Slawomir Golda

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.

Heme Oxygenase-1 Prevents Airway Mucus Hypersecretion Induced by Cigarette Smoke in Rodents and Humans

We investigated the role of heme oxygenase-1 (HO-1), a powerful anti-inflammatory and anti-oxidant enzyme, in modulating cigarette smoke (CS)-induced mucus secretion. In both rats and mice, 5-day CS exposure increased HO-1 expression and activity, mucus secretion, MUCIN 5AC (MUC5AC) gene and protein expression, and local inflammation, along with up-regulation of dual oxidase 1 gene expression and both the activity and phosphorylation of the epidermal growth factor receptor, which is involved in MUC5AC induction. Pharmacological induction of HO-1 prevented these actions and inhibition of HO-1 expression by a specific siRNA potentiated them. In French participants to the European Community Respiratory Health Survey II (n = 210, 30 to 53 years of age, 50% males) exposed to CS, a significant increase in the percentage of participants with chronic sputum was observed in those harboring at least one allele with a long (GT)(n) in the HO-1 promoter gene (>33 repeats), which is associated with a low level of HO-1 protein expression, compared with those with a short number of (GT)n repeats (21.7% versus 8.6%, P = 0.047). No such results were observed in those who had never smoked (n = 297). We conclude that HO-1 has a significant protective effect against airway mucus hypersecretion in animals and humans exposed to CS.

Novelty-Seeking Behaviors and the Escalation of Alcohol Drinking After Abstinence in Mice Are Controlled by Metabotropic Glutamate Receptor 5 on Neurons Expressing Dopamine D1 Receptors

BACKGROUND Novel experiences activate the brains reward system in a manner similar to drugs of abuse, and high levels of novelty-seeking and sensation-seeking behavior have been associated with increased susceptibility to alcohol and drug abuse. Here, we show that metabotropic glutamate receptor 5 (mGluR5) signaling on dopaminoceptive neurons is necessary for both novelty-seeking behavior and the abstinence-induced escalation of alcohol drinking. METHODS Mice harboring a transgene expressing microRNA hairpins against mGluR5 messenger RNA under the control of the D1 dopamine receptor gene promoter (mGluR5(KD-D1)) were tested in a battery of behavioral tests measuring learning abilities, anxiety levels, reactions to novelty, operant sensation seeking, and alcohol sensitivity. In addition, we have developed a method to assess long-term patterns of alcohol drinking in mice housed in groups using the IntelliCage system. RESULTS mGluR5(KD-D1) mice showed no behavioral deficits and exhibited normal anxiety-like behaviors and learning abilities. However, mGluR5(KD-D1) animals showed reduced locomotor activity when placed in a novel environment, and exhibited decreased interaction with a novel object. Moreover, unlike control animals, mutant mice did not perform instrumental responses under the operant sensation-seeking paradigm, although they learned to respond for food normally. When mGluR5(KD-D1) mice were provided access to alcohol, they showed similar patterns of consumption as wild-type animals. However, mutant mice did not escalate their alcohol consumption after a period of forced abstinence, but control mice almost doubled their intake. CONCLUSIONS These data identify mGluR5 receptors on D1-expressing neurons as a common molecular substrate of novelty-seeking behaviors and behaviors associated with alcohol abuse.

Astrocytes are a neural target of morphine action via glucocorticoid receptor‐dependent signaling

Chronic opioid use leads to the structural reorganization of neuronal networks, involving genetic reprogramming in neurons and glial cells. Our previous in vivo studies have revealed that a significant fraction of the morphine‐induced alterations to the striatal transcriptome included glucocorticoid (GC) receptor (GR)‐dependent genes. Additional analyses suggested glial cells to be the locus of these changes. In the current study, we aimed to differentiate the direct transcriptional effects of morphine and a GR agonist on primary striatal neurons and astrocytes. Whole‐genome transcriptional profiling revealed that while morphine had no significant effect on gene expression in both cell types, dexamethasone significantly altered the transcriptional profile in astrocytes but not neurons. We obtained a complete dataset of genes undergoing the regulation, which includes genes related to glucose metabolism (Pdk4), circadian activity (Per1) and cell differentiation (Sox2). There was also an overlap between morphine‐induced transcripts in striatum and GR‐dependent transcripts in cultured astrocytes. We further analyzed the regulation of expression of one gene belonging to both groups, serum and GC regulated kinase 1 (Sgk1). We identified two transcriptional variants of Sgk1 that displayed selective GR‐dependent upregulation in cultured astrocytes but not neurons. Moreover, these variants were the only two that were found to be upregulated in vivo by morphine in a GR‐dependent fashion. Our data suggest that the morphine‐induced, GR‐dependent component of transcriptome alterations in the striatum is confined to astrocytes. Identification of this mechanism opens new directions for research on the role of astrocytes in the central effects of opioids.

Common transcriptional effects in the mouse striatum following chronic treatment with heroin and methamphetamine

The molecular alterations that underlie the long‐lasting behavioural effects of drugs of abuse, such as psychomotor sensitization and physical dependence, are still not known. Moreover, it is not known which molecular effects are similar for addictive drugs from various pharmacological classes. In this study, we utilized whole‐genome microarray profiling to evaluate the detailed time‐course of transcriptional alterations in the mouse striatum during chronic treatment with heroin (HER) and methamphetamine (METH) and after period of spontaneous withdrawal. We identified 27 genes regulated by chronic drug administration. The overlap between lists of HER‐ and METH‐induced genes was highly significant. The most substantial impact on the gene expression profile was observed for circadian genes (Per1, Per2 and Nr1d1). However, changing the treatment scheme from diurnal to nocturnal was sufficient to attenuate the drug‐induced changes in circadian gene mRNA levels. Both of the drugs caused a dose‐dependent induction in glucocorticoid‐dependent genes with relatively long mRNA half‐lives (Fkbp5, Sult1a1 and Plin4). The analysis also showed a drug‐regulated group of transcripts enriched in the nucleus accumbens and includes well known (Pdyn, Cartpt and Rgs2) as well as new (Fam40b and Inmt) candidate genes. All identified alterations in the striatal transcriptome were transient and persisted up to 6 days after withdrawal. Behavioural sensitization, however, was maintained throughout the 12‐day withdrawal period for both HER and METH. We suggest that transient gene expression alterations during drug treatment and in the early period of withdrawal are involved in the establishment of persistent neuroplastic alterations responsible for the development of drug addiction.

Behavioral and molecular alterations in mice resulting from chronic treatment with dexamethasone: relevance to depression.

Chronic stress, the administration of glucocorticoids and the prolonged activation of glucocorticoid receptors (GRs) are reported to induce affective changes in humans and rodents that resemble a depressive state. However, data concerning the behavioral and molecular effects of the selective activation of specific GRs are limited, and the conclusions derived remain debatable. In this study, our goal was to investigate the behavioral and molecular changes following the prolonged activation of GRs in mice via exposure to the specific agonist dexamethasone (DEX). C57BL/6J mice were injected daily with DEX (4 mg/kg, i.p.) or saline, and the behavior of the animals was assessed in the following paradigms: the forced swimming test (FST), the light-dark box test, the saccharin preference test and activity boxes. The mRNA expression levels of the corticosteroid receptors mineralocorticoid (MR, Nr3c2) and glucocorticoid (GR, Nr3c1), selected stress dependent genes and glial markers were analyzed in the prefrontal cortex, hippocampus and striatum. DEX-treated mice exhibited a variety of depression-like behaviors: increased time of immobility in the FST, a reduced preference for saccharin consumption and increased anxiety-like behavior. Behavioral alterations were accompanied by a decrease in the mRNA expression of GR and the increased expression of Fkbp5 and Sgk1 in the prefrontal cortex, hippocampus and striatum of DEX-treated mice. Furthermore, our results indicate a decrease in the mRNA expression of glutamate aspartate transporter (GLAST, Slc1a3), an astroglial cell marker, in the hippocampus and prefrontal cortex. These results demonstrate that the prolonged activation of GR receptors induced a depression-like state in mice, activated stress-related genes and induced a decrease in the mRNA expression of GLAST, an astroglial marker, in the prefrontal cortex and hippocampus. Together, the results reported here challenge several hypotheses concerning the role of GRs in the development of behavioral and molecular alterations relevant to stress-related disorders, such as depression, under the same experimental conditions.

Genotype-dependent consequences of traumatic stress in four inbred mouse strains

Post‐traumatic stress disorder (PTSD) is an anxiety disorder that develops in predisposed individuals following a terrifying event. Studies on isogenic animal populations might explain susceptibility to PTSD by revealing associations between the molecular and behavioural consequences of traumatic stress. Our study employed four inbred mouse strains to search for differences in post‐stress response to a 1.5‐mA electric foot shock. One day to 6 weeks after the foot shock anxiety, depression and addiction‐like phenotypes were assessed. In addition, expression levels of selected stress‐related genes were analysed in hippocampus and amygdala. C57BL/6J mice exhibited up‐regulation in the expression of Tsc22d3, Nfkbia, Plat and Crhr1 genes in both brain regions. These alterations were associated with an increase of sensitized fear and depressive‐like behaviour over time. Traumatic stress induced expression of Tsc22d3, Nfkbia, Plat and Fkbp5 genes and developed social withdrawal in DBA/2J mice. In 129P3/J strain, exposure to stress produced the up‐regulation of Tsc22d3 and Nfkbia genes and enhanced sensitivity to the rewarding properties of morphine. Whereas, SWR/J mice displayed increase only in Pdyn expression in the amygdala and had the lowest conditioned fear. Our results reveal a complex genetic background of phenotypic variation in response to stress and indicate the SWR/J strain as a valuable model of stress resistance. We found potential links between the alterations in expression of Tsc22d3, Nfkbia and Pdyn, and different aspects of susceptibility to stress.

Overexpression of biliverdin reductase enhances resistance to chemotherapeutics

Biliverdin reductase (BVR) converts biliverdin to bilirubin. Additionally, acting as a transcription factor and possessing a capacity of a serine/threonine kinase, it may modulate signaling pathways. In order to gain better understanding of BVR functions, we used genetically modified line of mouse fibroblasts with reversible overexpression of BVR. Current study revealed that enhanced activity of BVR may protect cells in stressful conditions arising from anti-cancer drugs, cisplatin and doxorubicin, the effect most probably related to PKC α/β activity, as its inhibition reversed BVR action. Therefore activity of BVR may be of significance in tumors and may influence the effectiveness of therapies.

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB.

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-1β, 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.