The dietary histone deacetylase inhibitor sulforaphane induces human beta-defensin-2 in intestinal epithelial cells

Abstract

Antimicrobial peptides like human beta-defensin-2 (HBD-2) play an important role in the innate immune system protecting the intestinal mucosa against bacterial invasion. The dietary histone deacetylase (HDAC) inhibitors sulforaphane (SFN) and butyrate have received a great deal of attention because of their ability to simultaneously modulate multiple cellular targets involved in cellular protection. In this study the influence of SFN and butyrate on HBD-2 expression as well as the molecular pathways involved in SFN-mediated induction of HBD-2 were scrutinized. Treatment of Caco-2, HT-29 and SW480 cells with SFN led to a time- and dose-dependent upregulation of HBD-2 mRNA expression as determined by semi-quantitative reverse transcription-polymerase chain reaction. Moreover, HBD-2 protein production increased in response to SFN, measured by enzyme-linked immunosorbent assay. Induction of HBD-2 was also observed in response to butyrate. Immunofluorescence analysis revealed that the protein was localized in the cytosol. Coincubation of SFN with a vitamin D receptor (VDR), or an extracellular-regulated kinase 1/2 or a nuclear factor-kappaB inhibitor all reduced HBD-2 mRNA upregulation. In contrast, transfection of cells with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutant vector to inhibit PPARgamma wild-type action and inhibition of p38 mitogen-activated protein kinase (MAPK) signalling did not affect SFN-mediated upregulation of HBD-2 mRNA. Moreover, SFN induced the expression of VDR, PPARgamma and phosphorylated ERK1/2 but did not affect p38 MAPK activation. The data clearly demonstrate for the first time that the dietary HDAC inhibitor SFN is able to induce antimicrobial peptides in colonocytes. In this process HBD-2 expression is regulated via VDR, mitogen-activated protein kinase kinase/extracellular-regulated kinase and nuclear factor-kappaB signalling.

Figure 1

Dose-dependent effect of sulforaphane (1–20 μm) and butyrate (1–5 mm) on β-defensin-2 messenger RNA (mRNA) expression in HT-29 cells after 24 hr of treatment. Moreover the induction of β-defensin-2 mRNA expression in Caco-2 and SW480 cells in response to sulforaphane (20 μm) was demonstrated. The β-defensin-2 mRNA expression was measured by semi-quantitative reverse transcription–polymerase chain reaction using the fluorescent dye Pico Green. All values for mRNA levels are normalized to corresponding mRNA amounts for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). One representative gel of three independent experiments is shown. Induction of β-defensin-2 mRNA is displayed as relative percentage to solvent treated control cells. ***P < 0·001, NS, not significant.

Figure 2

(a) β-defensin-2 protein expression in Caco-2 cells measured by enzyme-linked immunosorbent assay (ELISA). Cells were starved for 72 hr and then stimulated with sulforaphane (10–20 μm) or butyrate (3 mm) for 24 and 48 hr. Proteins were harvested and β-defensin-2 was measured by ELISA. The concentration range of all experiments was in ng/ml. Induction of β-defensin-2 protein is displayed as fold induction relative to solvent-treated control cells. ***P < 0·001, *P < 0·05. (b) Representative immunofluorescence assays for β-defensin-2 in Caco-2 cells. Confluent cells were challenged with sulforaphane (20 μm) for 48 hr (III) or grown in culture medium as controls (I). (II, IV) Nuclear counterstaining with 4′,6-diamidino-2-phenylindol (DAPI) mounting medium for each of the adjacent panels. Magnification, × 400.

Figure 3

(a) Time-dependent effect of sulforaphane (10 μm) on β-defensin-2 messenger RNA (mRNA) expression in wild-type, empty-vector and peroxisome proliferator-activated receptor γ (PPARγ) mutant HT-29 cells after 24 and 48 hr of treatment. β-Defensin-2 mRNA was measured by semi-quantitative reverse transcription polymerase chain reaction with the fluorescent dye Pico Green. All values for mRNA levels are normalized to corresponding mRNA amounts for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Induction of β-defensin-2 mRNA is displayed as a relative percentage to solvent-treated control cells; NS, not significant. (b) Western blot for PPARγ expression after treatment of HT-29 wild-type cells with sulforaphane (10 μm) for 48 hr. One representative blot of three independent experiments is shown.

Figure 4

(a) β-defensin-2 messenger RNA (mRNA) expression in Caco-2 cells. Medium was supplemented with the solvent, sulforaphane (10 μm), the vitamin D receptor (VDR) inhibitor ZK191732 (10 μm) or a combination of sulforaphane and ZK191732 for 24 hr. Semi-quantitative analysis of polymerase chain reaction products was performed using Pico Green. All values for mRNA levels are normalized to corresponding mRNA amounts of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Induction of β-defensin-2 mRNA is displayed as relative percentage to solvent-treated control cells. ***P < 0·001, *P < 0·05, NS, not significant. (b) Western blot for VDR expression after treatment of HT-29 and Caco-2 cells with sulforaphane (10–20 μm) for 24 hr. One representative blot of three independent experiments is shown.

Figure 5

(a) Influence of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (20 μm) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (40 μm) on sulforaphane-induced (10 μm) β-defensin-2 messenger RNA (mRNA) expression after 24 hr of treatment in Caco-2 cells. β-defensin-2 expression was analysed by semi-quantitative reverse transcription–polymerase chain reaction with the fluorescent dye Pico Green. All values for mRNA levels are normalized to corresponding mRNA amounts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). One representative gel of three independent experiments is shown. Induction of β-defensin-2 mRNA is displayed as relative percentage to solvent-treated control cells. ***P < 0·001, NS, not significant. (b) Western blot for ERK1/2, phospho-ERK1/2, p38 MAPK and phospho-p38 MAPK expression after treatment of Caco-2 cells with sulforaphane (10–20 μm) for 8 hr. One representative blot of three independent experiments is shown.