Resveratrol promotes lysosomal function via ER calcium-dependent TFEB activation to ameliorate lipid accumulation.

Abnormal lipid accumulation is associated to the development of metabolic diseases such as hepatic steatosis and lipid storage diseases. Pharmacological agents that can attenuate lipid accumulation therefore have therapeutic potentials for these diseases. Resveratrol (RSV), a natural active substance found in fruits and nuts, has been reported to effectively reduce the intracellular lipid accumulation, but the underlying mechanisms of RSV remain elusive. Here, we show that RSV triggers an endoplasmic reticulum (ER)- Ca2+ signaling that activates transcriptional factor EB (TFEB), a master transcriptional regulator of autophagic and lysosomal biogenesis. Moreover, RSV activates protein phosphatase 2A (PP2A), which binds and dephosphorylates TFEB, promoting its nuclear translocation and the expression of TFEB target genes required for autophagosome and lysosomal biogenesis. Notably, genetic inhibition of TFEB significantly ameliorates RSV-mediated lipid clearance. Taken together, these data link RSV-induced ER calcium signaling, PP2A and TFEB activation to promote autophagy and lysosomal function, by which RSV may trigger a cellular self-defense mechanism that effectively mitigate lipid accumulation commonly associated with many metabolic diseases.


Introduction
Macro-autophagy is a cellular process involving lysosomal degradation of cytosolic components through formation of a double membrane structure (autophagosome) and its fusion to lysosome (autophagolysosome) [1]. Amino acids and other small molecules produced by lysosome degradation can be reused or generate energy. One of the main functions of autophagy is to maintain cell survival when cells are threatened by stressful death, and plays an important role in maintaining the metabolic homeostasis of cells [2]. Impairment of autophagy-lysosome pathway is associated with metabolic disorders and aging.
In metabolic syndromes such as obesity and fatty liver disease [3,4], excessive nutrients challenge the adaptive response capacity of degradative autophagy-lysosome machinery.
Undigested macromolecules (ie. lipids, proteins) and impaired organelle turnover compromise metabolic activity, provoking intracellular stresses, and exacerbate collateral defects in insulin action or other metabolic pathologies. Therefore, pharmacological interventions that enhance autophagic and lysosomal function are emerging as a promising strategy to ameliorate metabolic symptoms and promote longevity.
The transcriptional factor EB (TFEB) is a major regulator of autophagic and lysosomal biogenesis [5,6]. TFEB belongs to MITF/TFE family of basic helix-loop-helix/leucine zipper transcriptional factors and binds to the "coordinated lysosomal expression and regulation (CLEAR)" motifs located in the promoter region of a variety of autophagic and lysosomal genes [6,7]. TFEB activity is regulated by phosphorylation [8][9][10], which keeps TFEB inactive in the cytoplasm; in contrast, dephosphorylated TFEB travels to the nucleus to activate transcriptional target genes [11].
Several pathways reportedly control TFEB activity under different conditions. Under starvation, the phosphorylation of TFEB is suppressed by mammalian target of rapamycin kinase (mTOR) inhibition [12]. Under oxidative stress, TFEB is activated via a lysosomal Ca 2+ -dependent mechanism independent of mTOR [13]. Notably, overexpression of TFEB homolog HLH-30 in Caenorhabditis elegans can increase lifespan through possible induction of autophagy [14]. Hence, TFEB agonists are potential therapeutic intervention for diseases in which dysfunctional autophagy and lysosome has been implicated. 4 Natural compounds in food are known to be good potential drug targets, but the underlying mechanisms are not clear [15]. In the present study, we demonstrate that resveratrol (RSV) (Fig. 1a), an active natural substance from fruits and peanuts, induces a Ca 2+ signaling from endoplastic recticulum (ER) and promotes autophagic and lysosomal function through protein phosphatase 2A (PP2A) -mediated TFEB activation. Moreover, genetic inactivation of TFEB impairs RSV function of ameliorating lipid accumulation, suggesting that TFEB is required in RSV-mediated lipid clearance.

Ca 2+ is required for RSV-induced TFEB nuclear translocation
First, we investigated the effect of RSV on TFEB activation in HeLa cells stably expressing GFP-TFEB [16]. At the concentration of 300 µM, but not at a lower concentration (100 µM), RSV treatment for 4 h induced a five-fold increase of TFEB translocation from the cytosol to the nucleus (Fig. 1b, c). Consistently, RSV (300 µM, 4 h) induced endogenous TFEB nuclear translocation in HeLa wild-type (WT) cells (Fig. 1d, e).
We next analyzed the mRNA expression of TFEB target genes, including a selected list of genes involved in autophagy and lysosome using quantitative real time PCR (Q-PCR).
RSV is a known antioxidant and transcriptional factor -nuclear factor -E2-related factor 2 (NRF2) inducer [17]. Hence, the transcriptional expression levels of HO-1 and NQO1 gene (NRF2 downstream genes) were used as a positive control. Upon RSV treatment (300 µM, 12 h), the expression levels of lysosomal genes (CTSD, NEU1 and LAMP1) and autophagic genes (WIPI) were significantly increased in HeLa cells (Fig. 1f). Collectively, in agreement with recent studies [18], RSV induces TFEB nuclear translocation and the expression of TFEB target genes.
As intracellular Ca 2+ reportedly involves in TFEB activation [11,13], we then assessed the dependence of Ca 2+ in TFEB activation by RSV using specific Ca 2+ chelators.

ER calcium store contributes to TFEB activation by RSV
Our data indicate that RSV-induced TFEB nuclear translocation requires cytosolic calcium.
As known that lysosomes, mitochondria and ER are the major compartmentalized Ca 2+ stores in cells [20], the mechanistic links between chemically induced cytosolic Ca 2+ and TFEB activation lead us to consider the discrete Ca 2+ sources as a potential specificity determinant of RSV-induced TFEB activation. We next investigated the determinant Ca 2+ sources in RSV-induced TFEB activation. In HeLa GFP-TFEB cells, RSV-induced TFEB nuclear translocation was blocked by pretreatment with thapsigargin (TG) (300 nM, 30 min), a specific inhibitor of ER Ca 2+ ATPase SERCA pump that is commonly used to deplete ER Ca 2+ stores [21] (Fig. 3a, b). In contrast, RSV-induced TFEB nuclear translocation was not affected by 30 min pretreatment of glycyl-L-phenylalanine 2-napthylamide (GPN) (200 µM), a lysosome-disrupting agent to deplete lysosome-specific Ca 2+ stores [22] (Fig. 3a, b). To further confirm that lysosomal Ca 2+ did not involve in RSV-mediated TFEB activation, we examined the TFEB localization by RSV in mucolipidosis IV patient-derived TRPML1 KO (ML-IV) fibroblasts [13]. TRPML1/ MCOLN1 is the principle Ca 2+ -permeant channel on the lysosomal membrane [23]. Hence, as shown in Fig. 3c, d, RSV can induce TFEB nuclear translocation in both WT and ML-IV cells, suggesting that lysosomal Ca 2+ is not required in RSV-mediated TFEB activation. Torin 1, a potent inhibitor of mTOR that is commonly used to induce autophagy, was used as a control. Taken together, these results suggest that the Ca 2+ signaling induced by RSV is from ER, but not from lysosome. We also measured the direct Ca 2+ efflux by RSV with Ca 2+ imaging. Acute application of RSV significantly increased cytosolic Ca 2+ release in HeLa cells and this effect was dramatically reduced by a 30 min pretreatment of TG (Fig. 3e, f), but not by GPN pretreatment (Fig. 3g, h).
Collectively, these data suggested that RSV promotes autophagic flux.
We then explored the effects of RSV on lysosomal function. Lysosomal associated membrane protein 1 (LAMP1) is a marker of late endosomes and lysosomes (referred to as "lysosomes" for simplicity hereafter). Western blot analyses showed gradual increases in the expression of LAMP1 proteins upon RSV (300 µM, 3-9 h) treatment in a time dependent manner in HeLa cells (Fig. 6a, b). Consistently, RSV (300 µM, 6 h) significantly increased the immunofluorescence intensity of LAMP1 in HeLa cells (Fig. 6c,   d). Lysosomal enzymes operate better under acidic conditions, and the degradation-active lysosomes can be tracked using LysoTracker, a fluorescent acidotropic probe [27].
Significant increases of LysoTracker staining were observed in HeLa cells following treatment with RSV (300 µM, 3-9 h) (Fig. 6e, f). Collectively, these results suggest that RSV also promotes lysosomal function and biogenesis.

Discussion
In this study we identify RSV, a natural polyphenol compound, induces a calcium signaling mechanism that originates from the ER and activates phosphatase PP2A to dephosphorylate TFEB. Activated TFEB then regulates the expression of genes that are required for lysosomal biogenesis and autophagosome formation, promoting lysosomal function (Fig. 8). Moreover, genetic inhibition of TFEB ameliorates the lipid clearance function of RSV, suggesting that TFEB plays an essential role in RSV-mediated lipid clearance. Collectively, our study has providing the underlying mechanism of RSV to promote lysosomal function and proposing RSV as a therapeutic candidate to treat or prevent diseases in which lysosome dysfunction has been implicated.   [13]. In this study we found that ROS was not involved in RSV-mediated TFEB activation and RSV-induced TFEB activation through an ER Ca 2+ release. This finding reveals a unique mechanism of RSV. In addition, the mechanism of different ROS induction and source of Ca 2+ presents as an intriguing opportunity for future investigation. We also found that the ER Ca 2+ controls the activities of phosphatase PP2A, which is consistent with previous reports that ER stress regulates PP2A activity [41]. However, the exact mechanism of this process is still unclear.
Lysosomes are the cell's recycling center, which degrades biomolecules from endocytic and autophagic pathways [42]. In addition to the cellular degradation pathways, recent studies have revealed that lysosomes play active roles in nutrient sensing, adaption to multiple cellular stress, plasma membrane repair, cell signaling, and membrane trafficking [42]. Hence, lysosomal dysfunction has been associated to many diseases ie. lysosomal storage diseases (LSDs) [43]. As the role of lysosomes and autophagy in macromolecule metabolism becomes clearer, boosting this innate cellular clearance machinery, such as the TFEB agonists, offers a new therapeutic strategy for treatment of these diseases. In fact, pharmacological intervention of targets upstream of the TFEB, such as mTOR inhibitors and AMPK activators has been applied [44]. Our findings show that RSV can promote lysosomal function, which proposes RSV as the potential new therapeutic candidate in lysosomal dysfunction diseases.

Methods and materials
Mammalian cell culture. HeLa cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, 11195-065) with 10% fetal bovine serum (Thermo Fisher Scientific, 10091148). All cell cultures were maintained at 37 °C in a humidified 5% CO 2 incubator.  Statistical analysis. Data are presented as mean ± s.e.m. from at least 3 independent experiments. Statistical comparisons were performed with analyses of variance (ANOVA) or two tailed Student's t-test with paired or unpaired wherever appropriate. A P value < 0.05 was considered statistically significant. 13 publish, or preparation of the manuscript. We are grateful to Dr. Shawn M. Ferguson for the GFP-TFEB stable cells, to Dr. David Rubinsztein for the mRFP-GFP-LC3 cells and to Dr.
Haoxing Xu for the TFEB-KO stable cells.

Disclosure statement
No potential conflict of interest was reported by the authors.