https://orcid.org/0000-0001-9198-5236
Figures
Abstract
The membrane peroxisomal proteins PEX11, play a crucial role in peroxisome proliferation by regulating elongation, membrane constriction, and fission of pre-existing peroxisomes. In this study, we evaluated the function of PEX11B gene in neural differentiation of human embryonic stem cell (hESC) by inducing shRNAi-mediated knockdown of PEX11B expression. Our results demonstrate that loss of PEX11B expression led to a significant decrease in the expression of peroxisomal-related genes including ACOX1, PMP70, PEX1, and PEX7, as well as neural tube-like structures and neuronal markers. Inhibition of SIRT1 using pharmacological agents counteracted the effects of PEX11B knockdown, resulting in a relative increase in PEX11B expression and an increase in differentiated neural tube-like structures. However, the neuroprotective effects of SIRT1 were eliminated by PPAR inhibition, indicating that PPARɣ may mediate the interaction between PEX11B and SIRT1. Our findings suggest that both SIRT1 and PPARɣ have neuroprotective effects, and also this study provides the first indication for a potential interaction between PEX11B, SIRT1, and PPARɣ during hESC neural differentiation.
Citation: Esmaeili M, Nasr-Esfahani MH, Shoaraye Nejati A, Safaeinejad Z, Atefi A, L. Megraw T, et al. (2024) PPARgamma dependent PEX11beta counteracts the suppressive role of SIRT1 on neural differentiation of HESCs. PLoS ONE 19(5): e0298274. https://doi.org/10.1371/journal.pone.0298274
Editor: Peng Gao, Army Medical University, CHINA
Received: August 20, 2023; Accepted: January 18, 2024; Published: May 16, 2024
Copyright: © 2024 Esmaeili et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: hESC, human embryonic stem cell; PBDs, peroxisomal biogenesis disorders; SIRT, sirtuins; PPARγ, PGC1α, PPARɣ coactivator-1α;Peroxisome proliferator-activated receptor γ; NF-κβ, nuclear factor kappa β; LXR, liver X receptor; PPRE, peroxisomal proliferator response element; mESC, mouse embryonic stem cell; DMEM/F12, Dulbecco’s modified Eagle medium /Ham’s-Nutrient Mixture F-12; KSR, ; ITS, insulin-transferrin-selenite; shRNA, Short hairpin RNA; shCtrl, Control shRN; MOI, multiplicity of infection; Dox, Doxycycline; DMSO, dimethyl-sulfoxide; RT-qPCR, real-time quantitative PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PVDF, olyvinylidenedifluoride; HRP, horseradish peroxidase; ANOVA, one-way analysis of variance; Acox1, acyl-coenzyme A oxidase 1; NPCs, Neural precursor cells; Pax6, paired box gene 6; PBS, phosphate-buffered saline; PMPs, peroxisomal membrane proteins; SEM, standard error of mean; Sox1, SRY-box containing gene 1; VLCFA, very long chain fatty acid
Introduction
Peroxisomes are a versatile class of ubiquitous membrane-bound organelles that respond dynamically to metabolic, developmental, and environmental cues by altering their morphology, abundance, enzyme content, and shape in eukaryotic organisms [1–5]. In mammals, peroxisomes harbor approximately 50 matrix enzymes involved in a diverse range of metabolic reactions, including very long chain fatty acid degradation, plasmalogen biosynthesis, glycolysis, and reactive oxygen species elimination [2,5,6]. Peroxisome biogenesis requires the coordinated action of over 30 peroxins, a group of peroxisomal proteins, among which the PEX11 family has been shown to play a direct role in peroxisome proliferation in yeast, plants, and mammals [1,3,7]. Mutations in PEX11B have been associated with peroxisome biogenesis disorders (PBDs) [1], and studies on animal models lacking either PEX11A or PEX11B have provided significant insights into peroxisome biology [3]. The spectrum of PBDs, such as Zellweger syndrome, highlights the critical role of peroxisomes in human health [8,9]. In mouse brain tissue, Pex11b deletion results in oxidative stress and neural phenotypes, although the severity is less pronounced than in homozygous animals [10]. Manipulation of PEX11B expression levels, either through overexpression or knock-down, alters the expression of peroxisomal-related genes and thus impacts peroxisome proliferation [11].
Sirtuins (SIRTs) are deacetylases that belong to the class III histone deacetylase family [12]). These enzymes, including seven isoforms (SIRT1 to SIRT7), vary in subcellular localization, substrate specificity, and response to stimuli [13]. SIRT1, the most well-characterized isoform, is expressed in the nucleus and regulates a variety of biological processes, such as gene expression control, oxidative stress, genome stability, homeostasis, aging, cellular proliferation, and metabolism [14], modulation of synaptic plasticity and memory formation in the adult brain [15]. Sirtuins, particularly SIRT1, possess neuroprotective characteristics [16] by deacetylating various substrates including tumor suppressor P53 [17], peroxisome proliferator-activated receptor gamma (PPARɣ), PPARɣ coactivator-1α (PGC1α) [18], nuclear factor kappa β (NF-κβ), and liver X receptor (LXR) [19].
PPARɣ, one of three significant isoforms in the nuclear receptor superfamily [20], is present in high levels in brain tissue [21,22] and with lower expression in heart, liver, and skeletal muscle [21]. PPARɣ activates transcription of target genes by binding to specific DNA regions called peroxisome proliferation hormone response elements (PPREs). Through transcriptional activation, PPARɣ regulates adipogenesis, energy balance, lipid biosynthesis [23], and neuroprotective mechanisms in neurophysiology [21].
In this study, we aimed to investigate the relationship between SIRT1, PEX11B, and PPARɣ, considering the established neuroprotective roles of PPARɣ [24,25], the ability of pioglitazone, as a PPARɣ agonist, to alleviate the effects of PEX11B knockdown [26], and the proposed direct substrate role of PPARɣ for SIRT1 as well as indirect effect of PPARɣ on SIRT1 via SIRT1-PGC1α axis [18,35]. Previously, we demonstrated that PEX11B knockdown reduced the expression of neuronal markers and peroxisomal-related genes during neural differentiation of mouse embryonic stem cell (mESCs) [26]. Here, we replicated and expanded on these findings by performing experiments using hESC culture and neural differentiation. To explore the interplay between PEX11B, SIRT1, and PPARɣ, we employed a SIRT1 inhibitor (EX-527) and observed that SIRT1 inhibition alleviated the blockade of neural differentiation caused by PEX11B knockdown. Moreover, the inhibitor of PPARɣ (GW9662) reversed the effect of SIRT1 inhibition, confirming that the influence of SIRT1 inhibition on neural tube formation was mediated by PPARɣ.
Materials and methods
Cell culture and neural differentiation
The human embryonic stem cell line RH6 [25] was cultured on Matrigel (Sigma-Aldrich, E127) and maintained in specific hESC medium consisting of Dulbecco’s modified Eagle medium /Ham’s-Nutrient Mixture F-12 (DMEM/F12, Gibco, 21331–020), supplemented with 20% knock-out serum (KSR, Gibco, 10828–028), 100 units/mL penicillin, 100 ng/mL bFGF (Royan Institute), 2 mM L-glutamine (Gibco, 25030–024), 1% insulin-transferrin-selenite (ITS, Gibco, 41400–045),100 μg/mL streptomycin (Gibco, 15070063),1% nonessential amino acids (Gibco, 11140–035), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, M7522). Cells were maintained at 37°C with 5% CO2 and passaged using accutase (Millipore, SCR005). Neural differentiation was performed using a previously described protocol [26] with some modifications. The culture medium was changed every other day after day 4, and neural tube-like structures were manually selected on day 14 and cultured in adherent Matrigel-coated dishes. After 7 days, neurite outgrowth was observed.
Vector construction
We employed lentiviral vectors, including pLVTHM and pLVPT-tTR-KRAB (Addgene), to introduce short hairpin RNA (shRNA) targeting PEX11B into the genome of hESCs, following the supplier’s protocol (Addgene, Cambridge, MA, USA; Table 1). Initially, the shRNA sequence directed towards PEX11B was integrated downstream of the tetO-H1 region in pLVTHM vector. Subsequently, the cassette was excised from pLVTHM containing shRNA and sub-cloned into pLVPT-tTR-KRAB, an expression vector that regulates transcription in a doxycycline-dependent manner. As such, two vectors were generated: pLVPT-tTRKRAB/ShPEX11B and pLVPT-tTR-KRAB/ShCtrl.
Generation of stable cell line using lentiviruses
Lentiviral particles were generated by transiently transfecting HEK293T cells with 2nd generation packaging vectors (PsPAX2 and pMD2G) along with the inducible vector pLVPT-tTR-KRAB containing shPEX11B or shCtrl. The transfection was carried out using lipofectamine LTX (Invitrogen) as previously described. After 72 h, the supernatant was collected, filtered, and concentrated. The titration of the lentiviral particles was carried out via FACS analysis (Becton Dickinson, Franklin Lakes, NJ, USA) according to a previously reported method [27]. Next, hESCs were seeded into 6-well plates and transduced with freshly prepared lentiviral particles at a multiplicity of infection (MOI) level of 10. Two days after transduction, the cells were treated with blasticidin (6 μg/mL) (Gibco, Grand Island, NY, USA) for two weeks until stable colonies appeared.
Dox, EX-527 and GW9662 treatments
To induce the expression level of shPEX11B and shCtrl 750 ng/mL doxycycline (Dox) we used (Dox, Clontech) during neural differentiation of hESCs. Following Dox treatment, the inhibitory effects of tTR-KRAB on nearby promotor activity was eliminated and shRNA was expressed [28]. EX-527 (6-chloro-2, 3, 4, 9-tetrahydro-1H-carbazole-1- carboxamide; Sigma-Aldrich, E7034) and GW9662 (Sigma-Aldrich, M6191) were dissolved in dimethyl sulfoxide (DMSO) with final concentrations of 5 mM and 10μM respectively. In all experiments, equal amount of solvent (vehicle) was used for controls.
RNA isolation and RT-qPCR analysis
Total RNA was isolated using the RNeasy Kit (Qiagen, 74004) and treated with DNaseI (Thermo Scientific, EN0521) to synthesize cDNA from 1 μg of total RNA using cDNA synthesis Kit (Thermo Fisher Scientific, K1622). real-time quantitative PCR (RT-qPCR) was performed using SYBR green Gene Expression Master Mix (TaKaRa, RR820Q) and 25 ng of cDNA on a Rotor-Gene 6000 thermal cycler (Corbett). The expression levels of all target genes were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene in three independent experiments. The data were analyzed using the ΔΔCt method [29]. The primer pairs for each gene were designed using Beacon Designer software (Version 7.2, USA) and synthesized by Metabion Company (Germany). The primer sequences for each gene are provided in Table 2.
Western blot analysis
Three separate cell cultures of hESCs were washed with PBS and directly lysed in TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA, catalog no: 93289) according to the manufacturer’s instructions. The protein content of the lysed cells was estimated using the Bradford method. Equal amounts of protein (30 μg) from each lysate were separated by SDS-12% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 162–0176). The primary antibodies were PEX11b (Assay Biotech, C17630, dilution 1:1000), SIRT1 (Abcam, AB110304, dilution 1:4000), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich, A2228, dilution 1:5000). The secondary antibody was HRP-conjugated goat anti-mouse IgG (Dako, P0447, dilution 1:5000). Protein bands were visualized using an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK). Finally, the intensity of each band was quantified using ImageJ software, and the results were normalized against the GAPDH band.
Results
Establishment of transduced stable hESCs lines using lentiviral vectors
hESCs were transduced with a lentiviral construct expressing shRNA against PEX11B (pLVPT-tTR-KRAB) and a control vector as previously described. Lentiviral particles, obtained from the supernatant fraction of HEK293T cell culture, were used for transduction of hESCs, with transfection and transduction efficiencies estimated at 99.8% and 60.1%, respectively, by flow cytometry (data not shown). Positive colonies were selected after treatment with blasticidin (6 μg/mL) and were confirmed by genomic insert check PCR before being treated with doxycycline (750 ng/mL) for two days. The stemness characteristics of stable cell lines were evaluated based on the expression levels of NANOG and OCT4, and no significant changes were observed (S1 Fig). The induced expression of shRNA with doxycycline resulted in a significant reduction in PEX11B expression compared to the control shRNA, and was further confirmed by a decrease in PEX11B protein levels via Western blot analysis (Fig 1).
A) During neural differentiation of transduced stable hESCs lines, the RNA level of PEX11B decreased significantly after Dox treatment. B) Relative protein expression was quantified and normalized with GAPDH. Displayed value bars are the mean of duplicate independent experiments± SEM. * indicates a significant difference at P <0.05.
Knock-down of PEX11B reduces expression of neural tube and neuronal markers and peroxisomal-related genes
Stable lines of hESCs containing PEX11B and control shRNAs were differentiated into neural cells, as shown in Fig 2A, through serum reduction. The relative expression levels of neural progenitor markers SOX1 and PAX6, as well as the neuronal marker TUJ1, were reduced upon knock-down of PEX11B, as illustrated in Fig 2B. The reduction in SOX1 and NESTIN expression levels was observed only on day 14, during neural tube formation, while PAX6 expression continued to decrease throughout the differentiation period (day 20). Additionally, transcript levels of peroxisomal-related genes were analyzed pre- and post-Dox treatment. Dox-induced knock-down of PEX11B significantly decreased the expression levels of PEX1, PEX3, PMP70, PEX7, and ACOX1, but not PEX13, as presented in Fig 2C. No changes in the expression profiles of these genes were observed in the control or untransfected lines.
A) schematic diagram of neural differentiation and Dox treatment (day 0). B) RT-qPCR for evaluation of SOX1, PAX6, NESTIN and TUJ1 RNA level before and after Dox treatment. C) RT-qPCR for estimation peroxisomal-related genes PEX1, PEX3, PMP70, PEX13, PEX7 and ACOX1 before and after Dox treatment. Relative expression was quantified and normalized with GAPDH. Displayed value bars are the mean of triplicate independent experiments± SEM. * represents significant difference at P <0.05.
SIRT1 inhibition enhances expression of markers of neural tube-like structures and peroxisomal-related genes in hESCs lines with PEX11B knocked-down
Considering the increased expression of SIRT1 during the time course of in vitro neural development of hESCs lines in the presence of Dox (Fig 3A), to explore the potential association between SIRT1 and PEX11B (Fig 3B and 3C), we used EX-527 and resveratrol to activate and inhibit SIRT1, respectively. Intriguingly, inhibiting SIRT1 with EX-527 and activating it with resveratrol caused an increase and decrease in the expression of PEX11B, respectively. When Dox-induced shPEX11B lines were treated with EX-527, the relative expression of markers for neural tube-like structures, including SOX1, PAX6, NESTIN, and the neuronal marker TUJ1 were increased (Fig 3D). Moreover, the number of neural tube-like structures also increased in Dox-induced shPEX11B cell lines treated with EX-527 (Fig 3E). Additionally, the mRNA levels of the peroxisomal-related genes ACOX1 and PMP70 were significantly increased after EX-527 treatment in shPEX11B cell lines during neural differentiation. These findings collectively suggest that SIRT1 has a negative regulatory effect on peroxisomal biogenesis genes during neural development in shPEX11B lines.
A) relative mRNA expression of SIRT1 before and after Dox treatment during neural differentiation. In neural precursor cells the expression level of SIRT1 was increased after Dox treatment. B) and C) mRNA and protein level of SIRT1 was decreased after Ex-257 treatment. D) relative expression of SOX1, PAX6, NESTIN and TUJ1 were increased after EX-527 and Dox treatment compared to Dox treatment alone. E) number of neural tube-like structure was increased after Ex-257 treatment. F) RT-qPCR for evaluation of peroxisomal-related genes PEX11B, ACOX1 and PMP70. Relative expression was quantified and normalized with GAPDH. Displayed value bars are the mean of triplicate independent experiments± SEM. * represents significant difference at P <0.05.
PPARɣ mediates the interaction between SIRT1 and PEX11B
To investigate whether PPARɣ mediates the relationship between SIRT1 and PEX11B, we employed GW9662, a PPARɣ antagonist, during neural differentiation of Dox-induced shPEX11B cell lines. Surprisingly, we observed no changes in the expression levels of neural tube-like markers and peroxisomal genes upon treatment with EX-527 after knockdown of PEX11B (Fig 4), suggesting that PPARɣ may not play a mediating role in this relationship. The western blot analysis of PEX11B (Fig 5) also validated the RT-qPCR results presented in Fig 4.
A) relative expression of PPARɣ before and after GW9662 treatment. B), C) and D) relative expression of neural progenitor cells and neuronal markers, number of neural tubes and peroxisomal-related genes during neural differentiation were decreased after GW treatment and EX-527 (EX) did not ameliorate the effects of PEX11B knock-down (Dox).
Discussion
The conserved regulatory function of the PEX11 protein family has been widely identified among the genes and proteins involved in peroxisome biogenesis [30]. PEX11B, in particular, has been identified as a key regulator of peroxisome elongation and division, as overexpression of PEX11B alone can induce these processes [31]. In contrast, a reduction in PEX11B gene expression has been shown to lead to a decrease in peroxisomal abundance [32]. Given the pivotal role of PEX11B in peroxisome biogenesis, we investigated the effects of PEX11B knockdown on neural differentiation of hESCs. Our findings demonstrate that knockdown of PEX11B expression during neural differentiation significantly reduces the expression of neural tube-like structure and mature neuronal markers, as well as the mRNA levels of peroxisomal-related genes such as PMP70, ACOX1, PEX1, PEX3, and PEX7 in neural tube-like and neuronal cells. However, the relative expression of PEX13 was not significantly change during neural differentiation. Taken together, our data, in conjunction with our previous study [26], suggest that neural precursor cells are particularly sensitive to the loss of PEX11B, resulting in a reduction in the size of embryoid bodies (EBs) from mESCs and a decline in the number of neural tube-like structures from hESCs, ultimately leading to a reduction in neurogenesis during in vitro neural differentiation. In mouse embryonic fibroblasts and undifferentiated muscle cells, SIRT1 has been reported to control the proliferation, cell cycle arrest, and differentiation as a redox sensor [33]. Furthermore, SIRT1 expression was shown to decrease during the differentiation and maturation of embryonic cortical neurons [34,35]. Building on these findings, we evaluated the expression levels of SIRT1 during neural differentiation of hESCs and shPEX11B lines. Our results revealed that the mRNA levels of SIRT1 in neural tube-like structures increased after PEX11B knockdown. We then investigated the effects of increased expression levels of SIRT1 in neural progenitors by using EX-527 as a SIRT1 inhibitor. The expression levels of neural tube and neural markers, as well as peroxisomal-related genes, were increased following SIRT1 inhibition with EX-527 treatment. Moreover, the abundance of neural tube-like structures was significantly increased. We propose that inhibition of SIRT1 improves neural differentiation by increasing the expression levels of PEX11B, which enhances peroxisomal biogenesis through the expression of peroxisomal-related genes. consequently, we hypothesized that PPARɣ, which activates the transcription of PEX11B [26] while is also considered as an inhibitory substrate of SIRT1 [18], may serve as the interface mediator between PEX11B and SIRT1. To test this hypothesis, we used GW9662 as an antagonist of PPARɣ. Interestingly, the effect of SIRT1 inhibition on elevating PEX11B expression level was eliminated by treatment with GW9662.
While GW9662 itself was found to reduce biomarkers expression of neural precursor and neuron cells, no additional effect was observed when it was combined with Dox. Our results suggest that the inhibition of SIRT1 may partially compensate for the effects of PEX11B knockdown, and that PPARɣ is likely the mediator between PEX11B and SIRT1. These findings confirm the neuroprotective effects of SIRT1 and PPARɣ previously reported in the literature, and this is the first report that demonstrates a potential interaction between PEX11B and SIRT1, which warrants further investigation such as immunofluorescence localization detection of PEX11B-SIRT1-PPARɣ axis (Fig 6).
A) normal neural differentiation. B) PEX11B knock-down caused to decrease of number of neural tubes and neurogenesis. C) amelioration of damages caused by PEX11B knock-down through inhibition of SIRT1. D) inhibition of PPARɣ reverses the ameliorative effects of inhibition of SIRT1. Accordingly, it seems that the interplay between PEX11B and SIRT1 is mediated by PPARɣ.
Supporting information
S1 Fig. mRNA level of NANOG and OCT4 before and after transfection.
Original western blot figures related to Fig 1, Fig 3 and Fig 5.
https://doi.org/10.1371/journal.pone.0298274.s001
(DOCX)
Acknowledgments
We are grateful to all colleagues who helped us in conducting and completing this study.
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