Astroglial Knockout of Glucocorticoid Receptor Attenuates Morphine Withdrawal Symptoms, but Not Antinociception and Tolerance in Mice.

The development of tolerance and drug dependence limit the clinical application of opioids for the treatment of severe pain. Glucocorticoid receptors (GRs) are among molecular substrates involved in these processes. Most studies focus on the role of neuronal GR, while the involvement of GR on glial cells is not fully understood. To address this issue, we used a transgenic model of conditional GR knockout mice, targeted to connexin 30-expressing astrocytes, treated with repeated doses of morphine. We observed no difference between control mice and astrocytic GR knockouts in the development of antinociceptive tolerance. Nevertheless, when animals were subjected to precipitated withdrawal, knockouts presented some attenuated symptoms, including jumping. Taken together, our data suggest that hippocampal and spinal astrocytic GRs appear to be involved in opioid withdrawal, and drugs targeting the GR may relieve some symptoms of morphine withdrawal without influencing its antinociceptive properties.

Knockdown of the astrocytic glucocorticoid receptor in the central nucleus of the amygdala diminishes conditioned fear expression and anxiety.

The amygdala is a key structure involved in both physiological and behavioural effects of fearful and stressful stimuli. The central stress response is controlled by the activity of the hypothalamic-pituitary-adrenal (HPA) axis via glucocorticoid hormones, acting mainly through glucocorticoid receptors (GR), widely expressed among different brain regions, including the central nucleus of the amygdala (CeA). Although to date, neuronal GR was postulated to be involved in the mediating stress effects, increasing evidence points to the vital role of glial GR. Here, we aimed to evaluate the role of astrocytic GR in CeA in various aspects of the stress response. We used a lentiviral vector to disrupt an astrocytic GR in the CeA of Aldh1l1-Cre transgenic mice. Astrocytic GR knockdown mice (GR KD) exhibited an attenuated expression of fear-related memory in the fear conditioning paradigm. Interestingly, the consolidation of non-stressful memory in the novel object recognition test remained unchanged. Moreover, GR KD group presented reduced anxiety, measured in the open field test. However, knockdown of astrocytic GR in the CeA did not affect an acute response to stress in the tail suspension test. Taken together, obtained results suggest that astrocytic GR in the CeA promotes aversive memory consolidation and some aspects of anxiety behaviour.

Astrocytes determine conditioned response to morphine via glucocorticoid receptor-dependent regulation of lactate release.

To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.

Glucocorticoid receptor signaling in astrocytes is required for aversive memory formation.

Stress elicits the release of glucocorticoids (GCs) that regulate energy metabolism and play a role in emotional memory. Astrocytes express glucocorticoid receptors (GR), but their contribution to cognitive effects of GC's action in the brain is unknown. To address this question, we studied how astrocyte-specific elimination of GR affects animal behavior known to be regulated by stress. Mice with astrocyte-specific ablation of GR presented impaired aversive memory expression in two different paradigms of Pavlovian learning: contextual fear conditioning and conditioned place aversion. These mice also displayed compromised regulation of genes encoding key elements of the glucose metabolism pathway upon GR stimulation. In particular, we identified that the glial, but not the neuronal isoform of a crucial stress-response molecule, Sgk1, undergoes GR-dependent regulation in vivo and demonstrated the involvement of SGK1 in regulation of glucose uptake in astrocytes. Together, our results reveal astrocytes as a central element in GC-dependent formation of aversive memory and suggest their relevance for stress-induced alteration of brain glucose metabolism. Consequently, astrocytes should be considered as a cellular target of therapies of stress-induced brain diseases.

An endogenous and ectopic expression of metabotropic glutamate receptor 8 (mGluR8) inhibits proliferation and increases chemosensitivity of human neuroblastoma and glioma cells.

The present study aimed to determine the role of metabotropic glutamate receptor 8 (mGluR8) in tumor biology. Using various molecular approaches (RNAi or GRM8 cDNA), cell clones with downregulated (human neuroblastoma SH-SY5Y and human glioma LN229) or overexpressed (human glioma U87-MG and LN18 cell lines) mGluR8 were generated. Next, comparative studies on cell proliferation and migration rates, induction of apoptosis and chemosensitivity were performed among these clones. The mGluR8-downregulated SH-SY5Y clones proliferated faster and were more resistant to cytotoxic action of staurosporine, doxorubicin, irinotecan and cisplatin when compared to control cells. Moreover, these clones were characterized by a lower activity of caspases, calpains and some kinases (GSK-3ß, Akt and JNK). The mGluR8-downregulated LN229 clones migrated faster and were less prone to cell-damaging effect of staurosporine and irinotecan when compared with relevant control cells. In contrast, in GRM8-overexpressing U87-MG and LN18 clones, a decreased cell proliferation, increased apoptosis and elevated vulnerability to some cytotoxic agents were found. Altogether, our in vitro data for the first time evidenced a tumor suppressor and chemosensitizing role of mGluR8.

Nrf2-heme oxygenase-1 axis in mucoepidermoid carcinoma of the lung: Antitumoral effects associated with down-regulation of matrix metalloproteinases.

Lung mucoepidermoid carcinoma (MEC) is a very poorly characterized rare subtype of non-small-cell lung cancer (NSCLC) associated with more favorable prognoses than other forms of intrathoracic malignancies. We have previously identified that heme oxygenase-1 (HO-1, encoded by HMOX1) inhibits MEC tumor growth and modulates the transcriptome of microRNAs. Here we investigate the role of a major upstream regulator of HO-1 and a master regulator of cellular antioxidant responses, transcription factor Nrf2, in MEC biology. Nrf2 overexpression in the NCI-H292 MEC cell line mimicked the phenotype of HO-1 overexpressing cells, leading to inhibition of cell proliferation and migration and down-regulation of oncogenic miR-378. HMOX1 silencing identified HO-1 as a major mediator of Nrf2 action. Nrf2- and HO-1 overexpressing cells exhibited strongly diminished expression of multiple matrix metalloproteinases and inflammatory cytokine interleukin-1ß, which was confirmed in an NCI-HO-1 xenograft model. Overexpression of HO-1 altered not only human MMP levels in tumor cells but also murine MMP levels within tumor microenvironment and metastatic niche. This could possibly contribute to decreased metastasis to the lungs and inhibitory effects of HO-1 on MEC tumor growth. Our profound transcriptome analysis and molecular characterization of the mucoepidermoid lung carcinoma helps to understand the specific clinical presentations of these tumors, emphasizing a unique antitumoral role of the Nrf2-HO-1 axis.

Regulation and novel action of thymidine phosphorylase in non-small cell lung cancer: crosstalk with Nrf2 and HO-1.

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1ß and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1ß and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

Heme oxygenase-1 is required for angiogenic function of bone marrow-derived progenitor cells: role in therapeutic revascularization.

AIMS: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. RESULTS: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin(-)CD45(-)cKit(-)Sca-1(+)VEGFR-2(+)) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1(-/-) BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1(+/-) diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1(+)CXCR-4(+)) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. INNOVATION: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. CONCLUSIONS: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.

Hypoxia-regulated overexpression of soluble VEGFR2 controls angiogenesis and inhibits tumor growth.

VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo: Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel-controlled normalization and the design of adjuvants for combined cancer therapies.

Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasis.

AIMS: Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. RESULTS: Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. INNOVATION AND CONCLUSION: In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers: James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.

Opposite effects of HIF-1α and HIF-2α on the regulation of IL-8 expression in endothelial cells.

Recently we have shown that hypoxia as well as overexpression of the stable form of hypoxia-inducible factor-1α (HIF-1α) diminished the expression of interleukin-8 (IL-8) by inhibition of the Nrf2 transcription factor in HMEC-1 cells. Because HIF isoforms may exert different effects, we aimed to examine the influence of HIF-2α on IL-8 expression in endothelial cells. In contrast to HIF-1α, overexpression of HIF-2α obtained by adenoviral transduction resulted in increased expression of IL-8 in an Nrf2-independent way. Importantly, HIF-2α augmented the activity of SP-1, a transcription factor involved in IL-8 regulation and known coactivator of c-Myc. Additionally, HIF-1 decreased, whereas HIF-2 increased, c-Myc expression, and silencing of Mxi-1, a c-Myc antagonist, restored IL-8 expression downregulated by HIF-1α or hypoxia. Accordingly, binding of c-Myc to the IL-8 promoter was abolished in hypoxia. Importantly, both severe (0.5% O(2)) and mild (5% O(2)) hypoxia diminished IL-8 expression despite the stabilization of both HIF-1 and HIF-2. This study reveals the opposite roles of HIF-1α and HIF-2α in the regulation of IL-8 expression in endothelial cells. However, despite stabilization of both isoforms in hypoxia the effect of HIF-1 is predominant, and downregulation of IL-8 expression in hypoxia is caused by attenuation of Nrf2 and c-Myc.

Oxidative stress in tumor angiogenesis- therapeutic targets.

Historically, oxidative stress was recognized to contribute to cancer development uniquely by induction of genomic instability. However, recent research has provided multiple evidence that reactive oxygen species and other free radicals, such as nitric oxide, often produced at elevated levels within tumor tissue, may function as signaling molecules that initiate and/or modulate the different regulatory pathways involved in tumorigenesis and metastasis. This review will focus on the complex role of oxidative stress and redox signaling in cancer neovascularization, a process without which the tumor is unable to grow beyond few millimeters in size. Reactive oxygen species and nitric oxide affect cell responses to hypoxia, a major trigger of angiogenic switch in tumors and are important upstream regulators as well as downstream mediators of action of the most potent proangiogenic factor - vascular endothelial growth factor. We will discuss targeting the redox-regulated mechanisms for antiangiogenic anticancer therapy and focus on recent developments in small-molecule agents that have either completed clinical trials or show a great promise to be subjected to them. Modulation of redox species production, signaling and metabolism and/or manipulating cellular antioxidant responses represents a multitargeted therapeutic approach which may possibly overcome the limitations of single-agent antiangiogenic treatments and potentiate effects of standard methods.

Nuclear delivery of NFkappaB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging.

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized kappaB motif to favour NFkappaB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was approximately 1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFkappaB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFkappaB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.