Michael Bliziotes

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.

The emerging role of serotonin (5-hydroxytryptamine) in the skeleton and its mediation of the skeletal effects of low-density lipoprotein receptor-related protein 5 (LRP5)

Novel molecular pathways obligatory for bone health are being rapidly identified. One pathway recently revealed involves gut-derived 5-hydroxytryptamine (5-HT) mediation of the complete skeletal effects of low-density lipoprotein receptor-related protein 5 (LRP5). Mounting evidence supports 5-HT as an important regulatory compound in bone with previous evidence demonstrating that bone cells possess functional pathways for responding to 5-HT. In addition, there is growing evidence that potentiation of 5-HT signaling via inhibition of the 5-HT transporter (5-HTT) has significant skeletal effects. The later is clinically significant as the 5-HTT is a popular target of pharmaceutical agents, such as selective serotonin reuptake inhibitors (SSRIs), used for the management of major depressive disorder and other affective conditions. The observation that 5-HT mediates the complete skeletal effects of LRP5 represents a significant paradigm shift from the traditional view that LRP5 located on the cell surface membrane of osteoblasts exerts direct skeletal effects via Wnt/beta-catenin signaling. This paper discusses the mounting evidence for skeletal effects of 5-HT and the ability of gut-derived 5-HT to satisfactorily explain the skeletal effects of LRP5.

Effects of selective serotonin reuptake inhibitors on bone health in adults: Time for recommendations about screening, prevention and management?

Evidence regarding a functional serotonin (5-hydroxytryptamine) signaling system in bone has generated considerable recent interest. The specific biochemical nature of serotoninergic pathways and their direct and/or indirect effects on bone metabolism are still unclear. Clinical evidence supports an effect of serotonin and altered serotonin signaling on bone metabolism. Serotonin is involved in the pathophysiology of depression, and therefore studies of depression and antidepressant treatments (as modulators of the serotonin system) are relevant with regard to bone outcomes. Studies on the effect of depression on bone mineral density (BMD) and fractures have been mixed. Studies on the associations between antidepressant use and BMD and/or fractures are more consistent. SSRIs have been associated with lower BMD and increased rates of bone loss, as well as increased rates of fracture after accounting for falls. These studies are limited by confounding because depression is potentially associated with both the outcome of interest (BMD and fracture) and the exposure (SSRIs). With mounting evidence for an effect on bone, this review considers the question of causality and whether selective serotonin reuptake inhibitors should be considered among those medications that contribute to bone loss, and therefore prompt clinicians to evaluate BMD proactively. Future research will be required to confirm the serotoninergic effects on bone and the biochemical pathways involved, and to identify clinical implications for treatment based on this novel pathway.

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.

Serotonin (5-hydroxytryptamine) transporter inhibition causes bone loss in adult mice independently of estrogen deficiency

Objective:Selective serotonin reuptake inhibitors (SSRIs) treat depression by antagonizing the serotonin (5-hydroxytryptamine) transporter (5-HTT). These drugs may also have skeletal effects given the presence of functional serotonergic pathways in bone and evidence demonstrating detrimental effects of SSRIs on postmenopausal bone changes. This study aimed to explore the influence of an SSRI (fluoxetine hydrochloride) on the bone changes associated with estrogen deficiency in adult mice. Design:Adult, female, Swiss-Webster mice underwent ovariectomy (OVX) or sham OVX and were treated daily for 4 weeks with either fluoxetine hydrochloride (5 or 20 mg/kg) or a vehicle solution (control). In vivo assessments of hindlimb areal and tibial volumetric bone mineral density were performed at baseline and after 4 weeks of intervention. Femurs and lumbar vertebrae were subsequently removed and assessed ex vivo for bone mineral density and trabecular bone architecture and turnover. Results:In vivo and ex vivo skeletal measures found no interactions between OVX (estrogen deficiency) and 5-HTT inhibition, indicating that the skeletal effects of these interventions were independent. 5-HTT inhibition had detrimental skeletal effects, with the fluoxetine-treated groups having reduced bone mineral density and altered trabecular architecture. These changes resulted from both a decrease in bone formation and increase in bone resorption. Conclusions:These data indicate that a commonly prescribed SSRI has a negative influence on the adult skeleton, independent of estrogen deficiency. This finding supports clinical data demonstrating SSRI use to be associated with accelerated bone loss after menopause and highlights a need for further research into the skeletal effects of SSRIs.

Update in Serotonin and Bone

CONTEXT Serotonin (5-HT) may be an important regulatory agent in bone, and agents that modify 5-HT signaling, such as selective serotonin reuptake inhibitors (SSRIs), are in widespread clinical use. EVIDENCE ACQUISITION Evidence was obtained by PubMed search and the authors knowledge of the field. EVIDENCE SYNTHESIS Recent data suggest that gut-derived 5-HT may mediate the skeletal effects of LDL receptor-related protein 5, stimulating intense interest in a novel mechanism for regulating bone mass. However, the specific biochemical nature of serotonergic pathways influencing bone and their direct and/or indirect effects on bone metabolism are still unclear. The weight of epidemiological evidence suggests that SSRIs are associated with reduced bone mass, increased bone loss, and increased risk of fractures. Interpretation of these studies is complicated by the confounding effects of depression, the usual indication for treatment with SSRIs. The mechanisms for putative SSRI-induced deleterious effects on the skeleton are unknown, and are likely multifactorial. CONCLUSIONS 5-HT may have regulatory effects on bone. Initial preclinical data suggest that its effects may be deleterious and may be regulated by low-density lipoprotein receptor-related protein 5. These studies need confirmation, as well as elucidation, of the biochemical pathways utilized and the feedback loops involved among bone, gut, and perhaps brain. Paradoxically, targeting of 5-HT synthesis and/or signaling in selective tissues may hold promise as an anabolic intervention for bone. Epidemiological data suggest that clinicians should be vigilant about detection of bone disease in patients who are using SSRIs.

Bone histomorphometric and biomechanical abnormalities in mice homozygous for deletion of the dopamine transporter gene

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 had shorter femur length and dry weight. Ash calcium content of the femur was 32% lower in the DAT-/- mice than in the wild-type animals. Cancellous bone volume in the proximal tibial metaphysis was significantly lower in the DAT-/- animals (p < 0.04). There was a 32% reduction in trabecular thickness (p = NS). For the vertebrae, cancellous bone volume was again lower in the DAT-/- animals compared with wild-type as a consequence of increased trabecular spacing (p < 0.05) and reduced trabecular number (p < 0.05). Cortical thickness and bone area in the femoral diaphysis were reduced in the DAT-/-animals. The ultimate bending load (femoral strength) for the DAT-/- mice was 30% lower than the wild-type mice (p = 0.004). Thus, deletion of the DAT gene results in deficiencies in skeletal structure and integrity.

Psychotropic drugs have contrasting skeletal effects that are independent of their effects on physical activity levels.

Popular psychotropic drugs, like the antidepressant selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs), and the mood stabilizer lithium, may have skeletal effects. In particular, preclinical observations suggest a direct negative effect of SSRIs on the skeleton. A potential caveat in studies of the skeletal effects of psychotropic drugs is the hypoactive (skeletal unloading) phenotype they induce. The aim of this study was to investigate the contribution of physical inactivity to the skeletal effects of psychotropic drugs by studying bone changes in cage control and tail suspended mice treated with either vehicle, SSRI, TCA or lithium. Tail suspension was used to control for drug differences on physical activity levels by normalizing skeletal loading between groups. The psychotropic drugs were found to have contrasting skeletal effects which were independent of drug effects on animal physical activity levels. The latter was evident by an absence of statistical interactions between the activity and drug groups. Pharmacological inhibition of the 5-hydroxytryptamine (5-HT) transporter (5-HTT) using a SSRI reduced in vivo gains in lower extremity BMD, and negatively altered ex vivo measures of femoral and spinal bone density, architecture and mechanical properties. These effects were mediated by a decrease in bone formation without a change in bone resorption suggesting that the SSRI had anti-anabolic skeletal effects. In contrast, glycogen synthase kinase-3[beta] (GSK-3[beta]) inhibition using lithium had anabolic effects improving in vivo gains in BMD via an increase in bone formation, while TCA-mediated inhibition of the norepinephrine transporter had minimal skeletal effect. The observed negative skeletal effect of 5-HTT inhibition, combined with recent findings of direct and indirect effects of 5-HT on bone formation, are of interest given the frequent prescription of SSRIs for the treatment of depression and other affective disorders. Likewise, the anabolic effect of GSK-3[beta] inhibition using lithium reconfirms the importance of Wnt/beta-catenin signaling in the skeleton and its targeting by recent drug discovery efforts. In conclusion, the current study demonstrates that different psychotropic drugs with differing underlying mechanisms of action have contrasting skeletal effects and that these effects do not result indirectly via the generation of animal physical inactivity.

Periosteal remodeling at the femoral neck in nonhuman primates

Periosteal bone turnover is poorly understood. We documented intramembranous periosteal bone turnover in the femoral neck in intact nonhuman primates and an increase in osteoclast numbers at the periosteal surface in sex steroid–deficient animals. Our studies are the first to systematically document periosteal turnover at the femoral neck.

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.