The dietary ingredient, genistein, stimulates cathelicidin antimicrobial peptide expression through a novel S1P-dependent mechanism - PubMed (original) (raw)

The dietary ingredient, genistein, stimulates cathelicidin antimicrobial peptide expression through a novel S1P-dependent mechanism

Kyungho Park et al. J Nutr Biochem. 2014 Jul.

Abstract

We recently discovered that a signaling lipid, sphingosine-1-phosphate (S1P), generated by sphingosine kinase 1, regulates a major epidermal antimicrobial peptide's [cathelicidin antimicrobial peptide (CAMP)] expression via an NF-κB→C/EBPα-dependent pathway, independent of vitamin D receptor (VDR) in epithelial cells. Activation of estrogen receptors (ERs) by either estrogens or phytoestrogens also is known to stimulate S1P production, but it is unknown whether ER activation increases CAMP production. We investigated whether a phytoestrogen, genistein, simulates CAMP expression in keratinocytes, a model of epithelial cells, by either a S1P-dependent mechanism(s) or the alternate VDR-regulated pathway. Exogenous genistein, as well as an ER-β ligand, WAY-200070, increased CAMP mRNA and protein expression in cultured human keratinocytes, while ER-β antagonist, ICI182780, attenuated the expected genistein- and WAY-200070-induced increase in CAMP mRNA/protein expression. Genistein treatment increased acidic and alkaline ceramidase expression and cellular S1P levels in parallel with increased S1P lyase inhibition, accounting for increased CAMP production. In contrast, siRNA against VDR did not alter genistein-mediated up-regulation of CAMP. Taken together, genistein induces CAMP production via an ER-β→S1P→NF-κB→C/EBPα- rather than a VDR-dependent mechanism, illuminating a new role for estrogens in the regulation of epithelial innate immunity and pointing to potential additional benefits of dietary genistein in enhancing cutaneous antimicrobial defense.

Keywords: Cathelicidin antimicrobial peptide; Estrogen receptor β; Genistein; Innate immunity; Keratinocytes.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Fig. 1

Genistein increases estrogen receptors mRNA expression. Primary cultured KC (CHK) were incubated with genistein (50 μM) for 24 h. ER-α and ER-β mRNA expression was determined by _q_RT-PCR. Similar results were obtained when the experiment was repeated (more than twice) using different cell preparations.

Fig. 2

Increased expression of CAMP mRNA and protein in CHK following genistein. Primary cultured KC (CHK) were treated for 24 h with the indicated concentrations of genistein. CAMP mRNA expression was determined by _q_RT-PCR (A). CAMP and LL-37 (an active form of CAMP) protein/peptide levels quantified by ELISA (B and C). Similar results were obtained when the experiment was repeated (more than twice) using different cell preparations. Values are means ± SD (n=3). One-way ANOVA coupled with Duncan’s multiple comparison test (A). Means with different letters are significantly different at p < 0.01 (B and C).

Fig. 3

ER-β is required for genistein-induced upregulation of CAMP expression. Both Primary cultured KC (CHK) and HaCaT were pretreated with or without ER-β antagonist, ICI (1 μM, 30 min), were incubated with genistein (50 μM) or ER-β agonist (WAY, 1 μM) for 24 h. CAMP mRNA expression was assessed by _q_RT-PCR (A and B). Protein expression of CAMP was determined by western immunoblot analysis (C) and immunohistochemistry (D). Similar results were obtained when the experiment was repeated (in triplicate) using different cell preparations. Letters with different superscripts are significantly different at p < 0.01.

Fig. 4

Genistein unlikely stimulates CAMP production through VDR mechanism. Primary cultured KC (CHK) were incubated as in Fig. 3. mRNA expressions were assessed by _q_RT-PCR. Similar results were obtained when the experiment was repeated (in triplicate) using different cell preparations. Letters with different superscripts are significantly different at p < 0.01 (D).

Fig. 5

Genistein could alter S1P metabolism. Primary cultured KC were incubated as in Fig. 3. mRNA expressions were assessed by _q_RT-PCR. Similar results were obtained when the experiment was repeated (in triplicate) using different cell preparations. Letters with different superscripts are significantly different at p < 0.01 (C and D).

Fig. 6

Proposed mechanism of genistein for CAMP expression. Genistein binds to estrogen receptor (ER) β to increase cellular S1P levels by upregulating acidic/alkaline ceramidase expression. Increased ceramidase leads to increased production of S1P. Simultaneously S1P lyase is inhibited to also decrease S1P hydrolysis, further stimulating the S1P signal, which increases CAMP production. Genistein also suppresses SPHK 2 expression, which decreases CAMP production [5]. The pool of S1P produced by SPHK 2 is hydrolyzed to sphingosine by S1P phosphatase [4] and sphingosine likely suppresses CAMP production [5].

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The dietary ingredient, genistein, stimulates cathelicidin antimicrobial peptide expression through a novel S1P-dependent mechanism - PubMed (original) (raw)