Cannabinoids have antiinflammatory and antitumorigenic properties. Some cannabinoids, such as O-1602, have no or only little affinity to classical cannabinoid receptors but exert cannabinoid-like antiinflammatory effects during experimental colitis. Here, we investigated whether O-1602 shows antitumorigenic effects in colon cancer cells and whether it could reduce tumorigenesis in the colon in vivo.
The colon cancer cell lines HT-29 and SW480 were used to study the effect of O-1602 on viability and apoptosis. The effect of O-1602 on tumor growth in vivo was studied in a colitis-associated colon cancer mouse model.
O-1602 decreased viability and induced apoptosis in colon cancer cells in a concentration-dependent manner (0.1-10 μM). In the mouse model, treatment with O-1602 (3 mg/kg, i.p.,12x) reduced tumor area by 50% and tumor incidence by 30%. Histological scoring revealed a significant decrease in tumor load. In tumor tissue, O-1602 decreased levels of proliferating cell nuclear antigen (PCNA), activation of oncogenic transcription factors STAT3 and NFκB p65 and expression of TNF-α while levels for proapoptotic markers, such as p53 and BAX increased. The in vivo effects of O-1602 on PCNA, BAX and p53 were also observed in colon cancer cells.
The data provide a novel insight into antitumorigenic mechanisms of atypical cannabinoids. O-1602 exerts antitumorigenic effects by targeting colon cancer cells as well as proinflammatory pathways known to promote colitis-associated tumorigenesis. Due to its lack of central sedation, O-1602 could be an interesting compound for the treatment of colon and possibly other cancers.
atypical cannabinoid; colitis-associated colon cancer; apoptosis; protumorigenic
Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2AF374Y (exon 9) mutation, inherited from his mother, who developed PHEO/PGL.
We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2AF374Y may be predisposing rather than causative of PHEO/PGL. This report, in view of 2 other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.
HIF2A; Paraganglioma; Familial erythrocytosis
The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature targeting ligand, anginex to improve the therapeutic ratio for strategies developed against solid tumors. Our findings indicate that Galectin-1 is (i) one of the major receptors for anginex (ii) over expressed by tumor neo-vasculature; and (iii) further specifically upregulated in endothelial cells in response to radiation exposure as low as 0.5 Gy. An investigation of -F-labeled anginex biodistribution in SCK tumors indicates that anginex is an effective targeting molecule for image and radiation-guided therapy of solid tumors. An anginex-conjugated liposome capable of being loaded with drug was shown to selectively target endothelial cells post-radiation. The presence of endothelial cells in a three dimensional co-culture system with tumor cells developed to study tumor/endothelial cell interactions in vitro led to higher levels of galectin-1 and showed a further increase in expression upon radiation exposure when compared to tumor cell spheroids alone. Similar increase in galectin-1 was observed in tumor tissue originating from the tumor-endothelial cell spheroids in vivo and radiation exposure further induced galectin-1 in these tumors. The overall results suggest feasibility of using a clinical or subclinical radiation dose to increase expression of the Galectin-1 receptor on the tumor microvasculature to promote delivery of therapeutics via the anginex peptide. This approach may reduce systemic toxicity, overcome drug resistance and improve the therapeutic efficacy of conventional chemo/radiation strategies.
Galectin-1; endothelial cells; tumor vasculature; anginex; tumor-endothelial cell spheroids
Glioblastoma Multiforme (GBM) is the most malignant brain tumor and highly resistant to intensive combination therapies. GBM is one of the most vascularized tumors and vascular endothelial growth factor (VEGF) produced by tumor cells is a major factor regulating angiogenesis. Successful results of preclinical studies of anti-angiogenic therapies using xenograft mouse models of human GBM cell lines encouraged clinical studies of anti-angiogenic drugs such as bevacizumab (Avastin), an anti-VEGF antibody. However, these clinical studies have shown that most patients become resistant to anti-VEGF therapy after an initial response. Recent studies have revealed some resistance mechanisms against anti-VEGF therapies involved in several types of cancer. In this review, we address mechanisms of angiogenesis, including unique features in GBMs, and resistance to anti-VEGF therapies frequently observed in GBM. Enhanced invasiveness is one such resistance mechanism, and recent works report the contribution of activated MET signaling induced by inhibition of VEGF signaling. On the other hand, tumor cell-originated neovascularization including tumor-derived endothelial cell-induced angiogenesis and vasculogenic mimicry has been suggested to be involved in the resistance to anti-VEGF therapy. Therefore, these mechanisms should be targeted in addition to anti-angiogenic therapies to achieve better results for patients with GBM.
glioblastoma; angiogenesis; invasion; MET; alternative neovascularization; vasculogenic mimicry; endothelial differentiation
Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors and proteases that remodel the tumor microenvironment. Invasion and metastasis requires neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment, and examines the factors allowing these cells to promote each stage of metastasis.
Tumor microenvironment; immune cell; metastasis; inflammation; stroma
Retinal and choroidal vascular diseases constitute the most common causes of moderate and severe vision loss in developed countries. They can be divided into retinal vascular diseases, in which there is leakage and/or neovascularization (NV) from retinal vessels, and subretinal NV, in which new vessels grow into the normally avascular outer retina and subretinal space. The first category of diseases includes diabetic retinopathy, retinal vein occlusions, and retinopathy of prematurity and the second category includes neovascular age-related macular degeneration (AMD), ocular histoplasmosis, pathologic myopia, and other related diseases. Retinal hypoxia is a key feature of the first category of diseases resulting in elevated levels of hypoxia-inducible factor-1 (HIF-1) which stimulates expression of vascular endothelial growth factor (VEGF), platelet-derived growth factor-B (PDGF-B), placental growth factor, stromal-derived growth factor-1 and their receptors as well as other hypoxia-regulated gene products such as angiopoietin-2. Although hypoxia has not been demonstrated as part of the second category of diseases, HIF-1 is elevated and thus the same group of hypoxia-regulated gene products plays a role. Clinical trials have shown that VEGF antagonists provide major benefits for patients with subretinal NV due to AMD and even greater benefits are seen by combining antagonists of VEGF and PDGF-B. It is likely that addition of antagonists of other agents listed above will be tested in the future. Other appealing strategies are to directly target HIF-1 or to use gene transfer to express endogenous or engineered anti-angiogenic proteins. While substantial progress has been made, the future looks even brighter for patients with retinal and choroidal vascular diseases.
Angiogenesis; age-related macular degeneration; diabetic retinopathy; hypoxia-inducible factor-1; vascular endothelial growth factor; platelet-derived growth factor
Pyruvate dehydrogenase kinase (PDK) is activated in right ventricular hypertrophy (RVH), causing an increase in glycolysis relative to glucose oxidation that impairs RV function. The stimulus for PDK upregulation, its isoform specificity and the long-term effects of PDK inhibition are unknown. We hypothesize that FOXO1-mediated PDK4 upregulation causes bioenergetic impairment and RV dysfunction, which can be reversed by dichloroacetate.
Adult male Fawn-Hooded rats (FHR) with pulmonary arterial hypertension (PAH) and RVH (age 6–12 months) were compared to age-matched controls. Cardiac glucose and fatty acid oxidation (GO, FAO) were measured at baseline and after acute dichloroacetate (1mM×40-minutes) in isolated working-hearts and in freshly dispersed RV myocytes. The effects of chronic dichloroacetate (0.75 g/L drinking water for 6 months) on cardiac output (CO) and exercise capacity were measured in vivo. Expression of PDK4 and its regulatory transcription factor, FOXO1, were also measured in FHR and RV specimens from PAH patients (n=10).
Microarray analysis of 168 genes related to glucose or FA metabolism showed >4-fold upregulation of PDK4, aldolase B and acyl-coenzyme A oxidase. FOXO1 was increased, in FHR RV whereas HIF-1α was unaltered. PDK4 expression was increased and the inactivated form of FOXO1 decreased in human PAH RV (P<0.01). PDH inhibition in RVH increased proton production and reduced GO’s contribution to the TCA cycle. Acutely, dichloroacetate reduced RV proton production and increased GO’s contribution (relative to FAO) to the TCA cycle and ATP production in FHR (P<0.01). Chronically dichloroacetate decreased PDK4 and FOXO1, thereby activating PDH and increasing GO in FHR. These metabolic changes increased CO (84±14 vs. 69±14 ml/min, P<0.05) and treadmill-walking distance (239±20 vs. 171±22 m, P<0.05).
Chronic dichloroacetate inhibits FOXO1-induced PDK4 upregulation and restores GO, leading to improved bioenergetics and RV function in RVH.
glycolysis; HIF-1α; Aldolase B; FOXO3; acyl-coenzyme A oxidase 2
Pulmonary hypertension is a complex, progressive condition arising from a variety of genetic and pathogenic causes. Patients present with a spectrum of histologic and pathophysiological features, likely reflecting the diversity in underlying pathogenesis. It is widely recognized that structural alterations in the vascular wall contribute to all forms of pulmonary hypertension. Features characteristic of the remodeled vasculature in patients with pulmonary hypertension include increased stiffening of the elastic proximal pulmonary arteries, thickening of the intimal and/or medial layer of muscular arteries, development of vaso-occlusive lesions and the appearance of cells expressing smooth muscle specific markers in normally non-muscular small diameter vessels, resulting from proliferation and migration of pulmonary arterial smooth muscle cells and cellular trans-differentiation. The development of several animal models of pulmonary hypertension has provided the means to explore the mechanistic underpinnings of pulmonary vascular remodeling, although none of the experimental models currently used entirely replicates the pulmonary arterial hypertension observed in patients. Herein, we provide an overview of the histological abnormalities observed in humans with pulmonary hypertension and in preclinical models and discuss insights gained regarding several key signaling pathways contributing to the remodeling process. In particular, we will focus on the roles of ion homeostasis, endothelin-1, serotonin, bone morphogenetic proteins, Rho kinase and hypoxia-inducible factor 1 in pulmonary arterial smooth muscle and endothelial cells, highlighting areas of cross-talk between these pathways and potentials for therapeutic targeting.
Recent evidence suggests that processes of inflammation and angiogenesis are interconnected, especially in human pathologies. Newly formed blood vessels enable the continuous recruitment of inflammatory cells, which release a variety of proangiogenic cytokines, chemokines, and growth factors and further promote angiogenesis. These series of positive feedback loops ultimately create a vicious cycle that exacerbates inflammation, transforming it into the chronic process. Recently, this concept of reciprocity of angiogenesis and inflammation has been expanded to include oxidative stress as a novel mechanistic connection between inflammation-driven oxidation and neovascularization. Production of reactive oxygen species (ROS) results from activation of immune cells by pro-inflammatory stimuli. As oxidative stress can lead to chronic inflammation by activating a variety of transcription factors including NF-κB, AP-1, and PPAR-γ, inflammation itself has a reciprocal relationship with oxidative stress. This review discusses the recent findings in the area bridging neovascularization and oxidation and highlights novel mechanisms of inflammation and oxidative stress driven angiogenesis.
Angiogenesis; Inflammation; Oxidative stress
The field of endothelial progenitor cell (EPC) biology is approaching a decade and a half since generating substantial promise as a potential reparative cell therapy for a spectrum of human clinical disorders. With considerable speed, scientists and clinicians moved from basic studies of isolating and characterizing the biologic properties of EPCs, to pre-clinical EPC treatment studies in rodent model systems of cardiovascular disease, and to the delivery of EPC or marrow-derived cells into selected human subjects (reviewed in [1, 2]). In some disease settings, patient benefits from the infused EPC or marrow-derived cells have been documented, though perhaps not to the extent hoped for or predicted by the results in the preclinical animal model systems . In most human clinical trials, autologous bone marrow mononuclear cells have been infused into patients with cardiovascular disease in an attempt to provide certain presumed EPC subsets to ameliorate ischemic insult [4-7]. To provide some perspective on the advances to date, this review will begin by highlighting the major clarifications in EPC definitions that have occurred over the past 10 years and how this information has instructed changes to the selection of bone marrow subsets for patient use [8-11]. To bring perspective to the increased appreciation of the roles played by hematopoietic cells in vascular repair, we will provide an overview of the hematopoietic hierarchy in mouse and man and identify those subsets that display proangiogenic activities. This perspective may help the reader consider crucial milestones in the discovery and application of HSC and progenitor cells as a cell therapeutic that have not been well explored in the EPC field. The review will conclude with a list of issues that need to be addressed to permit a more quantitative and definable nomenclature for the cells that participate in vascular endothelial repair and replacement. This review will not address the role of those EPC comprised of resident or circulating endothelial cells or endothelial colony forming cells involved in vascular repair and regeneration under normal or pathological conditions (reviewed in [8-15]).
Proopiomelanocortin (POMC) is a polypeptide hormone precursor that is expressed in the brain and in peripheral tissues such as in the pituitary gland, immune system, and skin. In the brain, POMC is processed to form several peptides including alpha-melanocyte stimulating hormone (α-MSH). alpha-MSH is expressed in the hypothalamic arcuate nucleus and in the nucleus tractus solitarius of the brainstem where it has a crucial role in the regulation of metabolic functions. Specifically, α-MSH is an anorexigenic peptide. Its production and maturation processes have been shown to be regulated according to the metabolic condition of the organism. This review summarizes our current knowledge on α-MSH processing including its maturation and degradation processes and pharmacological aspects of its manipulation.
Melanocortin system; Hypothalamus; alpha-Melanocyte stimulating hormone; Propylcarboxypeptidase
The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer.
Actin; Myosin; Cytoskeleton; MRCK; Kinase; Cancer
Nucelosides such as adenosine (Ado) influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides liberated at local sites of inflammation are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases including ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73), found on the surface of a variety of cell types. Once generated, Ado is made available to bind and activate one of four G-protein-coupled Ado receptors. Recent in vitro and in vivo studies implicate Ado in a broad array of tissue protective mechanisms that provide new insight into adenosine actions. Studies in cultured cells and murine tissues have indicated that Ado receptors couple to novel post-translational protein modifications, including Cullin deneddylation, as a new anti-inflammatory mechanism. Studies in Ado receptor-null mice have been revealing and indicate a particularly important role for the Ado A2B receptor in animal models of intestinal inflammation. Here, we review contributions of Ado to cell and tissue stress responses, with a particular emphasis on the gastrointestinal mucosa.
mucosa; inflammation; colitis; neutrophil; epithelium; endothelium; murine model
Inflammatory lesions, ischemic tissues or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors – particularly of hypoxia-inducible factor 1α (HIF1A) - results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors - the ADORA1, ADORA2A, ADORA2B and ADORA3 receptor. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides such as ATP or AMP. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors – particularly of the ADORA2B – results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia-tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.
adenosine; A1; A2A; A2B; A3; ischemia; cancer; hypoxia-inducible factor; HIF1; HIF2; equilibrative nucleoside transporters; ENT1; ENT2; adenosine kinase; adenosine deaminase; CD73; ecto-nucleotidase; CD39; apyrase; AMP; ATP; acute lung injury; colitis; inflammatory bowel disease; ischemia; sepsis
adenosine; A1; A2A; A2B; A3; ischemia; cancer; hypoxia-inducible factor; equilibrative nucleoside transporters; ENT1; ENT2; adenosine kinase; adenosine deaminase; CD73; ecto-nucleotidase; CD39; apyrase; AMP; ATP; acute lung injury; colitis; inflammatory bowel disease; ischemia; sepsis
The recent approval by the FDA of cancer vaccines and drugs that blockade immunological negative regulators has further enhanced interest in promising approaches of the immunotherapy of cancer. However, the disappointingly short life extension has also underscored the need to better understand the mechanisms that prevent tumor rejection and survival even after the blockade of immunological negative regulators. Here, we describe the implications of the “metabolism-based” immunosuppressive mechanism, where the local tissue hypoxia-driven accumulation of extracellular adenosine triggers suppression via A2 adenosine receptors on the surface of activated immune cells. This molecular pathway is of critical importance in mechanisms of immunosuppression in inflamed and cancerous tissue microenvironments. The protection of tumors by tumor-generated extracellular adenosine and A2 adenosine receptors could be the misguided application of the normal tissue-protecting mechanism that limits excessive collateral damage to vital organs during the anti-pathogen immune response. The overview of the current state of the art regarding the immunosuppressive effects of extracellular adenosine is followed by an historical perspective of studies focused on the elucidation of the physiological negative regulators that protect tissues of vital organs from excessive collateral damage, but, as a trade-off, may also weaken the anti-pathogen effector functions and negate the attempts of anti-tumor immune cells to destroy cancerous cells.
adenosine; A2A adenosine receptor; cyclic AMP; hypoxia; inflammation; tumor; cancer immunotherapy; adoptive immunotherapy; tumor microenvironment; immunosuppression; T lymphocytes; regulatory T cells; cytokines; cytotoxicity
Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis.
adenosine receptors; inflammation; fibrosis; vascular barrier function; CD73; ADORA2B; ADORA2A; ADORA3; ADORA1; acute lung injury; remodeling; anti-inflammatory
Though the existence of H2S in biological tissues has been known for over 300 years, it is the most recently appreciated of the gasotransmitters as a physiologic messenger molecule. The enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) had long been speculated to generate H2S, and inhibitors of these enzymes had been employed to characterize influences of H2S in various organs. Definitive evidence that H2S is a physiologic regulator came with the development of mice with targeted deletion of CSE and CBS. Best characterized is the role of H2S, formed by CSE, as an endothelial derived relaxing factor that normally regulates blood pressure by acting through ATP-sensitive potassium channels. H2S participates in various phases of the inflammatory process, predominantly exerting anti-inflammatory actions. Currently, the most advanced efforts to develop therapeutic agents involve the combination of H2S donors with non-steroidal anti-inflammatory drugs (NSAIDs). The H2S moiety provides cytoprotection to gastric mucosa normally adversely affected by NSAIDs, while the combination of H2S and inhibition of prostaglandin synthesis may afford synergistic anti-inflammatory influences.
sulfhydration; cardio protection; inflammation; cystathionine γ-lyase; cystathionine-β-synthase
In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel-Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzymes genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wildtype VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc but not Pdk2 was decreased and skeletal muscle expression of Glut1, Pdk1 and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.
VHL; hypoxia inducible factors; glucose; insulin; glycolysis; gluconeogenesis
Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptor death receptor 4 (DR4) have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation and leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MRs) clustering and formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, TRAIL triggered ASM translocation, ceramide production and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking and fusion with membrane and formation of MR redox signaling platforms, which may play an important role in DR4-mediated redox signaling in CAECs and consequent endothelial dysfunction.
TRAIL; lysosome fusion; acid sphingomyelinase; membrane raft; endothelial cell; vasorelaxation
A major hurdle for hematopoietic stem cell (HSC) gene therapy for inherited bone marrow disorders, including Fanconi anemia (FA), is adequate engraftment of gene-modified cells. A phenotypic defect in DNA repair renders FA patients sensitive to alkylating agents such as cyclophosphamide (Cy); however, at lower doses Cy is well tolerated in the FA transplant setting. We tested whether non-alkylating agents could replace Cy for pre-transplant conditioning to enhance engraftment of FANCA gene-modified hematopoietic cells. We compared Cy pre-conditioning with fludarabine (Flu) or cytarabine (AraC) or no conditioning as a control in fanca−/− mutant mice receiving gene-modified bone marrow cells. Only mice conditioned with Cy exhibited appreciable engraftment of gene-modified cells by PCR and resistance to mitomycin C (MMC). Cy administration following transplantation increased gene marking levels in all animals treated, but highest gene marking and corresponding MMC resistance were observed in mice receiving Cy pre- and post-transplantation. Importantly, no cytogenetic abnormalities were observed in Cy-treated mice. We conclude that Cy is an effective and superior preparative regimen with respect to engraftment of lentivirus-transduced cells and functional correction in fanca−/− mice. Thus, appropriately dosed Cy may provide a suitable conditioning regimen for FA patients undergoing HSC gene therapy.
gene therapy; Fanconi anemia; lentivirus vector; cyclophosphamide; autologous transplantation
Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of pro-inflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-γ signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression.
Combined analysis of genomic Stat occupancy and transcriptome revealed novel Stat target genes in LPS-induced microglia.Jmjd3 transcription factor is a novel transcriptional target of Stat1 and Stat3.Stat1 and Stat3 cooperate with Jmjd3 to induce the expression of pro-inflammatory genes.Constitutively active Stat1 and Stat3 fully mimic the LPS-induced upregulation of inflammatory genes and secretion of cytokines.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1090-5) contains supplementary material, which is available to authorized users.
Inflammation; Signal transducers and activators of transcription; ChIP–chip; Jmjd3 H3K27me3 histone demethylase; Brain macrophages
Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-β1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-β signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-β1-induced renal fibrosis. In the obstructed kidneys and TGF-β1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-β signaling by inhibiting N-linked glycosylation of the type II TGF-β receptor (TβRII), leading to an inefficient trafficking of TβRII to the membrane surface. Defective N-glycosylation of TβRII further suppressed the TGF-β1-binding to TβRII, thereby decreasing TGF-β signaling. Notably, GS-HCl treatment significantly reduced TGF-β1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-β signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-β1-induced renal fibrosis in kidney diseases.
Glucosamine-mediated attenuation of TGF-β signaling ameliorates renal fibrosis in vivoTGF-β1-induced fibrogenic action is reduced by glucosamine in vitroN-glycosylation of the type II TGF-β receptor is suppressed by glucosamineGlucosamine-induced defective N-glycosylation of TβRII decreases TGF-β signaling.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1086-1) contains supplementary material, which is available to authorized users.
Renal fibrosis; Glucosamine hydrochloride; TGF-β signaling; N-glycosylation; Type II TGF-β receptor
Human CD4+CD25highFOXP3+ T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4+CD25neg RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4+CD25highFOXP3+ Treg±150 or ±1,000 IU/ mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p<0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4+CD25high Treg and CD4+CD25neg RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme–perforin pathway depending on IL-2 concentrations.
Human CD4+CD25high Treg; Cell death; Granzymes; Perforin; Interleukin-2
Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin-receptor (IR) signaling and, when over-expressed, induces insulin resistance in vitro and in vivo. Understanding the regulation of ENPP1 expression may, thus, unravel new molecular mechanisms of insulin resistance. Recent data point to a pivotal role of the ENPP1 3’UTR, in modulating ENPP1 mRNA stability and expression. We sought to identify trans-acting proteins binding the ENPP1-3’UTR and to investigate their role on ENPP1 expression and on IR signaling. By RNA electrophoresis mobility shift analysis and tandem mass spectrometry, we demonstrated the binding of heat shock protein 70 (HSP70) to ENPP1-3’UTR. Through this binding, HSP70 stabilizes ENPP1 mRNA and increases ENPP1 transcript and protein levels. This positive modulation of ENPP1 expression is paralleled by a reduced insulin-induced IR and IRS-1 phosphorylation. Taken together these data suggest that HSP70, by affecting ENPP1 expression, may be a novel mediator of altered insulin signaling.
Inhibitors of insulin signaling; Insulin resistance; 3′untranslatedregions; Tyrosine-kinasereceptors