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1.  A serotonin-induced N-glycan switch regulates platelet aggregation 
Scientific Reports  2013;3:2795.
Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.
PMCID: PMC3786303  PMID: 24077408
2.  Serotonin 2B Receptor (5-HT2B R) Signals through Prostacyclin and PPAR-ß/δ in Osteoblasts 
PLoS ONE  2013;8(9):e75783.
Osteoporosis is due to an imbalance between decreased bone formation by osteoblasts and increased resorption by osteoclasts. Deciphering factors controlling bone formation is therefore of utmost importance for the understanding and the treatment of osteoporosis. Our previous in vivo results showed that bone formation is reduced in the absence of the serotonin receptor 5-HT2B, causing impaired osteoblast proliferation, recruitment, and matrix mineralization. In this study, we investigated the signaling pathways responsible for the osteoblast defect in 5-HT2BR−/− mice. Notably, we investigated the phospholipase A2 pathway and synthesis of eicosanoids in 5-HT2BR−/− compared to wild type (WT) osteoblasts. Compared to control osteoblasts, the lack of 5-HT2B receptors was only associated with a 10-fold over-production of prostacyclin (PGI2). Also, a specific prostacyclin synthase inhibitor (U51605) rescued totally osteoblast aggregation and matrix mineralization in the 5-HT2BR−/− osteoblasts without having any effect on WT osteoblasts. Prostacyclin is the endogenous ligand of the nuclear peroxisome proliferator activated receptor ß/δ (PPAR-ß/δ), and its inhibition in 5-HT2BR−/− cells rescued totally the alkaline phosphatase and osteopontin mRNA levels, cell-cell adhesion, and matrix mineralization. We conclude that the absence of 5-HT2B receptors leads to the overproduction of prostacyclin, inducing reduced osteoblast differentiation due to PPAR-ß/δ -dependent target regulation and defective cell-cell adhesion and matrix mineralization. This study thus reveals a previously unrecognized cell autonomous osteoblast defect in the absence of 5-HT2BR and highlights a new pathway linking 5-HT2B receptors and nuclear PPAR- ß/δ via prostacyclin.
PMCID: PMC3775737  PMID: 24069449
3.  Stimulating healthy tissue regeneration by targeting the 5-HT2B receptor in chronic liver disease 
Nature medicine  2011;17(12):1668-1673.
Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood1. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT2B) on HSCs by serotonin, which activates expression of transforming growth factor β1 (TGF-β1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT2B enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT2B or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT2B attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT2B is clinically safe in humans and may be therapeutic in chronic liver disease.
PMCID: PMC3428919  PMID: 22120177
4.  5-HT2B receptors are required for serotonin-selective antidepressant actions 
Molecular Psychiatry  2011;17(2):154-163.
The therapeutic effects induced by serotonin-selective reuptake inhibitor (SSRI) antidepressants are initially triggered by blocking the serotonin transporter and rely on long-term adaptations of pre- and post-synaptic receptors. We show here that long-term behavioral and neurogenic SSRI effects are abolished after either genetic or pharmacologic inactivation of 5-HT2B receptors. Conversely, direct agonist stimulation of 5-HT2B receptors induces an SSRI-like response in behavioral and neurogenic assays. Moreover, the observation that (i) this receptor is expressed by raphe serotonergic neurons, (ii) the SSRI-induced increase in hippocampal extracellular serotonin concentration is strongly reduced in the absence of functional 5-HT2B receptors, and (iii) a selective 5-HT2B agonist mimics SSRI responses, supports a positive regulation of serotonergic neurons by 5-HT2B receptors. The 5-HT2B receptor appears, therefore, to positively modulate serotonergic activity and to be required for the therapeutic actions of SSRIs. Consequently, the 5-HT2B receptor should be considered as a new tractable target in the combat against depression.
PMCID: PMC3381222  PMID: 22158014
8-Hydroxy-2-(di-n-propylamino)tetralin; adverse effects; Analysis of Variance; Animals; Bromodeoxyuridine; metabolism; Cell Differentiation; drug effects; Chromatography, High Pressure Liquid; Exploratory Behavior; Fluoxetine; pharmacology; Gene Expression Regulation; Hippocampus; Hypothermia; chemically induced; Ki-67 Antigen; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdialysis; Neurogenesis; Reaction Time; Receptor, Serotonin, 5-HT2B; deficiency; physiology; Serotonin; Serotonin Plasma Membrane Transport Proteins; Serotonin Receptor Agonists; adverse effects; Serotonin Uptake Inhibitors; Time Factors; Transcription Factors; serotonin levels; 5-HT2B receptors; antidepressants; neurogenesis
5.  Deconstructing Antiobesity Compound Action: Requirement of Serotonin 5-HT2B Receptors for Dexfenfluramine Anorectic Effects 
Neuropsychopharmacology  2010;36(2):423-433.
The now-banned anorectic molecule, dexfenfluramine, promotes serotonin release through a serotonin transporter-dependent mechanism, and it has been widely prescribed for the treatment of obesity. Previous studies have identified that 5-HT2B receptors have important roles in dexfenfluramine side effects, that is, pulmonary hypertension, plasma serotonin level regulation, and valvulopathy. We thus investigated a putative contribution of 5-HT2B receptors in dexfenfluramine-dependent feeding behavior in mice. Interestingly, the hypophagic response to dexfenfluramine (3–10 mg/kg) observed in wild-type mice (1–4 h) was eliminated in mice lacking 5-HT2B receptors (5-HT2B−/−). These findings were further validated by the lack of hypophagic response to dexfenfluramine in wild-type mice treated with RS127445, a highly selective and potent antagonist (pKi=8.22±0.24). Using microdialysis, we observed that in 5-HT2B−/− awake mice, the dexfenfluramine-induced hypothalamic peak of serotonin release (1 h) was strongly reduced (fourfold) compared with wild type. Moreover, using hypothalamic synaptosomes, we established the serotonergic neuron autonomous properties of this effect: a strong serotonin release was observed upon dexfenfluramine stimulation of synaptosome preparation from wild type but not from mice lacking active 5-HT2B receptors. These findings strongly suggest that activation of presynaptic 5-HT2B receptors is a limiting step in the serotonin transporter dependant-releasing effect of dexfenfluramine, whereas other serotonin receptors act downstream with respect to feeding behavior.
PMCID: PMC3055663  PMID: 20927048
behavior; feeding; receptor; releaser; serotonin; transporter; eating/metabolic disorders; transporters; psychopharmacology; serotonin; 5-HT2B receptor; dexfenfluramine
6.  Platelet-derived serotonin links vascular disease and tissue fibrosis 
Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.
Vascular damage and platelet activation are associated with tissue remodeling in diseases such as systemic sclerosis, but the molecular mechanisms underlying this association have not been identified. In this study, we show that serotonin (5-hydroxytryptamine [5-HT]) stored in platelets strongly induces extracellular matrix synthesis in interstitial fibroblasts via activation of 5-HT2B receptors (5-HT2B) in a transforming growth factor β (TGF-β)–dependent manner. Dermal fibrosis was reduced in 5-HT2B−/− mice using both inducible and genetic models of fibrosis. Pharmacologic inactivation of 5-HT2B also effectively prevented the onset of experimental fibrosis and ameliorated established fibrosis. Moreover, inhibition of platelet activation prevented fibrosis in different models of skin fibrosis. Consistently, mice deficient for TPH1, the rate-limiting enzyme for 5-HT production outside the central nervous system, showed reduced experimental skin fibrosis. These findings suggest that 5-HT/5-HT2B signaling links vascular damage and platelet activation to tissue remodeling and identify 5-HT2B as a novel therapeutic target to treat fibrotic diseases.
PMCID: PMC3092343  PMID: 21518801
Nature  2010;468(7327):1061-1066.
Impulsivity, describing action without foresight, is an important feature of several psychiatric diseases, suicidality and violent behavior. The complex origins of impulsivity hinder identification of the genes influencing both it and diseases with which it is associated. We performed exon-centric sequencing of impulsive individuals in a founder population, targeting fourteen genes belonging to the serotonin and dopamine domain. A stop codon in HTR2B that is common (MAF >1%) but exclusive to Finns was identified. Expression of the gene in the human brain was assessed, as well as the molecular functionality of the stop codon that was associated with psychiatric diseases marked by impulsivity in both population and family-based analyses. Knockout of Htr2b increased impulsive behaviors in mice, indicative of predictive validity. Our study shows the potential for identifying and tracing effects of rare alleles in complex behavioral phenotypes using founder populations, and suggests a role for HTR2B in impulsivity.
PMCID: PMC3183507  PMID: 21179162
8.  Lack of Serotonin 5-HT2B Receptor Alters Proliferation and Network Volume of Interstitial Cells of Cajal in Vivo 
Normal gastrointestinal motility requires intact networks of interstitial cells of Cajal (ICC). ICC numbers are maintained by a balance between cell loss factors and survival/trophic/growth factors. Activation of 5-HT2B receptors expressed on ICC increases ICC proliferation in vitro. It is not known whether 5-HT2B receptors on ICC are activated in vivo. The aims of this study were to investigate if adult ICC proliferate, whether the proliferation of ICC in vivo is affected by knocking out the 5-HT2B receptor, and if alterations in proliferation affect ICC networks.
Proliferating ICC were identified by immunoreactivity for Ki67 in both the myenteric and deep muscular plexus regions of the jejunum in mice with a targeted insertion of a neomycin resistance cassette into the second coding exon of the htr2b receptor gene.
Key Results
Adult ICC do proliferate. The number of proliferating ICC was lower in the myenteric plexus region of Htr2b−/− compared to Htr2b+/+ mice. The volume of Kit-positive ICC was 30% lower in the myenteric plexus region and 40% lower in the deep muscular plexus region in Htr2b−/− mice where the number of ICC was also reduced.
Conclusions & Inferences
ICC proliferate in adult mice and activation of 5-HT2B receptors results in increased proliferation of ICC in vivo. Furthermore, lack of 5-HT2B receptor signaling reduces the density of ICC networks in mature mice. These data suggest that 5-HT2B receptor signaling is required for maintenance of ICC networks, adding 5-HT to the growing number of factors shown to regulate ICC networks.
PMCID: PMC2852486  PMID: 19941613
Cellular plasticity; Kit; Gastrointestinal motility; Mouse jejunum; Serotonin receptors
9.  Role of Serotonin via 5-HT2B Receptors in the Reinforcing Effects of MDMA in Mice 
PLoS ONE  2009;4(11):e7952.
The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) reverses dopamine and serotonin transporters to produce efflux of dopamine and serotonin, respectively, in regions of the brain that have been implicated in reward. However, the role of serotonin/dopamine interactions in the behavioral effects of MDMA remains unclear. We previously showed that MDMA-induced locomotion, serotonin and dopamine release are 5-HT2B receptor-dependent. The aim of the present study was to determine the contribution of serotonin and 5-HT2B receptors to the reinforcing properties of MDMA.
We show here that 5-HT2B−/− mice do not exhibit behavioral sensitization or conditioned place preference following MDMA (10 mg/kg) injections. In addition, MDMA-induced reinstatement of conditioned place preference after extinction and locomotor sensitization development are each abolished by a 5-HT2B receptor antagonist (RS127445) in wild type mice. Accordingly, MDMA-induced dopamine D1 receptor-dependent phosphorylation of extracellular regulated kinase in nucleus accumbens is abolished in mice lacking functional 5-HT2B receptors. Nevertheless, high doses (30 mg/kg) of MDMA induce dopamine-dependent but serotonin and 5-HT2B receptor-independent behavioral effects.
These results underpin the importance of 5-HT2B receptors in the reinforcing properties of MDMA and illustrate the importance of dose-dependent effects of MDMA on serotonin/dopamine interactions.
PMCID: PMC2775951  PMID: 19956756

Results 1-9 (9)