A novel LXR-α activator identified from the natural product Gynostemma pentaphyllum

Abstract

Liver X receptors (LXR) play an important role in cholesterol homeostasis by serving as regulatory sensors of cholesterol levels in tissues. The present study reports a novel LXR-α activator, (20S)-2α, 3β, 12β, 24(S)-pentahydroxydammar-25-ene 20-O-β-d-glucopyranoside (TR1), a dammarane-type gynosaponin, isolated from the herbal medicine, Gynostemma pentaphyllum. Gynosaponin TR1 demonstrated greater selectivity toward activation of the LXR-α isoform than LXR-β in HEK293 cells. TR1 selectively enhanced LXR-mediated transcriptional activation and protein expression of ABCA1 and apoE gene expression and secretion in THP-1-derived macrophages. The selectivity of TR1 for LXR-α was consistent with ligand docking studies, which showed favourable interaction of TR1 in the LXR-α-binding domain, whereas the presence of the sugar substituent interfered with binding to the LXR-β site. Findings from the present study may provide insight into the development of pharmaceutical agents for treating atherosclerosis.

Introduction

A global research effort has led to significant advances in therapeutic treatments toward preventing or curing cardiovascular disease (CVD). Vascular biology has revealed much information on the pathophysiology of atherosclerosis [1], [2], from the first report of its association with cholesterol [3], to the current notion that inflammation and the immune response also contribute to atherogenesis [1], [4]. An important target for pharmacological intervention is through the up-regulation of reverse cholesterol transport (RCT), whereby cholesterol is removed from macrophages and other peripheral cells and transported to the liver by plasma lipoproteins for excretion as bile salts [5]. These systemic changes involve regulation of the expression of a myriad of genes through ligand-activated transcription factors called nuclear receptors. One particular therapeutic target, known as transcription factor liver X receptor (LXR), regulates RCT by functioning as a sensor of cholesterol levels in tissues [6], [7], [8].

At present, two LXR isoforms have been characterised. LXR-α (NR1H3) is primarily expressed in the liver, intestine, adipose tissue, spleen and macrophages, whilst LXR-β (NR1H2) is ubiquitously expressed [6]. LXR-α and LXR-β share considerable sequence homology (∼77% identity in DNA- and ligand-binding domains) and appear to respond to the same endogenous ligands [9]. Both receptors are activated by naturally occurring oxysterols, such as 22(R)- and 24(S)-hydroxycholesterol [10] and 24(S)- and 25-epoxycholesterol [9] and by synthetic agonists, such as T0901317 [11], [12], GW3965 [13] and acetyl-podocarpic dimmer [14].

Theoretical investigations of the known LXR agonists have provided much insight in the understanding of the LXR ligand-binding domain (LBD) [15], [16] and identification of the the specificity towards each LXR isoform [17], [18]. The recent reported X-ray crystal structures of human LXR-α and LXR-β LBDs complexed with 24(S), 25-epoxycholesterol, T0901317 and GW3965 provide insight into key elements of ligand recognition as well as the mechanisms of activation for each ligand [12]. The LBDs share a common, mainly α-helical fold that embeds a hydrophobic ligand-binding pocket. The structures have shown that ligands can affect the conformation of the ligand-dependent activation function 2 residing in the C-terminal H12. Therefore, an agonist allows H12 to cover the ligand-binding pocket, thereby providing one of the sides in the coactivator-binding pocket [19]. Furthermore, this ligand-activation of the LXR transcription factors results in the formation of obligate heterodimers with the retinoid X receptor. The complexes subsequently bind to the consensus response element sequence DR4 in the promoter regions of several target genes, including cholesterol 7α-hydroxylase, cholesterol ester-transfer protein, ATP-binding cassette (ABC) transporter proteins (ABCA1, ABCG5 and ABCG8), apolipoprotein E (apoE), lipoprotein lipase and sterol-response element-binding protein-1c [8], [20].

The ABCA1 protein is critical for the first step in the RCT pathway through its up-regulation in lipid-loaded macrophages and its involvement in the efflux of excess cellular cholesterol to apolipoprotein acceptors. Humans who are genetically deficient in ABCA1 due to mutations in both ABCA1 alleles (homozygous Tangier disease) lack high-density lipoprotein (HDL) cholesterol almost completely, resulting in the accumulation of cholesterol esters in peripheral macrophages, leading to a greater risk of atherosclerosis [21], [22]. Numerous studies have shown ABCA1 to be a definite LXR target gene both in vitro and in vivo. The increase in cholesterol efflux by LXR ligands was shown to be ABCA1-dependent, as the effect of ligands was lost in the ABCA1-deficient Tangier fibroblasts. The ability of LXRs to control ABCA1 expression appears to be a significant factor in the enhanced HDL levels in mice [23].

ApoE is another essential LXR target gene in cholesterol metabolism. ApoE mediates the hepatic uptake of very-low-density lipoprotein (VLDL) and chylomicron remnants and can serve as an extracellular acceptor for cholesterol in the ABCA1 efflux pathway [24]. In contrast to ABCA1, regulation of apoE by LXR is tissue-specific, occurring only in macrophages and adipocytes [13]. The beneficial nature of apoE production by macrophages is evident from several animal studies. Mice expressing apoE only in macrophages were protected against atherosclerosis, whereas those specifically lacking apoE expression in macrophages were more susceptible [9], [25], [26]. Therefore, induction of the ABCA1 and apoE gene by LXR agonists might lead to a decreased cholesterol burden in the arterial wall, as well as enhanced HDL levels [5], [27].

Identifying potential medicinal compounds from nature with increasing efficacy and safety has become important in the growing population of pre-dyslipidaemic patients, as well as patients suffering from related CVD events [28]. Recently, the first non-oxysterol natural product ligand of LXR from Penicillium paxilli was discovered. The indole alkaloid fungal metabolite, paxilline, functions as a ligand for both LXR-α and LXR-β. Unfortunately, the clinical application of paxilline is limited by its tremorgenic mycotoxin properties and antagonism of high conductance calcium-activated K channels [29]. To date, there has been no report of LXR agonists from plant-derived material.

Gynostemma pentaphyllum (Cucurbitaceae), known as Jiagulan in Chinese herbal medicine, is a perennial vine endemic to southern China, Japan, India and Korea [30]. The pharmacological studies of G. pentaphyllum have illustrated a variety of biological activities, including anti-inflammatory, anti-tumour, immunopotentiating, anti-ulcer, anti-oxidant and retardation of aging [30], [31], [32]. G. pentaphyllum has been used clinically in China and has been described as having minimal toxicity [33], [34]. The dammarane saponins, namely gypenosides or gynosaponins, isolated from G. pentaphyllum are believed to be the active principles responsible for its biological activities and reported clinical effect of the G. pentaphyllum extract in the treatment of CVDs and related disorders [31], [35]. However, the mode of action of gypenosides has not been well-defined. Due to their similarity in structure to the oxysterols (Fig. 1), we postulated that G. pentaphyllum may contain novel LXR agonist(s) with similar biological and ligand-binding properties.

In the present study, we demonstrate that the new dammarane saponin, gynosaponin TR1 ((20S)-2α, 3β, 12β, 24(S)-pentahydroxydammar-25-ene 20-O-β-d-glucopyranoside), isolated from G. pentaphyllum, is an agonist of LXR, with selectivity for LXR-α over LXR-β. Furthermore, it was also found to be an inducer of ABCA1 and apoE gene expression in vitro. Theoretical investigations were also employed to rationalise the selectivity of TR1 to LXR-α through an understanding of its potential binding interactions.