Lysophosphatidic Acid Receptor 3 Promotes Mitochondrial Homeostasis against Oxidative Stress: Potential Therapeutic Approaches for Hutchinson–Gilford Progeria Syndrome

Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activation of multiple LPA G protein-coupled receptors. We previously reported that LPA suppresses oxidative stress in premature aging Hutchinson-Gilford progeria syndrome (HGPS) patient fibroblasts via its type 3 receptor (LPA3). Mitochondria have been suggested to be the primary origin of oxidative stress via the overproduction of reactive oxygen species (ROS). Mitochondria are responsible for producing ATP through oxidative phosphorylation (OXPHOS) and have a calcium buffering capacity for the cell. Defects in mitochondria will lead to declined antioxidant capacity and cell apoptosis. Therefore, we aim to demonstrate the regulatory role of LPA3 in mitochondrial homeostasis. siRNA-mediated depletion of LPA3 leads to the depolarization of mitochondrial potential (ΔΨm) and cellular ROS accumulation. In addition, the depletion of LPA3 enhances cisplatin-induced cytochrome C releasing. This indicates that LPA3 is essential to suppress the mitochondrial apoptosis pathway. LPA3 is also shown to improve mitochondrial ADP-ATP exchange by enhancing the protein level of ANT2. On the other hand, LPA3 regulates calcium uptake from the ER to mitochondria via the IP3R1-VDAC1 channel. Moreover, activation of LPA3 by selective agonist OMPT rescues mitochondrial homeostasis of H2O2-induced oxidative stress cells and HGPS patient fibroblasts by improving mitochondrial ΔΨm and OXPHOS. In summary, our findings imply that LPA3 acts as the gatekeeper for mitochondrial healthiness to maintain cell youth. Furthermore, LPA3 can be a promising therapeutic target to prevent mitochondrial oxidative stress in aging and HGPS.


Introduction
Lysophosphatidic acid (1-or 2-acyl-sn-glycerol 3-phosphate/radyl-glycerol-phosphate, LPA) is a bioactive lipid mediator produced both intracellularly and extracellularly from membrane phospholipids and is detected robustly in all eukaryotic tissues and blood plasma [1]. LPA evokes various cellular responses by activating six distinct G proteincoupled receptors (GPCRs) localized in the plasma membrane of various cells, mediating cell proliferation, migration, and cytoskeletal reorganization [2]. These receptors are subdivided into the endothelial cell differentiation gene family (Edg, LPA 1 -LPA 3 ) [3] and the P2Y purinergic receptor family (LPA 4 -LPA 6 ) [4]. Upon binding with LPA, conformational changes of LPA receptors allow them to act as guanine nucleotide exchange factors for one or more of the four classes of heterotrimeric G proteins, G 12/13 , G q/11 , G i/o , and G s , which initiate signaling cascades through downstream molecules such as Rho, IP3-DAG, adenylyl cyclase and Ras/mitogen-activated protein kinase (MAPK) pathways [5][6][7]. Studies have documented the wide range of LPA downstream signaling pathways involving diverse physiological and pathophysiological functions, including nervous/vascular system development, reproduction, angiogenesis, cancer progression, and hematopoiesis [8][9][10].
Moreover, we also showed that LPA can exert different functions according to its GPCRs expression pattern. For instance, our recent studies suggest that LPA 2 and LPA 3 expressed at different stages of hematopoietic progenitors decide the fate of blood cell differentiation [11,12]. In addition, studies proposed the concept that the profile of LPA receptors change during the cell aging process which affect activities of the downstream pathway [13,14]. Similarly, we also observed that LPA 3 decreases in the senescent and premature aging disease, Hutchinson-Gilford progeria symptom (HGPS) model [11,15]. LPA 3 knockout zebrafish larvae we established showed senescent phenotypes and lower locator activity, and the adult fish have a shorter life span than wild-type zebrafish [16]. LPA also protects cells from mitochondrial apoptosis by enhancing JNK-mediated phosphorylation of Bcl-2 and ERK1/2 pathways and by accumulating the anti-apoptotic protein, Mcl-1 [17,18]. These beneficial effects of LPA on mitochondria can align with our findings that activation of LPA 3 in HGPS cells rescued premature aging phenotype. Notably, our recently published studies showed that activation of LPA 3 stabilizes the protein of nuclear factor erythroid 2-related factor (Nrf2), consequently promoting the expression of antioxidants and reducing mitochondrial superoxide [15]. This evidence suggests that the potentiation of LPA 3 signaling may constitute a new approach against aging progress and mitochondrial oxidative stress.
Mitochondria are currently considered a critical component of intracellular signaling, immune response, and a modulator of cell replication, more than simple bioenergetic factories. The development of a wide range of aging-related diseases is highly associated with the decline of mitochondrial quality and activity. It is widely accepted that, during the aging process, the free radical (reactive oxygen species, ROS) leads to cumulative damage and irreversible cell damage. The mitochondria are considered the central regulators of redox activities and oxidative stress, called mitochondrial ROS (mtROS). Therefore, mitochondrial dysfunctions have been suggested to be the hallmark and driver of aging [19]. In addition, HGPS, which is severe premature aging, shows similar signaling pathway patterns as normal aging cells [20]. This syndrome is typically caused by a silent mutation (c. 1824C>T; p. Gly608Gly) of LMNA that activates an alternative pre-mRNA cryptic splicing site and causes a 150-nucleotide, 50 amino acid deletion. The missing sequence includes a recognition site for ZMPSTE24 endoprotease, leading to the accumulation of an un-cleaved Lamin A isoform, named progerin [21]. HGPS cells display significant dysregulation of oxidative phosphorylation (OXPHOS), mitochondrial activities, and ROS scavenging [22,23]. Treatments aimed to improve the mitochondrial homeostasis were shown to ameliorate the cell senescence of HGPS cells [24]. Therefore, we aim to explore whether LPA 3 signaling may constitute a new approach to maintain mitochondrial homeostasis and alleviate mitochondrial oxidative stress during the aging process. The outcomes of this investigation will advance our understanding of the molecular mechanisms leading to mitochondrial homeostasis in aging and novel intervention strategies against these pathological conditions.

Cell Culture and Pharmacological Reagents
Human cervical cancer HeLa and Human embryonic kidney HEK293 cells were obtained from ATCC and maintained in Dulbecco's modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Pen/Strep, Lonza, Gampel-Bratsch, Switzerland). Human erythroleukemia K562 cells were obtained from ATCC and cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS and Pen/Strep. Hutchinson-Gilford progeria syndrome (HGPS) fibroblast lines AG11513F and age-matched control fibroblast line AG08470B were received from the Coriell Institute (Camden, NJ, USA) and were maintained in minimum essential medium Eagle-alpha modification (Alpha MEM, Thermo Fisher Scientific) supplemented with 15% FBS. Normal human skin fibroblasts (HSF, passage 9) were maintained in the same culture condition. HSF cells were derived from neonatal foreskin and used in our previous work [15]. Cells were cultured at 37 • C in a humidified atmosphere of 5% CO 2 .

Plasmid Construct, siRNA and Transfection
The PCR products of the LMNA and progerin gene were cloned into a pAS 2 V 1 vector, which used in our previous study [15]. The vectors were then transfected into HEK293 cells with Lipofectamine 2000 (Thermo Fisher Scientific) and selected by puromycin at 2 ug/mL for positive clone.

Analysis of Cellular ROS and Mitochondrial Membrane Potential
For the detection of cellular ROS, exponentially growing cells were washed twice with PBS and then incubated with 20 µM 2 ,7 -dichlorofluorescin diacetate (DCFDA, Thermo Fisher Scientific) in a 37 • C incubator for 30 min. For the detection of mitochondria membrane potential (MMP, ∆Ψm), exponentially growing cells were incubated with culture medium containing 200 nM of tetramethylrhodamine ethyl ester (TMRE, Thermo Fisher Scientific) in a 37 • C incubator for 30 min. All experiments were then detected by Amnis FlowSight imaging flow cytometry (Luminex, Austin, TX, USA), Keyence BZ-X700-Allin-one Fluorescence microscope (Keyence, Osaka, Japan) or Cytation 5 Multi-Mode Cell Imaging Reader (BioTek, Winooski, VT, USA). The mean fluorescent intensity analyzed in each treatment group was quantified and normalized to the control group.

Cell Proliferation Assay
1000 cells were plated in 96-well microplate overnight, followed by treatment with OX-PHOS inhibitors, 0.01-1 µM antimycin (Sigma) or 0.01-1 µM rotenone (Sigma), for 3 days. Cells were then stained with DAPI and the cell number were analyzed using a Cytation 5 Multi-Mode Cell Imaging Reader (BioTek). The mean fluorescent intensity analyzed was quantified and normalized to the fluorescent intensity of DAPI and control group.
2.6. In Situ Proximity Ligation Assay (PLA) and Immunofluorescence Analysis Cells were harvested, followed by fixation with 4% PFA and permeabilization by 0.5% Triton X. in situ Proximity ligation assay (PLA) was performed by using Duolink PLA reagents (MilliporeSigma, Burlington, MA, USA) according to the manufacturer's instructions. Cells were blocked with Duolink in situ blocking solution for 1 h before being probed with primary antibodies in Duolink in situ antibody diluent overnight at 4 • C. Cells were then incubated with oligonucleotides-conjugates secondary antibodies (PLA probe anti-rabbit PLUS and anti-mouse MINUS), followed by ligation and amplification with the fluorophore-labeled oligonucleotide probe (excitation = 495 nm, emission = 527 nm). For immunofluorescence staining, cells were incubated with primary antibodies overnight and then labeled with secondary antibodies for 1 h. Cells were stained with DAPI and the fluorescent images were developed by Keyence BZ-X700-All-in-one Fluorescence microscope (Keyence). Primary antibodies for PLA and immunofluorescent staining were as follows: mouse anti-VDAC1 (820701, BioLegend, San Diego, CA, USA), rabbit anti-IP3R1 (A302-158, Bethyl, Montgomery, TX, USA), mouse anti-Cytochrome c (612503, BioLegend), and rabbit anti-HSP60 (12165, CellSignaling).

ADP-ATP Exchange Assay
The ADP-ATP exchange assay in microplates was performed following previous studies [25,26] with modification. Cells were resuspended in Kreb medium (10 mM HEPES pH = 7.4, 145 mM NaCl, 5 mM KCl, 2.6 mM CaCl 2 , 5.6 mM glucose, 5 mM glutamate, and 5 mM malate) containing 1.1 µM MgGreen 5K+ (Thermo Fisher Scientific), 1 µM Na 3 VO 4 , 1 µM NaF, 1 µM β-glycerophosphate and protease inhibitors, followed by permeabilization by 375 µM digitonin. Cells were then loaded in 96-well solid black polystyrene microplate (Thermo Fisher Scientific). The fluorescence signal was read by BioTek Cytation 5 Multi-Mode Reader with the absorption/emission wavelength at 506/531 nm. After 10 min of reading the basal level of MgGreen 5K+ fluorescence, 5 mM of ADP was added to initiate ADP-ATP exchange. 5 mM EDTA were added at the end of the experiment for minimum fluorescence (F min ), then recorded the maximum fluorescence (F max ) by adding 10 ] t is the concentration of total free Mg 2+ contained in the reaction solution before adding ADP.
[ADP] t (t = 0) and [ATP] t (t = 0) are the concentrations of ADP and ATP, respectively, at time zero, which is 0 for both. To calculate [ATP] e , we applied K d values from references due to similar experimental conditions (K ADP = 0.906 mM and K ATP = 0.114 mM).

Cellular Bioenergetics Measurements by Seahorse Assay
To measure the rate of oxidative phosphorylation (OXPHOS), a Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA, USA) was applied, according to manufacturer's instructions. For measuring the oxygen consumption rate (OCR), 5 × 10 4 cells were seeded in an XF assay plate overnight. Cells were incubated in a non-CO 2 incubator at 37 • C for 1 h before experiments, and the culture medium was replaced with assay medium (medium without HEPES nor sodium bicarbonate). In addition, the cartridge was activated by calibration buffer overnight and calibrated by the XF24 analyzer before experiments. OCR was determined under basal conditions, then 0.25 µM oligomycin, 1 µM FCCP, and 1 µM rotenone plus antimycin A were added sequentially to estimate individual parameters of mitochondria.

Statistical Analysis
Each result represents at least three independent experiments. The data are presented as mean ± SD. Differences between control and experimental groups were determined with t-test or one-way analysis of variance (ANOVA). Results with p * ≤ 0.05, p ** ≤ 0.01, p *** ≤ 0.001 were considered statistically significant.

LPA 3 Signaling Is Crucial to Maintain Mitochondria Homeostasis and ROS Accumulation
To delineate the role of LPA 3 and its underlying mechanism in regulating mitochondrial activity, we subjected the siRNA control and LPA 3 siRNA-treated human cervical cancer HeLa cells to a cell proliferation assay with electron transport chain (ETC) complex I inhibitor, Rotenone, and complex III inhibitor, Antimycin A ( Figure 1A). Two LPA 3 siRNA were used in this study ( Figure S1). LPA 3 siRNA knockdown cells were susceptible to the two ETC inhibitors, suggesting that that LPA 3 was required to counter mitochondrial stress. Furthermore, the mitochondrial membrane potential (∆Ψm) was measured by tetramethylrhodamine ethyl ester, perchlorate (TMRE) staining. TMRE is a cell-permeant that shows the level of negative charge across the healthy inner mitochondrial membrane to the matrix. ETC complex I, III, and IV serve as proton pumps to generate a proton gradient (∆Ψm) across the inner mitochondrial membrane as energy storage for driving OXPHOS. Thus, mitochondrial ∆Ψm is a remarkable indicator for mitochondrial healthiness. We found significantly depolarized mitochondrial ∆Ψm in LPA 3 -depleted cells ( Figure 1B). Since superoxide can leak out from mitochondria to contribute endogenous ROS, the cellular ROS was detected by 2 ,7 -dichlorofluorescein diacetate (DCFDA) staining. It showed that the cellular ROS accumulation increased in LPA 3 -depleted cells ( Figure 1C).
Moreover, cytochrome c release was monitored to detect the healthiness of mitochondria. In healthy mitochondria, cytochrome c locates at the mitochondrial intermembrane space and functions as an electron shuttle in the ETC. The mitochondrial damage induces the outer membrane's permeabilization, facilitating cytochrome c releasing from the mitochondria to cytosol. The released cytochrome c in the cytosol further mediates the activation of caspase-3 dependent apoptosis [27]. LPA 3 siRNA knockdown cells displayed an increased ratio of cytochrome c release. Moreover, cisplatin as a mitochondrial DNA damaging reagent was treated to induce cytochrome c releasing [28]. Relative to siRNA control HeLa cells, LPA 3 depletion cells were more sensitive to cisplatin-induced cytochrome c releasing ( Figure 2). These results highlight the crucial role of LPA 3 in mitochondria homeostasis, evidenced by the cytochrome c releasing, mitochondrial ∆Ψm depolarization, and cellular ROS accumulation in LPA 3 -depletion cells.
increased ratio of cytochrome c release. Moreover, cisplatin as a mitochondrial DNA damaging reagent was treated to induce cytochrome c releasing [28]. Relative to siRNA control HeLa cells, LPA3 depletion cells were more sensitive to cisplatin-induced cytochrome c releasing ( Figure 2). These results highlight the crucial role of LPA3 in mitochondria homeostasis, evidenced by the cytochrome c releasing, mitochondrial ΔΨm depolarization, and cellular ROS accumulation in LPA3-depletion cells. Figure 1. Impairment of mitochondrial homeostasis in LPA3-deficient cells. (A) Control (siRNA control) and two LPA3 siRNA knockdown HeLa cells were subjected to a cell proliferation assay against mitochondrial electron transport chain (ETC) inhibitors, complex-III inhibitor Antimycin A and complex-I inhibitor Rotenone, at indicated doses for 72 hr. LPA3 siRNA knockdown cells were sensitive to inhibitors against ETC. (B) Mitochondrial ΔΨm of siRNA control and two LPA3 siRNA knockdown cells were analyzed with TMRE dye by imaging flow cytometry. TMRE fluorescence was reduced in two LPA3 siRNA knockdown cells. Representative images of TMRE stained cells were shown in the right panel. (C) Cellular ROS of siRNA control and two LPA3 siRNA knockdown cells were analyzed by DCFDA dye. Cells were stained with 20 μM of DCFDA at 37 °C for 30 min, followed by imaging flow cytometry analysis. DCFDA fluorescence showed increasing in two LPA3 siRNA knockdown cells. Representative images of DCFDA stained cells were shown in the right panel. The bar graphs were generated from three independent analyses. * p < 0.05; ** p < 0.01; *** p < 0.001. Representative images of DCFDA stained cells were shown in the right panel. The bar graphs were generated from three independent analyses. * p < 0.05; ** p < 0.01; *** p < 0.001.

LPA3 Signaling Is Involved in Mitochondrial ADP-ATP Exchange
Previous research has shown that low expression of ANT2 is linked to mitochondrialinherent to cell senescence [29]. Therefore, we aimed to test if LPA3 signaling regulates protein level and activity of ANT2. The Western blot analysis of mitochondrial proteins showed that the protein level of ANT2 was significantly reduced by siLPA3, whereas ANT1 and the mitochondrial matrix chaperone HSP60 were not ( Figure 3A). Adenine nucleotide translocase 2 (ANT2) is the crucial regulator for mitochondrial homeostasis by exchanging ADP and ATP across the mitochondrial inner membrane. In most physiological conditions, electron transport through the ETC is tightly coupled with the phosphorylation of ADP to ATP by F0F1-ATPase. Impairing the ADP supply to mitochondria will slow down OXPHOS, increase the redox status of mitochondria, and thereby drive excess ROS production [30]. Thus, we measured the mitochondrial ADP-ATP exchange using an Mg 2+ sensitive fluorescent probe Magnesium Green 5K+ (abbreviated as MgGreen) [25,26]. Since biologically active ATP and ADP are bound with Mg 2+ and have different affinity (KADP = 0.906 mM and KATP = 0.114 mM). The dynamics of free extra-mitochondrial Mg 2+ can be measured and determine ADP-ATP exchange activity in cells. We observed that the LPA3 knockdown cells by siRNA showed a significant attenuation in ADP-ATP exchange ( Figure 3B). Moreover, activation of LPA3 with its highly selective agonist, OMPT,

LPA 3 Signaling Is Involved in Mitochondrial ADP-ATP Exchange
Previous research has shown that low expression of ANT2 is linked to mitochondrialinherent to cell senescence [29]. Therefore, we aimed to test if LPA 3 signaling regulates protein level and activity of ANT2. The Western blot analysis of mitochondrial proteins showed that the protein level of ANT2 was significantly reduced by siLPA 3 , whereas ANT1 and the mitochondrial matrix chaperone HSP60 were not ( Figure 3A). Adenine nucleotide translocase 2 (ANT2) is the crucial regulator for mitochondrial homeostasis by exchanging ADP and ATP across the mitochondrial inner membrane. In most physiological conditions, electron transport through the ETC is tightly coupled with the phosphorylation of ADP to ATP by F0F1-ATPase. Impairing the ADP supply to mitochondria will slow down OXPHOS, increase the redox status of mitochondria, and thereby drive excess ROS production [30]. Thus, we measured the mitochondrial ADP-ATP exchange using an Mg 2+ sensitive fluorescent probe Magnesium Green 5K+ (abbreviated as MgGreen) [25,26]. Since biologically active ATP and ADP are bound with Mg 2+ and have different affinity (K ADP = 0.906 mM and K ATP = 0.114 mM). The dynamics of free extra-mitochondrial Mg 2+ can be measured and determine ADP-ATP exchange activity in cells. We observed that the LPA 3 knockdown cells by siRNA showed a significant attenuation in ADP-ATP exchange ( Figure 3B). Moreover, activation of LPA 3 with its highly selective agonist, OMPT, increased the ANT2 protein expression in both Hela and human skin fibroblast (HSF) cells ( Figure 3C) and the activity of ADP-ATP exchange ( Figure 3D). These results suggest that LPA 3 signaling is crucial to regulate the mitochondrial ADP-ATP exchange. increased the ANT2 protein expression in both Hela and human skin fibroblast (HSF) cells ( Figure 3C) and the activity of ADP-ATP exchange ( Figure 3D). These results suggest that LPA3 signaling is crucial to regulate the mitochondrial ADP-ATP exchange.

LPA3 Signaling Is Involved in the Regulation of Mitochondrial Ca 2+ Influx from ER to Mitochondria
On the other hand, the mitochondrial electrochemical proton gradient (ΔμH + ) is the major component of the mitochondria ΔΨ. ΔμH + drives the flow of H + through ATP synthase in a reaction coupled to the generation of ATP from ADP [31] and represents a substantial driving force for Ca 2+ accumulation [32]. Mitochondrial Ca 2+ traffics from ER to mitochondria in response to physiological stimuli and stresses [33,34]. The protein channel complex IP3R1-VDAC1 was suggested to transfer the mitochondrial Ca 2+ influx from the ER to mitochondrial intermembrane space. Therefore, the interacting structure of IP3R1-VDAC1 was detected with in situ proximity ligation assay (PLA) by probing IP3R1 and VDAC1 antibodies. The hybridization with PLA secondary antibodies coupled to fluorescent oligonucleotides hybridizes if the distance is <40 nm [35]. The interactions between IP3R1-VDAC1 were decreased in LPA3 depleted cells ( Figure 4A). To verify the Ca 2+ transport efficiency from the ER to mitochondria, the mitochondrial Ca 2+ was measured by mitochondria-specific Ca 2+ indicator Rhod-2 AM with live cell image analysis. ATP was used to stimulate P2Y receptors-dependent intracellular Ca 2+ releasing from the ER. By measuring the dynamics and the peaks of Rhod-2 AM fluorescence, we demonstrated that

LPA 3 Signaling Is Involved in the Regulation of Mitochondrial Ca 2+ Influx from ER to Mitochondria
On the other hand, the mitochondrial electrochemical proton gradient (∆µH + ) is the major component of the mitochondria ∆Ψ. ∆µH + drives the flow of H + through ATP synthase in a reaction coupled to the generation of ATP from ADP [31] and represents a substantial driving force for Ca 2+ accumulation [32]. Mitochondrial Ca 2+ traffics from ER to mitochondria in response to physiological stimuli and stresses [33,34]. The protein channel complex IP3R1-VDAC1 was suggested to transfer the mitochondrial Ca 2+ influx from the ER to mitochondrial intermembrane space. Therefore, the interacting structure of IP3R1-VDAC1 was detected with in situ proximity ligation assay (PLA) by probing IP3R1 and VDAC1 antibodies. The hybridization with PLA secondary antibodies coupled to fluorescent oligonucleotides hybridizes if the distance is <40 nm [35]. The interactions between IP3R1-VDAC1 were decreased in LPA 3 depleted cells ( Figure 4A). To verify the Ca 2+ transport efficiency from the ER to mitochondria, the mitochondrial Ca 2+ was measured by mitochondria-specific Ca 2+ indicator Rhod-2 AM with live cell image analysis. ATP was used to stimulate P2Y receptors-dependent intracellular Ca 2+ releasing from the ER. By measuring the dynamics and the peaks of Rhod-2 AM fluorescence, we demonstrated that the rate and amount of Ca 2+ efflux into mitochondria following administration of ATP treatment were significantly attenuated in LPA 3 depleted cells ( Figure 4B-D). The results highlight that LPA 3 signaling is important to support the structure of the Ca 2+ channel complex and Ca 2+ trafficking into the mitochondria from ER. the rate and amount of Ca 2+ efflux into mitochondria following administration of ATP treatment were significantly attenuated in LPA3 depleted cells ( Figure 4B-D). The results highlight that LPA3 signaling is important to support the structure of the Ca 2+ channel complex and Ca 2+ trafficking into the mitochondria from ER. The average Rhod-2 AM peak fluorescence following administration of ATP was decreased in LPA3 knockdown cells. The graphs were generated from three independent analyses. *** p < 0.001.

LPA3 Signaling Participates in the Regulation of Mitochondrial Stress Response
Since we have shown that LPA3 plays a crucial role as regulator of mitochondria, we next aimed to discuss whether LPA3 signaling may constitute a new approach against oxidative stress-induced mitochondrial dysfunction. Activation of LPA3 with its highly selective agonist, OMPT, showed potent induction of mitochondrial antioxidants, glutathione peroxidase 1 (Gpx1), and superoxide dismutase type 1 and type 2 (SOD1 and SOD2) protein expression in human cervical cancer HeLa cells ( Figure 5A). To further delineate the role of LPA3 in regulating the mitochondrial oxidative stress response, we subjected HeLa cells with treatment of hydrogen peroxide (H2O2), followed by analysis of the mitochondrial membrane potential (ΔΨm) by TMRE staining. We found significantly depolarized mitochondrial ΔΨm in H2O2-induced oxidative stress and rescued with OMPT treatment (Figures 5B,C). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used as a negative control to depolarize mitochondria. The antioxidant N-acetylcysteine (NAC) was used as the positive control to rescue ΔΨm. Human erythroleukemic K562 cells were also used to confirm the protection effect of OMPT ( Figure S2). The results also suggested similar antioxidant function of The average Rhod-2 AM peak fluorescence following administration of ATP was decreased in LPA 3 knockdown cells. The graphs were generated from three independent analyses. *** p < 0.001.

LPA 3 Signaling Participates in the Regulation of Mitochondrial Stress Response
Since we have shown that LPA 3 plays a crucial role as regulator of mitochondria, we next aimed to discuss whether LPA 3 signaling may constitute a new approach against oxidative stress-induced mitochondrial dysfunction. Activation of LPA 3 with its highly selective agonist, OMPT, showed potent induction of mitochondrial antioxidants, glutathione peroxidase 1 (Gpx1), and superoxide dismutase type 1 and type 2 (SOD1 and SOD2) protein expression in human cervical cancer HeLa cells ( Figure 5A). To further delineate the role of LPA 3 in regulating the mitochondrial oxidative stress response, we subjected HeLa cells with treatment of hydrogen peroxide (H 2 O 2 ), followed by analysis of the mitochondrial membrane potential (∆Ψm) by TMRE staining. We found significantly depolarized mitochondrial ∆Ψm in H 2 O 2 -induced oxidative stress and rescued with OMPT treatment (Figure 5B,C). The mitochondrial uncoupler carbonyl cyanide ptrifluoromethoxyphenylhydrazone (FCCP) was used as a negative control to depolarize mitochondria. The antioxidant N-acetylcysteine (NAC) was used as the positive control to rescue ∆Ψm. Human erythroleukemic K562 cells were also used to confirm the protection effect of OMPT ( Figure S2). The results also suggested similar antioxidant function of OMPT. In addition, we further subjected HeLa cells to a cell proliferation assay with inhibitors against OXPHOS. The respiratory complex I (NADH: ubiquinone oxidoreductase) and respiratory complex III (ubiquinone: cytochrome c reductase) are recognized to produce mtROS relevant to pathological aged diseases [36]. While highly mtROS is presented, the respiratory complexes are likely to be attacked and drive reverse electron transfer (RET), which at the same time leads to electron leak to damage cell compartments [37]. The OMPT treated cells were highly resistant to ETC inhibitors Rotenone (complex I) and Antimycin A (complex III) induced oxidative stress ( Figure 5D). These findings suggested the protective functions of LPA 3 to alleviate mitochondrial function during oxidative stress. hibitors against OXPHOS. The respiratory complex I (NADH: ubiquinone oxidoreduc-tase) and respiratory complex III (ubiquinone: cytochrome c reductase) are recognized to produce mtROS relevant to pathological aged diseases [36]. While highly mtROS is presented, the respiratory complexes are likely to be attacked and drive reverse electron transfer (RET), which at the same time leads to electron leak to damage cell compartments [37]. The OMPT treated cells were highly resistant to ETC inhibitors Rotenone (complex I) and Antimycin A (complex III) induced oxidative stress ( Figure 5D). These findings suggested the protective functions of LPA3 to alleviate mitochondrial function during oxidative stress. (D) HeLa cells were subjected to a cell proliferation assay against mitochondrial electron transport chain (ETC) inhibitors, complex-III inhibitor Antimycin A (left), and complex-I inhibitor Rotenone (right), at indicated doses for 72 hr. Cells showed protective effects against ETC inhibitors in 50 nM of OMPT treatment. The bar graphs were generated from three independent analyses. * p < 0.05; ** p < 0.01; *** p < 0.001. FCCP was used as a positive control for loss of mitochondrial ∆Ψm. (D) HeLa cells were subjected to a cell proliferation assay against mitochondrial electron transport chain (ETC) inhibitors, complex-III inhibitor Antimycin A (left), and complex-I inhibitor Rotenone (right), at indicated doses for 72 h. Cells showed protective effects against ETC inhibitors in 50 nM of OMPT treatment. The bar graphs were generated from three independent analyses. * p < 0.05; ** p < 0.01; *** p < 0.001.

LPA 3 Activation Rescues Mitochondrial Activity in Hutchinson-Gilford Progeria Syndrome Cells
The ROS leads to cumulative damage during the aging process and results in irreversible cell damage. The mitochondria are considered the central regulators of redox activities, and oxidative stress is referred to as mitochondrial ROS (mtROS). Therefore, the alterations in mitochondrial functions have been suggested to be the hallmark and the drivers of aging. Our previous publication indicated that the expression level of LPA 3 declined in the aging disease Hutchinson-Gilford progeria syndrome (HGPS). Enhancement of LPA 3 signaling by agonist is suggested to ameliorate premature aging [11,15]. HGPS also displayed the dysregulation of OXPHOS and mitochondrial activity [22]. Therefore, we aimed to explore whether the protective effects of LPA 3 on mitochondria and against oxidative stress can constitute a new approach to HGPS treatment. We first showed that OMPT treatment protected normal fibroblast against H 2 O 2 induced oxidative stress ( Figure S3). Next, we analyzed the mitochondrial ∆Ψm in HGPS patient fibroblast AG11 and agematched control fibroblast line AG08 cells by TMRE staining. Relative to the control AG08 cells, HGPS AG11 cells displayed depolarized mitochondrial ∆Ψm. Activation of LPA 3 signaling had no effect on control cells but rescued depolarized mitochondrial ∆Ψm in HGPS cells ( Figure 6A). Moreover, we subjected lamin A control and HGPS progerin expression HEK 293 cells to the Agilent Seahorse XFe24 analyzer to determine mitochondrial OXPHOS by measuring the oxygen consumption rate (OCR). The OCR was determined under basal conditions followed by the addition of oligomycin (ATP synthase inhibitor), FCCP (mitochondrial uncoupler), and rotenone plus antimycin A (respiratory complex I and III inhibitors) to estimate individual parameters of mitochondria. Seahorse XF Cell Mito Stress assays revealed that the OXPHOS was significantly disrupted in HGPS cells ( Figure 6B,C). OMPT treatment rescued the OCR level of basal respiration, ATP production, and maximal respiration ( Figure 6B,C). Together, we conclude that LPA 3 acts as the upstream signal to maintain mitochondria homeostasis through regulating mitochondria Ca 2+ trafficking and ADP-ATP exchange. Loss of LPA 3 leads to dysregulation of mitochondria biogenesis, OXPHOS, and ROS control in HGPS and aging cells. Activating LPA 3 signaling by its agonist OMPT can rescue the mitochondrial defects from these pathological conditions. These results suggested that the potentiation of LPA 3 signaling may constitute a new approach against aging progress and mitochondrial oxidative stress.

Discussion
Mitochondria are considered as both the source and the target of ROS. ROS, such as superoxide and hydrogen peroxide, are the by-products of mitochondrial aerobic metabolism. Two different sites in the ETC, respiratory complex I (NADH dehydrogenase) and respiratory complex III (ubiquinone-cytochrome c reductase), are recognized as the primary sites of ROS production [38]. Under normal conditions, ROS are controlled at the physiological levels by several endogenous antioxidants and scavengers, including SODs, catalase, and glutathione peroxidase. Normal levels of ROS are important physiological regulators of the cellular signaling pathway [39]. However, down-regulation or defects in antioxidant enzymatic systems in the aging process contribute to the accumulation of high ROS (hi-ROS) [40]. When hiROS is presented, the respiratory complexes are likely to be attacked and drive reverse electron transfer (RET), which at the same time leads to electron leak to damaged cell compartments. Consequently, the OXPHOS activities will be prohibited to lower the production of ROS, whereas ATP production from mitochondria will also be reduced. On the other hand, mitochondrial membrane potential (MMP, ∆Ψm) established by the proton gradient through ETC is essential to drive both F0F1-ATPase to produce ATP and acts as quality control for mitochondrial maintenance. Due to the reduction of ATP production and lower mitochondrial ∆Ψm, the accumulated ROS drives the cell senescence [41]. Moreover, the ROS-induced oxidative stress further leads to modification of cellular proteins, lipids, and DNA that are generally more irreversible [42]. Therefore, the interplay between mitochondria ROS and protective antioxidant responses is essential for aging.
Mitochondrial defects and the perturbation of endoplasmic reticulum-mitochondria contact sites (MERC) are indicated as a hallmark in aging and many aging-associated neuronal degeneration diseases, including Alzheimer's disease (AD), Parkinson's disease, and amyotrophic lateral sclerosis [43][44][45]. The communication between ER and mitochondria regulates lipid metabolism, Ca 2+ homeostasis, unfolded protein response, ER stress, and mitochondrial quality control, all implicated in neurodegenerative diseases [46]. Furthermore, Studies unveil that the cerebral metabolic rate of oxygen is significantly decreased and correlated to the severity of dementia in the brain of AD patients [47]. Mitochondria are the primary source for energy generation in neuron cells through OXPHOS, which involves a flow of electrons through ETC and consumes oxygen. Therefore, reduced glucose usage, so-called "glucose hypo-metabolism," strongly implies mitochondrial dysfunction in the course of AD [48]. In addition, the failure of ETC leads to RET and constitutes a positive feedback loop for enhancing ROS production, inducing mitochondria dysfunction, and releasing more ROS. Excessive generation of ROS will harm all compartments of cells which drive cell death signaling. This study indicates that the reduction of MERC Ca 2+ IP3R-VDAC channel, and ANT2 activity in LPA 3 -deficient cells impairs the import of Ca 2+ and ADP into the mitochondrial matrix. These limit the efficiency of F0F1-ATPase in the final step of the OXPHOS reaction and consequently attenuate electron transfer through the ETC and increase the production of toxic ROS by RET. Notably, dysfunction of LPA pathways is also reported in AD [49]. Thus, we suspect that the absence of the LPA 3 leads to the dysregulation of MERC and ANT2 during progression of neuronal degeneration diseases. Since LPA 3 has a beneficial effect on mitochondrial health and protective antioxidant responses, the LPA 3 agonists hold value as potential drugs against the pathogenesis of neuronal degeneration diseases in the future.
On the other hand, LPA 3 has been reported to protect erythroid differentiation and aging-related anemia [11,50]. The regulation of mitochondrial biogenesis is also important in erythropoiesis since mitochondria is crucial for heme and iron metabolism. Extracellular Ferric iron (Fe 3+ ) binds to a carrier protein transferrin (Tf) and is transported into the cell by Tf receptor-mediated endocytosis [51]. A proton pump then acidifies the iron-loaded endosomes to change the conformation of the Tf and Tf receptor, followed by iron releasing [52]. Ferric iron is converted into ferrous iron (Fe 2+ ) in the endosomes. These released iron ions are transferred across the endosomal membrane to the cytoplasm by divalent metal trans-porter 1 (DMT1) [53]. Next, intracellular iron is further transported to mitochondria for heme synthesis and iron-sulfur cluster (Fe-S) biogenesis. In addition, heme and iron-sulfur clusters are crucial for erythropoiesis and function as an important cofactor of enzymes for various cellular physiologies [54]. Therefore, defective mitochondria could seriously impact erythropoiesis and systemic defect. Moreover, impaired iron metabolism will lead to iron overload in multiple organs, which is called hemochromatosis. Iron release and overload from damaged erythrocytes cause cell damage through ferroptosis, a recently identified iron-dependent form of programmed cell death [55]. The high redox potential of iron leads to the Fenton reaction, leading to accumulation of toxic oxidative stress, lipid peroxidation in the plasma membrane, and various organelle damage. The metabolic rate decreases with aging, leading to deficiency in molecules using iron as cofactors or working on iron metabolism [56]. Accordingly, iron would be in excess with aging progression, leading systemic ferroptosis. These findings align with our results and suggest that LPA 3 may provide beneficial effects against aging/anemia-induced ferroptosis in multiple organs.

Conclusions
This study demonstrated that the LPA 3 signal is crucial for mitochondrial biogenesis and oxidative stress response. siRNA-mediated depletion of LPA 3 led to the depolarization of mitochondrial ∆Ψm and ROS accumulation. In addition, depletion of LPA 3 by siRNA resulted in cisplatin-induced cytochrome c releasing, suggesting that LPA 3 is essential to suppress the mitochondrial apoptosis pathway. Moreover, LPA 3 was associated with the ANT2 protein expression and ADP-ATP exchange activities. Depletion of LPA 3 led to the destabilization of ANT2 and disrupted ADP-ATP exchange activities. In addition, LPA 3 knockdown cells showed decreased calcium uptake from ER to mitochondria via the IP3R1-VDAC1 channel, which contributes to increasing ROS accumulation and mitochondria dysfunction. Notably, the LPA 3 agonist OMPT protected mitochondrial functions against oxidative stress by H 2 O 2 and OXPHOS inhibitors. Moreover, treatment of OMPT rescued mitochondrial activities in HGPS premature aging cells. Based on our findings, we propose that LPA 3 acts as the gatekeeper for mitochondrial healthiness and protects cells from oxidative stress to maintain cell youth. Additionally, LPA 3 agonists can be a promising therapeutic target to prevent mitochondrial oxidative stress in aged cells.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11020351/s1, Figure S1: The knockdown efficiency of LPA 3 siRNA. LPA 3 mRNA expression level was measured using real-time PCR and relative to siRNA control. The expression level of GAPDH was used as a loading control. The bar graph was generated from three independent analyses. *** p < 0.001. Figure S2: LPA 3 protected against oxidative stress-induced mitochondrial ∆Ψm loss in K562 cells. K562 cells showed decreased mitochondria ∆Ψm under 500 µM of H 2 O 2 treatment. 50 nM of OMPT and 2 µM of NAC protected cells against H 2 O 2 -induced mitochondrial ∆Ψm loss. FCCP was used as a positive control for loss of mitochondrial ∆Ψm. The bar graphs were generated from three independent analyses. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure S3: LPA 3 improved cell viability from H 2 O 2 induced oxidative stress. HSF cells were subjected to a cell proliferation assay under 50 µM of H 2 O 2 for 72 h. 50 nM of OMPT showed protective effects against H 2 O 2 . The bar graphs were generated from three independent analyses. **, p < 0.01.