1. Introduction
The retinal pigment epithelium (RPE) is a monolayer of cells that performs several crucial functions vital for maintaining retinal homeostasis. RPE cells are responsible for the proper functioning of photoreceptor cells by phagocytosing 10% of their volume daily [
1]. RPE cells are the most active phagocytic cells in the body as they phagocytose hundreds of thousands of outer segments in an individual’s lifetime [
1]. Since, the RPE cells are post-mitotic, they are highly vulnerable to age-related accumulations of metabolic byproducts. Autophagy, a cellular degradation pathway, is crucially important to remove damaged structures and metabolic byproducts and reduce cellular accumulation in the RPE [
2]. Dysfunctional autophagy process is known to result in impairment of RPE homeostasis, leading to age-related macular degeneration (AMD) [
2]. During autophagy, cellular material and damaged organelles are sequestered in a double membrane organelle called autophagosome [
3]. A crucially important stage in the autophagy process is the fusion of autophagosomes with lysosomes to form autophagolysosomes, where the autophagy substrates are degraded by the lysosomal hydrolases [
4]. Since lysosomes are important in the terminal stages of the autophagy process, impaired lysosomal function can have significant implications on the autophagy pathway [
3]. The effective clearance of the autophagy substrates is highly dependent on the functional ability of the lysosomal degradation pathway. In addition to being terminal organelles in the autophagy pathway, lysosomes are also known to directly regulate the autophagy pathway by influencing the activity of mTORC1 [
5]. Several studies have implicated lysosomal dysfunction in the pathogenesis of AMD [
6,
7]. Age-related decline in lysosomal function is known to result in accumulation of phagocytosed outer segments and autophagic cellular substrates within the RPE, leading to impaired RPE function [
6,
7]. Acidification of the phagosome is also important for its proper degradation [
8]. A2E, the major component of lipofuscin, is known to decrease the activity of the lysosomal proton pump and elevate lysosome pH [
9]. Furthermore, oxidized lipoproteins contribute to a decline in lysosomal function by inhibiting the activity of lysosomal proteolytic enzymes [
10].
Since, lysosomes and autophagy play an undeniably interdependent role in the RPE, strategies to induce both lysosomal function and autophagy can have tremendous implications in enhancing cellular clearance in the RPE. Hence, it is important to identify cellular mediators that coordinately regulate autophagy and lysosomal function in the RPE. Recent studies have identified transcription factor EB (TFEB) as a cellular regulator of both lysosomal and autophagy pathway [
11]. During stress, activation and nuclear shuttling of TFEB results in transcriptional induction of genes involved in several stages of the autophagy and lysosomal biogenesis pathway [
12]. In addition to transcriptional activators of autophagy and lysosomal function, autophagy-mediated cellular clearance functions are also under the control of transcriptional repressors. ZKSCAN3 (Zinc finger protein with KRAB and SCAN domains 3) is a member of a family of proteins containing zinc finger, Kruppel-associated box (KRAB), and SCAN domains [
13]. ZKSCAN3 possesses the C2H2 zinc finger motif and a KRAB domain [
13]. The KRAB domain is a highly potent transcriptional repressor module [
14]. The KRAB domain in ZKSCAN3 is responsible for the transcription repression functions by binding to repressor proteins, whereas the C2H2 zinc finger motifs confer DNA binding ability [
14]. The notion that proteins containing KRAB domains are transcriptional suppressors has been recently challenged by studies identifying some KRAB domain proteins such as ZKSCAN3, which can also stimulate colorectal tumor progression [
15]. ZKSCAN3 also possess a highly conserved leucine rich 84 residue SCAN domain at the amino-terminus. The SCAN domain mediates protein–protein interactions either by self-association or by binding to other proteins [
16].
ZKSCAN3 was recently implicated as a critical player in the transcriptional repression of autophagy and lysosomal function [
13]. Gene expression profiling studies show that ZKSCAN3 acts as a transcriptional suppressor of about 60 genes involved in the lysosome biogenesis/function and autophagy pathway [
13]. In addition, silencing ZKSCAN3 is known to increase the number of lysosomes in the cell [
13]. Previous studies have shown that starvation-induced activation of protein kinase C-mediated signaling pathway phosphorylates ZKSCAN3, leading to its translocation out of the nucleus and subsequent de-repression of ZKSCAN3-regulated genes [
17]. Unbiased screening studies have identified ZKSCAN3-binding motifs in genes regulating angiogenesis, cell migration, and growth, including vascular endothelial growth factor (VEGF) and integrin β4 [
18]. These studies, taken together, suggest that ZKSCAN3 belongs to a small subset of transcription factors that co-regulate autophagy and angiogenic pathways. The biological significance of ZKSCAN3 in the RPE is largely unknown. In our studies, we aimed to determine the role of ZKSCAN3 in transcriptional regulation of lysosomal and autophagy pathways in response to cellular stress in the RPE.
In this report, we investigated the role of ZKSCAN3 in the RPE and the underlying regulatory mechanisms that contribute to the de-repression of ZKSCAN3 target genes in response to cellular stress. Our results indicated that nutrient deprivation diminishes nuclear levels of ZKSCAN3 resulting in de-repression and activation of ZKSCAN3 target genes, leading to induction of lysosomal function and autophagy pathway. Our results also show that in the absence of cellular stress, silencing of ZKSCAN3 induces lysosomal function and autophagy. Our findings provide insights into ZKSCAN3-regulated transcriptional program in the RPE which could be explored to induce cellular clearance in the RPE.
2. Materials and Methods
2.1. Cell Culture and Transfection
Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells were cultured in the presence of DMEM/F12 with L-Glutamine and 15 mM HEPES (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) along with 10% Fetal Bovine Serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% Antibiotic-Antimitotic (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA). ARPE-19 cells are widely used as in vitro models to study the function of the RPE. The data obtained from ARPE-19 cells was further substantiated in primary RPE cells, isolated from C57BL6/J and ATP-binding cassette, sub-family A, member 4 (ABCA4) knockout mice as described previously [
6]. Primary mouse RPE cells and RPE choroid extracts were isolated from C57BL/6J mice according to the protocol approved by the Institutional Animal Care and Use Committee, Indiana University/School of Optometry and conformed to the ARVO Statement for the Use of Animals in Ophthalmologic and Vision Research. For starvation, the cells were cultured in Earle’s Balanced Salt Solution (EBSS) (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) with calcium and magnesium for 24–48 h.
2.3. Cell Viability Assay
ARPE-19 cells were seeded in 96-well plates and treated with various concentrations of SMARTPOOL ZKSCAN3 siRNA (25, 50, 100, μM) (Dharmacon, Lafayette, CO, USA) for 24, 48, 72, and 96 h. We used a range of concentrations and time points to evaluate dose and time-dependent effects of the siRNA on the viability of the RPE. The medium with different concentrations was then removed and cells were incubated with 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) for 4 h at 37 °C. The optical density was measured at 490 nm using a Microplate Reader (BMG Labtechnologies, Cary, NC, USA) after dissolving the formazan with 0.5% DMSO.
2.4. Immunoblotting
ARPE-19 cells were treated with EBSS (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) and ZKSCAN3 siRNA (Dharmacon, Lafayette, CO, USA) at 100 nM for 48 h. Total cell lysates were obtained by adding Radioimmunoprecipitation assay buffer (RIPA) lysis buffer. Protein concentration was measured by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). The Sodium dodecyl sulfate polyacrylamide gel electrophoresis (National Diagnostics, Atlanta, GA, USA) was used to detect protein and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Burlington, MA, USA). The membranes were block with 5% milk made by Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) at room temperature for an hour and incubated with primary antibodies overnight at 4 °C followed by incubating in secondary. The image Lab software (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect the bands.
2.5. Nuclear and Cytoplasmic Extraction
ARPE-19 cells were seeded in 10 cm petri dish and cultured in the presence of EBSS (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA). Cells were transfected with ZKSCAN3 siRNA (Dharmacon, Lafayette, CO, USA) at 100 nM for 48 h. The cells were harvested using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Grand Island, NY, USA) following the manufacturer’s instructions. The α-tubulin antibody was developed by Walsh, C and was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.
2.6. Immunostaining and Microscopy
ARPE-19 and mouse primary RPE cells were seeded in 8 chamber slide and treated with Earle’s Balanced salt solution (EBSS)(Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) and SMARTPOOL ZKSCAN3 siRNA (Dharmacon, Lafayette, CO, USA) at a concentration of 100 nM for 48 h. After 48 h, the cells were fixed by 4% paraformaldehyde, permeabilized with 0.5% triton-x diluted in PBS, and blocked with blocking buffer (5% BSA and 0.5% Tween-20 in 1× PBS and 10% goat serum (MP biomedicals, Irvine, CA, USA) The primary antibody was incubated overnight at 4 °C. Next day, cells were treated with secondary antibody, which was added for 1 h at room temperature. The slide was mounted by Prolong Gold antifade reagent with DAPI (Life technologies, Carlsbad, CA, USA). Cells were imaged under a Zeiss microscope equipped with a camera (Axiocam Mrm; Carl Zeiss, Oberkochen, Germany).
2.7. Quantitative Real Time-PCR
RNeasy Mini Kit (QIAGEN, Hilden, Germany) was used to isolate RNA from ARPE-19 cells 48 h after transfection with the ZKSCAN3 siRNA. Then, 400 ng of RNA was converted to cDNA by RNA-to-cDNA Kit (Applied Biosystems, Waltham, MA, USA) using SsoAdvanced™ SYBR
® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Primer sequences are provided in
Supplementary Table S2.
2.8. Cathepsin B Activity Assay
ARPE-19 cells were seeded on 8-well chamber slides and treated with EBSS (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) and ZKSCAN3 siRNA (Dharmacon, Lafayette, CO, USA) at 100 nM for 48 h. Cathepsin B activity was determined using the Magic Red Cathepsin B Assay Kit (Immunochemistry). Briefly, Cells transfected with ZKSCAN3 siRNA for 48 h were incubated with 1× Magic Red dye for 1 h at 37 °C, rinsed with PBS and mounted. Cells were imaged by a Zeiss LSM800 microscope (20× objective). Images were quantified using the ImageJ software (NIH, Bethesda, MD, USA).
2.9. Lysosomal Function Assay
ARPE-19 cells were seeded in 8 chamber slide and treated with EBSS (Gibco; Thermo Fisher Scientific, Grand Island, NY, USA) and ZKSCAN3 siRNA (Dharmacon, Lafayette, CO, USA) at 100 nM for 48 h. LysoTracker Green DND-26 (Invitrogen, Waltham, MA) was used for lysosomal function activity examination. After 48 h, the cell medium was replaced with prewarmed (37 °C) probe-containing medium and incubated for 1 h in a 37 °C incubator. The image was examined by EVOS FL Auto (Life Technologies, Carlsbad, CA, USA). The slide was later mounted by Prolong Gold antifade reagent with DAPI (Life Technology, Carlsbad, CA, USA) examined by Zeiss microscope equipped with a camera (Axiocam Mrm; Carl Zeiss, Oberkochen, Germany) again.
2.10. Quantification and Statistical Analysis
Quantification of LC3 puncta was performed using ImageJ software (NIH, Bethesda, MD, USA). Briefly, the corrected total fluorescence intensity was calculated after background subtraction and the puncta were quantified using analyze particles plugin on ImageJ. Data is represented as mean LC3 puncta ± standard deviation of three independent experiments. Cathepsin B immunostaining was quantified by measuring corrected total cell fluorescence (CTCF), which was obtained by subtracting the product of area of selected cell and mean fluorescence of background readings from integrated density of each cell using ImageJ. CTCF is represented as mean fluorescence intensity ± standard deviation. Densitometry was measured for each band and represented relative to the corresponding loading control using ImageJ software. qRT-PCR data is quantified using the comparative or ΔΔCt method. qRT-PCR data is represented as fold change in the expression of a gene of interest relative to the reference gene. All data is presented as mean ± standard deviation. For statistical analysis, student’s t-test (two tailed) and one-way ANOVA with Tukey post hoc multiple comparison was used. Statistical analysis was performed using the Graphpad prism software.
4. Discussion
In recent years, remarkable progress has been made in our understanding of the molecular events that coordinately regulate the transcription of genes involved in the lysosomal and autophagy pathway. It is also well known that lysosomes are crucial for the proper completion of the autophagy process, and unsurprisingly lysosomal function and autophagy are co-coordinately regulated by two major transcription factors; ZKSCAN3 (zinc-finger protein with KRAB and SCAN domains 3) and transcription factor EB (TFEB) [
22,
23]. TFEB and ZKSCAN3 are known to possess opposing actions, suggesting that they function as on and off switches, respectively, to regulate lysosome biogenesis/function and autophagy in response to cellular stress [
23,
24].
Age-related decline in lysosomal enzyme activity due to accumulation of A2E contributes to the progressive accumulation of cellular material in the RPE [
7,
9]. Autophagy serves as a protective mechanism against cellular stresses such as oxidative stress, lipofuscin accumulation, and mitochondrial dysfunction [
4,
25]. In this manuscript, we elucidated the protective role of lysosomal and autophagic enhancement mediated by ZKSCAN3 suppression in the RPE. ZKSCAN3 is a transcription suppression factor that exerts its biological effects by binding to repressor domains in target genes and inhibiting both autophagy and lysosomal processes [
13]. Our studies show that in normal cells, ZKSCAN3 is predominantly nuclear and represses its target genes to maintain only basal levels of autophagy. When cells are stressed, nuclear levels of ZKSCAN3 are reduced, causing de-repression and induction of ZKSCAN3 target genes. SiRNA-mediated knockdown of ZKSCAN3 induces starvation-induced autophagy responses in ARPE-19 cells. We also observed an increase in acidic lysosomes and lysosomal Cathepsin B activity.
Furthermore, identifying the mechanisms that regulate the activity of ZKSCAN3 is critical to target ZKSCAN3 in order to induce autophagy and lysosomal function in the RPE. Previous studies showed that protein kinase C δ (PKCδ) acting through P38MAPK (Mitogen-activated protein kinase) phosphorylates ZKSCAN3, resulting in its nuclear export and alleviation of ZKSCAN3-mediated repression [
17]. Our results show that ARPE-19 cells treated with the P38MAPK inhibitor, SB203580, retains ZKSCAN3 in the nucleus even under conditions of nutrient deprivation. In addition to the PKC pathway, the mTOR-signaling pathway is known to be associated with autophagy. mTOR is a key regulator of the autophagy pathway and is known to affect the activity of several autophagy pathway kinases by phosphorylation [
26]. Inhibition of mTOR signaling by rapamycin has been used as a strategy to induce autophagy in order to promote cellular clearance in various aggregation-prone degenerative diseases [
27]. In addition, PKCδ-dependent and mTOR-independent mechanisms of autophagy and lysosomal regulation are explored. Recent studies have also shown that PKC activators induce PKC-dependent export of ZKSCAN3 from the nucleus and also promote clearance of amyloid β (Aβ) plaques in mouse models of Alzheimer’s disease [
17]. Our results support previous studies showing that activation of PKCδ through P38MAPK pathway can be used as a strategy to inhibit endogenous ZKSCAN3 in the RPE.
In summary, our studies have elucidated a role of the transcription suppressor, ZKSCAN3 in regulating autophagy and lysosomal function in the RPE. Our results have shown that knockdown of ZKSCAN3 induces autophagy and lysosomal function in the RPE. These studies have significant therapeutic potential as they lead to the identification of novel molecular targets that could be harnessed to induce cellular clearance processes in the RPE and protect RPE cell death. However, a recent study using a mouse model lacking ZKSCAN3 has shown that the expression of ZKSCAN3-regulated autophagy and lysosomal genes was not affected
in vivo [
14]. In mouse embryonic fibroblasts, loss of ZKSCAN3 did not result in an induction of autophagy upon nutrient withdrawal [
14]. These conflicting results could be due to the differences in ZKSCAN3 amino acid composition between human and mouse proteins, which likely contributes to the functional differences in protein function. These responses could also be specific to our siRNA-mediated knockdown approach.
Since, TFEB and ZKSCAN3 have opposing functions [
23], our future studies are directed towards elucidating reciprocal regulation of both the transcription factors under conditions of nutrient stress. We will also study whether changes in the expression of one transcription factor affects the expression of the other factor in the RPE. Furthermore, previous studies have shown that P38 MAPK regulates autophagy via the glycogen synthase kinase 3β (GSK3β) signaling pathway [
28]. Since GSK3β also effects TFEB signaling [
28], future studies are directed towards investigating the role of P38 MAPK pathway in co-regulation of TFEB and ZKSCAN3 in the RPE.