Regulation of intestinal serotonin transporter expression via epigenetic mechanisms: role of HDAC2

Abstract

The serotonin (5-HT) transporter (SERT) facilitates clearance of extracellular 5-HT by its uptake and internalization. Decreased expression of SERT and consequent high 5-HT levels have been implicated in various diarrheal disorders. Thus, appropriate regulation of SERT is critical for maintenance of 5-HT homeostasis in health and disease. Previous studies demonstrated that SERT is regulated via posttranslational and transcriptional mechanisms. However, the role of epigenetic mechanisms in SERT regulation is not known. Current studies investigated the effects of histone deacetylase (HDAC) inhibition on SERT expression and delineated the mechanisms. Treatment of Caco-2 cells with the pan-HDAC inhibitors butyrate (5 mM) and trichostatin (TSA, 1 μM) decreased SERT mRNA and protein levels. Butyrate- or TSA-induced decrease in SERT was associated with decreased activity of human SERT (hSERT) promoter 1 (upstream of exon 1a), but not hSERT promoter 2 (upstream of exon 2). Butyrate + TSA did not show an additive effect on SERT expression, indicating that mechanisms involving histone hyperacetylation may be involved. Chromatin immunoprecipitation assays demonstrated enrichment of the hSERT promoter 1 (flanking nt -250/+2) with tetra-acetylated histone H3 or H4, which was increased (~3-fold) by butyrate. Interestingly, specific inhibition of HDAC2 (but not HDAC1) utilizing small interfering RNA decreased SERT mRNA and protein levels. The decrease in SERT expression by HDAC inhibition was recapitulated in an in vivo model. SERT mRNA levels were decreased in the ileum and colon of mice fed pectin (increased availability of butyrate) compared with controls fed a fiber-free diet (~50-60%). Our results identify a novel role of HDAC2 as a regulator of SERT gene expression in intestinal epithelial cells.

Fig. 1.

Treatment of Caco-2 cells with histone deacetylase (HDAC) inhibitors decreases 5-HT transporter (SERT) mRNA expression. Caco-2 cells grown on plastic supports for 10–12 days were treated for 24 h with butyrate (But, 5–10 mM), or trichostatin A (TSA, 1 μM), or butyrate (5 mM) + TSA (1 μM) in culture medium supplemented with 0.1% FBS. Total RNA was extracted, and quantitative real-time RT-PCR was performed utilizing SYBR Green fluorescent dye. SERT mRNA levels were normalized to levels of β-actin mRNA. Values are means ± SE from ≥3 different experiments performed in triplicate. *P < 0.05 vs. control. Similar results were obtained from Caco-2 cells grown on Transwell inserts for 12 days (data not shown).

Fig. 2.

Butyrate or TSA decreases SERT protein expression. Fully differentiated Caco-2 cells (grown for 10–12 days) were treated for 24 h with 5 mM butyrate, 1 μM TSA, or 5 mM butyrate + 1 μM TSA in cell culture medium supplemented with 0.1% FBS. Protein lysates were prepared, run on SDS-PAGE, and transblotted, and Western blotting was performed utilizing anti-SERT antibody. A: representative blot of 3 different experiments. B: densitometric analysis showing relative expression of SERT normalized to GAPDH. *P < 0.05 vs. control.

Fig. 3.

Effects of butyrate or TSA on human SERT (hSERT) promoter (hSERTp1 and hSERTp2) activities in Caco-2 cells. A: effect of 5 mM butyrate or 1 μM TSA on hSERTp1 activity. Caco-2 cells were transiently cotransfected with the hSERTp1 fragment, along with β-galactosidase vector to correct for transfection efficiency. At 24 h posttransfection, cells were incubated with butyrate, TSA, or butyrate + TSA for another 24 h. Butyrate or TSA decreased hSERTp1 activity, with no additive effect on simultaneous addition. Values are means ± SE of 4–5 different experiments performed in triplicate. *P < 0.001 vs. control. B: effect of 5 mM butyrate on hSERTp2 activity. Caco-2 cells were transiently cotransfected with the hSERTp2 fragment, along with β-galactosidase vector. At 24 h posttransfection, cells were incubated with butyrate for another 24 h. Values are means ± SE of 3 different experiments performed in triplicate.

Fig. 4.

Butyrate- or TSA-responsive regions of hSERTp1 (hSERTp1_B and hSERTp1_C). A: progressive 5′ deletions of hSERTp1 treated with butyrate. Different promoter constructs of hSERTp1 were treated with 5 mM butyrate for 24 h. At 24 h posttreatment, cells were harvested for measurement of promoter activity by luciferase assay. Values were normalized to β-galactosidase to adjust for transfection efficiency. Inhibitory effect of butyrate was retained in all constructs. Values are means ± SE from ≥3 different experiments performed in triplicate. *P < 0.001 vs. control. B: progressive 5′-deletion constructs of hSERTp1 treated with TSA. Similar to butyrate, TSA-responsive region predominantly spans nucleotides −272/+2 of hSERTp1. Values are means ± SE from ≥3 different experiments performed in triplicate. *P < 0.001 vs. control.

Fig. 5.

Butyrate alters histone status at hSERTp1. Control cells or cells treated with butyrate (5 mM) were fixed in formaldehyde, chromatin was sonicated, and immunoprecipitated with specific antibodies against tetra-acetylated histone H3 or H4. After reverse cross-linking and DNA extraction, immunoprecipitated chromatin was used as the template for real-time quantitative PCR utilizing primers spanning nucleotides −872/+2 of hSERTp1. A: PCR products at the end of amplification separated on 1% agarose gel containing ethidium bromide. B: enrichment of acetylated histone H3 or H4 with hSERTp1 at nucleotides −250/+2 by butyrate. Values are means ± SE from 3 different experiments. *P < 0.01 vs. control.

Fig. 6.

Expression of HDAC1–11 in Caco-2 cells. Total RNA was extracted from Caco-2 cells, and quantitative real-time RT-PCR was performed utilizing primers specific for human HDAC1–11. HDAC mRNA levels were normalized to levels of GAPDH mRNA. Results are expressed as relative expression compared with levels of HDAC1 mRNA taken as 1. Values are means ± SE from ≥3 different experiments performed in triplicate.

Fig. 7.

Small interfering RNA (siRNA) silencing of HDAC2 inhibits SERT mRNA expression. mRNA levels of HDAC1 (A), HDAC2 (B), or SERT (C) were measured and normalized to GAPDH in Caco-2 cells treated with scrambled, HDAC1, or HDAC2 siRNA for 48 h. Values are means ± SE of values obtained from ≥3 different experiments performed in triplicate. *P < 0.01 vs. control.

Fig. 8.

siRNA silencing of HDAC2 inhibits SERT protein levels. A: representative gel showing protein levels of HDAC1, HDAC2, SERT, and GAPDH in Caco-2 cells treated with scrambled, HDAC1, or HDAC2 siRNA for 72 h. B: densitometric analysis of HDAC2 after siRNA knockdown. C: densitometric analysis of SERT protein levels after siRNA knockdown of HDAC1 or HDAC2. Values are means ± SE of ≥3 different experiments performed in triplicate. *P < 0.01; **P < 0.001 vs. control (scrambled).

Fig. 9.

Pectin feeding decreases SERT mRNA in native mouse intestine. Total RNA was extracted from different regions of intestine isolated from control and pectin-fed mice. SERT mRNA levels were normalized to levels of β-actin/GAPDH mRNA. **P < 0.001; *P < 0.05 vs. control.

Fig. 10.

Schematic of proposed model of butyrate- or TSA-mediated effects on SERT. HDAC inhibitors butyrate and TSA decrease SERT expression by increasing association of acetylated histone H3 or H4 with hSERTp1 (upstream of exon 1a), resulting in decrease in promoter activity. Specific inhibition of HDAC2 (but not HDAC1) mimicked effects of butyrate or TSA in decreasing SERT expression.