Understanding the addiction cycle: a complex biology with distinct contributions of genotype vs. sex at each stage.

Ethanol abuse can lead to addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. There is evidence for contributions of both genotype and sex to alcoholism, but an understanding of the biological underpinnings is limited. Utilizing both sexes of genetic animal models with highly divergent alcohol withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) mice, the distinct contributions of genotype/phenotype and of sex during addiction stages on neuroadaptation were characterized. Transcriptional profiling was performed to identify expression changes as a consequence of chronic intoxication in the medial prefrontal cortex. Significant expression differences were identified on a single platform and tracked over a behaviorally relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more sensitive to ethanol with higher fold expression differences. Bioinformatics showed a strong effect of sex on the data structure of expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, differences were observed instead between the lines/phenotypes irrespective of sex. Confirmation of identified pathways showed distinct inflammatory signaling following intoxication at peak withdrawal, with a pro-inflammatory phenotype in females but overall suppression of immune signaling in males. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of stage- and sex-specific therapies for alcohol withdrawal and the maintenance of sobriety.

The effect of a corticosteroid cream and a barrier-strengthening moisturizer in hand eczema. A double-blind, randomized, prospective, parallel group clinical trial.

BACKGROUND: Hand eczema is a common and persistent disease with a relapsing course. Clinical data suggest that once daily treatment with corticosteroids is just as effective as twice daily treatment. OBJECTIVES: The aim of this study was to compare once and twice daily applications of a strong corticosteroid cream in addition to maintenance therapy with a moisturizer in patients with a recent relapse of hand eczema. METHODS: The study was a parallel, double-blind, randomized, clinical trial on 44 patients. Twice daily application of a strong corticosteroid cream (betamethasone valerate 0.1%) was compared with once daily application, where a urea-containing moisturizer was substituted for the corticosteroid cream in the morning. The investigator scored the presence of eczema and the patients judged the health-related quality of life (HRQoL) using the Dermatology Life Quality Index (DLQI), which measures how much the patient's skin problem has affected his/her life over the past week. The patients also judged the severity of their eczema daily on a visual analogue scale. RESULTS: Both groups improved in terms of eczema and DLQI. However, the clinical scoring demonstrated that once daily application of corticosteroid was superior to twice daily application in diminishing eczema, especially in the group of patients with lower eczema scores at inclusion. CONCLUSIONS: Twice daily use of corticosteroids was not superior to once daily use in treating eczema. On the contrary, the clinical assessment showed a larger benefit from once daily treatment compared with twice daily, especially in the group of patients with a moderate eczema at inclusion.

Importance of genetic background for risk of relapse shown in altered prefrontal cortex gene expression during abstinence following chronic alcohol intoxication.

Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.

Genetically correlated effects of selective breeding for high and low methamphetamine consumption.

Improved prevention and treatment of drug addiction will require deeper understanding of genetic factors contributing to susceptibility to excessive drug use. Intravenous operant self-administration methods have greatly advanced understanding of behavioral traits related to addiction. However, these methods are not suitable for large-scale genetic experiments in mice. Selective breeding of mice can aggregate 'addiction alleles' in a model that has the potential to identify coordinated effects of multiple genes. We produced mouse lines that orally self-administer high (MAHDR) or low (MALDR) amounts of methamphetamine, representing the first demonstration of selective breeding for self-administration of any psychostimulant drug. Conditioned place preference and taste aversion results indicate that MAHDR mice are relatively more sensitive to the rewarding effects and less sensitive to the aversive effects of methamphetamine, compared to MALDR mice. These results validate the oral route of self-administration for investigation of the motivational effects of methamphetamine and provide a viable alternative to intravenous self-administration procedures. Gene expression results for a subset of genes relevant to addiction-related processes suggest differential regulation by methamphetamine of apoptosis and immune pathways in the nucleus accumbens of MAHDR and MALDR mice. In each line, methamphetamine reduced an allostatic state by bringing gene expression back toward 'normal' levels. Genes differentially expressed in the drug-naï ve state, including Slc6a4 (serotonin transporter), Htr3a (serotonin receptor 3A), Rela [nuclear factor kappaB (NFkappaB)] and Fos (cFos), represent candidates whose expression levels may predict methamphetamine consumption and susceptibility to methamphetamine reward and aversion.

Treatment with a barrier-strengthening moisturizing cream delays relapse of atopic dermatitis: a prospective and randomized controlled clinical trial.

BACKGROUND: Standard treatment of atopic dermatitis (AD) is based on topical glucocorticosteroids or calcineurin inhibitors to treat flares combined with moisturizer treatment to alleviate dry skin symptoms. Patients with AD have an abnormal skin barrier function, and strategies for reducing the risks for eczema would be to repair the barrier or prevent barrier dysfunction. OBJECTIVES: The objective of this study was to explore the time to relapse of eczema during a 26-week maintenance treatment with a urea containing moisturizer compared to no treatment (neither medical nor non-medicated preparations) after successful clearing of atopic lesions. The moisturizer has previously been shown to improve skin barrier function. METHODS: Patients applied betamethasone valerate (0.1%) on eczematous lesions during a 3-week period. Those with cleared eczema entered a 26-week maintenance phase, applying the moisturizer or left the previously affected area untreated. Upon eczema relapse, patients were instructed to contact the clinic and to have the relapse confirmed by the investigator. RESULTS: Fifty-five patients entered the study and 44 patients were included in the maintenance phase (22 using moisturizer twice daily and 22 using no treatment). Median time to relapse for patients treated with moisturizer was > 180 days (duration of the study) compared with 30 days for the no-treatment group. Sixty-eight per cent of the patients treated with the moisturizer and 32% of the untreated patients remained free from eczema during the observation period. CONCLUSIONS: Maintenance treatment with a barrier-improving urea moisturizer on previous eczematous areas reduced the risk of relapse to approximately one third of that of no treatment.

Enhancement of bioavailability by lowering of fat content in topical formulations.

BACKGROUND: The cosmetic properties of topical formulations are important parameters for the adherence to treatment, where modern oil-in-water emulsions are considered more acceptable compared with ointments. After application of an emulsion to the skin, the concentration of active ingredients in the formulation residue on the skin will increase, due to evaporation of volatile ingredients. OBJECTIVES: The aim of the present study was to investigate the effect of changes in vehicle fatty content on the skin penetration of two active ingredients: benzyl nicotinate (BN) and betamethasone valerate (BV). METHODS: Formulations containing 0.5% BN and 0.3% BV in vehicles with different lipid content (10-80%) were applied in a randomized and double-blind manner to the forearm of healthy volunteers. The changes in skin colour (erythema and blanching) were then monitored visually and with a new noninvasive instrument. RESULTS: The BN formulation containing 10% fat induced erythema more rapidly and with higher intensity than the formulations with higher fat content. Increased efficacy was also observed from the low-fat content formulation of BV, which gave more blanching than the formulations with high fat content. CONCLUSIONS: The rate of penetration of the active ingredients was inversely related to the lipid content, i.e. simple changes of the cosmetic properties by modifications of the lipid content may affect the efficacy of a formulation.

Sex differences in the effect of finasteride on acute ethanol withdrawal severity in C57BL/6J and DBA/2J mice.

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of GABAA receptors that can modulate ethanol (EtOH) withdrawal. The 5alpha-reductase inhibitor finasteride can block the formation of ALLO and other GABAergic neurosteroids and also reduce certain effects of EtOH. Treatment with finasteride during chronic EtOH exposure decreased EtOH withdrawal severity and blood EtOH concentrations (BECs), suggesting an additional effect of finasteride on EtOH pharmacokinetics. Thus, the purpose of the present study was to determine the effect of finasteride on acute EtOH withdrawal severity, to minimize the effect of finasteride on EtOH metabolism. Male and female C57BL/6J and DBA/2J mice received a pretreatment of finasteride (50 mg/kg i.p.) or vehicle 24 h prior to an injection of EtOH (4 g/kg i.p.) or saline. Handling-induced convulsions (HICs) were scored at baseline, and then over a 24 h period after EtOH or saline injection. In another experiment, plasma estradiol and corticosterone levels were assessed at selected time points (0, 2, 8, and 24 h). In a final study, retro-orbital blood samples were collected at 30, 60, 120, and 240 min post-EtOH administration to access finasteride's effects on EtOH clearance parameters. Pretreatment with finasteride increased acute EtOH withdrawal severity in female C57BL/6J and DBA/2J mice but decreased withdrawal severity in male mice of both strains. Finasteride did not alter BECs, EtOH clearance, estradiol, or corticosterone concentrations in a manner that appeared to contribute to the sex difference in finasteride's effect on acute EtOH withdrawal severity. These findings suggest that male and female C57BL/6J and DBA/2J mice differ in their sensitivity to changes in ALLO or other GABAergic neurosteroid levels during acute EtOH withdrawal. Sex differences in the modulation of GABAergic 5alpha-reduced steroids may be an important consideration in understanding and developing therapeutic interventions in alcoholics.

Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

Anticonvulsive effects of kappa-opioid receptor modulation in an animal model of ethanol withdrawal.

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.

Androgen inhibition of MAP kinase pathway and Elk-1 activation in proliferating osteoblasts.

Non-aromatizable androgens have significant beneficial effects on skeletal homeostasis independently of conversion to estradiol, but the effects of androgens on bone cell metabolism and cell proliferation are still poorly understood. Using an osteoblastic model with enhanced androgen responsiveness, MC3T3-E1 cells stably transfected with androgen receptor (AR) under the control of the type I collagen promoter (colAR-MC3T3), the effects of androgens on mitogenic signaling were characterized. Cultures were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) and the effects on osteoblast viability were determined as measured by an MTT assay. A complex response was observed in that continuous short-term DHT treatment enhanced osteoblast viability, but with longer-term DHT treatment inhibition was observed. The inhibition by DHT was prevented by the specific AR antagonist hydroxyflutamide, and was also observed in primary cultures of normal rat calvarial osteoblasts. In order to identify potential mediators of this effect, mitogenic pathway-specific cDNA microarrays were interrogated. Reduced hybridization of several genes important in MAP kinase-mediated signaling was observed, with the most dramatic effect on Elk-1 expression. Analysis of phosphorylation cascades demonstrated that DHT treatment inhibited phosphoERK1/2 levels, MAP kinase activation of Elk-1, Elk-1 protein and phosphoElk-1 levels, and downstream AP-1/luciferase reporter activity. Together, these data provide the first evidence that androgen inhibition of the MAP kinase signaling pathway is a potential mediator of osteoblast growth, and are consistent with the hypothesis that the MAP cascade may be a specific downstream target of DHT.

Osteoblast differentiation influences androgen and estrogen receptor-alpha and -beta expression.

Significant levels of estrogen and androgens circulate in men and women, and both play an important role in bone metabolism. While it is well established that either estrogen or androgen replacement therapy is effective at ameliorating bone loss associated with hypogonadism, recent evidence nevertheless suggests that estrogen and androgens have distinct molecular actions on the skeleton. In this study, we have employed normal rat calvarial osteoblast cultures to characterize relative expression profiles of estrogen (ERalpha and ERbeta) and androgen receptors (AR) during osteoblast differentiation. Normal osteoblast cultures can proceed through in vitro differentiation with distinct stages of proliferation, matrix maturation and mineralization in the appropriate differentiation medium containing ascorbic acid. Expression profiles of AR, ERalpha and ERbeta in primary cultures during osteoblast differentiation were characterized both by semi-quantitative relative RT-PCR and by Western analysis. In cultures induced to differentiate by growth in the presence of ascorbic acid, the expression profile for each receptor was unique during the course of differentiation. ERalpha levels were elevated during matrix maturation and then declined during mineralization. ERbeta expression was relatively constant throughout differentiation, exhibiting more constitutive expression. In contrast, AR levels were lowest during proliferation, and then increased throughout differentiation with highest levels in the most mature mineralizing cultures. Since steroid hormone action is generally mediated by specific cognate receptors, these results suggest that androgen actions may target cells during the mineralization stage of osteoblast differentiation, while estrogen action through either receptor isoform is more likely to affect osteoblasts earlier during matrix maturation. Interestingly, sex steroid receptor expression profiles did not exhibit the same patterns of regulation if osteoblast cultures were grown without ascorbic acid in medium that did not support extracellular matrix deposition. Thus, sex steroids may distinctly influence skeletal health by differential modulation of function during osteoblast differentiation.

The role of dopamine and serotonin in regulating bone mass and strength: studies on dopamine and serotonin transporter null mice.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. Dopamine (DA) has been reported to have effects on calcium and phosphorus metabolism. The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released DA by rapid uptake of the neurotransmitter into presynaptic terminals. We have evaluated the histologic and biomechanical properties of the skeleton in mice homozygous for deletion of the DA transporter gene (DAT (-/-)) to help delineate the role of DA in bone biology. We have demonstrated that DAT (-/-) mice have reduced bone mass and strength. DAT (-/-) animals have shorter femur length and dry weight, and lower ash calcium content. Cancellous bone volume in the DAT (-/-) proximal tibial metaphysis is significantly decreased with reduced trabecular thickness. DAT (-/-) vertebrae have lower cancellous bone volume as a consequence of increased trabecular spacing and reduced trabecular number, and cortical thickness and bone area in the femoral diaphysis are reduced. The ultimate bending load (femoral strength) for the DAT (-/-) mice is 30% lower than the wild-type mice. Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity. Since serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we explored the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines including ROS 17/2.8, UMR 106-H5 and Py1a show mRNA expression for the 5-HTT, and the 5-HT(1A), 5-HT(1D), 5-HT(2A) and 5-HT(2B) receptors by RT-PCR analysis and immunoblot. A relatively high density of nanomolar affinity 5-HTT binding sites is present in ROS 17/2.8 and UMR 106-H5 cells. The maximal [(3)H]5-HT uptake rate in ROS cells was 110 pmol/10 min/well, with a K(m) value of 1.13 microM. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased by almost eight-fold at day 31. In mature rOB cultures, the estimated density of [(125)I]RTI-55 binding sites was 600 fmol/mg protein. PMA treatment caused a significant 40% reduction in the maximal uptake rate of [(3)H]5-HT, an effect prevented by pretreatment with staurosporine. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR 106-H5 cells. In 5-HTT (-/-) animals, cancellous bone volume (BV/TV) in the lumbar vertebrae is reduced, with a trend toward decreased trabecular thickness and trabecular number. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT, and disruption of the 5-HTT gene may cause osteopenia.

Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Ethanol-regulated gene expression of neuroendocrine specific protein in mice: brain region and genotype specificity.

Neuroendocrine specific protein or reticulon 1 (NSP/RTN1) was identified as a putative ethanol-regulated gene using mRNA differential display in mice genetically selected for severe ethanol withdrawal (withdrawal seizure-prone, WSP). One transcript of RTN1 (3.0 kb) showed a statistically significant increase (13%) in relative abundance in whole brain of ethanol-treated WSP mice but not in mice selected for resistance to ethanol withdrawal convulsions (WSR). We hypothesized that ethanol-induced regulation of gene expression of mRTN1 is specific to mice predisposed to exhibit severe ethanol withdrawal and that the gene might be regulated differentially in specific brain regions. WSP and WSR selected lines and DBA/2J and C57BL/6J inbred strains of mice were exposed to ethanol vapor or air for 72 h. mRNA steady-state expression of RTN1 was assessed in hippocampus, cortex, and cerebellum. Results indicated that the pattern of ethanol-induced changes in gene expression was dependent upon transcript size, brain region, and genotype. Modest increases in the relative abundance of both transcripts of RTN1 were observed in the hippocampus and cortex of all ethanol-treated mice. Results from cerebellum showed a moderate decrease in expression of RTN1 (3.0 kb transcript) in WSP and DBA/2J mice, but not in the mice resistant to ethanol withdrawal (C57BL/6J and WSR). These results suggest a genotype-specific effect of chronic ethanol exposure on steady-state mRNA levels of RTN1 in the cerebellum. Overall, the results indicate a complex pattern of ethanol-induced regulation of the putative mouse homologue of RTN1 and suggest that specific brain regional changes may be involved in the expression of physical dependence.

Regulated nuclear-cytoplasmic localization of CCAAT/enhancer-binding protein delta in osteoblasts.

Insulin-like growth factor I (IGF-I) plays a central role in skeletal growth by promoting bone cell replication and differentiation. Prostaglandin E2 (PGE2) and parathyroid hormone enhance cAMP production in cultured rat osteoblasts and stimulate IGF-I expression through a transcriptional mechanism mediated by cAMP-dependent protein kinase (PKA). We previously showed that PGE2 activated the transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta) in osteoblasts and induced its binding to a DNA element within the IGF-I promoter. We report here that a PKA-dependent pathway stimulates nuclear translocation of C/EBPdelta. Under basal conditions, C/EBPdelta was cytoplasmic but rapidly accumulated in the nucleus after PGE2 treatment (t(1/2) < 30 min). Nuclear translocation occurred without concurrent protein synthesis and was maintained in the presence of hormone. Nuclear localization required PKA and was blocked by a dominant-interfering regulatory subunit of the enzyme, even though C/EBPdelta was not a PKA substrate. Upon removal of hormonal stimulus, C/EBPdelta quickly exited the nucleus (t(1/2) < 12 min) through a pathway blocked by leptomycin B. Mutagenesis studies indicated that the basic domain of C/EBPdelta was necessary for nuclear localization and that the leucine zipper region permitted full nuclear accumulation. We thus define a pathway for PKA-mediated activation of C/EBPdelta through its regulated nuclear import.

Reduced G-protein-coupled-receptor kinase 2 activity results in impairment of osteoblast function.

Rapid phosphorylation of many G-protein-coupled receptors (GPCRs) by G-protein-coupled receptor kinases (GRKs) accompanies stimulus-driven desensitization. Recent evidence suggests that GRKs and their associated arresting proteins, beta-arrestins, function as essential elements in the GPCR-mediated mitogen-activated protein (MAP) kinase signaling cascade. We investigated the interaction between GRKs and MAP kinase activation by growth factors in UMR 106-H5 osteoblastic cells stably expressing a dominant negative mutant of GRK2 (K220R). Expression of K220R in osteoblastic cells results in reduced cellular proliferation, both basally and in response to insulin-like growth factor-1 (IGF-1), and blunting of IGF-1- and EGF-induced MAP kinase activation. Reduced MAP kinase activation is not associated with alterations in IGF-1-receptor autophosphorylation. Both a constitutively active Ras mutant and PMA fully activate MAP kinase in K220R cells. We found that disruption of the GRK2 gene results in: (1) reduced osteoblast proliferation in response to growth factors, and (2) impaired receptor tyrosine kinase activation of mitogenic signaling pathways. Thus, GRK2 may regulate growth factor responsiveness in osteoblasts by modulating multiprotein complex formation following receptor tyrosine kinase activation.

Elevated prodynorphin expression associated with ethanol withdrawal convulsions.

The hypothesis that kappa-opioid system activity may in part mediate convulsions exhibited during ethanol withdrawal was tested by exposing Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice to chronic ethanol. Whole brain was harvested for RNA isolation and prodynorphin mRNA steady-state levels in whole brain were examined using Northern blot analysis. The data revealed significantly increased levels of prodynorphin mRNA expression in mice susceptible to ethanol withdrawal convulsions after withdrawal, with no corresponding increase in prodynorphin steady-state levels in mice resistant to ethanol withdrawal convulsions. These findings were not due to basal differences in prodynorphin expression between the WSP and WSR mice. To verify that the differences observed were not due to an ethanol-induced global alteration in gene transcription, mRNA levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were measured. Glyceraldehyde-3-phosphate dehydrogenase expression was unchanged following both chronic exposure to ethanol and chronic exposure followed by withdrawal. These results extend our understanding of prodynorphin's role in generalized seizure activity to include ethanol withdrawal-induced convulsions. Our findings suggest that prodynorphin expression is modulated during ethanol withdrawal convulsions, or alternatively, prodynorphin may mediate the severity of ethanol withdrawal convulsions.

Homologous androgen receptor up-regulation in osteoblastic cells may be associated with enhanced functional androgen responsiveness.

Although androgens have myriad effects on the skeleton, the regulation of androgen action in bone is not well understood. Androgen receptors (ARs) are known to play an important role in mediating androgen action. We have examined the effects of androgens and other sex steroids on AR levels in osteoblastic cells in vitro using two clonal human cell lines, SaOS-2 and U-2 OS. AR protein levels were quantitated both by specific androgen binding studies and Western analyses, and AR messenger RNA was measured with RNase protection assays. Potential changes in AR functionality was assessed by reporter assays. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) increased specific androgen binding 2-to 4-fold. Similar increases in AR protein levels were documented by Western analysis in both cell lines. The androgen-mediated increase in receptor levels was time and dose dependent as well as androgen specific. Steady-state AR messenger RNA levels were also increased by DHT. When AR concentrations in osteoblastic cells were elevated with exogenous receptor, there was an enhancement of DHT responsiveness, measured by increased trans-activation of an androgen-responsive promoter. Thus, androgen exposure increased androgen receptor protein levels and specific androgen binding in osteoblastic cells. Androgen action as measured by androgen-mediated transcriptional activation is enhanced in the presence of elevated AR levels. Consequently, these studies have revealed an additional means by which androgens may modulate skeletal metabolism.

Identification of neuroendocrine-specific protein as an ethanol-regulated gene with mRNA differential display.

Chronic ethanol exposure produces changes in behavior that may result from effects of ethanol on gene expression. To identify potentially ethanol-regulated genes, mRNA differential display was used to screen the expressed genes in whole brain of mice chronically exposed to ethanol vapors. Mice genetically selected for susceptibility (Withdrawal Seizure-Prone; WSP) or resistance (Withdrawal Seizure-Resistant; WSR) to ethanol withdrawal convulsions were exposed to either ethanol vapor (ETOH group) or air (CTL group) for 72 h. A putative ethanol-regulated product was isolated; nucleotide sequence analysis of this product revealed >85% nucleotide identity to human neuroendocrine-specific protein (NSP) gene. Northern analysis of the expression of this product revealed hybridization to two transcripts ( approximately 3.0 kb and 1.4 kb) on blots containing whole brain RNA, consistent with the transcript sizes of hNSP. Ethanol-induced regulation of mNSP was suggested in whole brain of WSP mice, but not in WSR mice, by Northern blot analysis. One transcript (3.0 kb) suggests a 26% increase in relative abundance in whole brain of ethanol-exposed WSP mice, while there was no effect of ethanol on abundance of the 1.4-kb transcript in WSP mice. No effects of ethanol were observed for WSR mice. These preliminary findings suggest that mNSP represents a novel ethanol-induced gene in mice selected for genetic susceptibility to severe ethanol withdrawal.

Transcriptional up-regulation of the human androgen receptor by androgen in bone cells.

Androgen regulation of androgen receptor (AR) expression has been observed in a variety of tissues, generally as inhibition, and is thought to attenuate cellular responses to androgen. AR is expressed in osteoblasts, the bone-forming cell, suggesting direct actions of androgens on bone. Here we characterized the effect of androgen exposure on AR gene expression in human osteoblastic SaOS-2 and U-2 OS cells. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone increased AR steady state messenger RNA levels in a time- and dose-dependent fashion. Reporter assays with 2.3 kilobases of the proximal 5'-flanking region of the human AR promoter linked to the chloramphenicol acetyltransferase gene in transfected cultures showed that up-regulation of AR promoter activity by androgen was time and dose dependent. Treatment with other steroid hormones, including progesterone, 17beta-estradiol, and dexamethasone, was without effect. The antiandrogen hydroxyflutamide completely antagonized androgen up-regulation. Thus, in contrast to many other androgen target tissues, androgen exposure increases steady state AR messenger RNA levels in osteoblasts. This regulation occurs at least partially at the level of transcription, is mediated by the 5'-promoter region of the AR gene, and is dependent on functional AR. These results suggest that physiological concentrations of androgens have significant effects on AR expression in skeletal tissue.