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Serotonin (5-HT) is a monoamine neurotransmitter and plays important roles in several of the human body’s systems. AcknowledgmentsThis work was financially supported by a new researcher grant from the Thailand Research Fund (TRF)-the Commission on Higher Education (CHE), a grant for development of new faculty staffs, Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University, a graduate thesis grant from Graduate School, Chulalongkorn University and the Chulalongkorn University Centenary Academic Development Project. We will be provided with an authorization token (please note: passwords are not shared with us) and will sync your accounts for you.
Pea3 subfamily of E–twenty six transcription factors consist of three major -exhibit branching morphogenesis, the function of Pea3 family in nervous system development and regeneration is only beginning to unfold.
E–twenty six domain proteins have been shown to be targets for signaling pathways, particularly the MAPK pathway, during development (rev. In this study, we wished to address if Pea3 was capable of differentiation of neuronal model cells, such as PC12, SH-SY5Y, or NSC-34, and even in adult DRG cells, and if so which target genes it may be regulating. Cloning of wild type NFL and NFM promoters were cloned and mutations of the putative Pea3 binding regions on these promoters were introduced by using primers in Table 1. Serine residues on mouse Pea3 protein that are potential targets for Proline-directed phosphorylation were mutated by two-step PCR-based site directed mutagenesis method. PC12 pheochromacytoma cells were maintained in DMEM supplemented with 10% Horse serum and 5% Fetal Bovine serum in the presence of antibiotics, penicillin and streptomycin, and L-glutamine. Mouse Motor Neuron (NSC-34) cell line and SH-SY5Y neuroblastoma cell line were grown in the high glucose DMEM (Gibco, 1129855) supplemented with 10% Fetal Bovine serum in the presence of penicillin, streptomycin, L-Glutamine and amphotericin B (Biological Industries, 03-033-1B) and primocin (Invivogen, ant-pm-1). In order to study the effects of Pea3 directly in differentiation, a chimeric protein with full length mouse Pea3 and the potent acidic activation domain of VP16 transactivator of the Herpes Simplex Virus (HSV) was constructed. Although a constitutively active Pea3-VP16 fusion protein is useful in studying the effects of an activated Pea3, it does not represent a physiologically relevant situation. In order to further analyze whether Pea3 would have the same effect on axonal outgrowth in adult neurons, we have studied adult DRG cultures transfected with mPea3 expression vectors, in combination with EGF, bFGF, and IGF-1. NSC-34 is a commercially available motor neuron cell line (produced through the fusion of embryonic mouse spinal cord cells with neuroblastoma cells); they are capable of differentiation as readily monitored by extensive axonal outgrowth, and are readily transfected, making them valuable developmental models. Since in experimental model systems Pea3 overexpression lead to morphological changes similar to differentiation, the next question was identifying which target genes would be responsible for this transformation. Upon initial bioinformatic analysis, two neuron-specific promoters were particularly striking with respect to their putative binding motifs: neurofilament-L and neurofilament-M.
Known as a primary target for psychoactive drug development, the 5-HT transporter (5-HTT, SERT) plays a critical role in the regulation of serotonergic function by reuptaking 5-HT.
IntroductionSerotonin (5-Hydroxytryptamine, 5-HT) is most frequently thought of as a monoamine neurotransmitter in the central nervous system (CNS).
AP was party supported by a graduate scholarship and a teaching assistant fellowship from the Faculty of Allied Health Sciences, Chulalongkorn University. This means that you will not need to remember your user name and password in the future and you will be able to login with the account you choose to sync, with the click of a button.
This page doesn't support Internet Explorer 6, 7 and 8.Please upgrade your browser or activate Google Chrome Frame to improve your experience. In this study, we provide evidence that Pea3 can directs neurite extension and axonal outgrowth in different model systems, and that Serine 90 is important for this function. We demonstrate that Pea3 can indeed induce neurite extension in these various systems, indicating that it may prove a useful target towards neuroregeneration, and we present evidence that MAPK signaling may be important for this process, as a transcriptionally inactive Serine90-to-Alanine mutant of Pea3 is incapable of differentiating NSC-34 cells in vitro. Essentially, forward and reverse primers were designed to replace the codon for serine with codon for either alanine or glutamic acid (Table 2).
Bacterial transformation was done for the amplification of resultant plasmid constructs and for further validation analysis. Differentiation experiments were performed in collagen-coated plates, unless otherwise stated (Collagen Type IV, Sigma C0543).
Differentiation experiments were performed in collagen-coated plates (Collagen I, Gibco A10483-01). They were then transferred to RPMI medium containing 10% horse serum and 5% fetal bovine serum and gently triturated by 5-10 passages through a fire-polished Pasteur pipette. For differentiation experiments, cells were commonly co-transfected with GFP plasmid and live cells were routinely scored at 48 and 96 h after transfection under inverted microscope (Nikon ECLIPSE TE200). 24 h prior to transfection, cells were seeded into 24-well plates with the density of 50 thousand cells per well. The fusion protein was confirmed to activate from an artificial luciferase reporter (data not shown). Either a constitutively active Pea3-VP16 fusion or Pea3 induced with EGF can induce neurite formation in PC12 cells. Therefore, PC12 cells were next transfected with mouse Pea3, either in the presence or absence of Epidermal Growth Factor (EGF). Adult DRG neurons co-transfected with Pea3 expression vector and DsRed and stimulated with bFGF, EGF, or IGF-1.
The known transcriptional targets of Pea3 mostly include matrix metalloproteases, such as MMP-2 and MMP-9, identified in the context of breast or prostate cancer models. The promoter sequences for these genes were obtained through the Transcriptional Regulatory Element Database (TRED) Cold Spring Harbor Laboratory3, and thereafter analyzed by ALGGEN PROMO4, a virtual laboratory for the study of transcription factor binding sites in DNA sequences, to identify potential Pea3 binding motifs.
Neurofilament-L and –M promoters contain several ets motifs with significant similarity to Pea3 consensus binding site.
The allelic variation of 5-HTT expression is caused by functional gene promoter polymorphism with two principal variant alleles, 5-HTT gene-linked polymorphic region (5-HTTLPR).
In fact, it plays numerous critical roles in several human body’s systems, especially gastrointestinal (GI) system [1,2]. The authors gratefully acknowledge the generous gifts of the SW480 cell line from Apiwat Mutirangura, the HT-29 cell line from Weerah Wongkham, and plasmid vectors from Robert K.
We have also identified neurofilament-L and neurofilament-M as two putative novel targets for Pea3. ER81 loss-of-function mutants have provided supporting evidence for its role in the establishment of sensory-motor circuits at the limb level (Arber et al., 2000).
ERM and Pea3 were implicated in nasal development in response to FGF signaling (Firnberg and Neubuser, 2002).
We further identify two presumptive novel targets of Pea3 to be neurofilament-L and neurofilament-M.
The validation of the cloning was done by the colony PCR where positive bacterial colonies were directly used as a PCR template, then restriction enzyme digestion was done with Hind III-XhoI for further confirmation of the presence of Pea3 coding sequence.
Transient transfections were carried out with Effectene reagent (Qiagen 301425), following manufacturer’s instructions.
When exogenous Pea3-VP16 fusion was expressed in PC12 cells, it induced neurite formation within 2 days, regardless of collagen-coating (Figure 1A). In contrast to Nerve Growth Factor (NGF), which results in sustained MAPK activation and neuronal differentiation, EGF is known to lead to proliferation in these cells through transient activation of the MAPK pathway (Marshall, 1995; Aksan and Kurnaz, 2003).


Therefore, in an attempt to identify more neurologically relevant targets of Pea3, we have initially started investigating genes that are correlated with neuronal differentiation and whose promoters contained putative Pea3-binding motifs. It has been demonstrated that 5-HTTLPR is associated with numerous neuropsychiatric disorders. Strong erm expression was seen in the caudal region, for example, with restricted expression in the cephalic region, prospective midbrain and ventral forebrain (Firnberg and Neubuser, 2002).
In spite of all these pieces of data on the function of Pea3 subfamily at various levels of nervous system development, and its interaction with various upstream signals such as FGF, NGF, or GDNF, detailed molecular mechanisms of upstream signaling and downstream transcriptional targets are still far from understood. Plasmid clones from positive bacterial colonies were then purified for sequence analysis for final confirmation of presence of Pea3 sequence with desired mutation (data not shown). After 24 h, the cells were treated with 10 μM retinoic acid (RA, Sigma R2625) to induce neuronal differentiation. Differentiation was scored as the percentage of neurite-bearing cells (at least two cell body length was taken as the cut-off point) among the GFP-expressing cell population. Essentially, plasmids were transfected per well in the following amounts: 100 ng Renilla Luciferase (Promega), 200 ng reporter construct (NFM-Luc or NFL-Luc) and wild type Pea3 expression plasmid with varying concentrations as indicated in the text (usually 5, 25, 50, 100, 150, and 200 ng, completed up to 200 ng DNA in total with pCMV empty vector). Pea3 protein bound DNA was then eluted by reverse crosslink reaction in high salt environment enhanced with heat treatment.
The percentage of neurite-bearing cells was significantly increase, up to around 25%, when Pea3-VP16 was transfected to PC12 cells (Figure 1B).
Interestingly, Pea3-EN, a dominant negative form of Pea3 in fusion with the repression domain of Engrailed transcription factor, also showed some enhanced differentiation (around 15%), although this was lower than differentiation induced by either Pea3 or Pea3VP16. Human NF-M promoter, on the other hand, contained four ets motifs, only one of which shared significant homology to consensus Pea3-binding motif (1% dissimilarity; Figure 4A).
The functional roles of 5-HTTLPR have been reported in human choriocarcinoma (JAR), lymphoblast and raphe cells. The predominant site of 5-HT synthesis, storage, and release is the enterochromaffin (EC) cells of the intestinal mucosa, and virtually all of the 5-HT in the blood is derived from the GI tract [3].
Unfortunately, since members of the PEA3 family of transcription factors have overlapping expression patterns, the full picture of their function in the organism may not be revealed until double or triple knockouts are established.
Most of the studies on Pea3 are carried out with respect to breast tumorigenesis, and several targets such as matrix metalloproteases have been identified with respect to metastasis (de Launoit et al., 2006), however how exactly Pea3 is involved in neuronal differentiation has remained unclear.
Pea3 phospho-mutant plasmids were further analyzed for proper gene expression and translation (data not shown).
Cultures were maintained in RPMI medium with B27 supplement (Gibco Invitrogen) and antibiotic-antimycotic at 37°C in a humidified atmosphere with 5% CO2. At least three different fields per well, and at least two different wells per transfection were scored, and average and standard deviation calculated using MS Excel software. To date, the significance of 5-HTTLPR in gastrointestinal tract-derived cells has never been elucidated.
Within the GI system, 5-HT plays a critical role as a paracrine messenger and a neurotransmitter in the regulation of GI motility, secretion [3,4] and sensation [5,6] via secretion from EC cells. In adults, ER81 transcript was found to be present in a variety of tissues, including heart, brain, lung, liver, pancreas, spleen, testis, and intestines (Monte et al., 1995). Pea33-Luciferase reporter construct, where 5 × Pea3 optimal sites upstream of the TK minimal promoter drives the expression of the luciferase reporter gene.
The results were represented as the average of duplicate wells (three fields per well) in at least two independent experiments. While the motor neuron-derived cells normally appear round in morphology, in Pea3- or Pea3-VP16-transfected cells rather extensive axon-like neurite outgrowths of more than 2 cell body length are readily observed and scored as differentiation.
The ets motifs predicted on the Neurofilament-H promoter yielded much lower similarities (not shown), hence not included in this study.
Thus, the impact of 5-HTTLPR on 5-HTT transcription was studied in SW480 human colon carcinoma cells, which were shown to express 5-HTT. These particular cells function as sensory transducers in response to mechanical pressure and nutrients such as glucose and short-chain fatty acids, and secrete 5-HT from their basolateral surfaces into the wall of the bowel [2]. The DRGs used for cell culture were obtained from young adult rats immediately after exsanguinations.
Luciferase activity was measured with Luminoskan Ascent (ThermoLab Systems) for 10 s for each substrate. Percent Input was calculated so as to normalize qPCR data, and essentially signal intensities obtained from the ChIP samples were divided by signal intensities obtained from the Input sample. We have therefore proceeded with our analyses, focusing on the only 2 ets motifs in the Neurofilament-L promoter, and the highest scoring ets motif in the Neurofilament-M promoter, in order to estimate whether these promoters indeed would respond to Pea3-induced transcriptional regulation. Then, the released 5-HT acts as a crucial signaling molecule, which initiates responses as diverse as nausea, vomiting, peristaltic and secretory reflexes [7] through a variety of 5-HT receptors located on the mucosal processes of both intrinsic and extrinsic primary afferent neurons that project to the lamina propria. Furthermore, regulation of Pea3 through FGF18 signaling was shown to be important for the laminar positioning of developing neocortex (Hasegawa et al., 2004). Nevertheless, the transcriptional effect of estrogen and glucocorticoid on 5-HTT expression via 5-HTTLPR was not found in this cell line. However, mucosal 5-HT molecules have to be very rapidly metabolized, mainly as a result of the activity of monoamine oxidase by an inactivating mechanism, a transporter-mediated uptake, in the gut because circulating free 5-HT is toxic and passive diffusion is too slow to prevent the accumulation of 5-HT in contact with its receptors [8].
However, ER81 was not found to be regulated by FGF in Xenopus, indicating a divergence in signal response of Pea3 family members (Roussigne and Blader, 2006). Our study was the first to demonstrate the molecular role of this allelic variation in gastrointestinal tract cells. More recently, Pea3 was shown to be upregulated in DRGs upon NGF signaling during target innervation (Fontanet et al., 2013).
The products were resolved in 2.5 % Nu-Sieve agarose gels and were analyzed using QuantiOne imaging software (BioRad).
A polymorphism has been identified in the region for transcriptional control of SLC6A4, termed the 5-HTT gene-linked polymorphic region (5-HTTLPR), which consists of different lengths of the repetitive GC-rich sequence containing 20–23 bp located approximately 1.4 kb upstream of the 5-HTT gene transcription site [9]. The experiment was repeated with two different DNA preparations for pCDNA3 Pea3, and at least 3 fields per well were counted. The control plasmid pCDNA3 yielded no differentiation (not shown), while NGF in a similar time scale (2–4 days after transfection) resulted in around 12% differentiation (not shown). As far as the significance of this particular allelic variation is concerned, 5-HTTLPR has been demonstrated to affect the expression and function of 5-HTT. Studies using reporter gene assays revealed that the S variant reduced transcriptional efficiencies of the SLC6A4 in a human placental choriocarcinoma cell line (JAR) [11], lymphoblast cell lines [12] and in the raphe nucleus-derived cell line (RN46A) [13].
The L variant was also correlated with higher rates (Vmax) of 5-HT uptake in platelets [14], suggesting that the S variant may act as a dominant allele. In addition, the alteration of the serotonergic activity has been shown to be involved in several diseases. Although the results of these studies have been somewhat inconsistent, this polymorphism has been reported to associate with not only psychiatric disorders such as depression [15–18], schizophrenia [19], anxiety [20], suicide [21,22] and autism [23], but also certain GI syndromes such as irritable bowel syndrome (IBS) [24,25].


Thus, 5-HTT has also shown itself to be an effective pharmacological target in the treatment of IBS [26,27].While the previous studies provided evidence for allele-dependent differential 5-HTT promoter activity in cells from blood, placenta and brain, information regarding the significance of 5-HTTLPR in cells originated from the GI tracts has never been elucidated.
Therefore, the impact of 5-HTTLPR on 5-HTT transcription was studied in human epithelial-like colon carcinoma cells using functional reporter gene assays in this study. We also intended to investigate the effects of steroid hormones including glucocorticoid and estrogen on 5-HTT expression because the binding of these hormones to their receptors can trigger conformational changes enabling the receptor dimer to interact with DNA-associated regulatory elements. The physiological response is induced by activation or deactivation of an adjoining gene promoter which influences transcriptional efficiencies.
Recently, it has been reported that 5-HTT expression is modulated by glucocorticoid administration in the placental (JAR) cell line with allele-dependent effects [28]. For example, significant decrease in 5-HTT mRNA was observed upon estrogen treatment in rhesus macaques [29,30] and rat brain cells [31], but no study regarding the involvement of 5-HTTLPR has been performed.
Thus, we hypothesized that these hormonal effects with respect to 5-HTTLPR may be similar in GI tract-derived cells as compared to other cell types in the previous studies. In this article, in addition to studying the allele-dependent role of 5-HTTLPR in GI cells, we investigated whether 5-HTT expression is modulated by estrogen or glucocorticoid administration via 5-HTTLPR in cellular model system.
Therefore, prior to reporter gene assays, we examined whether mRNA transcripts for 5-HTT, glucocorticoid receptor (GR) and estrogen receptor (ER) are expressed in human colon carcinoma cell lines, SW480 and HT-29, using the reverse transcription-polymerase chain reaction (RT-PCR) method for evaluating an appropriate cell model.
Serotonin receptors and transporters—roles in normal and abnormal gastrointestinal motility. Expression of 5-HTT, GR? and ER? in Human Colon Carcinoma CellsWe evaluated two human colon carcinoma cell lines, SW480 and HT-29, using RT-PCR approach in order to select the most suitable in vitro model. All mRNA transcripts examined were found to express in the two cell lines, but GR? transcripts were not detected in HT-29 (Figure 3).
Therefore, SW480 was a cellular model chosen in this study as both estrogen and glucocorticoid receptors were expressed, indicating that this cell line could be employed for testing the effects of respective steroid hormones via 5-HTT. Antidepressant- and cocaine-sensitive human serotonin transporter: Molecular cloning, expression, and chromosomal localization. Effect of Steroids on 5-HTT Promoter ActivityIn addition, we also investigated whether the steroid hormones, glucocorticoid and estrogen, regulate 5-HTT expression by employing the synthetic glucocorticoid dexamethasone, which binds specifically to the GR, and ?-estradiol, which binds specifically to the ER. SW480 cell line was a proper model because it was demonstrated to express not only 5-HTT transcripts, but also transcripts for steroid hormone receptors, GR? and ER?. No difference was found at 10?3, 10?6 and 10?9 M concentration when compared to 0 M dexamethasone or ?-estradiol for the same allele. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells. Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets.
Allelic variation of serotonin transporter expression is associated with depression in Parkinson’s disease. Subthreshold depression is linked to the functional polymorphism of the 5HT transporter gene.
Serotonin transporter and tryptophan hydroxylase gene polymorphisms in Chinese patients with generalized anxiety disorder.
PCR Analysis of the 5-HTT Gene-Linked Polymorphic RegionDNA was isolated from whole blood using FlexiGene DNA kit (Qiagen, Hilden, Germany), and subsequently screened by polymerase chain reaction (PCR) using Taq PCRx DNA polymerase (Invitrogen, Carlsbad, CA, USA) and primers specific to the 5-HTTLPR [11] as shown in Table 1. The serotonin transporter gene: Polymorphism and haplotype analysis in Russian suicide attempters. Transcription Factor Binding Site AnalysisThe sequencing data of S allele was compared to L allele by MegAlign program (DNASTAR, Madison, WI, USA). Transcription factor binding sites of the different sequences were analyzed by Promo program [32,33]. Cell CultureThe SW480 and HT-29 human colon carcinoma cell lines were kindly provided by Dr. Apiwat Mutirangura (Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand) and Dr.
A controlled crossover study of the selective serotonin reuptake inhibitor citalopram in irritable bowel syndrome. Weerah Wongkham (Faculty of Science, Chiang Mai University, Chiang Mai, Thailand), respectively. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and cells were maintained at 37 °C in a humidified atmosphere at 5% CO2. Transient Transfection and Luciferase Reporter Gene AssayTransient transfections were performed using Lipofectamine™ 2000 (Invitrogen) following a procedure described by the manufacturer.
Yu), which expresses Renilla luciferase under the control of the CMV promoter, to monitor the transfection efficiency. The cultured medium was changed in the following day, and cells were analyzed for the firefly and Renilla luciferase activities after an additional 24 h in culture. After an additional 24 h in culture, cells were analyzed for the firefly and Renilla luciferase acivities using Dual-Glo™ luciferase assay system (Promega, Madison, WI, USA) following the manufacturer’s instructions, and chemiluminescence was read using a VICTOR3 luminometer (PerkinElmer, Waltham, MA, USA). In each transfection experiment, untransfected cells were included as controls for a background level of firefly and Renilla luciferase.
ConclusionsIn this study, we discovered a differential regulation of 5-HTT expression by 5-HTTLPR in human cell line derived from the GI tract.
This finding is in agreement with studies conducted using other cell lines including placental cells, lymphocytes and neuronal cells. Nevertheless, investigation using cells with no 5-HTT expression should be conducted to witness if this particular differential regulation by 5-HTTLPR is cell-specific. In addition, measurement of serotonin content or 5-HTT level actually present in cells is recommended to experimentally confirm the serotonin reuptake mechanism. Unfortunately, we did not observe an effect of glucocorticoid and estrogen regulating the 5-HTT expression in conjunction with these allelic variants. However, it is interesting to investigate the molecular interactions between estrogen and serotonin because their interactions and related mechanisms have not been extensively elucidated to date. As far as disease susceptibility genes and pharmacogenomics are concerned in this post-genomic era, studies performed using individual ethnic samples are of critical importance.



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