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Keywords:

  • anxiety;
  • glucocorticoid hormone;
  • serotonin;
  • serotonin transporter;
  • stress;
  • promoter

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

Mood, emotion and cognition are modulated by serotonergic neurotransmission, while the physiological function of serotonergic synapses depends on serotonin reuptake, which is mediated by the serotonin transporter (5-HTT). Allelic variation of 5-HTT expression in humans is caused by a functional gene-promoter polymorphism with two predominant variant alleles, which are associated with variations in anxiety measures as previously reported. Here we report that administration of dexamethasone, a potent glucocorticosteroid hormone, results in an increase in 5-HTT expression in immortalized human B-lymphoblastoid cells, which express the human 5-HTT. Functional reporter gene assays as well as 5-HT uptake and inhibitor binding measures revealed a genotype-dependent dose–response to glucocorticosteroid administration, which was antagonized by RU 38486, a non-specific glucocorticosteroid hormone antagonist. The allele-specific differences after administration of dexamethasone depended on the repetitive GC-rich sequence located approximately 1.4 kb upstream of the 5-HTT gene transcription site because of absence of a significant steroid effect after transfecting a deletional mutant reporter gene construct, which lacks this repetitive promoter sequence. Our findings may contribute to explain the vulnerability to stress-related disorders in susceptible individuals, in whom further clinical studies should follow up on these in vitro findings.

Abbreviations used
5-HT

serotonin

5-HTT

serotonin transporter

5-HTTLPR

5-HTT gene-linked polymorphic region

5-HTT/P-HBluc+

HindIII/BamHI-restriction site fragment of the human 5-HTT gene promoter, ligated into the promoterless luciferase expression vector

5-HTT/P-EcoRIluc+

EcoRI deletional mutant reporter gene construct

CRH

corticotropin-releasing hormone

GR

glucocorticoid receptor

HPA axis

hypothalamic–pituiary–adrenal axis

SSRI

selective serotonin reuptake inhibitors

EBV

Epstein–Barr virus

L5-HTTP, S 5-HTTP

long and short variant of the 5-HTT gene promoter

MR

mineralcorticoid receptor

A large body of converging evidence indicates that adrenocortical steroids and serotonergic neurones exert reciprocal modulatory actions (Holmes et al. 1995a), although the site of interaction and functional significance of the effects of corticosteroids and serotonin (5-HT), particularly with regard to the pathophysiology of anxiety and depression, have been controversial (DeKloet et al. 1982; DeKloet et al. 1986). 5-HT is an important regulator of morphogenetic activities during early CNS development, including cell proliferation, migration and differentiation as well as synaptogenesis (Lauder 1990, 1993). Serotonergic raphe neurones diffusely project to a variety of brain regions (e.g. cortex, amygdala, hippocampus; Chen et al. 1992; Hensler et al. 1994) and play known roles in integrating emotion, cognition and motor function as well as in food intake, sleep, pain and sexual activity (Lesch 1997). This diversity of physiologic functions is due to the fact that 5-HT acts as a master control neurotransmitter within a highly complex system of neural communication mediated by multiple pre- and post-synaptic 5-HT receptors, thus orchestrating the activity and interaction of several other neurotransmitter systems (Blakely et al. 1994; Uhl and Johnson 1994).

5-HT acts directly on corticotropin-releasing hormone (CRH)-synthesizing neurones via 5-HT1A, 5-HT2A or 5-HT2C receptors. CRH-producing neurones are primarily located in the hypothalamic paraventricular nucleus (PVN) which receives serotonergic projections originating in the raphe nuclei (Holmes et al. 1995a, 1995b; Zhong and Ciaranello 1995). Distribution and arborization of the serotonergic axons is prominent in the parvocellular subnuclei of the PVN and ultrastructural analysis demonstrates that 5-HT containing terminals form axo-dendritic and somatic synapses with CRH-immunoreactive neurones (Liposits et al. 1987). Because selective lesions of ascending serotonergic fibres do not prevent the hypothalamic–pituitary–adrenal (HPA) response to 5-HT-releasing compounds or 5-HT agonists in rats, the 5-HT receptor subtypes mediating the 5-HT-evoked HPA axis activation are likely to be located post-synaptically on the CRH-synthesizing neurones (Van de Kar et al. 1985).

Corticosteroids are secreted by the adrenal gland in a circadian pattern and in response to stress and exert their effects in various tissues, including the brain, through two types of receptors, the high-affinity mineralocorticoid receptor (MR) and the lower-affinity glucocorticoid receptor (GR). Binding of corticosteroids to their receptors triggers 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 efficiency. By this mechanism, corticosteroids control expression of genes involved in the regulation of the 5-HT/HPA circuit in a tissue-specific fashion. While the hippocampus and hypothalamus are principal targets of corticosteroid-dependent feedback regulation in the brain, expression of corticosteroid receptors in 5-HT neurones located in the raphe complex further supports the bidirectionality of the 5-HT/HPA system. Differences in affinity and localization of these receptors result in a complex corticosteroid-induced modulation of 5-HT synthesis and metabolism as well as 5-HT receptor and 5-HTT function.

The 5-HTT provides the primary mechanism for inactivation of 5-HT after its release into the synaptic cleft (Amara and Arriza 1993). By conferring allele-specific transcriptional activity on the 5-HTT gene promoter in humans, the 5-HTT gene-linked polymorphic region (5-HTTLPR) influences a constellation of personality traits related to anxiety and depression (Lesch et al. 1996). Moreover, allelic variation in 5-HTT function may increase the risk for various neurodevelopmental, neurodegenerative and affective spectrum disorders (Collier et al. 1996).

In this study we investigated whether 5-HTT expression is modulated by glucocorticoid administration in a cellular model system. We focussed on glucocorticoid effects by employing the synthetic glucocorticoid dexamethasone, which binds specifically to the glucocorticoid receptor and lacks the mineralocorticoid activity of cortisol. Furthermore, we determined whether there are allele-dependent effects of glucocorticoid administration on 5-HTT expression.

Cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

JAR human placental choriocarcinoma cells (ATCC HTB 144) which constitutively express the human 5-HTT (Cool et al. 1991; Ramamoorthy et al. 1993a, 1993b) were grown in RPMI-1640 supplemented with 10% newborn calf serum at 37°C in a humidified atmosphere at 5% CO2. Before transfection, cells were washed twice with sterile phosphate-buffered saline (PBS), trypsinized and 2 × 105 cells were grown in 35-mm plates. For Epstein–Barr virus (EBV)-transformation of B-lymphoblastoid cells, 3-mL blood samples from donors with a PCR-verified homozygous genotype were separated by a Ficoll gradient, and lymphocytes were washed twice with sterile PBS. Cells were incubated 1 h in an EBV-containing medium at 37°C. T cell separation was carried out by supplementing the 15% newborn calf serum–RPMI-1640 medium with 1 µg/mL cyclosporine (Lesch et al. 1996). Three cell lines of each genotype were established and 5-HTT activity and density was determined by [3H]5-HT uptake and [125I]RTI-55 binding.

Antagonization of the glucocorticoid effect was carried out with RU 38486, a non-specific glucocorticosteroid hormone antagonist [17β-hydroxy-11β-(4-dimethylaminophenyl-17α(prop-1-ynyl)-estra-4,9-dien-3-one] (Roussel, Paris, France) in 10% methanol under equimolar conditions.

PCR analysis of the 5-HTT gene-linked polymorphic region

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

DNA was isolated from blood, and subsequently oligonucleotide primers flanking the 5-HTTLPR and corresponding to the nucleotide positions ranging from −1416 to −1397 (stpr5, 5′-GGCGTTGCCGCTCTGAATTGC) and from −910 to −888 (stpr3, 5′-GAGGGACTGAGCTGGACAACCAC) of the h5-HTT gene regulatory region were used to generate a 484/528 bp fragment. PCR amplification was carried out in a final volume of 30 µL consisting of 50 ng genomic DNA, 2.5 mm deoxyribonucleotides, 0.1 µg sense and antisense primers, 10 mm Tris–HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 5% dimethyl sulphoxide (DMSO) and 1 U Taq DNA polymerase. Annealing was carried out at 61°C for 30 s, extension at 72°C for 1 min and denaturation at 95°C for 30 s for 35 cycles.

Transfection and luciferase reporter gene assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

The long (bp −1428 to + 217 with respect to the transcription initiation site) and short promoter alleles were ligated into the promoterless luciferase expression vector pGL3 basic (Promega, Madison, WI, USA) as previously described (Heils et al. 1996). For transient expression, 5 µg of these allelic variant 5-HTT/P-HBluc+ constructs as well as of the 5-HTT/P-EcoRIluc+ deletional mutant were complexed with 5 µL of Transfectam lipofectin reagent (Promega) in 5-µL defined and serum-free RPMI-1640 medium. Luciferase gene expression was studied in comparison to the promoterless vector pGL3 basic. Transfection efficiency was assessed by cotransfection with the pSV-βGal (Promega).

24 h after transfection dexamethasone (Sigma, St Louis, MO, USA) was added within a concentration range between 0 and 25 nm. After an additional 24 h, cells were harvested in 1 mL of luciferase lysis buffer (Promega). Extracts were assayed for luciferase activity by addition of 20 µL cell lysate samples in 15-s intervals to 100 µL luciferin reagent (Promega). Chemiluminescence was counted for 15 s at a constant time (90 s) following reagent mixing in a liquid scintillation spectrometer (Beckman, Palo Alto, CA, USA). Luciferase activity was expressed as cpm/mg protein.

[125I]RTI-55 binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

RTI-55 [3β-(4-iodophenyl)tropan-2β-carboxyl acid methyl ester tartrate] is a cocaine analogue that potently inhibits 5-HT uptake and binds to the serotonin transporter with high affinity. Lymphoblastoid cells were washed twice with PBS and desintegrated by ultrasound in a membrane preparation buffer containing 10 mm MOPS, 1 mm sodium EDTA, 0.1 mm benzethoniumchloride, 1 mm benzamidine, 0.1% aprotinin (Trasylol®) and 8% saccharose at 4°C. The homogenate was centrifuged at 3000 g and the resulting supernatant centifuged again at 60 000 g. Equilibrium binding of [125I]RTI-55 to the lymphoblastoid cell plasma membranes was assayed by incubating 50 mg of the protein preparation within a concentration range of the radioligand from 0.05 to 1 nm for 1 h at room temperature in triplicate. Non-specific binding was determined in the presence of 5 µm paroxetine. Kd-values and Bmax, expressed as fmol/mg protein, were obtained by Scatchard plot analysis.

[3H]5-HT uptake

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

5-HT uptake of 107 B-lymphoblastoid cells, washed twice with PBS, was determined by incubating the suspended cells with 0.1–1 µm[3H]serotonin in triplicate for 10 min at 37°C in the presence or absence of 0.1 mm imipramine in 0.9% sodium-chloride. [3H]5-HT uptake measurements were assumed to measure initial rates of uptake within 10 min. Incubation was stopped by 0.9% sodium chloride/1.5% formaldehyde with a cell harvester and Whatman GF/B filters. Bound radioactivity was measured in a Beckman liquid scintillation counter. Kinetic analysis revealedKm- and Vmax-values as pmol/107 cells × 10 min.

Effect of dexamethasone on 5-HTT promotor activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

The organization of the human serotonin transporter gene and its promoter region is shown in Fig. 1. The basal luciferase activity of the short variant promoter construct was about 20.5% of the long variant, as described previously (Heils et al. 1996). Dexamethasone stimulation resulted in a maximum 10-fold increase of the activity of the long promoter construct at 10 nm, whereas a 10.4-fold rise with the short variant was observed after administration of 25 nm dexamethasone (Fig. 2). Allele-dependent differences were statistically significant ( p < 0.001 by Student's t-test) at all concentrations apart from the 25-nm dexamethasone concentration. The dose–response in luciferase activity of the short construct was shifted towards higher dexamethasone concentrations compared with the long construct, but remained below the activity of the long construct. Equimolar RU 38486 administration led to an almost complete antagonization of the glucocorticosteroid. Transfection of the EcoRI deletional mutant, which lacks the tandem repeats of the promoter sequence and starts at base pair −734 with respect to the 5-HTT gene transcription initiation site (Fig. 2a), did not reveal any statistically significant dexamethasone effect in reporter gene activity (Fig. 2c).

image

Figure 1. Organization of the human serotonin transporter gene and its 5′-flanking regulatory promoter region. (a) Solid and hatched boxes represent coding and non-coding regions. (b) The serotonin transporter gene promoter is defined by a TATA-like motif and several potential transcription factor binding sites. EcoRI represents a deletional restriction site. (c) The polymorphic tandem repeat of the serotonin transporter gene promoter is defined by a length variation of a repetitive sequence comprising GC-rich, 20–23 bp-long repeat elements. The polymorphism is generated by a deletion with an overall length of 44 bp.

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image

Figure 2. Effect of dexamethasone on 5-HTT promotor activity. (a) Overview of the 5-HTT promoter–luciferase constructs. (b) Reporter gene activity expressed as luciferase activity in cpm/µg protein after transfection of the long and short variant promoter constructs into JAR cells. Stimulation with dexamethasone in a defined concentration range was done 24 h after transfection. After another 24 h, cells were harvested and luciferase activity was determined. All experiments were done five times. Results are expressed as mean ± SEM. Black columns represent reporter gene activity of the long variant allele, white columns represent reporter gene activity of the short allele. Hatched columns illustrate the results of RU 38486-antagonization of the long variant activity as well as of the short allele. *p < 0.05, **p < 0.01, ***p < 0.001 compared to 0 nm dexamethasone for the same allele. In comparisons between alleles, genotype-dependent differences were significant for a concentration range of 0–20 nm dexamethasone (p < 0.001 by Student's t-test) but not for 25 nm dexamethasone (not indicated by asterisks in this diagram). (c) Absence of a dexamethasone effect in the 5-HTTP-EB construct. Results are expressed as mean ± SD. Note the different scale of the luciferase activity axis, compared to (b).

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Effects of dexamethasone on the expression of the 5-HTT

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

As shown in Fig. 3, basal 5-HT uptake capacity of B-lymphoblastoid cell lines homozygous for the short variant of the promoter was about 66% of uptake capacity of cell lines homozygous for the long allele, as previously reported (Lesch et al. 1996), and thus consistent with the reporter gene results. The magnitude of difference between the alleles was less in the uptake experiments than in the reporter assays. Dexamethasone administration resulted in a dose-dependent increase in uptake capacity in both genotypes. Similar to the observation in reporter gene assays, the increase was shifted to higher dexamethasone concentrations in the short genotype (basal: 66%; 15 nm: 61%; 25 nm: 83%; comparing the short with the long genotype). Allele-dependent differences were significant for a concentration range of 0–20 nm dexamethasone (p < 0.05 by Student's t-test) but not for 25 nm dexamethasone.

image

Figure 3. Basal and dexamethasone induced [3H] 5-HT uptake. Uptake parameters were determined in three B-lymphoblastoid cell lines of each genotype in triplicate. Vmax is given in pmol/107 cells × 10 min and displayed as mean ± SEM. *p < 0.05, **p < 0.01 compared to 0 nm dexamethasone in the same genotype. In comparisons between genotypes, genotype-dependent differences were significant for a concentration range of 0–20 nm dexamethasone (p < 0.05 by Student's t-test) but not for 25 nm dexamethasone (not indicated by asterisks in this diagram).

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Km-values of between 149 and 176 nm for the long genotype and 156 and 184 nm for the short genotype did not reflect any dexamethasone-induced changes in transport activity. Differences in Km-values between the genotypes were not significant. Equimolar administration of RU 38486 resulted in an almost complete antagonization of the dexamethasone-induced increase in transport capacity in both groups (not shown).

Effects of dexamethasone on [125I]RTI-55 binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

[125I]RTI-55 binding represents 5-HTT protein density in human B-lymphoblastoid cells. Figure 4 shows that Bmax of cell lines homozygous for the short allele are about 70% of those homozygous for the long allele, similar to the results of functional 5-HT uptake measures. This difference between the long and short genotype is clearly evident in the absence of dexamethasone (0 nm dexamethasone), demonstrating that the 5-HTT promoter polymorphism influences 5-HTT expression at the protein level. The Kd-values were between 0.23 and 0.31 nm for the long and 0.21 and 0.27 nm for the short genotype.

image

Figure 4. Basal and dexamethasone induced [125I]RTI-55 binding to the 5-HTT. Binding to the 5-HTT was determined in three B-lymphoblastoid cell lines of each genotype in triplicate. Bmax is given in fmol/mg protein and displayed as mean ± SEM. Bmax- and Kd-values were determined by Scatchard plot analysis and all experiments were done in triplicate. Kd = 0.23–0.31 nm for the long genotype, Kd = 0.21–0.27 nm for the short genotype. #p = 0.07, **p < 0.01 compared to 0 nm dexamethasone in the same genotype. In comparisons between genotypes, genotype-dependent differences were significant for a concentration range of 0–20 nm dexamethasone (p < 0.05 by student's t-test) but not for 25 nm dexamethasone (not indicated by asterisks in this diagram).

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Generally, dexamethasone administration led to an increase of Bmax in both genotypes (Fig. 4). This dose-dependent effect of dexamethasone is particularly evident at 25 nm dexamethasone: 5-HTT protein density is increased by +26.8% for the L genotype, compared to 0 nm dexamethasone (p = 0.07). Similarly, 25 nm dexamethasone increases 5-HTT protein density by +48.3% in the S genotype, compared to 0 nm dexamethasone (p < 0.01). This dexamethasone effect of increased binding was abolished by RU 38486-antagonization (not shown). As in the uptake experiments, the effect of 25 nm dexamethasone was greater in the S genotype (+48.3%) than in the L genotype (+26.8%). As a consequence, 5-HTT protein density at 25 nm dexamethasone was only 17.8% less in the S genotype than in the L genotype. Thus, allele-dependent differences between the L and S genotype were significant for a concentration range of 0–20 nm dexamethasone (p < 0.05 by Student's t-test) but not for 25 nm dexamethasone.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References

Serotonin and 5-HT-receptors have been implicated in mechanisms of adaptation to chronic and unavoidable stress (McEwen 1993). Previous results of studies in animal models demonstrated the involvement of serotonergic neurotransmission in stress-related responses for example as higher levels of 5-hydroxyindoleacetic acid (5-HIAA) in subordinates than in dominant animals (McKittrick et al. 1995). These higher 5-HIAA levels in limbic brain areas suggest an increase in stress-related serotonin activity and turnover, implicating serotonin clearance mechanisms such as the serotonin transporter as directly involved in these synaptic processes.

Glucocorticosteroid hormones represent a class of endocrine stress-related mediators which follow a circadian rhythm of liberation. Because serotonergic neurotransmission is related to the normal HPA function as well as being involved in reactions to stressful life events and depression-related conditions, we investigated the acute effects of glucocorticosteroid hormone administration on 5-HTT gene activity, employing a reporter gene system and determining functional 5-HT-uptake- and inhibitor binding parameters.

Genotype-dependent results were obtained by transfection studies with the two allelic variant reporter gene constructs in the human choriocarcinoma cell line JAR as well as by investigating functional 5-HTT expression in human B-lymphoblastoid cells.

As we used a defined and hormone-free culture medium within these stimulation experiments and as all effects were completely abolished by RU 38486 administration, our findings describe glucocorticosteroid hormone-specific cellular effects.

Generally, dexamethasone administration resulted in an increase in 5-HTT gene activity, demonstrated as an increase in 5-HT uptake capacity and inhibitor binding in B-lymphoblastoid cells homozygous for the long or the short variant promoter allele, which was most evident at 20 and 25 nm dexamethasone. The dexamethasone concentrations employed in the present study are comparable to the cortisol concentrations in depression. In severely depressed patients, diurnal plasma cortisol levels of 252 ± 113 nm (mean ± SD) were found, while control subjects had cortisol levels of 160 ± 97 nm (Weber et al. 2000). Given the 30- to 40-fold higher glucocorticoid potency of dexamethasone compared to cortisol (Fauci et al. 1998), a dexamethasone concentration range of 10–15 nm corresponds to the elevated cortisol levels in depressed patients. This further underscores the pathophysiological importance of the effect of glucocorticoids in this concentration range on 5-HTT gene activity. Elevated glucocorticoid concentrations causing an acute increase in the number of 5-HTT may thus be involved in the onset of depression, by way of decreasing synaptic 5-HT concentrations as a consequence of increased 5-HT uptake.

Differentiating post-transcriptional and post-translational regulatory effects, the allele-dependent increase in 5-HTT gene activity was clearly seen in experiments of reporter gene activity comparing the luciferase expression of the short and long promoter constructs. We have previously described differences in basal activity of reporter gene activity and functional uptake parameters between the two variant alleles (Lesch et al. 1996). Administration of dexamethasone resulted in clear differences in luciferase activity between both alleles within a concentration range of dexamethasone approximately representing physiological glucocorticosteroid hormone levels during circadian rhythms of HPA function and stress-related situations.

These functional promoter assays could theoretically give spurious results because of not representing a closed loop of gene regulation. Effects in luciferase promoter assays generally tend to be large due to the lack of negative feedback, as the product of the reaction, the fluorescent protein luciferase, does not activate the negative feedback loops limiting its transcription in the way the protein product 5-HTT would do. It is therefore important to look also at physiological cellular systems where these feedback mechanisms operate. Hence we investigated 5-HTT protein expression in a cellular system. The results of functional 5-HTT parameters including uptake measurements and inhibitor binding supported the allele-specific and dexamethasone-dependent findings of the luciferase promoter analyses. These dexamethasone effects on 5-HT uptake and 5-HTT binding were less marked than the dexamethasone effect on the luciferase promoter assays, as expected.

Because the genotype of the two variant promoter alleles is associated with anxiety-related traits in humans, contributing approximately 4% to the total population variation of anxiety-related traits (Lesch et al. 1996), our findings suggest the described genetic promoter polymorphism might also contribute to individual differences in reaction to stressful and unavoidable life events as well as to depression-related states by variations of 5-HTT expression. Clinical association studies should follow-up on these results and further elucidate the role of glucocorticoid-dependent and allelic variation of 5-HTT regulation in acute stress-related serotonergic neurotransmission.

Our findings of glucocorticosteroid-dependent increase in 5-HTT expression seem to be contradictory to early findings of decreased platelet 5-HTT inhibitor binding in post-traumatic stress disorder (Arora et al. 1993; Fichtner et al. 1995). However, subsequent studies failed to replicate decreased 5-HTT inhibitor binding in post-traumatic stress disorder (Weizman et al. 1996; Maes et al. 1999), and 5-HT uptake measurements were also found to be unchanged in post-traumatic stress disorder compared to controls (Mellmann and Kumar 1994; Cicin-Sain et al. 2000). Moreover, cortisol levels are actually lower in post-traumatic stress disorder patients than in normal comparison groups (reviewed in Yehuda 2001). Thus, these findings represent a chronic state of reaction and not an acute effect of HPA activation as we imitated in our experiments. Within our experimental setting, chronic dexamethasone administration failed because of the loss of reporter gene activity 72 h after transient transfection and of 5-HTT expression in B-lymphoblastoid cells cultured in a serum-free defined medium longer than 48 h (data not shown).

In analogy, previous studies with laboratory stressors investigating hippocampal 5-HT1A receptor binding in the rat demonstrated an initial transient increase, followed by a chronic decrease of 5-HT1A receptor expression (Mendelson and McEwen 1991; Watanabe et al. 1993). In addition, regulation of 5-HTT expression also depends on 5-HT1A autoreceptor activity, which might facilitate a long-term decrease in transcriptional activity of the 5-HTT gene under these conditions.

Previous reports on 5-HTT expression under different hormonal conditions either revealed no effect (Kuroda et al. 1994) or remained controversial (Modai et al. 1992; Watanabe et al. 1993). Most of these data were collected in experiments with rodent animal models. Removing the tandemly repeated human promoter element (EcoRI deletional mutant) led to a loss of responsitivity in reporter gene activity to glucocorticosteroid hormone administration in our experiments. Because this polymorphic sequence is not represented in the rodent genome (Bengel et al. 1997) but only in higher primates (Lesch et al. 1997), we suggest that rodent animal models might not be as useful in exploring stress-related effects on 5-HTT expression as primates.

Furthermore, we conclude the tandemly repeated polymorphic promoter element, which is involved in allele-dependent population variation of anxiety measures, is also responsible for allelic variation of glucocorticosteroid hormone-induced regulation of 5-HTT expression in humans and perhaps in non-human primates.

Taken together, we demonstrated the acute glucocorticosteroid hormone-dependent increase in 5-HTT expression as an allele-specific promoter activation in humans, which might contribute to individual differences in stress-related responses to HPA activation and depressive states. These differences strictly depend on the polymorphic 5-HTT gene promoter repeat, which is only represented in humans and higher primates but not in rodents.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Cell culture
  5. PCR analysis of the 5-HTT gene-linked polymorphic region
  6. Transfection and luciferase reporter gene assay
  7. [125I]RTI-55 binding
  8. [3H]5-HT uptake
  9. Results
  10. Effect of dexamethasone on 5-HTT promotor activity
  11. Effects of dexamethasone on the expression of the 5-HTT
  12. Effects of dexamethasone on [125I]RTI-55 binding
  13. Discussion
  14. References
  • Amara S. G. and Arriza J. L. (1993) Neurotransmitter transporters: three distinct gene families. Curr. Opin. Neurobiol. 3, 337344.
  • Arora R. C., Fichtner C. G., O'Connor F. and Crayton J. W. (1993) Paroxetine binding in the blood platelets of post-traumatic stress disorder patients. Life Sci. 53, 919928.
  • Bengel D., Heils A., Petri S., Seemann M., Glatz K., Andrews A., Murphy D. L. and Lesch K. P. (1997) Gene structure and 5′-flanking regulatory region of the murine serotonin transporter. Brain Res. Mol. Brain Res. 44, 286292.
  • Blakely R. D., De Felice L. J. and Hartzell H. C. (1994) Molecular physiology of norepinephrine and serotonin transporters. J. Exp. Biol. 196, 263281.
  • Chen H. T., Clark M. and Goldman D. (1992) Quantitative autoradiography of 3H-paroxetine binding sites in rat brain. J. Pharmacol. Toxicol. Methods 27, 209216.
  • Cicin-Sain L., Mimica N., Hranilovic D., Balija M., Ljubin T., Makaric G., Folnegovic-Smalc V. and Jernej B. (2000) Post-traumatic stress disorder and platelet serotonin measures. J. Psychiatr. Res. 34, 155161.
  • Collier D. A., Stober G., Li T., Heils A., Catalano M., Di Bella D., Arranz M. J., Murray R. M., Vallada H. P., Bengel D., Muller C. R., Roberts G. W., Smeraldi E., Kirov G., Sham P. and Lesch K. P. (1996) A novel functional polymorphism within the promoter of the serotonin transporter gene: possible role in susceptibility to affective disorders. Mol. Psychiatry 1, 453460.
  • Cool D. R., Leibach F. H., Bhalla V. K., Mahesh V. B. and Ganapathy V. (1991) Expression and cyclic AMP-dependent regulation of a high-affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR). J. Biol. Chem. 266, 1575015757.
  • De Kloet E. R., Sybesna H. and Reul H. M. (1986) Selective control by corticosterone of serotonin I receptor capacity in raphe-hippocampal system. Neuroendocrinology 42, 513521.
  • De Kloet E. R., Kovacs G. L., Szabo G., Telegdy G., Bohus B. and Versteeg D. H. (1982) Decreased serotonin turnover in the dorsal hippocampus of rat brain shortly after adrenalectomy: selective normalization after corticosterone substitution. Brain Res. 239, 659663.
  • Fauci A. S., Braunwald E., Isselbacher K. J., Wilson J. D., Martin J. B., Kasper D. L., Hauser S. L. and Longo D. L. (1998) Harrison's Principles of Internal Medicine. McGraw-Hill, New York.
  • Fichtner C. G., O'Connor F. L., Yeoh H. C., Arora R. C. and Crayton J. W. (1995) Hypodensity of platelet serotonin uptake sites in post-traumatic stress disorder: associated clinical features. Life Sci. 57, PL3744.
  • Heils A., Teufel A., Petri S., Stober G., Riederer P., Bengel D. and Lesch K. P. (1996) Allelic variation of human serotonin transporter gene expression. J. Neurochem. 66, 26212624.
  • Hensler J. G., Ferry R. C., Labow D. M., Kovachich G. B. and Frazer A. (1994) Quantitative autoradiography of the serotonin transporter to assess the distribution of serotonergic projections from the dorsal raphe nucleus. Synapse 17, 115.
  • Holmes M. C., French K. L. and Seckl J. R. (1995a) Modulation of serotonin and corticosteroid receptor gene expression in the rat hippocampus with circadian rhythm and stress. Brain Res. Mol. Brain Res. 28, 186192.
  • Holmes M. C., Yau J. L., French K. L. and Seckl J. R. (1995b) The effect of adrenalectomy on 5-hydroxytryptamine and corticosteroid receptor subtype messenger RNA expression in rat hippocampus. Neuroscience 64, 327337.
  • Kuroda Y., Watanabe Y., Albeck D. S., Hastings N. B. and McEwen B. S. (1994) Effects of adrenalectomy and type I or type II glucocorticoid receptor activation on 5-HT1A and 5-HT2 receptor binding and 5-HT transporter mRNA expression in rat brain. Brain Res. 648, 157161.
  • Lauder J. M. (1990) Ontogeny of the serotonergic system in the rat: serotonin as a developmental signal. Ann. NY Acad. Sci. 600, 297313.
  • Lauder J. M. (1993) Neurotransmitters as growth regulatory signals: role of receptors and second messengers. Trends Neurosci. 16, 233240.
  • Lesch K. P. (1997) Molecular biology, pharmacology, and genetics of the serotonin transporter: psychobiological and clinical implications. In: Serotonergic Neurons and 5-HT Receptors in the CNS (BaumgartenH. G., GöthertM., eds), pp. 671705. Springer, New York.
  • Lesch K. P., Bengel D., Heils A., Sabol S. Z., Greenberg B. D., Petri S., Benjamin J., Muller C. R., Hamer D. H. and Murphy D. L. (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 15271531.
  • Lesch K. P., Meyer J., Glatz K., Flugge G., Hinney A., Hebebrand J., Klauck S. M., Poustka A., Poustka F., Bengel D., Mossner R., Riederer P. and Heils A. (1997) The 5-HT transporter gene-linked polymorphic region (5-HTTLPR) in evolutionary perspective: alternative biallelic variation in rhesus monkeys. J. Neural Transm. 104, 12591266.
  • Liposits Z., Uht R. M., Harrison R. W., Gibbs F. P., Paull W. K. and Bohn M. C. (1987) Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF). Histochemistry 87, 407412.
  • McEwen B. S. (1993) Effects of stress on the neurochemistry and morphology of the brain: counterregulation versus damage. Handbook of Stress: Theoretical and Clinical Aspects. Free Press, New York, pp. 101126.
  • McKittrick C. R., Blanchard D. C., Blanchard R. J., McEwen B. S. and Sakai R. R. (1995) Serotonin receptor binding in a colony model of chronic social stress. Biol. Psychiatry 37, 383393.
  • Maes M., Lin A., Verkerk R., Delmeire L., Gastel A. V., Planken M. V. and Scharpe S. (1999) Serotonergic and noradrenergic markers of post-traumatic stress disorder with and without major depression. Neuropsychopharmacology 20, 188197.
  • Mellmann T. A. and Kumar A. M. (1994) Platelet serotonin measures in post-traumatic stress disorder. Psychiatry Res. 53, 99101.
  • Mendelson S. D. and McEwen B. S. (1991) Autoradiographic analyses of the effects of restraint-induced stress on 5-HTIA, 5-HTIC and 5-HT2 receptors in the dorsal hippocampus of male and female rats. Neuroendocrinology 54, 454461.
  • Modai I., Malmgren R., Wetterberg L., Eneroth P., Valevski A. and Asberg M. (1992) Blood levels of melatonin, serotonin, cortisol, and prolactin in relation to the circadian rhythm of platelet serotonin uptake. Psychiatry Res. 43, 161166.
  • Ramamoorthy S., Bauman A. L., Moore K. R., Han H., Yang-Feng T., Chang A. S., Ganapathy V. and Blakely R. D. (1993a) Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc. Natl Acad. Sci. USA 90, 25422546.
  • Ramamoorthy S., Cool D. R., Mahesh V. B., Leibach F. H., Melikian H. E., Blakely R. D. and Ganapathy V. (1993b) Regulation of the human serotonin transporter. Cholera toxin-induced stimulation of serotonin uptake in human placental choriocarcinoma cells is accompanied by increased serotonin transporter mRNA levels and serotonin transporter-specific ligand binding. J. Biol. Chem. 268, 2162621631.
  • Uhl G. R. and Johnson P. S. (1994) Neurotransmitter transporters: three important gene families for neuronal function. J. Exp. Biol. 196, 229236.
  • Van de Kar L. D., Karteszi M., Bethea C. L. and Ganong W. F. (1985) Serotonergic stimulation of prolactin and corticosterone secretion ist mediated by different pathways from the mediobasal hypthalamus. Neuroendocrinology 41, 380384.
  • Watanabe Y., Sakai R. R., McEwen B. S. and Mendelson S. (1993) Stress and antidepressant effects on hippocampal and cortical 5-HT1A and 5-HT2 receptors and transport sites for serotonin. Brain Res. 615, 8794.
  • Weber B., Lewicka S., Deuschle M., Colla M., Vecsei P. and Heuser I. (2000) Increased diurnal plasma concentrations of cortisone in depressed patients. J. Clin. Endocrinol. Metab. 85, 11331136.
  • Weizman R., Laor N., Schujovitsky A., Wolmer L., Abramovitz-Schnaider P., Freudstein-Dan A. and Rehavi M. (1996) Platelet imipramine binding in patients with posttraumatic stress disorder before and after phenelzine treatment. Psychiatry Res. 63, 143150.
  • Yehuda R. (2001) Biology of posttraumatic stress disorder. J. Clin. Psychiatry 62, 4146.
  • Zhong P. and Ciaranello R. D. (1995) Transcriptional regulation of hippocampal 5-HT1a receptors by corticosteroid hormones. Brain Res. Mol. Brain Res. 29, 2334.