GPR183 Regulates 7α,25-Dihydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell

7α,25-dihydroxycholesterol (7α,25-DHC) is an oxysterol synthesized from 25-hydroxycholesterol by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) and is a monooxygenase (oxysterol-7α-hydroxylase) expressed under inflammatory conditions in various cell types. In this study, we verified that 7α,25-DHC-induced oxiapoptophagy is mediated by apoptosis, oxidative stress, and autophagy in L929 mouse fibroblasts. MTT assays and live/dead cell staining revealed that cytotoxicity was increased by 7α,25-DHC in L929 cells. Consequentially, cells with condensed chromatin and altered morphology were enhanced in L929 cells incubated with 7α,25-DHC for 48 h. Furthermore, apoptotic population was increased by 7α,25-DHC exposure through the cascade activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in the intrinsic pathway of apoptosis in these cells. 7α,25-DHC upregulated reactive oxygen species (ROS) in L929 cells. Expression of autophagy biomarkers, including beclin-1 and LC3, was significantly increased by 7α,25-DHC treatment in L929 cells. 7α,25-DHC inhibits the phosphorylation of Akt associated with autophagy and increases p53 expression in L929 cells. In addition, inhibition of G-protein-coupled receptor 183 (GPR183), a receptor of 7α,25-DHC, using GPR183 specific antagonist NIBR189 suppressed 7α,25-DHC-induced apoptosis, ROS production, and autophagy in L929 cells. Collectively, GPR183 regulates 7α,25-DHC-induced oxiapoptophagy in L929 cells.


Introduction
Oxysterols are cholesterol derivatives with a steroid backbone composed of seven carbon molecules with methylheptyl side chains [1]. They are synthesized from cholesterol by enzymatic and/or non-enzymatic pathways through encounters with reactive oxygen species (ROS) during sterol metabolism [2,3]. Oxysterols are bioactive molecules that are closely associated with the regulation of cholesterol homeostasis, lipid metabolism, and intermediates in the synthesis of bile acid and steroid hormones, and they act as ligands for nuclear and G-protein-coupled receptors (GPCR) [3]. Although oxysterols are involved in various physiological processes including apoptosis [4], autophagy [5], and prenylation of proteins [6], their effects remain largely unknown [7]. Previous studies have reported that certain oxysterols are physiological risk factors associated with age-related disorders, including cardiovascular and neurodegenerative diseases, osteoarthritis, and tumorigenesis [8][9][10].  is an oxysterol with a hydroxyl group at the 25th carbon of cholesterol that is mediated by cholesterol-25-hydroxylase, which is produced by ROS [11]. As shown in Figure 1, 7α,25-dihydroxycholesterol (7α,25-DHC) is an oxysterol formed from 25-HC by cytochrome P450 family 7 subfamily B member 1 (CYP7B1), also known as steroid 7α-hydroxylase [12]. In particular, 7α,25-DHC has been identified as a potential natural ligand for GPR183 (Epstein-Barr virus-induced gene 2) [13]. Recent studies have reported that the 7α,25-DHC-GPR183 axis is closely associated with immune regulation, including innate and adaptive immunity [13,14], regulation of neuropathic pain in the spinal cord [15], and regulation of cytokine release through modulation of proinflammatory signaling in astrocytes [16]. Furthermore, studies have also reported that 7α,25-DHC participates in chemokine-induced migration of B, T, and dendritic cells [13]. However, the physiological and biological roles of 7α,25-DHC are poorly understood compared with those of 25-HC. Although many studies reported that 25-HC, an oxysterol precursor of 7α,25-DHC, induced apoptosis in various types of cell including human aortic smooth muscle cells [17], human keratinocytes [18], human colorectal cancer DLD-1 cells [19], adult rat Leydig cells [20], mouse macrophages [21], and rat chondrocytes [10], the cytotoxic properties of 7α,25-DHC remain largely unknown. Furthermore, Bartlett et al. reported that the 7α,25-DHC receptor regulates interferons and autophagy in primary human monocytes during Mycobacterium tuberculosis infection [22]. Recently, cytotoxic dosages of oxysterols have been shown to induce cell death with complex apoptotic characteristics, oxidative stress, and autophagy, defined as oxiapoptophagy [23]. We hypothesized that 7α,25-DHC, a downstream oxysterol of 25-HC that is cytotoxic to various cell types, might induce apoptotic cells involved in oxidative stress and autophagy. Fibroblasts are the most common cells to compose the connective tissues through the formation of stroma in animals [24]. Hence, an in vitro cytotoxicity assay was developed to verify the toxicity of reagents or materials based on fibroblasts [25]. Especially, the L929 fibroblast cell line is specialized to use the toxicity assay [25].
Therefore, this study aimed to evaluate 7α,25-DHC-induced oxiapoptophagic cell death that is regulated by GPR183 in the L929 mouse fibroblast cell line.

Cell Death Is Mediated by the Intrinsic Pathway of Apoptosis in L929 Cells Incubated with 7α,25-DHC
Western blot was performed using antibodies against pro-or anti-apoptotic molecules in L929 cells incubated with 1, 5, 10, or 25 µg/mL 7α,25-DHC for 48 h (Figure 5a,b). Antiapoptotic molecules, such as B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), were decreased by 7α,25-DHC in L929 cells. In contrast, BH3 interacting-domain death agonist (Bid), a pro-apoptotic molecule belonging to the intrinsic apoptosis pathway, was decreased in L929 cells incubated with 7α,25-DHC. These data indicate that Bid was cleaved into a membrane-targeted death ligand, truncated Bid (tBid). Sequentially, the expression of Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist killer (Bak), and Bcl-2 associated agonist of cell death (Bad) induced the activation of caspase-9 in L929 cells incubated with 7α,25-DHC ( Figure 5a). Furthermore, activated caspase-9 induced the cascade activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) to induce cell death (Figure 5b,c). Our results demonstrate 7α,25-DHC-induced intrinsic apoptosis in L929 cells.  . 7α,25-DHC-induced cell death was involved with the intrinsic apoptosis pathway in L929 cells. L929 cells were incubated with 1, 5, 10, and 25 µg/mL 7α,25-DHC for 48 h. Total proteins were extracted using cell lysis buffer and then quantified by BCA assay kit. Sequentially, equal concentrations of protein samples were loaded onto SDS-PAGE gels to perform Western blotting using antibodies against pro-and antiapoptotic molecules. Thereafter, immunoreactive bands were exposed by ECL solution and acquired under microChemi 4.2. Results are presented as the relative ratio of target protein/β-actin, ** p < 0.01. β-actin was used as an internal control.

Discussion
Cholesterol, being a sterol, is not only an essential organic component that maintains the integrity and fluidity of mammalian cell membranes, but it is also involved in cell growth and proliferation through the biogenesis and functions of the cell membrane [26][27][28]. Furthermore, cholesterol not only serves as a precursor of bile acids, steroid hormones, and vitamin D [29], but it also acts as a ligand for various receptors, including nuclear receptors and GPCRs [30][31][32]. However, cholesterol, which is highly susceptible to oxidation, tends to form various oxysterol derivatives with polar groups such as hydroxy, keto, hydroperoxyl, epoxy, or carboxyl groups through either enzymatic activity, auto-oxidation, or both [33].
The enzymatic synthesis of oxysterol is carried out by oxidoreductases, hydrolases, and transferases. Oxysterols synthesized by these processes generally contain oxidized side chains [34]. Hence, genetic alterations in these enzymes are closely involved in carcinogenesis through abnormal synthesis of oxysterols from cholesterol [35,36]. Oxysterols synthesized by non-enzymatic processes associated with auto-oxidation of cholesterol have various types of molecules such as singlet oxygen, hydrogen peroxide, hydroxyl radicals, and ozone in their sterol rings. Furthermore, certain oxysterols, including 7-ketocholesterol (KC), 7α-HC, 7β-HC, and 25-HC, are synthesized via both enzymatic activity and ROS production pathway [37]. Although oxysterols are associated with the regulation of various biological processes, such as lipid and cholesterol metabolism, apoptosis, autophagy, and prenylation of proteins, their physiological action is still largely unknown.
Apoptosis is generally mediated by two apoptotic cellular signaling pathways: extrinsic and intrinsic [33]. The extrinsic pathway of apoptosis is initiated by the binding of death receptors with extracellular signaling molecules such as tumor necrosis factor (TNF), CD95ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL) [33]. Thereafter, death receptor-ligand binding induces the cleavage of inactive pro-caspase-8 into its active form. Subsequently, activated caspase-8 induces the activation of caspase-3 and PARP [33]. Intrinsic apoptosis is mediated by the reduction of mitochondrial membrane potential (∆Ψm), which results in the downregulation of anti-apoptotic molecules related to the maintenance of mitochondrial outer membrane integrity (such as Bcl-2 and Bcl-xL), and the upregulation of pro-apoptotic molecules (such as Bid, Bad, Bim, and Bax) related to the enhancement of mitochondrial outer membrane permeabilization [33,43]. Subsequently, cytochrome c released from the mitochondrial outer membrane with a decreasing ∆Ψm induces the formation of an apoptosome composed of apoptotic protease-activating factor-1 (Apaf-1), pro-caspase-9, and ATP. Thereafter, cleaved caspase-9 induces cell death by sequential activation of caspase-3 and PARP, leading to oligonucleosomal DNA fragmentation [43].
As shown in Figure 5a, 7α,25-DHC decreased Bid expression, indicating that an increase in Bid, a mitochondrial membrane-targeted death ligand, induces Bak oligomerization [44]. 7α,25-DHC suppressed antiapoptotic molecules including Bcl-2 and Bcl-xL and upregulated pro-apoptotic molecules including Bax, Bad, and Bak. Consequently, activated caspase-9 was increased in L929 cells incubated with 7α,25-DHC. These data indicate that 7α,25-DHC released cytochrome c from the mitochondrial outer membrane into the cytosol through a decrease in ∆Ψm in L929 cells. Finally, activated caspase-9 induced the activation of caspase-3 and PARP (Figure 5b,c). Collectively, these data indicate that 7α,25-DHC induces intrinsic apoptosis in L929 cells. However, intrinsic apoptosis is linked to mitochondrial dysfunction, which results in oxidative stress associated with ROS, including hydroxyl radicals (OH − ), hydrogen peroxide (H 2 O 2 ), and superoxide anions (O 2 − ) [33]. Furthermore, ROS induces oxidative damage to mitochondrial lipids, DNA, and proteins [33]. Additionally, previous studies on oxysterol-induced cell death reported that ROS production is upregulated in various types of cells incubated with oxysterols including 5,6-epoxycholesterol isomers, 7-KC, and 7β-HC [40,41]. In our previous study, ROS production was significantly increased in L929 cells incubated with 25-HC [39]. Moreover, ROS production is closely associated with the progression of inflammation [45]. Similar to 25-HC, ROS production was enhanced by 7α,25-DHC in the L929 cells ( Figure 6). Our results indicate that 7α,25-DHC-induced intrinsic apoptosis is involved in oxidative stress caused by enhanced ROS in L929 cells.
In the present study, autophagy staining showed that L929 cells with autophagic status were increased by 7α,25-DHC (Figure 7a). Furthermore, beclin-1 and LC3 expressions were significantly upregulated in L929 cells incubated with 7α,25-DHC (Figure 7b). Moreover, 7α,25-DHC suppressed Akt phosphorylation, whereas p53 phosphorylation was enhanced in L929 cells incubated with 7α,25-DHC (Figure 7c). The Akt cellular signaling pathway is closely associated with cell survival, proliferation, and growth during development and carcinogenesis through phosphorylation of forkhead box O (FOXO), a downstream target molecule of Akt [46]. Furthermore, FOXO mediates the role of p53, a tumor suppressor [46]. Hence, downregulation of Akt phosphorylation suppresses cell survival, proliferation, and growth through the upregulation of p53. p53 is a well-known transcription factor that regulates the expression of various pro-apoptotic genes, including cell cycle inhibitors, oxidative stress mediators, signaling molecules associated with death receptors, caspase activators, and the pro-apoptotic Bcl-2 family, which are related to mitochondrial outer membrane permeabilization [47]. 7α,25-DHC-induced apoptosis is involved in the Akt/p53 crosstalk axis in L929 cells. In addition, Akt/p53 crosstalk is closely associated with autophagy, known as autophagocytosis, which is a natural cellular degradation process that removes unnecessary or dysfunctional cellular components such as organelles, proteins, and lipids by autophagosome-containing lysosomes [33,39,48]. Generally, the phosphorylation of Akt, a vital messenger in the phosphoinositide 3-kinase (PI3K) pathway, induces mTOR Ser2448 phosphorylation to directly activate mTORC1, a master regulator of unc-51-like kinase 1 (ULK1), which is associated with the initiation of autophagy [47,49]. However, mTORC1 chemical inhibitors, such as rapamycin, upregulate ULK1 kinase activity [50]. In contrast, mTORC1 activation mediated by the Ras homolog enriched in the brain (Rheb) potently suppresses the kinase activity of ULK1 [51]. Therefore, Akt phosphorylation suppresses autophagy by suppressing ULK1 function to initiate the biogenesis of autophagosomes composed of beclin-1, LC3, and autophagy-related proteins (ATGs), including ATG5, ATG12, and ATG16L. Our data indicate that 7α,25-DHC-induced apoptosis is involved in autophagy via the Akt/p53 crosstalk axis in L929 cells.
GPR183 is a receptor of 7α,25-DHC [52]. Sun and Liu have reported that the 7α,25-DHC-GPR183 axis contributes to inflammation and various diseases, including cancer, autoimmune diseases, cardiovascular diseases, and neurodegenerative diseases, through the regulation of various physiological properties such as migration, activation, and function in B cells, T cells, dendritic cells, and immune cells [13]. Furthermore, Bartlett et al. reported that the inhibition of GPR183 using GSK682753, a chemical antagonist of GPR183, decreased the expression of autophagy biomarker LC3 and autophagic flux in 7α,25-DHC stimulated Bacille de Calmette-Guerin vaccine (BCG)-infected THP-1 cells [22]. In this study, we showed that inhibition of GPR183 using NIBR189 significantly suppressed 7α,25-DHC-induced apoptosis, ROS production, and autophagy in L929 cells (Figure 8). Our results indicate that the 7α,25-DHC-GPR183 axis regulated oxiapoptophagy in L929 cells.

Cell Live/Dead Staining
L929 cells (1 × 10 5 cells/mL) were cultured in 8-well chamber slides (Nunc ® Lab-Tek ® Chamber Slide™ system, Sigma-Aldrich) for 24 h and then treated with 0, 1, 5, 10, and 25 µg/mL 7α,25-DHC for 48 h. Thereafter, cell survival was verified by a LIVE/DEAD™ cell assay kit (Thermo Fisher Scientific, Rockford, IL, USA) composed of calcein green AM to stain live cells with green fluorescence and ethidium homodimer-1 to stain dead cells with red fluorescence, according to the manufacturer's instructions. Images were acquired under an inverted fluorescence microscope (Eclipse TE2000; Nikon Instruments, Inc., Melville, NY, USA). Histogram presented the relative % of total cells including live and dead cells. In addition, relative % of dead cells per total cells was calculated by (number of dead cells/(number of live cells + number of dead cells) × 100). Results were presented as mean ± standard deviation of three independent experiments, * p < 0.05 and ** p < 0.01.

DAPI Staining
Chromatin condensation was observed using DAPI staining to stain the nucleus of cells. Briefly, L929 cells (1 × 10 5 cells/mL) were cultured in 8-well chamber slides for 24 h and then treated with 0, 1, 5, 10, or 25 µg/mL 7α,25-DHC for 48 h. After cultivation, the cells were rinsed with phosphate-buffered saline (PBS; Sigma-Aldrich), fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X-100, and then stained with DAPI. Cells with condensed chromatin were taken under an inverted fluorescence microscope. Relative % of total cells was counted by cells with condensed chromatin and intact chromatin. Thereafter, relative % of cells with condensed chromatin was calculated by (number of cells with condensed chromatin/(number of cells with condensed chromatin + number of cells with intact chromatin) × 100). Results were presented as mean ± standard deviation of three independent experiments, * p < 0.05 and ** p < 0.01.

H&E Staining
Morphological alteration as cell shrinkage is a representative characteristic of apoptosis. Hence to observe the morphological alteration, L929 cells (1 × 10 5 cells/mL) were cultured in 8-well chamber slides and allowed to adhere for 24 h. The cultured L929 cells were then treated with 0, 1, 5, 10, or 25 µg/mL 7α,25-DHC for 48 h at 37 • C. After cultivation, the cells were rinsed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at 4 • C. The morphology of L929 cells incubated with 7α,25-DHC was observed by H&E staining. Images of these cells were captured by a microscope (Leica DM750; Leica Microsystems, Heerbrugg, Switzerland). Relative % of total cells was counted by cells with both altered and intact morphologies. Thereafter, relative % of cells with altered morphologies was calculated by (number of cells with altered morphology/(number of cells with altered morphology + number of cells with intact morphology) × 100). Results were presented as mean ± standard deviation of three independent experiments, * p < 0.05 and ** p < 0.01.

Flow Cytometry Determination of Apoptosis
The apoptotic population was determined using FACS analysis. Briefly, L929 cells (1 × 10 5 cells/mL) were cultured in 6-well culture plates and treated with 0, 1, 5, 10, and 25 µg/mL 7α,25-DHC for 48 h. Thereafter, the cells were rinsed with ice-cold PBS resuspended in binding buffer (BD Biosciences, San Diego, CA, USA), and then incubated with annexin V-fluorescein isothiocyanate (annexin V-FITC) and PI (Cell Signaling Technology, Danvers, MA, USA) for 15 min at 37 • C to perform FACS analysis using an automated high-speed cytometer sorting system (BD Biosciences).

Western Blotting Analysis
L929 cells (1 × 10 5 cells/mL) were cultured in 6-well plates and treated with 0, 1, 5, or 10, and 25 µg/mL 7α,25-DHC for 48 h. The concentrations of total proteins extracted from the cells by cell lysis buffer (Cell Signaling Technology) were quantified using the bicinchoninic acid protein assay (Thermo Fisher Scientific). The same concentration of each protein sample was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA) at 4 • C. Subsequently, the PVDF membrane was blocked using 5% (v/v) bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline with Tween-20 (TBS-T; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then incubated with primary antibodies at 4 • C for 12 h. . The membranes were rinsed three times with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 2 h. Immunoreactive bands visualized using an ECL System (Amersham Biosciences, Piscataway, NJ, USA) were taken by MicroChemi 4.2 (Dong-Il Shimadzu Corp., Seoul, Korea). Thereafter, densitometric analysis was performed using ImageJ bundled with Java 1.8.0_172 software.

Cellular ROS Staining
After incubation under previously described cell culture conditions, ROS was detected by 5 µM DCFDA staining at 37 • C for 30 min. The image of cells was taken by a laser confocal scanning microscope (Leica Microsystems). ROS intensity was verified using an automated high-speed cytometer sorting system with BD Cell Quest version 3.3. Relative ROS production was measured using a fluorescence microplate reader (Bio Tek). Fluores-cent was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Detection of Autophagy
L929 cells (1 × 10 5 cells/mL) were cultured in 8-well chamber slides and allowed to adhere for 24 h. Cultured cells were treated with 0, 1, 5, 10, or 25 µg/mL 7α,25-DHC for 48 h at 37 • C. Thereafter, autophagy was detected using an autophagy detection kit (ab139484, Abcam, Waltham, MA, USA), according to the manufacturer's protocol. The image of cells was taken using a laser confocal scanning microscope.

Statistical Analysis
Statistical differences were calculated using ANOVA and Tukey's multiple comparison test. All data are presented as mean ± standard deviation. Statistical significance was set at p < 0.05.

Conclusions
This study demonstrated that 7α,25-DHC induced cell death through oxiapoptophagy accompanied with apoptosis, oxidative stress and autophagy by the crosstalk of Akt and p53 cellular signaling pathways in L929 fibroblast cells. Hence, our study suggests that the synthesis of 25-HC-7α,25-DHC from cholesterol by the enhancement of inflammation is closely associated with progressive degeneration of fibroblastic tissues through the induction of oxiapoptophagic cell death. Furthermore, our study suggests that the inhibition of GPR183, a receptor of 7α,25-DHC, might suppress or retard 7α,25-DHC-induced fibroblastic tissue degeneration.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.