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. 2024 Mar 3;38(2):567–573. doi: 10.21873/invivo.13475

Exosome Secretion and Cellular Signaling Change in a Fabry Disease Cell Model Induced by Gene-silencing

SANG HYUN PARK 1, DAE HAN LEE 2, SOON AE KIM 2
PMCID: PMC10905446  PMID: 38418159

Abstract

Background/Aim

Fabry disease (FD) is caused by α-galactosidase A (AGA) deficiency, which ultimately leads to the intracellular accumulation of globotriaosylceramide (Gb3). Exosomes play a role in maintaining cellular homeostasis by clearing damaged or toxic materials, including proteins. In the process of excessive accumulation of intracellular Gb3 in Fabry disease, it may be suggested that exosomal secretion of Gb3 increases to preserve cell homeostasis. This study sought to determine how exosomal secretion and cell signaling change in an FD cell model produced by gene silencing.

Materials and Methods

HEK293T cells were transfected with plasmids carrying shRNA against the GLA gene to produce the FD cell model. A recombinant AGA, agalsidase-beta, was used to evaluate the effect of enzyme replacement therapy (ERT) on exosomal secretion and cell signaling.

Results

Exosome secretion was significantly increased in the Fabry disease cell model compared to the control vector cell model, and significantly decreased after agalsidase-beta treatment. The FD cell model showed higher reactive oxygen species (ROS) production and p53 protein expression compared to the control vector cell model.

Conclusion

Increased exosomal secretion in Fabry disease may be a cellular mechanism to avoid excessive and cytotoxic accumulation of Gb3 in lysosomes through intracellular signaling, including increased p53 expression.

Keywords: Fabry disease, HEK293T cell, gene silencing, exosome, oxidative stress, p53

Fabry disease (FD) is an X-linked inherited lysosomal storage disease (LSD) induced by decreased activity of the enzyme α-galactosidase A (AGA), which is encoded by the galactosidase alpha (GLA) gene (1,2). Absent or markedly reduced AGA activity leads to a progressive intracellular accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in the plasma and in various cell types through the body, resulting in multisystem disease (3).

The exact molecular mechanism by which the Gb3 accumulation gives rise to cellular damage is not yet known. Recent studies have suggested that Gb3 accumulation may play a key role in the pathogenesis of FD. In brief, it includes altered turnover of the extracellular matrix, inflammatory cytokine production, myocyte hypertrophy, and interference with secretion. Other pathologies, defined at the molecular and biochemical levels, include the induction of oxidative stress and up-regulation of cell adhesion molecule by Gb3 deposition in the FD endothelial cells (4), and impairment of autophagy (5).

The current standard treatment for FD is enzyme replacement therapy (ERT) with recombinant human AGA, which has been approved since 2001. ERT aims at maintaining organ function and/or ameliorating organ dysfunction. ERT has been shown to significantly alleviate the cardiac, renal, and neuropathic effect of FD (6).

Exosomes are small membrane microvesicles with a diameter of 50-100 nm that are actively secreted by most cell types. They are produced by inward budding of the early endosomal membrane, resulting in intraluminal vesicles within multivesicular bodies (7), which are either routed to lysosomes for the degradation of proteins and lipids or fuse with the plasma membrane leading to the secretion of their intraluminal vesicles as exosomes into the extracellular environment. Because multivesicular bodies are components of the lysosome-endosomal system, the lysosomal dysfunction in LSDs like FD could promote an increased exosomal secretion. Once the exosomes are secreted to the extracellular space, they are likely absorbed by the neighboring cells or transferred to circulating fluids (8-12).

Exosomes serve many functions, such as intercellular communication and secretion of unnecessary membranes and cytosol (13-19). A previous in vitro study suggested that exosomal cholesterol secretion may serve as a cellular mechanism to partially bypass the late endosomal trapping of cholesterol observed in Niemann-Pick type C disease, a type of LSD like FD (20). It is still unknown whether exosomal Gb3 secretion is increased in patients with FD and thus whether the accumulated Gb3 can be removed from the cell via exosomes to avoid Gb3 toxicity under lysosomal dysfunction.

There are several lines of evidence showing that oxidative stress may be involved in the pathophysiology of FD (21-23). In addition, a previous study provided direct evidence that excessive intracellular Gb3 induces oxidative stress via the increased production of reactive oxygen species (ROS) in vascular endothelial cells (24). It is well known that ROS can attack proteins, lipids, and DNA. A recent human study revealed that patients with FD have a higher level of basal DNA damage than healthy controls and that this damage includes oxidation of purines (25). In general, after DNA damage, the transcription factor p53 is activated to enhance or repress the transcription of many genes (26,27). Yu et al. reported that the p53 pathway regulates the production of exosomes and their secretion from the cells into the medium (28). A p53-regulated gene product, TSAP6, was shown to be sufficient to induce the secretion of exosomes in cells undergoing a p53 response to the stress. It can be suggested that in FD, exosome secretion may be increased via the p53 response to oxidative stress induced by intracellular Gb3 accumulation.

So far, there is no reported in vivo or in vitro study that has addressed exosome secretion or exosomal Gb3 secretion in FD. We postulated that such secretions to the extracellular milieu would be increased in this disease. We also sought to determine how oxidative stress-mediated cell signaling would change in FD. To study these issues, we used gene silencing with short-hairpin RNA (shRNA) to generate an in vitro cell model of AGA knockdown in the human embryonic kidney (HEK) 293T cell line. The HEK293 and HEK293T cell lines have been used as models of epithelial kidney cells in studies on renal physiology.

Materials and Methods

Materials. Fetal bovine serum (FBS) was purchased from Alphabioregen (Boston, MA, USA) Phosphate-buffered saline (PBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Penicillin-streptomycin (10,000 U/ml) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The MUSE™ Oxidative Stress Kit was purchased from Merck Millipore (Darmstadt, Germany). The Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MN, USA). The RIPA Lysis and Extraction Buffer was purchased from Thermo Fisher Scientific. The Bradford assay solution was purchased from Bio-Rad (Hercules, CA, USA). The alpha galactosidase antibody was purchased from Abcam (Cambridge, MA, USA) and the goat anti-rabbit IgG antibody from Santa Cruz Biotechnology (Dallas, TX, USA). Agalsidase-beta (Fabagal®) was purchased from ISU ABXIS (Seongnam, Republic of Korea). Ultracentrifuge tubes were purchased from Hitachi Koki (Tokyo, Japan).

Cell culture. The HEK293T cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Before culturing, the cells were tested for mycoplasma contamination. The cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. To prepare the complete medium, the FBS was first depleted of exosomes by ultracentrifugation at 100,000×g for 1 h at 4˚C.

Cell viability assay. HEK293T cells (2×104 cells/well) were seeded into a 96-well plate and cultured for 24 h before the addition of the human recombinant AGA (agalsidase-beta) in complete medium containing 5% FBS. Cell viability was measured using the Cell Counting Kit-8 according to the manufacturer’s instructions. Absorbance was measured using a Multiscan FC microplate photometer (Thermo Fisher Scientific). All experiments were performed in triplicate.

Gene silencing by shRNA-mediated knockdown. The shRNA plasmid TRCN0000299915, carrying an shRNA sequence (5’-CCGGCGCTCTTATACCATCGCAGTTCTCGAGAACTGCGATGGTATAAGAGCGTTTTTG-3’) targeting the galactosidase alpha (GLA) gene (NM_000169.2-1197s21c1) at nucleotide 549, was designed for the transient silencing of AGA. The glycerol stock solution used was from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

HEK293T cells in the log phase of growth were seeded into 6-well plates in growth medium. After overnight incubation, the cells were transfected with 2.5 μg of the purified shRNA plasmid using 1.5 μl of Omni Ultra-Transfect™ Transfection Reagent and Opti-MEM® I reduced Serum Media (Life Technology, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, the cells were trypsinized and reseeded in a 100-mm dish in antibiotics-free growth medium supplemented with 0.6 μg/ml puromycin (selection medium) for six days. Selection in medium containing 1.0 μg/ml puromycin was carried out for an additional seven days, during which the cells were re-fed with this selection medium every other day. The surviving colonies were expanded in growth medium without puromycin and tested for AGA activity. The colony with the lowest activity was subcloned by serial dilution, and colonies were expanded for 13 days in selection medium containing 3.0 μg/ml puromycin.

Four additional shRNA plasmids (TRCN0000299979, TRCN0000303790, TRCN0000310775, and TRCN0000083591) were designed for the shRNA-mediated silencing of AGA. Four HEK293T cell lines transfected with the above plasmids were then incubated and expanded as described above.

To control for nonspecific effects, HEK293T cells were also transfected with a control shRNA plasmid that encoded a scrambled shRNA sequence (also referred as an empty vector) that would not lead to the specific degradation of any known cellular mRNA (Control shRNA Plasmid-A, Santa Cruz Biotechnology), using the same procedure as used for the other shRNA plasmids. The cell line created with the control shRNA plasmid was designated as the control vector (CV) cell model in this study.

Protein isolation. The transfected cells (1×106) were lysed with 500 μl of RIPA buffer containing 5 μl of protease inhibitors, for 30 min on ice with vortexing every 10 min. After sonication, the lysates were centrifuged at 14,000×g for 15 min at 4˚C and the supernatant was used for further analyses.

Western blot analysis. The protein content of the cell extracts was determined using Bradford assay (Bio-Rad). The Proteins (30 μg per lane) and molecular weight markers (Thermo Scientific) were separated in SDS-PAGE using 10% Bis-Tris polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and probed with rabbit anti-alpha-galactosidase antibodies at a dilution of 1:5,000 in 5% bovine serum albumin (BSA). The protein bands were then labeled with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies and visualized using the enhanced chemiluminescence substrate (Merck Millipore). Chemiluminescence was detected using a ChemiDoc™ imaging system (Bio-Rad). Rabbit anti-actin antibody (Cell Signaling Technology, Danvers, CO, USA) was used as the positive control.

Immunocytochemistry. The control HEK293T cells and FD model cells were dispensed (at 2×105 cells) onto cell culture chamber slides (Merck Millipore) and fixed with 2% formaldehyde and 2.5% glutaraldehyde in PBS for 10 min at room temperature. After blocking with 0.1% Tween 20 in 3% BSA in PBS, the cells were incubated with the rat anti-human CD77/Gb3 monoclonal antibodies (diluted 1:1,000, Abcam), followed by the Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibody (diluted 1:1,000, Abcam) in 3% BSA-containing PBS for 1 h. All cells were counterstained with 4’,6-diamidino-2-phenylindole and observed under a KI2000 F fluorescence microscope (OPTINITY, Korea Lab Tech, Seongnam, Republic of Korea) using a 40× objective lens.

Exosome isolation. Exosomes from the media collected from the FD model cell cultures were isolated through differential centrifugation (at 270×g for 3 min to eliminate cells and large cellular debris) and filtration (0.22-μm syringe filter). The exosomes were pelleted by centrifuging the supernatant at approximately 100,000×g for 1 h and the supernatant was removed. The pellets were resuspended in exosome-free DMEM.

Size distribution and zeta-potential of exosomes. The size and zeta potential of the exosomes were measured using the ZetaView® nanoparticle tracking analyzer (Particle Metrix GmbH, Meerbusch, Germany).

Exosomal Gb3 analysis. The cells (1×107 cells/well) were seeded into a T-175 flask with 25 ml of exosome-free DMEM and incubated for 24 h to obtain a stable culture. After three days, the cell culture medium was harvested and the cells were replenished with fresh exosome-free DMEM. Thereafter, every 6 days, the same medium harvesting and replenishing procedures were repeated until the total volume of harvested culture media was 1 l. The culture media were centrifuged at 270×g for 3 min to eliminate cells and large cellular debris and filtered through 0.22-μm bottle-top filters. The exosomes were pelleted by centrifuging the supernatant at approximately 100,000×g for 1 h and the supernatant was removed. The pellets were resuspended in 1 ml of exosome-free DMEM, sonicated 10 times at 20 kHz on the ice (for 2 s each time), and stored at 4˚C. Exosomal Gb3 analysis was performed using an AB SCIEX API 4000™ liquid chromatography tandem mass spectrometer (AB SCIEX, Toronto, ON, Canada). The exosome pellet and cell buffer were prepared in a total volume of 1 ml. The mixture was stirred for 30 s using a vortex mixer and for 30 s using ultrasonic wave. Then, after centrifugation at 100,000×g for 5 min, the supernatant was transferred to a glass vial for analysis on a FAMOS autosampler. A 20-μl volume was introduced into the tandem mass spectrometer fitted with a C8 guard column for tandem mass spectrometry analysis. The analytical data were processed using Analyst software (version 1.4.2) from Applied Biosystems/MDS Sciex (Applied Biosystems, Foster City, CA, USA).

Treatment of the FD model cells with agalsidase-beta. Agalsidase-beta was freshly reconstituted with RPMI medium and then diluted further with complete RPMI medium before use. Cultured FD model cells were exposed to variable concentrations of the diluted agalsidase-beta. The influence of agalsidase-beta on oxidative stress and exosome count was assessed using a single dose for a shorter period of incubation.

Oxidative stress analysis. Cell samples were harvested with trypsin and prepared at 1×106/ml in 1× Assay Buffer in Museâ Oxidative Stress Kit (Merck Millipore). Prepared Muse Oxidative Stress Reagent working solution in the Kit were added to each tube and incubation was carried out for 30 min at 37˚C. Oxidative stress was analyzed by fluorescence-activated cell sorting (MUSE, Merck Millipore).

Statistical analysis. The Mann-Whitney U-test was used to evaluate the difference among means. Comparison of ratio among models in exosome count, ROS generation ad p53 expression were also performed using Mann-Whitney U-test. All statistical analyses were performed using SPSS version 22.0 (IBM, Armonk, NY, USA) and Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at a probability level of p<0.05.

Results

Validation of the FD cell models after shRNA-mediated GLA silencing. Five different cell lines were created using five different shRNA sequences and further processed to obtain colonies in order to develop the best cell model of FD. Figure 1A presents the western blot measurements of AGA activity in the five different colonies. Among them, one colony (F1) that showed the lowest AGA activity was selected as the most suitable FD cell model (FC) and used in this study. The HEK293T cell line that was not transfected with shRNA was named as the control (C) cell model. And the HEK293T cell line that was transfected with the empty vector was named as the control vector (CV) cell model.

Figure 1. Expression of α-galactosidase A (AGA) and Gb3 in cultured control, control vector, and Fabry disease cell models. A) Western blot for AGA in control (C), control vector (CV), and Fabry disease cell models (F1 to F5). The control (C) cell model comprised HEK293T cells that were not transfected with shRNA or the empty vector. The control vector (CV) cell model comprised HEK293T cells that were transfected with the empty vector without shRNA. Five colonies of HEK293T cells were generated by transfection with different shRNA plasmids, corresponding to five Fabry disease cell models (numbered from F1 to F5). B) Immunofluorescence staining for Gb3 in control (C), control vector (CV), and Fabry disease cell (FC) models. The FC model by F1 cells shows bright labeling within the cell body, indicating immunostained Gb3 molecules.

Figure 1

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Assessment of Gb3 accumulation using immunofluorescence staining in the different cell models. Figure 1B presents the Gb3 immunostaining results in the various cell models. The Gb3 staining intensity was obviously higher in the FD model cells than in the C and CV model cells. This indicated significant lysosomal accumulation of Gb3 in the FD model cells but not in the control cells.

Exosome size and number in the cultured FD cell model. The size and number of exosomes were measured in the various cell models as described in the Materials and Methods section. The exosome secretion level in the FD cell model was significantly higher than that in the CV cell model, but it decreased to the level of the CV cell model after ERT (Figure 2). There was no difference between the CV and FD cell models with respect to exosome size (Data not shown).

Figure 2. Relative exosome secretion by Fabry disease model cells (FC) and by Fabry disease model cells treated with enzyme replacement therapy (FC-ERT), compared with that by control vector (CV) model cells. The data are presented as the mean±SEM. The Mann-Whitney Utest was used to calculate the statistical significance of differences between the models (*p<0.05 for CV vs. FC and FC vs. FC-ERT).

Figure 2

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Oxidative stress in cultured Fabry cells. We initially evaluated ROS production in the CV and FD cell models in order to determine whether FD would lead to greater oxidative stress in the HEK293T cells. In addition, we evaluated whether ERT by agalsidase-beta could reduce the oxidative stress. The results are presented in Figure 3. Intracellular ROS generation was significantly increased in the FD model cells compared with that in the CV model cells (**p<0.001). After ERT, ROS generation in the FD model cells was significantly decreased compared with that before ERT, but it was not normalized to the CV cell level (*p<0.05 for Fabry disease Cell vs. Fabry disease Cell-ERT).

Figure 3. Relative reactive oxygen species (ROS) generation in Fabry disease model cells (FC) and in Fabry disease model cells treated with enzyme replacement therapy (FC-ERT), compared with that in control vector (CV) model cells. The data are presented as the mean±SEM. The Mann-Whitney U-test was used to calculate the statistical significance of differences between the models (*p<0.05, **p<0.001).

Figure 3

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Cell signaling related to exosome secretion in the FD cell model. Western blotting showed a remarkably lower level of AGA expression in the FD cell model than in the CV cell model (Figure 4). p53 expression was significantly increased in the FD model cells compared with that in the CV cells (*p<0.05). After treatment with agalsidase-beta, the AGA level increased albeit the difference was not statistically significant.

Figure 4. Western blot analysis of the alpha-galactosidase A (AGA) and p53 protein levels in control vector (CV) model cells, Fabry disease model cells (FC), and Fabry disease model cells treated with enzyme replacement therapy (FC-ERT). A) Representative western blot of AGA & p53 expression in the indicated cell models. B) Quantification of the p53 protein expression levels in the indicated models. The data in the graphs are the mean±SEM. The Mann-Whitney U-test was used to calculate the statistical significance of differences between the models (*p<0.05).

Figure 4

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Exosomal Gb3 secretion in cultured FD cell model. The measurement of exosomal Gb3 in exosomes obtained from the media harvested for two months was not successful. The measured values of exosomal Gb3 were lower than the detection limit of liquid chromatography tandem mass spectrometry analysis.

Discussion

Our study showed that exosome secretion was increased in the FD cell model relative to that in the CV cell model. To the best of our knowledge, no specific research has been undertaken into the exosome dynamics using in vivo or in vitro models of FD. Our study is the first to show the altered exosome secretion in the FD cell model compared with the control cell model. Exosome secretion was significantly increased in the FD cells compared with that in the wild-type cells, with no difference in exosome size. This result infers that Gb3 secretion would be increased in the FD cell model if it is excreted through exosomes. In the FD cell model, the increased exosome secretion may be a cellular mechanism to avoid the toxic effects caused by excessive Gb3 accumulation.

A previous study reported that accumulated Gb3 induced oxidative stress in a dose-dependent manner in an endothelial cell model of FD (29), although the mechanism was unclear (4). In addition to the in vitro data, previous clinical studies have observed excessive ROS formation in patients with FD (21,22,30). At the cellular level, ROS overproduction damages DNA, lipids, and proteins through oxidation and leads to cellular dysfunction (31). A recent human study reported that the patients with FD presented significantly higher levels of basal DNA damage and oxidative damage to the purines (25). Another recent in vitro study using HEK293T cells showed that globotriaosylsphingosine (lyso-Gb3)—as an additional accumulating glycosphingolipid in FD—induced DNA damage through oxidation of purines and pyrimidines (32). Our study also showed that oxidative stress was higher in the FD cell model than in the CV cell model, and decreased after ERT.

The factors associated with exosome secretion are not well understood, although roles have been reported for p53 (28,33), ceramide synthesis (34), calcium (35), and acidosis (36). Among them, the p53 protein responds to stress signals, such as oxidative stress, by regulating the transcription of various genes (26). A previous study suggested that exosome production by cells was regulated by the p53 pathway (28). The study showed that the p53-regulated gene product TSAP6 increased exosome production in cells undergoing a p53 response to stress. Our study showed that the p53 expression level was higher in the FD cell model than in the CV cell model. The increased exosomal secretion in the FD cell model in our study might have been induced by the increased p53 expression resulting from increased oxidative stress.

The HEK293T cell line that derives from the HEK293 cell line expresses a mutant version of the SV40 large T antigen. This cell line allows for the amplification of transfected plasmids and the extended temporal expression of a desired gene product. It is very commonly used in studies on the biology of protein expression. HEK293T cells are appropriate for studies of Gb3 accumulation in FD cells that are transfected with an shRNA plasmid. However, HEK293T cells may be inappropriate for the cell biology study of topics such as p53 cellular signaling, since expression of the tumor suppressor genes Rb and p53 is suppressed by the expression of the E1A and E1B oncoproteins of adenovirus. In this study, we focused on the exosome secretion changes in the FD cell model, where it was necessary to select the HEK293T cell line owing to its ease of genetic modification manipulation and study of exosome secretion. However, various additional in vitro cell line models for FD that originate from organs such as the heart and brain may be needed.

We tried to assess whether there would be a difference in exosomal Gb3 secretion between the FD and CV cell models. However, despite collecting a large amount of media, the Gb3 concentration in the exosomes was lower than the range detectable by high-performance lipid chromatography tandem mass spectrometry. Thus, it was difficult to conclude whether exosomal Gb3 release was increased in the FD cell model compared with its level in the C or CV cell models, although exosome release was definitely higher in the FD cell model. However, if intracellular Gb3 is released to the extracellular milieu via exosomes, it is reasonable to speculate that the increased exosome release in the FD cell model will result in increased exosomal Gb3 secretion. Further studies on exosomal Gb3 secretion are required to investigate this Gb3 pathway. Therefore, additional in vitro and in vivo research studies are needed to investigate the differences of Gb3 accumulation in patients with FD with the same genetic defect.

Conclusion

In summary, a cell model of FD, produced by gene silencing, showed that exosome secretion was significantly increased in the FD cell model compared with control cell models, and decreased after treatment with agalsidase-beta, the current standard treatment for FD. The expression of p53, which responds to oxidative stress and is known to activate exosome secretion, was higher in the FD cell model than in the CV cell model. Our results suggest that the increased exosome secretion in FD may have resulted from p53 cellular signaling, and it may be a salvage mechanism to remove the accumulated Gb3 within storage cells under lysosomal dysfunction. Further studies on exosomal Gb3 secretion are required to understand this Gb3 secretion pathway.

Funding

This research was supported by EMBRI Grants 2016EMBRIDJ0004 from Eulji University.

Conflicts of Interest

The Authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors’ Contributions

S.H.P: Conceptualization, Methodology, Writing-Original Draft, Writing-Review & Editing. D.H.L: Investigation, Data Curation, Writing-Review & Editing. S.A.K: Conceptualization, Methodology, Resources, Writing-Review and Editing & Supervision.

Acknowledgements

The Authors are grateful Prof. Myoung-Shin Lee gave a lot of help and guide to build Fabry cell model and methodology for the exosome study.

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