Leah M. Wuescher

Insulin regulates menin expression, cytoplasmic localization, and interaction with FOXO1.

Menin is the ubiquitously expressed nuclear protein product of the MEN1 gene, which interacts with PKB/Akt in the cytoplasm to inhibit its activity. This study describes a novel insulin-dependent mechanism of menin regulation and interaction with other metabolic proteins. We show that insulin downregulated menin in a time-dependent manner via the human insulin receptor. Inhibition analysis indicated a critical role for the protein kinase Akt in regulation of menin expression and localization. Insulin-mediated decrease in menin expression was abrogated by the PI3K/Akt inhibitor LY-294002 at early time points, from 2 to 7 h. Furthermore, exposure to insulin resulted in the cytoplasmic localization of menin and increased interaction with FOXO1. Fasting followed by refeeding modulates serum insulin levels, which corresponded to an increase in menin interaction with FOXO1 in the liver. Liver-specific hemizygous deletion of menin resulted in increased expression of FOXO1 target genes, namely IGFBP-1, PGC-1α, insulin receptor, Akt, and G-6-Pase. This study provides evidence that menin expression and localization are regulated by insulin signaling and that this regulation triggers an increase in its interaction with FOXO1 via Akt with metabolic consequences.

Cancer and Thrombosis: The Platelet Perspective

Platelets are critical to hemostatic and immunological function, and are key players in cancer progression, metastasis, and cancer-related thrombosis. Platelets interact with immune cells to stimulate anti-tumor responses and can be activated by immune cells and tumor cells. Platelet activation can lead to complex interactions between platelets and tumor cells. Platelets facilitate cancer progression and metastasis by: (1) forming aggregates with tumor cells; (2) inducing tumor growth, epithelial-mesenchymal transition, and invasion; (3) shielding circulating tumor cells from immune surveillance and killing; (4) facilitating tethering and arrest of circulating tumor cells; and (5) promoting angiogenesis and tumor cell establishment at distant sites. Tumor cell-activated platelets also predispose cancer patients to thrombotic events. Tumor cells and tumor-derived microparticles lead to thrombosis by secreting procoagulant factors, resulting in platelet activation and clotting. Platelets play a critical role in cancer progression and thrombosis, and markers of platelet-tumor cell interaction are candidates as biomarkers for cancer progression and thrombosis risk.

A novel conditional platelet depletion mouse model reveals the importance of platelets in protection against Staphylococcus aureus bacteremia

Platelets are critical cells for maintaining vascular hemostasis, but their activities in other processes are becoming apparent. Specifically, the ability of platelets to recognize and respond to infectious agents is an important area of investigation. To understand the physiologic roles of platelets in vivo, most researchers have used antibody‐mediated platelet depletion, which has certain limitations.

Glucocorticoid receptor β stimulates Akt1 growth pathway by attenuation of PTEN.

Background: The glucocorticoid receptor β (GRβ) is a positive regulator of growth. Results: GRβ suppression of PTEN resulted in enhanced phosphorylation of Akt and growth. Conclusion: GRβ enhances insulin-induced proliferation by suppressing PTEN and activating Akt1. Significance: GRβ suppression of PTEN indicates that it has an important role in growth factor signaling and potentially cancer. Glucocorticoids (GCs) are known inhibitors of proliferation and are commonly prescribed to cancer patients to inhibit tumor growth and induce apoptosis via the glucocorticoid receptor (GR). Because of alternative splicing, the GR exists as two isoforms, GRα and GRβ. The growth inhibitory actions of GCs are mediated via GRα, a hormone-induced transcription factor. The GRβ isoform, however, lacks helix 12 of the ligand-binding domain and cannot bind GCs. While we have previously shown that GRβ mRNA is responsive to insulin, the role of GRβ in insulin signaling and growth pathways is unknown. In the present study, we show that GRβ suppresses PTEN expression, leading to enhanced insulin-stimulated growth. These characteristics were independent of the inhibitory qualities that have been reported for GRβ on GRα. Additionally, we found that GRβ increased phosphorylation of Akt basally, which was further amplified following insulin treatment. In particular, GRβ specifically targets Akt1 in growth pathways. Our results demonstrate that the GRβ/Akt1 axis is a major player in insulin-stimulated growth.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Platelets Mediate Host Defense against Staphylococcus aureus through Direct Bactericidal Activity and by Enhancing Macrophage Activities.

Platelets are the chief effector cells in hemostasis. However, recent evidence suggests they have multiple roles in host defense against infection. Reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse platelets to participate in host defense against S. aureus infection was determined by assessing two possibilities. First, we determined the ability of platelets to kill S. aureus directly; and, second, we tested the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus. In this study we report evidence in support of both mechanisms. Platelets effectively killed two different strains of S. aureus. A clinical isolate of methicillin-resistant S. aureus was killed by platelets (>40% killing in 2 h) in a thrombin-dependent manner whereas a methicillin-sensitive strain was killed to equal extent but did not require thrombin. Interestingly, thrombin-stimulated platelets also significantly enhanced peritoneal macrophage phagocytosis of both methicillin-resistant S. aureus and methicillin-sensitive S. aureus by >70%, and restricted intracellular growth by >40%. Enhancement of macrophage anti-S. aureus activities is independent of contact with platelets but is mediated through releasable products, namely IL-1β. These data confirm our hypothesis that platelets participate in host defense against S. aureus both through direct killing of S. aureus and enhancing the antimicrobial function of macrophages in protection against S. aureus infection.

Menin liver-specific hemizygous mice challenged with high fat diet show increased weight gain and markers of metabolic impairment.

OBJECTIVE:The menin tumor suppressor protein is abundantly expressed in the liver, although no function has been identified because of lack of tumor development in multiple endocrine neoplasia type 1 (Men1) null livers. We examine the phenotype of mice lacking one functional allele of Men1 (consistent with the phenotype in humans with MEN1 syndrome) challenged with high fat diet (HFD) to elucidate a metabolic function for hepatic menin.METHODS:In this study, we challenged mice harboring a liver-specific hemizygous deletion of Men1 (HETs) alongside wild-type (WT) counterparts with HFD for 3 months and monitored the severity of metabolic changes. We demonstrate that the HET mice challenged with HFD for 3 months show an increased weight gain with decreased glucose tolerance compared with WT counterparts. Along with these changes, there was a more severe serum hormone profile involving increased serum insulin, glucose and glucagon, all hallmarks of the type 2 diabetic phenotype. In concert with increased serum hormones, we found that these mice have significantly increased liver triglycerides coupled with increased liver steatosis and inflammatory markers. Quantitative real-time PCR and western blotting studies show increases in enzymes involved with lipogenesis and hepatic glucose production.CONCLUSION:We conclude that hepatic menin is required for regulation of diet-induced metabolism, and our studies indicate a protective role for the Men1 gene in the liver when challenged with HFD.

Loss of menin mediated by endothelial cells treated with CoPP is associated with increased maturation of adipocytes

Oxidative stress is caused by an increase in reactive oxygen species (ROS) relative to the antioxidant defense system. An increase in ROS is known to decrease vascular function, increase inflammatory cytokines, and promote adipocyte hypertrophy. A known regulator of the oxidative stress response is the heat shock protein, heme-oxygenase 1 (HO-1), which is induced by cobalt protoporphyrin IX (CoPP). Menin was recently found to promote the sustained expression of heat shock proteins and is implicated in the regulation of oxidative stress. In this study, we investigated how changes in menin expression affected adipogenesis via the interaction between endothelial cells and adipocytes in response to CoPP treatment during oxidative stress. Using angiotensin II (Ang II) to induce oxidative stress in endothelial cells and adipocytes, we observed the induction of various cytokines including EGF, VEGF, angiogenin, IL-6, and MCP-1. Preadipocytes cultured in endothelial cell conditioned media treated with Ang II showed no changes in differentiation markers. Preadipocytes treated with the endothelial cell-conditioned media pretreated with CoPP resulted in an increase in the number of adipocytes, which expressed higher levels of adipocyte differentiation markers in direct correlation with the complete downregulation of the stress response regulator, menin. This change was not detected in adipocytes directly treated with CoPP alone. Therefore, we concluded that loss of menin is associated with the maturation of adipocytes induced by conditioned media from endothelial cells treated with CoPP.

Menin and GIP are inversely regulated by food intake and diet via PI3/AKT signaling in the proximal duodenum.

Background and Aims:Ingestion of food stimulates the secretion of incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 to ensure the proper absorption and storage of nutrients. Menin is the 67 kDa protein product of the MEN1 gene recently reported to have a role in metabolism. In this study, we will determine the regulation of menin in the proximal duodenum by food intake and diet in correlation with GIP levels in the proximal duodenum of mice after an 18 h fast followed by 4 and 7 h refeeding and 3 months of high-fat diet.Methods:A dual luciferase assay was used to determine GIP promoter activity and ELISA was used to measure the levels of GIP after inhibition of menin through small interfering RNA (siRNA) and exposure to MAPK and AKT inhibitors. Colocalization of menin and GIP were determined by immunofluorescence.Results:Menin and GIP expression are regulated by fasting, refeeding and diet in the proximal duodenum. Overexpression of menin in STC-1 cells significantly inhibited GIP mRNA and promoter activity, whereas menin siRNA upregulated GIP levels. Inhibition of GIP expression by the PI3/AKT inhibitor, LY294002, was abrogated in STC-1 cells with reduced menin levels, whereas the MAPK inhibitor, UO126, inhibited the expression of GIP independent of menin. Exposure of STC-1 cells to GIP reduced menin expression in a dose-dependent manner via PI3K-AKT signaling.Conclusion:Feeding and diet regulates the expression of menin, which inversely correlates with GIP levels in the proximal duodenum. In vitro assays indicate that menin is a negative regulator of GIP via inhibition of PI3K-AKT signaling. We show menin colocalizing with GIP in K cells of the proximal gut and hypothesize that downregulation of menin may serve as a mechanism by which GIP is regulated in response to food intake and diet.

Platelets enhance dendritic cell responses against S. aureus through CD40-CD40L interactions.

Staphylococcus aureus is a major human pathogen that can cause mild to severe life-threatening infections in many tissues and organs. Platelets are known to participate in protection against S. aureus by direct killing and by enhancing the activities of neutrophils and macrophages in clearing S. aureus infection. ABSTRACT Staphylococcus aureus is a major human pathogen that can cause mild to severe life-threatening infections in many tissues and organs. Platelets are known to participate in protection against S. aureus by direct killing and by enhancing the activities of neutrophils and macrophages in clearing S. aureus infection. Platelets have also been shown to induce monocyte differentiation into dendritic cells and to enhance activation of dendritic cells. Therefore, in the present study, we explored the role of platelets in enhancing bone marrow-derived dendritic cell (BMDC) function against S. aureus. We observed a significant increase in dendritic cell phagocytosis and intracellular killing of a methicillin-resistant Staphylococcus aureus (MRSA) strain (USA300) by thrombin-activated platelets or their releasates. Enhancement of bacterial uptake and killing by DCs is mediated by platelet-derived CD40L. Coculture of USA300 and BMDCs in the presence of thrombin-activated platelet releasates invokes upregulation of the maturation marker CD80 on DCs and enhanced production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), and IL-6. Overall, these observations support our hypothesis that platelets play a critical role in the host defense against S. aureus infection. Platelets stimulate DCs, leading to direct killing of S. aureus and enhanced DC maturation, potentially leading to adaptive immune responses against S. aureus.