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

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.

Introduction

Antimicrobial peptides (AMPs) are innate immune elements that protect their host against pathogenic microbes [1]. A major epithelial AMP, cathelicidin antimicrobial peptide (CAMP), displays antimicrobial activity against a broad range of pathogens, including Gram-negative and Gram-positive bacteria, fungi and certain viruses. CAMP displays not only antimicrobial activities, but it also modulates multiple cellular functions; for example, cytokine expression/production, angiogenesis, wound healing and adaptive immune responses [2]. CAMP is an inducible AMP whose levels increase at both a transcriptional and posttranscriptional level in response to external perturbations; for example, pathogen exposure, wounding, ultraviolet irradiation and barrier abrogation [3] that in turn increase endoplasmic reticulum stress [4].

We recently discovered that a signaling lipid, sphingosine-1-phosphate (S1P), up-regulates a NF-κB-C/EBPα-dependent [but vitamin D receptor (VDR)-independent] transcriptional pathway for CAMP in epithelial cells, in mouse epidermis, human epidermal keratinocytes (KCs) and HeLa cells [4], [5]. Importantly, this mechanism operates only after cells are exposed to external perturbations that induce endoplasmic reticulum stress, while VDR transactivity instead is suppressed under these conditions [4], suggesting that these two mechanisms adjust CAMP levels in epithelial tissues under circumstances that predominate under stress versus basal conditions. Moreover, we more recently demonstrated a dietary plant-derived stilbenoid, resveratrol, stimulates CAMP production through our identified S1P-mediated pathway. Yet, the effects of dietary compounds on innate immunity (in particular AMP, including CAMP) are incompletely understood.

Estrogens and a phytoestrogen, the soy-derived isoflavone, genistein, alter the expression of certain genes, leading to diverse alterations in cellular functions, including increased proliferation, differentiation, apoptosis and innate immunity [6]. The genomic mechanism classically involves ligand binding to one or both of two estrogen receptors (ERs), ER-α (ER-α) and ER-β (ER-β), forming homo- or heterodimers, followed by their translocation and binding to an ER (ER)-binding element (ERE) in nuclear DNA [7], [8]. Estrogen and estradiol preferentially bind to ER-α and ER-β, respectively, while estradiol exhibits equivalent affinity toward both receptors [7], [8].

Estrogens can also modulate cell functions by nongenomic mechanisms through binding to the G protein-coupled receptor (GPR30) which is widely distributed on cellular membranes. This mechanism then modulates cellular functions through the Mitogen-activated protein (MAP) kinase pathway [9], [10], among others. Though genistein exhibits a higher binding affinity for ER-β than for ER-α [11], it also binds to GPR30 [12]. Of potential pertinence to CAMP expression, genistein increases CYP27B1 expression, the enzyme that generates 1α, 25-dihydroxy vitamin D3 [13], the natural ligand of the VDR. But recent studies in breast cancer cells show that genistein also induces both acidic ceramidase expression, resulting in increased catalytic activity, and sphingosine kinase (SPHK) 1 mRNA expression, which likely increases S1P production (ceramide→sphingosine→S1P) [14]. Although S1P stimulates tumor growth in breast cancer cells [14], the impact of S1P appears to depend upon cell and tissue types. In KCs, S1P stimulates differentiation rather than increasing cell proliferation [15].

Because either an ER and/or GPR30 activation could increase VDR ligand production, and increased ceramidase expression/activation could also lead to enhanced S1P production, genistein could up-regulate CAMP expression via either a VDR- and/or S1P-dependent mechanism(s). Our present studies indicate that genistein stimulates CAMP production via ER-β-activated (but not stimulated by G protein-coupled receptor (GPR) activation) stimulation of S1P signal and does not involve a VDR-dependent mechanism. The insights from these studies illuminate a previously unidentified role for this phytoestrogen in regulating epithelial innate immunity.