVE: Low-grade inflammation from hepatocytes plays a causal role in hepatic and systemic insulin resistance (IR). We aimed to explore whether and how FOXO1 was involved in IR-related inflammation in hepatocytes. METHODS: We determined FOXO1 expression and activity, insulin and NF-κB signaling, and pro-inflammatory cytokine production in tumor necrosis factor-α (TNF-α)- or dexamethasone (DEX)-induced IR model in vitro and in high fat diet-induced obese or diabetic db/db mice in vivo with quantitative RT-PCR and Western blotting. RESULTS: We identified two different but physiologically relevant IR models characterized by attenuated insulin-induced phosphorylation of insulin receptor substrate-1 and AKT in TNF-α- or DEX-treated HepG2 cells. DEX largely increased FOXO1 expression in hepatocytes, while TNF-α did not. Notably, FOXO1 phosphorylation was attenuated in both models. TNF-α-stimulated nuclear translocation of NF-κB (p65) and mRNA levels of interleukin (IL)-1, IL-6 and monocyte attractant protein-1 were partly blocked, while the anti-inflammatory role of DEX was largely potentiated by insulin. FOXO1 knockdown by human-specific FOXO1 small interfering RNA exerted an identical role to insulin. Furthermore, augmented hepatic FOXO1 expression and decreased phosphorylation were found to be associated with elevated pro-inflammatory cytokine production in high fat diet-induced obese and db/db mice. CONCLUSION: FOXO1 potentiates pro-inflammatory cytokine production in insulin-resistant hepatocytes.

FOXO1 increases CCL20 to promote NF-κB-dependent lymphocyte chemotaxis.

Hepatic insulin resistance (IR) is associated with liver inflammatory diseases, but molecular mechanisms for the association remained elusive. IR is known to increase activity of forkhead box-containing protein O subfamily-1 (FOXO1), a transcription factor that was recently shown to enhance proinflammatory cytokine production in macrophages and adipocytes. Here we report that overexpression of constitutively active FOXO1 markedly increased chemokine ligand 20 (CCL20) expression and secretion in HepG2 hepatoma cells treated with TNF-α. The opposite was seen when endogenous FOXO1 was silenced. FOXO1 did not bind CCL20 promoter directly; instead, it potentiated CCL20 transcription through increasing the binding of p65/p50 heterodimer to a functional nuclear factor-κB site in the human CCL20 promoter. The conditional medium from TNF-α-treated HepG2 cells stimulated migration of human peripheral blood mononuclear cells. This stimulation was significantly enhanced when FOXO1 was overexpressed, and attenuated when FOXO1 was silenced. CCL20 antibody partly blocked the synergistic effect of FOXO1 and TNF-α on peripheral blood mononuclear cells migration. Additionally, TNF-α antagonizes the insulin/Akt signal transduction, thus leading to activation of FOXO1, which is capable of mediating a transcriptional activation role in response to TNF-α on CCL20 gene expression in HepG2 cells and promotes lymphocyte chemotaxis. Furthermore, we found that FOXO1 and CCL20 were coordinately up-regulated in the insulin resistant and inflammatory cell-infiltrated liver of db/db mice, an animal model that displayed hepatic and systemic low-grade inflammation. In conclusion, our data suggest that FOXO1 links IR to lymphocyte chemotaxis in the insulin-resistant hepatocytes and livers by amplifying nuclear factor-κB-dependent hepatic CCL20 production.

Peptide toxin components of Amanita exitialis basidiocarps.

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, ß-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.

Insulin response sequence-dependent and -independent mechanisms mediate effects of insulin on glucocorticoid-stimulated insulin-like growth factor binding protein-1 promoter activity.

IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor-gamma coactivator-1alpha, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in which insulin regulates IGFBP-1 expression through both IRS-dependent and IRS-independent mechanisms.

Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors.

FoxO1, a member of the FoxO subfamily of forkhead transcription factors, is an important target for insulin and growth factor signaling in the regulation of metabolism, cell cycle and proliferation, and survival in peripheral tissues. However, its role in the central nervous system is mostly unknown. In this study, we examined the effect of neurotrophic factors on nuclear/cytoplasmic shuttling of FoxO1. We showed that insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) potently induced the nuclear exclusion of FoxO1-green fluorescent protein (GFP) while neurotrophin (NT)-3 and NT-4 were much weaker and brain-derived neurotrophic factor (BDNF) failed to induce FoxO1 translocation in PC12 cells. FoxO1 translocation was inhibited by LY294002, a well-established PI3K/Akt kinase inhibitor. Moreover, FoxO1 was phosphorylated at Thr24 and Ser256 residues by the above neurotrophic factors, with the exception of BDNF. Triple mutant FoxO1, in which three Akt/PKB phosphorylation sites (Thr24, Ser256 and Ser319) were mutated to alanine, resulted in the complete nuclear targeting of the expressed FoxO1-GFP fusion protein in the presence of the above neurotrophic factors in both PC12 cells and cultured hippocampal and cortical neurons. Taken together, these findings demonstrate that neurotrophic factors are able to regulate nuclear/cytoplasmic shuttling of FoxO1 via the PI3K/Akt pathway in neuronal cells.

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

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.

Phosphorylation of serine 256 suppresses transactivation by FKHR (FOXO1) by multiple mechanisms. Direct and indirect effects on nuclear/cytoplasmic shuttling and DNA binding.

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. (2001) Biochem. J. 354, 605-612). Ser-256 is located in a basic region of the FKHR DNA binding domain where phosphorylation may have direct effects on DNA binding and/or nuclear targeting. Phosphorylation of Ser-256 may also be required for the phosphorylation of Thr-24 and Ser-319. Here, we provide the first direct evidence that basic residues in the FKHR DNA binding domain are critical for DNA binding and that Ser-256 phosphorylation alters binding activity. Ser-256 phosphorylation also is critical for regulating nuclear/cytoplasmic trafficking; however, this effect requires Thr-24/Ser-319 phosphorylation. Transient transfection studies with reporter gene constructs in 293 cells reveal that the phosphorylation of Ser-256 can inhibit the function of FKHR independent of Thr-24/Ser-319 phosphorylation. Studies with GFP(1) fusion proteins indicate that Ser-256 phosphorylation is critical for nuclear exclusion of FKHR. However, this effect is disrupted when Thr-24 and Ser-319 are replaced by alanine, indicating that nuclear exclusion of FKHR also requires Thr-24/Ser-319 phosphorylation. Gel shift and fluorescence anisotropy studies reveal that basic residues at the C-terminal end of the FKHR DBD are important for DNA binding, and the introduction of a negative charge at the site of Ser-256 limits binding activity. Binding is rapid and reversible, providing an opportunity for the phosphorylation of Ser-256 and subsequent phosphorylation of Thr-24 and Ser-319 and nuclear exclusion of FKHR.

Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors.

Transgenic mice expressing the homeobox gene Hoxa5 under the control of Hoxb2 regulatory elements present a growth arrest during weeks two and three of postnatal development, resulting in proportionate dwarfism. These mice present a liver phenotype illustrated by a 12-fold increase in liver insulin-like growth factor binding protein 1 (IGFBP1) mRNA and a 50% decrease in liver insulin-like growth factor 1 (IGF1) mRNA correlated with a 50% decrease in circulating IGF1. We show that the Hoxa5 transgene is expressed in the liver of these mice, leading to an overexpression of total (endogenous plus transgene) Hoxa5 mRNA in this tissue. We have used several cell lines to investigate a possible physiological interaction of Hoxa5 with the main regulator of IGFBP1 promoter activity, the Forkhead box transcription factor FKHR. In HepG2 cells, Hoxa5 has little effect by itself but inhibits the FKHR-dependent activation of the IGFBP1 promoter. In HuF cells, Hoxa5 cooperates with FKHR to dramatically enhance IGFBP1 promoter activity. This context-dependent physiological interaction probably corresponds to the existence of a direct interaction between Hoxa5 and FKHR and FoxA2/HNF3beta, as demonstrated by pull-down experiments achieved either in vitro or after cellular co-expression. In conclusion, we propose that the impaired growth observed in this transgenic line relates to a liver phenotype best explained by a direct interaction between Hoxa5 and liver-specific Forkhead box transcription factors, in particular FKHR but also Foxa2/HNF3beta. Because Hoxa5 and homeogenes of the same paralog group are normally expressed in the liver, the present results raise the possibility that homeoproteins, in addition to their established role during early development, regulate systemic physiological functions.