Modulation of human serotonin transporter expression by 5-HTTLPR in colon cells

Abstract

Serotonin (5-HT) is a monoamine neurotransmitter and plays important roles in several of the human body's systems. 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. 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). It has been demonstrated that 5-HTTLPR is associated with numerous neuropsychiatric disorders. The functional roles of 5-HTTLPR have been reported in human choriocarcinoma (JAR), lymphoblast and raphe cells. To date, the significance of 5-HTTLPR in gastrointestinal tract-derived cells has never been elucidated. 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. We found 42-bp fragment in long (L) allele as compared to short (S) allele, and this allelic difference resulted in 2-fold higher transcriptional efficiency of L allele (P < 0.05) as demonstrated using a functional reporter gene assay. Nevertheless, the transcriptional effect of estrogen and glucocorticoid on 5-HTT expression via 5-HTTLPR was not found in this cell line. Our study was the first to demonstrate the molecular role of this allelic variation in gastrointestinal tract cells.

Keywords: allelic variation; gastrointestinal tract cells; hormonal effect; promoter; serotonin transporter; transcriptional regulation.

Figure 1

Genotyping of 5-HTT using PCR and subjected to 3% agarose gel electrophoresis. From left to right, 100 bp DNA molecular weight marker (M); S/S homozygote (484 bp, 14 repeats), L/L homozygote (528 bp, 16 repeats).

Figure 2

Map of potential transcription factor binding sites on 42-bp 5-HTTLPR fragment analyzed using the Promo program.

Figure 3

Expression of mRNA transcripts for (A) β-actin; (B) 5-HTT; (C) ERα; and (D) GRα in human colon carcinoma cells, SW480 (Lane 1) and HT-29 (Lane 2) determined using RT-PCR. M represents the 100 bp DNA molecular weight marker. Arrows indicate positions of the specific bands of RT-PCR products.

Figure 4

The organization of the human 5-HTT gene promoter and map of 5-HTTLPR luciferase reporter gene constructs, p5HTT[S]Luc, p5HTT[L]Luc and p5HTT[L860]Luc. The nucleotide sequence of 42-bp length polymorphism is shown.

Figure 5

Transcriptional activity of the 5-HTTLPR luciferase reporter gene constructs, S, L and no 5-HTTLPR, respectively, in SW480 cells. Data are mean ±S.D. for triplicate determinations, *** P < 0.001 by student’s t-test.

Figure 6

Effects of (A) dexamethasone and (B) β-estradiol on 5-HTT promoter activity of the 5-HTTLPR luciferase reporter gene constructs, S, L and on 5-HTTLPR, respectively, in SW480 cells. Data are mean ± S.D. for triplicate determinations.