Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.


Introduction
Ferroptosis is a recently characterized programmed cell death process marked by the accumulation of iron and lipid peroxides, manifesting when the glutathione (GSH)dependent lipid peroxide repair system is compromised [1,2].The amino acid reverse transporter system Xc-, located in the plasma membrane, consists of two fundamental components: the light-chain subunit solute carrier family 7 member 11 (SLC7A11), and the heavy-chain subunit solute carrier family 3 member 2 (SLC3A2).This system regulates the balance of glutamate and cysteine by exchanging intracellular glutamate and extracellular cystine in a 1:1 ratio.Cystine is rapidly reduced to cysteine, a precursor essential for glutathione synthesis.On the other hand, GPX4 has been reported as a phospholipid hydroperoxidase [3], altering the state of phospholipid hydroperoxide from cytotoxic to non-toxic phospholipid alcohol.This prevents the formation and accumulation of lethal lipid reactive oxygen species (ROS) at the expense of GSH.Additionally, GPX4 exhibits antioxidant properties, shielding against cell membrane damage and lipid peroxidation during the ferroptosis process [4][5][6].
Iron is a crucial trace element in the human body, existing in two oxidation states: ferrous (Fe 2+ ) and ferric (Fe 3+ ).The iron redox cycle affects the sensitivity of cells to ferroptosis [7].In its Fe 3+ form, circulating iron is bound to transferrin and is imported into cells via the membrane protein transferrin receptor 1 (TFR1).Following absorption, STEAP3 metallo-reductase in the endosome reduces Fe 3+ to Fe 2+ , which is then released into the cytosol through solute carrier family 11 member 2 (SLC11A2).Excess cellular iron ions are stored in ferritin, an intracellular iron storage protein complex comprising ferritin light chain (FTL) polymer and ferritin heavy chain 1 (FTH1).Additionally, ferroxidases can reoxidize Fe 2+ to Fe 3+ , which is then exported into the extracellular space by the iron-efflux protein ferroportin (FPN) [8][9][10][11].These events regulate cellular iron uptake and are pivotal for ferroptosis induction [12].
In progerin-expressing K562 cells, LPA 3 protein expression levels were decreased [16,17], suggesting that LPA 3 signaling is inhibited during aging.Conversely, the activation of LPA 3 by OMPT resulted in the upregulation of erythrocyte markers, including glycophorin A (Gly A) and γ-globin.Interestingly, several studies indicated that red blood cell (RBC) numbers are decreased and that their function is impaired during the aging process due to a high level of oxidative stress [18][19][20].These findings suggest that LPA 3 may serves as an essential regulator both in preventing cellular aging by mitigating oxidative stress and by being a significant regulator during erythropoiesis.
Emerging research has demonstrated the relevance of ferroptosis in various physiological processes, including murine embryonic erythropoiesis and aging in different organs [21].However, the relationship between LPA 3 signaling and ferroptosis remains poorly understood.Therefore, the aim of this study was to investigate the impact of LPA 3 on ferroptosis and to establish the potential link among LPA 3, erythropoiesis, and aging.

An LPA 3 Agonist Mitigates Erastin-Induced Ferroptotic Death
Erastin, as a ferroptosis inducer, acts by inhibiting system Xc-and reducing intracellular GPX4 levels [8].To assess the toxicity of erastin in HT-1080 cells, varying erastin concentrations, ranging from 0 to 10 µM, were administered for 12 or 24 h.Cell viability was evaluated using the CCK-8 assay.The results demonstrated erastin-induced cytotoxicity in HT-1080 cells in a dose-and time-dependent manner (Figure 1A).The IC50 values were determined as 4.25 µM and 0.5549 µM at 12 and 24 h post-treatment, respectively.
We also assessed the levels of Fe 2+ , a trigger for Fenton's reaction leading to lipid peroxidation.The fluorescence intensity from the FerroOrange Fluorescent probe was utilized to measure Fe 2+ levels.HT-1080 cells were treated with erastin at various concentrations, ranging from 0 to 10 µM, for 12 or 24 h.The results revealed that erastin induced the dose-and time-dependent accumulation of Fe 2+ in HT-1080 cells (Figure 1B).The iron accumulation exhibited a significant increase at 10 µM for 12 h and 5 µM for 24 h (p-value < 0.01).Therefore, we employed a concentration of 5 µM for a duration of 24 h in subsequent experiments.

An LPA 3 Agonist Mitigates Erastin-Induced Lipid Peroxidation in HT-1080 Cells
Excessive ROS can target polyunsaturated fatty acids in cellular or organ membranes, resulting in lipid peroxidation [25].In our previous study, we proposed that lysophospha-tidic acid (LPA) modulates reactive oxygen species (ROS) levels and cellular senescence via LPA 3 , attenuating cellular aging in Hutchinson-Gilford progeria syndrome (HGPS) cells [14].Therefore, we aimed to validate the role of LPA 3 in stabilizing lipid peroxidation in erastin-induced cells.To assess the lipid oxidation level, we applied C11 BODIPY 581/591, a widely used indicator of lipid oxidation.The results indicated that erastin treatment increased the level of lipid ROS (Figure 2A, Gray area).However, the activation of LPA 3 mitigated erastin-induced lipid oxidation in HT-1080 cells (Figure 2A).Additionally, we investigated whether the activation of LPA 3 regulates lipid ROS levels through intracellular antioxidants.We measured SLC7A11 and GPX4 protein expression levels, which are important free radical scavengers during ferroptosis.The results showed that erastin treatment reduces the protein expression level of SLC7A11 and GPX4.However, the activation of LPA 3 rescued the expression level of SLC7A11 and GPX4 under erastin treatment (Figure 2B).To further address the role of LPA 3 in ferroptosis, we utilized siRNA to knock down LPAR3 in HT-1080 cells.The results showed that LPAR3 knockdown decreased SLC7A11 and GPX4 protein and mRNA levels (Figure 2C,D).Together, these data suggest that the activation of LPA 3 signaling protects cells from lipid peroxidation during erastin-induced ferroptosis.

An LPA3 Agonist Mitigates Erastin-Induced Lipid Peroxidation in HT-1080 Cells
Excessive ROS can target polyunsaturated fatty acids in cellular or organ membranes, resulting in lipid peroxidation [25].In our previous study, we proposed that lysophosphatidic acid (LPA) modulates reactive oxygen species (ROS) levels and cellular senescence via LPA3, attenuating cellular aging in Hutchinson-Gilford progeria syndrome (HGPS) cells [14].Therefore, we aimed to validate the role of LPA3 in stabilizing lipid peroxidation in erastin-induced cells.To assess the lipid oxidation level, we applied C11 BODIPY 581/591, a widely used indicator of lipid oxidation.The results indicated that erastin treatment increased the level of lipid ROS (Figure 2A, Gray area).However, the activation of LPA3 mitigated erastin-induced lipid oxidation in HT-1080 cells (Figure 2A).Additionally, we investigated whether the activation of LPA3 regulates lipid ROS levels through intracellular antioxidants.We measured SLC7A11 and GPX4 protein expression levels, which are important free radical scavengers during ferroptosis.The results showed that erastin treatment reduces the protein expression level of SLC7A11 and GPX4.However, the activation of LPA3 rescued the expression level of SLC7A11 and GPX4 under erastin treatment (Figure 2B).To further address the role of LPA3 in ferroptosis, we utilized siRNA to knock down LPAR3 in HT-1080 cells.The results showed that LPAR3 knockdown decreased SLC7A11 and GPX4 protein and mRNA levels (Figure 2C,D).Together, these data suggest that the activation of LPA3 signaling protects cells from lipid peroxidation during erastin-induced ferroptosis.

An LPA 3 Agonist Stabilizes Iron Homeostasis in Erastin-Induced HT-1080 Cells
Iron accumulation is also an important feature during ferroptosis.Next, we aimed to validate whether LPA 3 is involved in iron accumulation by analyzing the Fe 2+ level in erastin-induced cells.To determine this, we used the FerroOrange Fluorescent probe for detecting intracellular Fe 2+ [26].DFO, NAC, and Fer-1 served as inhibitors of ferroptosis and were used as positive controls.The results indicated that the Fe 2+ level was significantly increased with erastin treatment.However, the co-treatment of erastin with OMPT resulted in a significant decrease of Fe 2+ levels in erastin-induced cells (Figure 3A), suggesting that LPA 3 activation reduces Fe 2+ levels induced by erastin.pre-treated with erastin for 24 h, followed by treatment with or without OMPT for 5 min.Western blot analysis revealed that OMPT treatment increased p-MEK (N = 3) and p-ERK (N = 4) protein levels.HT-1080 cells were pre-treated with erastin for 2 h, followed by treatment with or without OMPT for an additional 22 h.Western blot analysis revealed that OMPT treatment increased the protein level of NRF2 (N = 4) in erastin-induced cells.Statistical analysis was performed using one-way ANOVA and Student's t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.not significant.
To further validate whether LPA 3 activation regulates iron metabolism and storage function during ferroptosis, we measured the protein expression levels of HO-1 and FTH1, which have been reported to be involved in cellular iron homeostasis control [27].Treatment with OMPT increased HO-1 and FTH1 protein expression levels in erastin-induced cells (Figure 3B), suggesting that LPA 3 activation promotes iron metabolism.We also applied siRNA to knock down LPAR3 in HT-1080 cells.The results showed that LPAR3 knockdown led to the downregulation of HO-1 and FTH1 protein and mRNA levels (Figure 3C).These findings indicate that LPA 3 has an important role in stabilizing Fe 2+ homeostasis by regulating the expression level of iron metabolism/storage proteins and thereby affects ferroptosis in cells.
Our prior study revealed that LPA 3 mitigates oxidative stress by upregulating NRF2 [14].Furthermore, MEK-ERK activation has been reported to be one of the major pathways in the activation of NRF2 [28].Thus, we proposed to explore whether LPA 3 regulates ferroptosis via MEK/ERK/NRF2-dependent oxidative stress.Western blotting was applied to measure the phosphorylation level of p-MEK and p-ERK in the MAPK pathway, along with NRF2 protein levels, which has been reported as a regulator of ROS in HPGS during OMPT treatment [14].The results showed that erastin-induced ferroptosis led to a reduction in NRF2 protein levels.However, treatment with OMPT significantly increased the protein levels of p-MEK, p-ERK, and NRF2 (Figure 3D).This indicates that erastin treatment effectively suppressed ROS production, but that LPA 3 activation restored NRF2 levels.This recovery appears to be dependent on the MAPK pathway.Taken together, these results suggest that LPA 3 might prevent cells from erastin-induced ferroptosis via the MEK/ERK/NRF2 signaling pathway.

LPA 3 Promotes Erythropoiesis in Erastin-Induced K562 Cells
In our previous study, we demonstrated that LPA 3 plays major roles in the regulation of myeloid differentiation [15].Therefore, we aimed to validate whether LPA 3 -dependent ferroptosis is involved in the differentiation of erythroblasts.To achieve this, we initially established a human erythroleukemic cell line, K562, expressing firefly luciferase driven by the GYPA promoter.Cells were treated with OMPT in the absence or presence of erastin, while 200 nM of hemin was used as the positive control.Treatment with both hemin and OMPT, respectively, increased luciferase activity, indicating enhanced GYPA transcriptional activity.Conversely, treatment with erastin significantly decreased GYPA luciferase signals, suggesting that ferroptosis induction disrupts the differentiation of red blood cells (RBCs).However, when OMPT was applied along with erastin, it increased Gly A transcriptional activity under the erastin-treated conditions (Figure 4B), which was consistent with our previous observations [15].
To further investigate the roles of LPA 3 in this signaling pathway, we used siRNA to knock down LPAR3 in K562 cells.The results indicated that LPAR3 knockdown decreased the transcription activity of Gly A. However, Gly A transcriptional activity did not respond to the treatment of OMPT (Figure 4C).Additionally, erastin treatment decreased Gly A gene expression both in control and LPA 3 -suppressed K562 cells (Figure 4D).Taken together, these data suggest that LPA 3 may play an important role in regulating erythropoiesis in erastin-induced K562 cells.
To further investigate the roles of LPA3 in this signaling pathway, we used siRNA to knock down LPAR3 in K562 cells.The results indicated that LPAR3 knockdown decreased the transcription activity of Gly A. However, Gly A transcriptional activity did not respond to the treatment of OMPT (Figure 4C).Additionally, erastin treatment decreased Gly A gene expression both in control and LPA3-suppressed K562 cells (Figure 4D).Taken together, these data suggest that LPA3 may play an important role in regulating erythropoiesis in erastin-induced K562 cells.

Discussion
Ferroptosis, characterized by lipid peroxidation and iron accumulation, is a recently identified programmed cell death pathway.Our study reveals that activating LPA 3 mitigates ferroptosis.Using the agonist OMPT, LPA 3 activation elevated anti-ferroptosis gene expression, reducing lipid peroxidation and intracellular iron in erastin-induced cells.Additionally, LPA 3 activation enhanced NRF2 expression, a key oxidative stress regulator.In human erythroleukemia K562 cells, LPA 3 activation rescued erastin-induced ferroptosis and erythropoiesis defects.Overall, our findings suggest that LPA 3 activation inhibits ferroptosis by suppressing lipid oxidation and iron accumulation, potentially linking LPA 3 to erythropoiesis and aging.
Our results propose a potential mechanism by which LPA 3 activation stabilizes intracellular Fe 2+ , a key factor contributing to ferroptosis [29].While the reduction in intracellular Fe 2+ levels upon OMPT treatment may not be as pronounced as with other known ferroptosis inhibitors, like DFO, NAC, and Fer-1, it still significantly attenuates Fe 2+ accumulation compared to that in cells treated solely with erastin.This suggests that the LPA 3 signaling pathway may be a damage response during ferroptosis.
The analyses of HO-1, pMEK, and pERK indicate that the combined treatment of OMPT and erastin produces a response similar to treatment with OMPT alone.This contrasts the responses observed for FTH1 and NRF2, where treatment with OMPT alone resulted in higher expression levels, as expected.This discrepancy suggests a likely indirect correlation among the variables under consideration.From our perspective, there is notable variability in outcomes with the co-treatment of OMPT and erastin across different groups, with distinct responses observed for HO-1, pMEK, and pERK compared to FTH1 and NRF2.However, when compared to exclusive treatment with erastin, each group subjected to co-treatment with OMPT and erastin demonstrates the restoration of approximately 20-30% of protein expression levels.Therefore, we propose that, in terms of efficacy, the outcomes show comparability across all groups.Nevertheless, these findings underscore the need for further investigation into the intricate molecular interactions involved in the LPA 3 -mediated inhibition of ferroptosis.
Previous studies have demonstrated that ferroptosis is involved in physiological processes, including embryonic erythropoiesis [30,31] and aging [32][33][34].By using the HNEJ-1 mouse monoclonal antibody, which specifically recognizes HNE-derived Michael adducts expressed in histidine, lysine, and cysteine residues, ferroptosis events were monitored throughout the entire life span of rats [21].A notable age-related increase in ferroptosis was observed in various organs, accompanied by iron accumulation.This phenomenon extended from embryonic stages to advanced age, implicating ferroptosis in both embryonic erythropoiesis and aging processes.
The prevalence of anemia In the elderly population is associated with declining hemoglobin levels [35].The level of hemoglobin, which is composed of more than 60% of iron, significantly decreases with aging [36].However, damaged erythrocytes under severe hemolytic anemia lead to more serious erythrophagocytosis by splenic red pulp macrophages (RPM) [30].Similarly, patients with liver cirrhosis were shown to have lower serum and hepatic transferrin, resulting in iron overloading and ferroptosis in multiple organs [37][38][39].Our recent findings on LPA 3 activation restoring erythrocyte numbers under acute hemolytic anemia conditions suggest a potential therapeutic avenue for reducing systemic ferroptosis in aging-induced anemia [15].To further investigate the connection between ferroptosis and erythropoiesis, we utilized a K562 cell model expressing firefly luciferase driven by the Gly A promoter, a marker of matured erythroid cells.Our results indicate that OMPT increases luciferase activity of the Gly A promoter during ferroptosis, supporting the link among LPA 3 activation, ferroptosis, and erythropoiesis.Knockdown experiments further strengthen this connection, emphasizing the role of LPA 3 in regulating these cellular processes.
In conclusion, our study substantiates that the activation of LPA 3 signaling indeed regulates ferroptosis and erythropoiesis.This finding not only enhances our understanding of the intricate molecular mechanisms governing these cellular processes but also holds promise for potential therapeutic strategies targeting hematopoiesis disorders and agerelated anemia.

Cell Viability Assay
The cell viability assay was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan).HT-1080 cells were seeded at 1000 cells per well in 96-well plates for 24 h.Cells were starved 4 h before reagent treatment.Next, the reagent was treated and incubated for 12 or 24 h.After reagent treatment, 10 µL of CCK-8 solution mixed with 90 µL of culture medium was added to each well, and the plates were incubated for an additional 1 h.Cell viability was evaluated by measuring the absorbance at a 450 nm wavelength using a SpectraMax i3X (Molecular Devices, San Jose, CA, USA).

Intracellular Ferrous Iron Content Detection
Intracellular ferrous iron content detection was determined using FerroOrange (Dojindo, Kumamoto, Japan).HT-1080 cells were seeded at a density of 1000 cells per well in 96-well plates for 24 h.Cells were starved 4 h before reagent treatment.Then, the reagent was treated and incubated for 12 or 24 h.Subsequently, reagent treatment was initiated, and cells were incubated for either 12 or 24 h.Following reagent treatment, the supernatant was discarded, and the cells were washed three times with DPBS.A 1 µM FerroOrange working solution with DPBS was then added to the cells, and they were incubated for 30 min.The intracellular ferrous iron content was quantified using a SpectraMax i3X (Molecular Devices), with excitation and emission bands set at 543 nm and 580 nm, respectively.

Measurement of Lipid ROS Level
The level of lipid reactive oxygen species (ROS) was quantified using the C11 BOD-IPY™ 581/591 Lipid Peroxidation Sensor (Thermo Fisher Scientific, Waltham, MA, USA).HT-1080 cells were seeded at a density of 50,000 cells per well in six-well plates, followed by incubation for 24 h.Cells were starved 4 h before reagent treatment.Subsequently, cells were treated with erastin (5 µM) in the presence or absence of OMPT (10 µM) for 24 h.Following reagent treatment, cells were washed with PBS.Cells were then stained with C11 BODIPY™ 581/591 for 20 min in a 37 • C incubator, followed by washing with PBS.Fluorescence signals were detected using the Guava ® Muse ® Cell Analyzer (Luminex Corp., Austin, TX, USA), with 10,000 cells measured and quantified for each experiment.

Luciferase Assay for GYPA Promoter Assay
The pGL4.32 [luc2P/NF-κB-RE/Hygro] plasmid (Promega, WI, USA) was utilized for luciferase assays.The luciferase plasmids were co-transfected with the GYPA promoter and CMV-RFP, serving as an internal control.The relative activities, calculated as the ratio of Firefly luciferase activity to RFP intensity, were quantified using the ONE-Glo™ Luciferase Assay system (Promega, WI, USA).

Statistical Analysis
The data are presented as the mean ± SD of a minimum of three independent experiments.Statistical analyses for multiple group comparisons of the mean ± SD were conducted using one-way analysis of variance (ANOVA).Significance levels were denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.Results with a p-value less than 0.05 were considered statistically significant.

Conclusions
In conclusion, our study establishes a crucial link between LPA 3 activation and the regulation of ferroptosis, a distinctive programmed cell death process associated with lipid peroxidation and iron accumulation.Utilizing the LPA 3 agonist OMPT, we demonstrated its potent impact on elevating anti-ferroptotic gene expression, reducing lipid peroxidation, and inhibiting intracellular ferrous iron accumulation in erastin-induced cells.Additionally, NRF2, a key oxidative stress regulator, was upregulated in OMPT-treated cells.Impor-

Figure 2 .
Figure 2.An LPA3 agonist mitigates erastin-induced lipid peroxidation in HT-1080 cells.(A) HT-1080 cells were starved 4 h before reagent treatment and pre-treated with erastin for 2 h, followed by treatment with or without OMPT for an additional 22 h.The fluorescence intensity of C11 BOD-IPY was measured via FACS, and the results showed that ROS levels decreased after OMPT treatment in erastin-induced cells.(B) HT-1080 cells were starved 4 h before reagent treatment and pretreated with erastin for 2 h, followed by incubation with or without OMPT for 22 h.Western blots revealed that OMPT treatment increased the protein level of SCL7A11 (N = 3) and GPX4 (N = 3) in erastin-induced cells.(C) Real-time PCR results showed the knockdown of LPAR3 mediated by siRNA (siLPA3) for 48 h (N = 3).(D) The knockdown of LPAR3 mediated by siRNA (siLPA3) for 48 h decreased the protein level of SLC7A11 (N = 4) and GPX4 (N = 3) and mRNA (N = 3) levels.Statistical analysis was performed using one-way ANOVA and Student's t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.not significant.

Figure 2 .
Figure 2.An LPA 3 agonist mitigates erastin-induced lipid peroxidation in HT-1080 cells.(A) HT-1080 cells were starved 4 h before reagent treatment and pre-treated with erastin for 2 h, followed by treatment with or without OMPT for an additional 22 h.The fluorescence intensity of C11 BODIPY was measured via FACS, and the results showed that ROS levels decreased after OMPT treatment in erastin-induced cells.(B) HT-1080 cells were starved 4 h before reagent treatment and pre-treated with erastin for 2 h, followed by incubation with or without OMPT for 22 h.Western blots revealed that OMPT treatment increased the protein level of SCL7A11 (N = 3) and GPX4 (N = 3) in erastin-induced cells.(C) Real-time PCR results showed the knockdown of LPAR3 mediated by siRNA (siLPA 3 ) for 48 h (N = 3).(D) The knockdown of LPAR3 mediated by siRNA (siLPA 3 ) for 48 h decreased the protein level of SLC7A11 (N = 4) and GPX4 (N = 3) and mRNA (N = 3) levels.Statistical analysis was performed using one-way ANOVA and Student's t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.not significant.

Figure 4 .
Figure 4. LPA3 promotes erythropoiesis in erastin-induced K562 cells.(A) Construction a luciferasebased screening system.(B) K562 cells were starved 4 h before reagent treatment and pre-treated with vehicle or erastin (5 µM) for 2 h, followed by treatment with or without hemin (200 nM) or OMPT (10 µM) for 22 h.The transcriptional activity of Gly A was decreased under erastin treatment, but was increased after OMPT treatment (N = 3).(C) Knockdown of LPAR3 mediated by siRNA for 48 h resulted in a decreased expression level of Gly A (N = 4).(D) LPAR3 knockdown mediated by

Figure 4 .
Figure 4. LPA 3 promotes erythropoiesis in erastin-induced K562 cells.(A) Construction a luciferasebased screening system.(B) K562 cells were starved 4 h before reagent treatment and pre-treated with vehicle or erastin (5 µM) for 2 h, followed by treatment with or without hemin (200 nM) or OMPT (10 µM) for 22 h.The transcriptional activity of Gly A was decreased under erastin treatment, but was increased after OMPT treatment (N = 3).(C) Knockdown of LPAR3 mediated by siRNA for 48 h resulted in a decreased expression level of Gly A (N = 4).(D) LPAR3 knockdown mediated by siRNA lasted for 48 h, followed by starvation for 4 h before pre-treatment with erastin for 2 h, and then treatment with or without OMPT for 22 h in K562 cells.Gly A expression level did not respond to OMPT under LPAR3-knockdown conditions in K562 cells (N = 4).Statistical analysis was performed using one-way ANOVA; ** p < 0.01, *** p < 0.001, n.s.not significant.