Kristine M. Wiren

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.

Comparison of RiboGreen and 18S rRNA quantitation for normalizing real-time RT-PCR expression analysis.

2001. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68:245-253. Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy. 1999. Effect of turkey (Meleagridis gallopavo) breeder hen age and egg size on poult development. 2. Intestinal villus growth, enterocyte migration and proliferation of the turkey poult. Comp. Bio. Physio. Real-time RT-PCR is becoming the method of choice for monitoring or confirming mRNA expression (1−3). With unparalleled sensitivity, the ability to use 100 to 1000 times less RNA than other methodologies, and high-throughput potential for gene discovery approaches, real-time RT-PCR gives researchers the ability to determine mRNA abundance quickly and efficiently (1,4). While Northern blot analysis remains the most accepted method for measuring mRNA abundance, there are drawbacks that limit its utility. First, Northern blot analysis requires several micrograms of total RNA for each lane, which precludes the analysis of microdissected samples, tissue from laser capture, or other valuable samples due to the high cost and/or inability to obtain sufficient amounts of RNA. Second, Northern analysis is limited by the number of samples that can be run simultaneously on a gel, or requires standard or control samples on each blot for normalization between other Northern blots. Finally, the limited sensitivity of Northern analysis, given the indirect nature of the measurement and the multiple steps required, necessitates a robust change in the abundance of a given transcript to obtain a statistically significant result. All methods of RNA quantification rely on normalization methods to quantitatively compare multiple disparate samples. Due to the sensitivity of the assay, real-time RT-PCR requires accurate methods for normalizing both for the amount of RNA added to each reaction and for reaction efficiency (1,4). Standard methods used for nor-malization of RNA amounts include analysis of absorbance at 260 nm, or real-time RT-PCR analysis of housekeeping genes or 18S rRNA abundance (1). Absorbance at 260 nm is the most problematic approach because a large amount of RNA is required, samples will necessarily be subsequently diluted , and contaminates that are present in the sample frequently adversely influence the accuracy of the quantifica-tions. Housekeeping genes are suitable for normalization in some cases, but require additional analysis to verify the specific treatment or experimental conditions do not alter their steady-state mRNA abundance (1). The use of 18S rRNA abundance for normalization in real-time RT-PCR analysis has been effective in our hands. Advantages include the …

Neural regulation of bone and the skeletal effects of serotonin (5-hydroxytryptamine).

There is increasing evidence for a contribution of the neural system to the regulation of bone metabolism. The skeleton is richly innervated by both sympathetic and sensory neurons. While these nerves serve sensory and vascular functions, they are also being found to influence bone cell activities. The most convincing evidence for this has been provided by studies into the skeletal effects of the hormone leptin, which has been shown to centrally regulate bone mass, and through studies into the skeletal effects of hypothalamic neuropeptide Y2 and Y4 receptors. This paper discusses recent evidence for the neural regulation of bone metabolism and, in particular, the potential role of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). Recent studies have demonstrated the presence of functional pathways in bone for both responding to and regulating the uptake of 5-HT. This is of high clinical relevance given the role of the serotonergic system in affective disorders, and the wide use of pharmacological agents that target the 5-HT system to manage these disorders. Initial data suggest that exposure to these agents at different stages during the lifespan may have significant effects on the skeleton.

Neurotoxic consequences of chronic alcohol withdrawal: expression profiling reveals importance of gender over withdrawal severity.

While women are more vulnerable than men to many of the medical consequences of alcohol abuse, the role of sex in the response to ethanol is controversial. Neuroadaptive responses that result in the hyperexcitability associated with withdrawal from chronic ethanol likely reflect gene expression changes. We have examined both genders for the effects of withdrawal on brain gene expression using mice with divergent withdrawal severity that have been selectively bred from a genetically heterogeneous population. A total of 295 genes were identified as ethanol regulated from each gender of each selected line by microarray analyses. Hierarchical cluster analysis of the arrays revealed that the transcriptional response correlated with sex rather than with the selected withdrawal phenotype. Consistent with this, gene ontology category over-representation analysis identified cell death and DNA/RNA binding as targeted classes of genes in females, while in males, protein degradation, and calcium ion binding pathways were more altered by alcohol. Examination of ethanol-regulated genes and these distinct signaling pathways suggested enhanced neurotoxicity in females. Histopathological analysis of brain damage following ethanol withdrawal confirmed elevated cell death in female but not male mice. The sexually dimorphic response was observed irrespective of withdrawal phenotype. Combined, these results indicate a fundamentally distinct neuroadaptive response in females compared to males during chronic ethanol withdrawal and are consistent with observations that female alcoholics may be more vulnerable than males to ethanol-induced brain damage associated with alcohol abuse.

Targeting of androgen receptor in bone reveals a lack of androgen anabolic action and inhibition of osteogenesis A model for compartment-specific androgen action in the skeleton

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

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 finasterides 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 finasterides 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.

Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation.

Androgens regulate body composition in youth and declining testosterone that occurs with aging is associated with muscle wasting, increased fat mass and osteopenia. Transgenic mice with targeted androgen receptor (AR) over‐expression in mesenchymal stem cells (MSC) were generated to explore the role of androgen signaling in the regulation of body composition. Transgenic males, but not females, were shorter and have reduced body weight and visceral fat accumulation. Dual‐energy X‐ray absorptiometry (DXA) revealed significant reductions in fat mass with a reciprocal increase in lean mass, yet no difference in food consumption or locomotor activity was observed. Adipose tissue weight was normal in brown fat but reduced in both gonadal and perirenal depots, and reduced hyperplasia was observed with smaller adipocyte size in visceral and subcutaneous white adipose tissue. Although serum leptin, adiponectin, triglyceride, and insulin levels were no different between the genotypes, intraperitoneal glucose tolerance testing (IPGTT) showed improved glucose clearance in transgenic males. High levels of the AR transgene are detected in MSCs but not in mature fat tissue. Reduced fibroblast colony forming units indicate fewer progenitor cells resident in the marrow in vivo. Precocious expression of glucose transporter 4 (GLUT4), peroxisome proliferator‐activated receptor γ (PPARγ), and CCAAT enhancer‐binding protein α (C/EBPα) was observed in proliferating precursor cultures from transgenic mice compared to controls. In more mature cultures, there was little difference between the genotypes. We propose a mechanism where enhanced androgen sensitivity can alter lineage commitment in vivo to reduce progenitor number and fat development, while increasing the expression of key factors to promote smaller adipocytes with improved glucose clearance. J. Cell. Biochem. 112: 1773–1786, 2011.

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.

Anticonvulsive effects of κ‐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 κ‐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 prevents hypogonadal bone loss via inhibition of resorption mediated by mature osteoblasts/osteocytes

Androgen receptor (AR) is expressed throughout the osteoblast lineage. Two different AR transgenic families (AR2.3-tg and AR3.6-tg mice) demonstrating overlapping and distinct expression profiles were employed to assess the effects of enhanced androgen sensitivity to ameliorate hypogonadal loss. Two different paradigms of steroid replacement following orchidectomy (ORX) were used as either preventative or therapeutic therapy. ORX was performed in male wild-type (WT), AR2.3-tg and AR3.6-tg mice at 5 months with immediate DHT replacement (prevention, higher turnover) or at 3 months with DHT treatment delayed for 2 months (therapeutic, lower turnover), both with treatment for the last 6 weeks. Dual energy X-ray absorptiometry (DXA), micro-computed tomography (μCT), and histomorphometry were performed. In the prevention model, ORX significantly reduced BMD and BMC in all genotypes compared to sham and DHT was effective at prevention of osteopenia. In the therapeutic model, all genotypes became osteopenic compared to sham, but after a prolonged hypogonadal period, delayed DHT treatment provided little benefit. μCT analysis of mid-shaft total bone in all genotypes generally showed reductions after ORX. Delayed DHT was ineffective at restoring bone volume in any genotype whereas immediate treatment prevented loss only in AR transgenic mice. Cortical thickness also decreased with ORX but immediate DHT treatment was effective to increase thickness only in WT mice, likely due to expansion of marrow volume in both AR-tg lines. In metabolically highly active cancellous bone, ORX resulted in lower bone volume/tissue volume (BV/TV) in all genotypes, consistent among 3 sites measured. Again with delayed treatment, there was little effect of DHT to restore BV/TV, but when administered at the time of ORX, DHT completely prevented the decrease in cancellous bone in all genotypes. Improvement in cancellous bone architecture was seen with immediate DHT replacement that was enhanced in AR transgenic lines compared to WT. In contrast, there were only modest changes in all genotypes using the delayed treatment paradigm. With ORX in both paradigms, trabecular number was decreased while spacing increased. Thus, androgen therapy is effective for the prevention of endosteal and cancellous osteopenia primarily through its anti-resorptive properties, but shows little anabolic action as a therapeutic strategy to restore bone. Given the similarity in response to androgen treatment in both AR transgenic lines, overlapping expression profiles suggest that the target cells mediating androgen action in vivo are mature osteoblast/osteocytes. Combined, these results demonstrate that in the adult mouse, androgen treatment can reduce bone resorption but has little overall anabolic activity.