HTR2B as a novel biomarker of chronic obstructive pulmonary disease with lung squamous cell carcinoma.

Chronic obstructive pulmonary disease (COPD) is often associated with lung squamous cell carcinoma (LUSC), which has the same etiology (smoking, inflammation, oxidative stress, microenvironmental changes, and genetics). Smoking, inflammation, and airway remodeling are the most important and classical mechanisms of COPD comorbidity in LUSC patients. Cancer can occur during repeated airway damage and repair (airway remodeling). Changes in the inflammatory and immune microenvironments, which can cause malignant transformation of some cells, are currently being revealed in both LUSC and COPD patients. We obtained the GSE76925 dataset from the Gene Expression Omnibus database. Screening for possible COPD biomarkers was performed using the LASSO regression model and a random forest classifier. The compositional patterns of the immune cell fraction in COPD patients were determined using CIBERSORT. HTR2B expression was analyzed using validation datasets (GSE47460, GSE106986, and GSE1650). HTR2B expression in COPD cell models was determined via real-time quantitative PCR. Epithelial-mesenchymal transition (EMT) marker expression levels were determined after knocking down or overexpressing HTR2B. HTR2B function and mechanism in LUSC were analyzed with the Kaplan‒Meier plotter database. HTR2B expression was inhibited to detect changes in LUSC cell proliferation. A total of 1082 differentially expressed genes (DEGs) were identified in the GSE76925 dataset (371 genes were significantly upregulated, and 711 genes were significantly downregulated). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs were mainly enriched in the p53 signaling and ß-alanine metabolism pathways. Gene Ontology enrichment analysis indicated that the DEGs were largely related to transcription initiation from the RNA polymerase I promoter and to the regulation of mononuclear cell proliferation. The LASSO regression model and random forest classifier results revealed that HTR2B, DPYS, FRY, and CD19 were key COPD genes. Immune cell infiltration analysis indicated that these genes were closely associated with immune cells. Analysis of the validation sets suggested that HTR2B was upregulated in COPD patients. HTR2B was significantly upregulated in COPD cell models, and its upregulation was associated with increased EMT marker expression. Compared with that in bronchial epithelial cells, HTR2B expression was upregulated in LUSC cells, and inhibiting HTR2B expression led to the inhibition of LUSC cell proliferation. In conclusions, HTR2B might be a new biomarker and therapeutic target in COPD patients with LUSC.

Serotonin Receptor HTR2B Facilitates Colorectal Cancer Metastasis via CREB1-ZEB1 Axis-Mediated Epithelial-Mesenchymal Transition.

A number of neurotransmitters have been detected in tumor microenvironment and proved to modulate cancer oncogenesis and progression. We previously found that biosynthesis and secretion of neurotransmitter 5-hydroxytryptamine (5-HT) was elevated in colorectal cancer cells. In this study, we discovered that the HTR2B receptor of 5-HT was highly expressed in colorectal cancer tumor tissues, which was further identified as a strong risk factor for colorectal cancer prognostic outcomes. Both pharmacological blocking and genetic knocking down HTR2B impaired migration of colorectal cancer cell, as well as the epithelial-mesenchymal transition (EMT) process. Mechanistically, HTR2B signaling induced ribosomal protein S6 kinase B1 (S6K1) activation via the Akt/mTOR pathway, which triggered cAMP-responsive element-binding protein 1 (CREB1) phosphorylation (Ser 133) and translocation into the nucleus, then the phosphorylated CREB1 acts as an activator for ZEB1 transcription after binding to CREB1 half-site (GTCA) at ZEB1 promoter. As a key regulator of EMT, ZEB1, therefore, enhances migration and EMT process in colorectal cancer cells. We also found that HTR2B-specific antagonist (RS127445) treatment significantly ameliorated metastasis and reversed EMT process in both HCT116 cell tail-vein-injected pulmonary metastasis and CT26 cell intrasplenic-injected hepatic metastasis mouse models. IMPLICATIONS: These findings uncover a novel regulatory role of HTR2B signaling on colorectal cancer metastasis, which provide experimental evidences for potential HTR2B-targeted anti-colorectal cancer metastasis therapy.

Contribution of the STAT Family of Transcription Factors to the Expression of the Serotonin 2B (HTR2B) Receptor in Human Uveal Melanoma.

Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5'-TTC (N)3 GAA3') located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the -280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

Colonic Motility Is Improved by the Activation of 5-HT2B Receptors on Interstitial Cells of Cajal in Diabetic Mice.

BACKGROUND & AIMS: Constipation is commonly associated with diabetes. Serotonin (5-HT), produced predominantly by enterochromaffin (EC) cells via tryptophan hydroxylase 1 (TPH1), is a key modulator of gastrointestinal (GI) motility. However, the role of serotonergic signaling in constipation associated with diabetes is unknown. METHODS: We generated EC cell reporter Tph1-tdTom, EC cell-depleted Tph1-DTA, combined Tph1-tdTom-DTA, and interstitial cell of Cajal (ICC)-specific Kit-GCaMP6 mice. Male mice and surgically ovariectomized female mice were fed a high-fat high-sucrose diet to induce diabetes. The effect of serotonergic signaling on GI motility was studied by examining 5-HT receptor expression in the colon and in vivo GI transit, colonic migrating motor complexes (CMMCs), and calcium imaging in mice treated with either a 5-HT2B receptor (HTR2B) antagonist or agonist. RESULTS: Colonic transit was delayed in males with diabetes, although colonic Tph1+ cell density and 5-HT levels were increased. Colonic transit was not further reduced in diabetic mice by EC cell depletion. The HTR2B protein, predominantly expressed by colonic ICCs, was markedly decreased in the colonic muscles of males and ovariectomized females with diabetes. Ca2+ activity in colonic ICCs was decreased in diabetic males. Treatment with an HTR2B antagonist impaired CMMCs and colonic motility in healthy males, whereas treatment with an HTR2B agonist improved CMMCs and colonic motility in males with diabetes. Colonic transit in ovariectomized females with diabetes was also improved significantly by the HTR2B agonist treatment. CONCLUSIONS: Impaired colonic motility in mice with diabetes was improved by enhancing HTR2B signaling. The HTR2B agonist may provide therapeutic benefits for constipation associated with diabetes.

Serotonin receptor type 2B activation augments TNF-α-induced matrix mineralization in murine valvular interstitial cells.

Calcification, fibrosis, and chronic inflammation are the predominant features of calcific aortic valve disease, a life-threatening condition. Drugs that induce serotonin (5-hydroxytryptamine [5-HT]) are known to damage valves, and activated platelets, which carry peripheral serotonin, are known to promote calcific aortic valve stenosis. However, the role of 5-HT in valve leaflet pathology is not known. We tested whether serotonin mediates inflammation-induced matrix mineralization in valve cells. Real-time reverse transcription-polymerase chain reaction analysis showed that murine aortic valve interstitial cells (VICs) expressed both serotonin receptor types 2A and 2B (Htr2a and Htr2b). Although Htr2a expression was greater at baseline, Htr2b expression was induced several-fold more than Htr2a in response to the pro-calcific tumor necrosis factor-α (TNF-α) treatment. 5-HT also augmented TNF-α-induced osteoblastic differentiation and matrix mineralization of VIC, but 5-HT alone had no effects. Inhibition of serotonin receptor type 2B, using specific inhibitors or lentiviral knockdown in VIC, attenuated 5-HT effects on TNF-α-induced osteoblastic differentiation and mineralization. 5-HT treatment also augmented TNF-α-induced matrix metalloproteinase-3 expression, which was also attenuated by Htr2b knockdown. Htr2b expression in aortic roots and serum levels of peripheral 5-HT were also greater in the hyperlipidemic Apoe-/- mice than in control normolipemic mice. These findings suggest a new role for serotonin signaling in inflammation-induced calcific valvulopathy.

Pathological Insight into 5-HT2B Receptor Activation in Fibrosing Interstitial Lung Diseases.

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)2 receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.

Sleep Deprivation Selectively Down-Regulates Astrocytic 5-HT2B Receptors and Triggers Depressive-Like Behaviors via Stimulating P2X7 Receptors in Mice.

Chronic loss of sleep damages health and disturbs the quality of life. Long-lasting sleep deprivation (SD) as well as sleep abnormalities are substantial risk factors for major depressive disorder, although the underlying mechanisms are not clear. Here, we showed that chronic SD in mice promotes a gradual elevation of extracellular ATP, which activates astroglial P2X7 receptors (P2X7Rs). Activated P2X7Rs, in turn, selectively down-regulated the expression of 5-HT2B receptors (5-HT2BRs) in astrocytes. Stimulation of P2X7Rs induced by SD selectively suppressed the phosphorylation of AKT and FoxO3a in astrocytes, but not in neurons. The over-expression of FoxO3a in astrocytes inhibited the expression of 5-HT2BRs. Down-regulation of 5-HT2BsRs instigated by SD suppressed the activation of STAT3 and relieved the inhibition of Ca2+-dependent phospholipase A2. This latter cascade promoted the release of arachidonic acid and prostaglandin E2. The depression-like behaviors induced by SD were alleviated in P2X7R-KO mice. Our study reveals the mechanism underlying chronic SD-induced depression-like behaviors and suggests 5-HT2BRs as a key target for exploring therapeutic strategies aimed at the depression evoked by sleep disorders.

Serotonin (5-HT) Shapes the Macrophage Gene Profile through the 5-HT2B-Dependent Activation of the Aryl Hydrocarbon Receptor.

Macrophages can either promote or resolve inflammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine [5-HT]). In fact, pro- and anti-inflammatory macrophages differ in the expression of serotonin receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF-primed monocyte-derived macrophages (M-MØ). 5-HT7 drives the acquisition of profibrotic and anti-inflammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT-related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antiparkinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT-mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B-specific antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics.

Serotonin receptors 5-HTR2A and 5-HTR2B are involved in cigarette smoke-induced airway inflammation, mucus hypersecretion and airway remodeling in mice.

BACKGROUND: Cigarette smoke plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Recently, elevated serotonin (5-HT) levels were found in the plasma of COPD patients. The role of 5-HT and its receptors in airway inflammation and remodeling induced by cigarette smoke is unclear. METHODS: BALB/c mice received the 5-HTR2A inhibitor ketanserin, the 5-HTR2B inhibitor RS-127445 or the natural 5-HTR2A/2B inhibitor quercetin intraperitoneally, then were exposed to cigarette smoke for 6 or 12 weeks. Control mice received placebo and were exposed to room air or cigarette smoke. Mice were sacrificed and bronchial alveolar lavage fluid (BALF) and lung tissue samples were collected. RESULTS: Immunohistochemistry and western blot confirmed an increase in both 5-HTR2A and 5-HTR2B expression in mouse lungs after exposure to cigarette smoke for 6 and 12 weeks. Cigarette smoke induced accumulation of macrophages and neutrophils and increased levels of inflammatory cytokines, including IL-1ß and TNF-ɑ, in BALF and lung tissue; these effects were inhibited by ketanserin, RS-127445 and quercetin. Pretreatment with 5-HT receptor antagonists suppressed the goblet cell hyperplasia induced by 6- or 12-week exposure to cigarette smoke, based on Alcian blue-periodic acid Schiff staining. After 12 weeks of cigarette smoke exposure, Masson's staining showed fibrosis surrounding the mouse airways, and inhibitor pretreatment significantly attenuated the thickening and collagen deposition around the small airways. CONCLUSIONS: Our results suggest that cigarette smoke-induced airway inflammation and small airway remodeling are partially mediated by 5-HTR2A and 5-HTR2B, which could be a new therapeutic target for airway remodeling in COPD.

Analysis of the proteasome activity and the turnover of the serotonin receptor 2B (HTR2B) in human uveal melanoma.

Uveal melanoma (UM), although a very rare disease, remains a particularly aggressive type of cancer as near 50% of the UM presenting patients will also develop liver metastases within 15 years from the initial diagnostic. One of the most reliable predictive markers of UM at risk of evolving toward the formation of liver lesions is an abnormally elevated level of expression of the transcript encoding the 5-Hydroxytryptamine (serotonin) receptor 2B (HTR2B). In our previous study, we demonstrated that transcription of the HTR2B gene was under the regulatory influences of two transcription factors (TFs), NFI and RUNX1. However, the action of these TFs was insufficient to explain the elevated level of the HTR2B protein in metastatic UM cells or the discrepancies we observed between its expression at the transcriptional and protein levels, therefore suggesting that additional post-translational modifications may also contribute to the altered expression of HTR2B in UM cells. In the present study, we investigated whether the turnover of HTR2B by the proteasome could account at least in part for its deregulated expression. Microarray analyses performed with UM cell lines derived from both non-metastatic and metastatic UM primary tumors revealed important alterations in the expression of some of the transcripts encoding both the E3 ubiquitin ligases and the various subunits of the proteasome, and these modifications were further exacerbated by cell passaging in culture. These alterations also correlated with significant changes in the enzymatic activity of the proteasome. However, the highest proteasome activity and amount of ubiquitinated HTR2B observed in the metastatic T142 cell line, as revealed by immunoprecipitation of ubiquitinated proteins and Western blotting using the HTR2B antibody, apparently had little impact on the total content of HTR2B protein. This contrasts with the near total disappearance of this receptor in the non-metastatic T108 cell line. Our study therefore suggests that the inability of the proteasome to degrade HTR2B in metastatic UM cells might rely on an increased stability of the ubiquitinated receptor in these cells.

Enhancement by Hydrogen Peroxide of Calcium Signals in Endothelial Cells Induced by 5-HT1B and 5-HT2B Receptor Agonists.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 µM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.

Effect of Selective Serotonin (5-HT)2B Receptor Agonist BW723C86 on Epidermal Growth Factor/Transforming Growth Factor-α Receptor Tyrosine Kinase and Ribosomal p70 S6 Kinase Activities in Primary Cultures of Adult Rat Hepatocytes.

Serotonin (5-hydroxytryptamine; 5-HT) can induce hepatocyte DNA synthesis and proliferation by autocrine secretion of transforming growth factor (TGF)-α through 5-HT2B receptor/phospholipase C (PLC)/Ca2+ and a signaling pathway involving epidermal growth factor (EGF)/TGF-α receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase 2 (ERK2)/mammalian target of rapamycin (mTOR). In the present study, we investigated whether 5-HT or a selective 5-HT2B receptor agonist BW723C86, would stimulate phosphorylation of TGF-α RTK and ribosomal p70 S6 kinase (p70S6K) in primary cultures of adult rat hepatocytes. Western blotting analysis was used to detect 5-HT- or BW723C86 (10-6 M)-induced phosphorylation of EGF/TGF-α RTK and p70S6K. Our results showed that 5-HT- or BW723C86 (10-6 M)-induced phosphorylation of EGF/TGF-α RTK peaked at between 5 and 10 min. On the other hand, 5-HT- or BW723C86 (10-6 M)-induced phosphorylation of p70S6K peaked at about 30 min. Furthermore, a selective 5-HT2B receptor antagonist LY272015, a specific PLC inhibitor U-73122, a membrane-permeable Ca2+ chelator BAPTA/AM, an L-type Ca2+ channel blocker verapamil, somatostatin, and a specific p70S6K inhibitor LY2584702 completely abolished the phosphorylation of p70S6K induced by both 5-HT and BW723C86. These results indicate that phosphorylation of p70S6K is dependent on the 5-HT2B-receptor-mediated autocrine secretion of TGF-α. In addition, these results demonstrate that the hepatocyte proliferating action of 5-HT and BW723C86 are mediated by phosphorylation of p70S6K, a downstream element of the EGF/TGF-α RTK signaling pathway.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

RATIONALE: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)-derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. OBJECTIVE: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT2B (serotonin 2B receptor)-mediated signaling. METHODS AND RESULTS: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. CONCLUSIONS: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT2B signaling-dependent manner.

Transcription of the Human 5-Hydroxytryptamine Receptor 2B (HTR2B) Gene Is under the Regulatory Influence of the Transcription Factors NFI and RUNX1 in Human Uveal Melanoma.

Because it accounts for 70% of all eye cancers, uveal melanoma (UM) is therefore the most common primary ocular malignancy. In this study, we investigated the molecular mechanisms leading to the aberrant expression of the gene encoding the serotonin receptor 2B (HTR2B), one of the most discriminating among the candidates from the class II gene signature, in metastatic and non-metastatic UM cell lines. Transfection analyses revealed that the upstream regulatory region of the HTR2B gene contains a combination of alternative positive and negative regulatory elements functional in HTR2B- but not in HTR23B⁺ UM cells. We demonstrated that both the transcription factors nuclear factor I (NFI) and Runt-related transcription factor I (RUNX1) interact with regulatory elements from the HTR2B gene to either activate (NFI) or repress (RUNX1) HTR2B expression in UM cells. The results of this study will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma.

Translational studies support a role for serotonin 2B receptor (HTR2B) gene in aggression-related cannabis response.

Cannabis use is increasing in the United States, as are its adverse effects. We investigated the genetics of an adverse consequence of cannabis use: cannabis-related aggression (CRA) using a genome-wide association study (GWAS) design. Our GWAS sample included 3269 African Americans (AAs) and 2546 European Americans (EAs). An additional 89 AA subjects from the Grady Trauma Project (GTP) were also examined using a proxy-phenotype replication approach. We identified genome-wide significant risk loci contributing to CRA in AAs at the serotonin receptor 2B receptor gene (HTR2B), and the lead SNP, HTR2B*rs17440378, showed nominal association to aggression in the GTP cohort of cannabis-exposed subjects. A priori evidence linked HTR2B to impulsivity/aggression but not to cannabis response. Human functional data regarding the HTR2B variant further supported our finding. Treating an Htr2b-/- knockout mouse with THC resulted in increased aggressive behavior, whereas wild-type mice following THC administration showed decreased aggression in the resident-intruder paradigm, demonstrating that HTR2B variation moderates the effects of cannabis on aggression. These concordant findings in mice and humans implicate HTR2B as a major locus associated with cannabis-induced aggression.

Interaction of hepatic stellate cells with neutrophils and macrophages in the liver following oncogenic kras activation in transgenic zebrafish.

Activation of hepatic stellate cells (HSC) plays a crucial role in the liver disease progression from liver fibrosis/cirrhosis to cancer. Here, we found a rapid change of microenvironment after kras V12 -induction in zebrafish liver with progressively increased stromal cell number and enlarged liver size. Neutrophils and macrophages exhibited a faster response than HSCs. By manipulating the numbers of neutrophils and macrophages through morpholino knockdown, we found that macrophages contributed to both HSC survival and activation while neutrophils appear to be only required for HSC activation. Serotonin, which is essential for HSC survival and activation, was found up-regulated in hepatocytes and macrophages, but not in neutrophils after kras V12 induction. Serotonin receptor was highly expressed in HSCs; increase of the receptor activity by an agonist stimulated HSCs and oncogenic growth of the liver while an opposite effect was observed with an antagonist. Activated HSCs promoted the pro-tumorigenesis functions of neutrophils and macrophages through secretion of Tgfb1. Overall, these observations elucidated a cellular interaction in microenvironment where that upregulated serotonin in hepatocytes and macrophages activated HSCs. Since the microenvironment crosstalk plays a vital role in manipulation of liver carcinogenesis, the underlying mechanism may provide potential therapeutic targets for liver diseases.

Expression of the serotonin receptor 2B in uveal melanoma and effects of an antagonist on cell lines.

Uveal melanoma (UM) is the most common primary tumor in the adult, and disseminates to the liver in half of patients. A 15-gene expression profile prognostic assay allows to determine the likelihood of metastasis in patients using their ocular tumor DNA, but a cure still remains to be discovered. The serotonin receptor 2B represents the discriminant gene of this molecular signature with the greatest impact on the prognosis of UM. However, its contribution to the metastatic potential of UM remains unexplored. The purpose of this study was to investigate the effects of a selective serotonin receptor 2B antagonist on cellular and molecular behaviours of UM cells. UM cell lines expressing high level of serotonin receptor 2B proteins were selected by Western blotting. The selective serotonin receptor 2B antagonist PRX-08066 was evaluated for its impact on UM cells using viability assays, phosphorylated histone H3 immunostainings, clonogenic assays, migration assays, invasion assays and membrane-based protein kinase phosphorylation antibody arrays. The pharmacological inhibition of the serotonin receptor 2B reduced the viability of UM cells and the population in mitosis, and impaired their clonogenicity and potential of migration. It also decreased the phosphorylation of kinases from signaling pathways classically activated by the serotonin receptor 2B, as well as kinases ß-catenin, Proline-rich tyrosine kinase 2, and Signal transducer and activator of transcription 5. Our findings support a role for the serotonin receptor 2B in the proliferation and migration of UM cells, through activation of many signaling pathways such as WNT, Focal adhesion kinase and Janus kinase/STAT.

Cross-talk inhibition between 5-HT2B and 5-HT7 receptors in phrenic motor facilitation via NADPH oxidase and PKA.

Intermittent spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of spinal respiratory motor plasticity. Episodic activation of either serotonin type 2 (5-HT2) or type 7 (5-HT7) receptors elicits pMF, although they do so via distinct cellular mechanisms known as the Q (5-HT2) and S (5-HT7) pathways to pMF. When coactivated, these pathways interact via mutual cross-talk inhibition. Although we have a rudimentary understanding of mechanisms mediating cross-talk interactions between spinal 5-HT2 subtype A (5-HT2A) and 5-HT7 receptor activation, we do not know if similar interactions exist between 5-HT2 subtype B (5-HT2B) and 5-HT7 receptors. We confirmed that either spinal 5-HT2B or 5-HT7 receptor activation alone elicits pMF and tested the hypotheses that 1) concurrent activation of both receptors suppresses pMF due to cross-talk inhibition; 2) 5-HT7 receptor inhibition of 5-HT2B receptor-induced pMF requires protein kinase A (PKA) activity; and 3) 5-HT2B receptor inhibition of 5-HT7 receptor-induced pMF requires NADPH oxidase (NOX) activity. Selective 5-HT2B and 5-HT7 receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) to anesthetized, paralyzed, and ventilated rats. Whereas integrated phrenic nerve burst amplitude increased after selective spinal 5-HT2B or 5-HT7 receptor activation alone (i.e., pMF), pMF was no longer observed with concurrent 5-HT2B and 5-HT7 receptor agonist administration. With concurrent receptor activation, pMF was rescued by inhibiting either NOX or PKA activity, demonstrating their roles in cross-talk inhibition between these pathways to pMF. This report demonstrates cross-talk inhibition between 5-HT2B- and 5-HT7 receptor-induced pMF and that NOX and PKA activity are necessary for that cross-talk inhibition.

Serotonin receptor 2B signaling with interstitial cell activation and leaflet remodeling in degenerative mitral regurgitation.

AIMS: Mitral valve interstitial cells (MVIC) play an important role in the pathogenesis of degenerative mitral regurgitation (MR) due to mitral valve prolapse (MVP). Numerous clinical studies have observed serotonin (5HT) dysregulation in cardiac valvulopathies; however, the impact of 5HT-mediated signaling on MVIC activation and leaflet remodeling in MVP have been investigated to a limited extent. Here we test the hypothesis that 5HT receptors (5HTRs) signaling contributes to MVP pathophysiology. METHODS AND RESULTS: Diseased human MV leaflets were obtained during cardiac surgery for MVP; normal MV leaflets were obtained from heart transplants. MV RNA was used for microarray analysis of MVP patients versus control, highlighting genes that indicate the involvement of 5HTR pathways and extracellular matrix remodeling in MVP. Human MV leaflets were also studied in vitro and ex vivo with biomechanical testing to assess remodeling in the presence of a 5HTR2B antagonist (LY272015). MVP leaflets from Cavalier King Charles Spaniels were used as a naturally acquired in vivo model of MVP. These canine MVP leaflets (N=5/group) showed 5HTR2B upregulation. This study also utilized CB57.1ML/6 mice in order to determine the effect of Angiotensin II infusion on MV remodeling. Histological analysis showed that MV thickening due to chronic Angiotensin II remodeling is mitigated by a 5HTR2B antagonist (LY272015) but not by 5HTR2A inhibitors. CONCLUSION: In humans, MVP is associated with an upregulation in 5HTR2B expression and increased 5HT receptor signaling in the leaflets. Antagonism of 5HTR2B mitigates MVIC activation in vitro and MV remodeling in vivo. These observations support the view that 5HTR signaling is involved not only in previously reported 5HT-related valvulopathies, but it is also involved in the pathological remodeling of MVP.

Cell proliferation and migration mechanism of caffeoylserotonin and serotonin via serotonin 2B receptor in human keratinocyte HaCaT cells.

Caffeoylserotonin (CaS), one derivative of serotonin (5-HT), is a secondary metabolite produced in pepper fruits with strong antioxidant activities. In this study, we investigated the effect of CaS on proliferation and migration of human keratinocyte HaCaT cells compared to that of 5-HT. CaS enhanced keratinocyte proliferation even under serum deficient condition. This effect of CaS was mediated by serotonin 2B receptor (5-HT2BR) related to the cell proliferation effect of 5-HT. We also confirmed that both CaS and 5-HT induced G1 progression via 5-HT2BR/ERK pathway in HaCaT cells. However, Akt pathway was additionally involved in upregulated expression levels of cyclin D1 and cyclin E induced by CaS by activating 5-HT2BR. Moreover, CaS and 5-HT induced cell migration in HaCaT cells via 5-HT2BR. However, 5-HT regulated cell migration only through ERK/AP-1/MMP9 pathway while additional Akt/NF-κB/MMP9 pathway was involved in the cell migration effect of CaS. These results suggest that CaS can enhance keratinocyte proliferation and migration. It might have potential as a reagent beneficial for wound closing and cell regeneration. [BMB Reports 2018; 51(4): 188-193].