LPA3 Receptor Phosphorylation Sites: Roles in Signaling and Internalization.

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with ß-arrestin 2 and LPA3 internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA3 receptor function.

LPA-induced expression of CCN2 in muscular fibro/adipogenic progenitors (FAPs): Unraveling cellular communication networks.

Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA1 receptor and, to a lower extent, by also the LPA6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs.

Deciphering a GPCR-lncrna-miRNA nexus: Identification of an aberrant therapeutic target in ovarian cancer.

Ovarian cancer ranks as a leading cause of mortality among gynecological malignancies, primarily due to the lack of early diagnostic tools, effective targeted therapy, and clear understanding of disease etiology. Previous studies have identified the pivotal role of Lysophosphatidic acid (LPA)-signaling in ovarian cancer pathobiology. Our earlier transcriptomic analysis identified Urothelial Carcinoma Associated-1 (UCA1) as an LPA-stimulated long non-coding RNA (lncRNA). In this study, we elucidate the tripartite interaction between LPA-signaling, UCA1, and let-7 miRNAs in ovarian cancer progression. Results show that the elevated expression of UCA1 enhances cell proliferation, invasive migration, and therapy resistance in high-grade serous ovarian carcinoma cells, whereas silencing UCA1 reverses these oncogenic phenotypes. UCA1 expression inversely correlates with survival outcomes and therapy response in ovarian cancer clinical samples, underscoring its prognostic significance. Mechanistically, UCA1 sequesters let-7 miRNAs, effectively neutralizing their tumor-suppressive functions involving key oncogenes such as Ras and c-Myc. More significantly, intratumoral delivery of UCA1-specific siRNAs inhibits the growth of cisplatin-refractory ovarian cancer xenografts, demonstrating the therapeutic potential of targeting LPAR-UCA1-let-7 axis in ovarian cancer. Thus, our results identify LPAR-UCA1-let-7 axis as a novel avenue for targeted treatment strategies.

LPA5-Dependent signaling regulates regeneration of the intestinal epithelium following irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.NEW & NOTEWORTHY We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells.

Lysophosphatidic acid (LPA) receptor-mediated signaling regulates hypoxia-induced biological functions of lymphatic endothelial cells.

The tumor microenvironment is an extremely complex composed of cancer cells and various non-cancer cells, including lymphatic endothelial cells. Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) activate a variety of malignant properties in human malignancies. In the present study, we examined the roles of LPA receptor-mediated signaling in biological responses of lymphatic endothelial SVEC4-10 cells induced by hypoxia. Lpar1, Lpar2 and Lpar3 expressions were decreased in SVEC4-10 cells cultured at hypoxic conditions (1 % O2). LPA had no impact on the cell growth activity of SVEC4-10 cells in 21 % O2 culture conditions. Conversely, the cell growth activity of SVEC4-10 cells in 1 % O2 culture conditions was reduced by LPA. The cell motile activity of SVEC4-10 cells was elevated by 1 % O2 culture conditions. GRI-977143 (LPA2 agonist) and (2S)-OMPT (LPA3 agonist) stimulated SVEC4-10 cell motility as well as AM966 (LPA1 antagonist). In tube formation assay, the tube formation of SVEC4-10 cells in 1 % O2 culture conditions was markedly increased, in comparison with 21 % O2. GRI-977143 and (2S)-OMPT elevated the tube formation of SVEC4-10 cells. Furthermore, the tube formation of SVEC4-10 cells was increased by AM966. These results suggest that LPA receptor-mediated signaling contributes to the modulation of hypoxic-induced biological functions of lymphatic endothelial cells.

Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center.

INTRODUCTION: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce. METHODS: In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome. RESULTS: The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression. CONCLUSION: Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1.

Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.

Phosphatidylethanolamine (18:2e/18:2) may inhibit adipose tissue wasting in patients with cancer cachexia by increasing lysophosphatidic acid receptor 6.

BACKGROUND: Cancer associated cachexia is characterized by the significant loss of adipose tissue, leading to devastating weight loss and muscle wasting in the majority of cancer patients. The effects and underlying mechanisms of degradation metabolites on adipocytes in cachectic patients remain poorly understood. To address this knowledge gap, we conducted a comprehensive study combining lipidomic analysis of subcutaneous and visceral adipose tissue with transcriptomics data from the database to investigate the mechanisms of lipid regulation in adipocytes. METHODS: We collected subcutaneous and visceral adipose tissue samples from cachectic and noncachectic cancer patients. Lipidomic analysis was performed to identify differentially expressed lipids in both types of adipose tissue. Additionally, transcriptomics data from the GEO database were analyzed to explore gene expression patterns in adipocytes. Bioinformatics analysis was employed to determine the enrichment of differentially expressed genes in specific pathways. Furthermore, molecular docking studies were conducted to predict potential protein targets of specific lipids, with a focus on the PI3K-Akt signaling pathway. Western blot analysis was used to validate protein levels of the identified target gene, lysophosphatidic acid receptor 6 (LPAR6), in subcutaneous and visceral adipose tissue from cachectic and noncachectic patients. RESULTS: Significant lipid differences in subcutaneous and visceral adipose tissue between cachectic and noncachectic patients were identified by multivariate statistical analysis. Cachectic patients exhibited elevated Ceramides levels and reduced CerG2GNAc1 levels (P < 0.05). A total of 10 shared lipids correlated with weight loss and IL-6 levels, enriched in Sphingolipid metabolism, GPI-anchor biosynthesis, and Glyceropholipid metabolism pathways. LPAR6 expression was significantly elevated in both adipose tissues of cachectic patients (P < 0.05). Molecular docking analysis indicated strong binding of Phosphatidylethanolamine (PE) (18:2e/18:2) to LPAR6. CONCLUSIONS: Our findings suggest that specific lipids, including PE(18:2e/18:2), may mitigate adipose tissue wasting in cachexia by modulating the expression of LPAR6 through the PI3K-Akt signaling pathway. The identification of these potential targets and mechanisms provides a foundation for future investigations and therapeutic strategies to combat cachexia. By understanding the underlying lipid regulation in adipocytes, we aim to develop targeted interventions to ameliorate the devastating impact of cachexia on patient outcomes and quality of life. Nevertheless, further studies and validation are warranted to fully elucidate the intricate mechanisms involved and translate these findings into effective clinical interventions.

Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

Network analysis-guided drug repurposing strategies targeting LPAR receptor in the interplay of COVID, Alzheimer's, and diabetes.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has greatly affected global health. Emerging evidence suggests a complex interplay between Alzheimer's disease (AD), diabetes (DM), and COVID-19. Given COVID-19's involvement in the increased risk of other diseases, there is an urgent need to identify novel targets and drugs to combat these interconnected health challenges. Lysophosphatidic acid receptors (LPARs), belonging to the G protein-coupled receptor family, have been implicated in various pathological conditions, including inflammation. In this regard, the study aimed to investigate the involvement of LPARs (specifically LPAR1, 3, 6) in the tri-directional relationship between AD, DM, and COVID-19 through network analysis, as well as explore the therapeutic potential of selected anti-AD, anti-DM drugs as LPAR, SPIKE antagonists. We used the Coremine Medical database to identify genes related to DM, AD, and COVID-19. Furthermore, STRING analysis was used to identify the interacting partners of LPAR1, LPAR3, and LPAR6. Additionally, a literature search revealed 78 drugs on the market or in clinical studies that were used for treating either AD or DM. We carried out docking analysis of these drugs against the LPAR1, LPAR3, and LPAR6. Furthermore, we modeled the LPAR1, LPAR3, and LPAR6 in a complex with the COVID-19 spike protein and performed a docking study of selected drugs with the LPAR-Spike complex. The analysis revealed 177 common genes implicated in AD, DM, and COVID-19. Protein-protein docking analysis demonstrated that LPAR (1,3 & 6) efficiently binds with the viral SPIKE protein, suggesting them as targets for viral infection. Furthermore, docking analysis of the anti-AD and anti-DM drugs against LPARs, SPIKE protein, and the LPARs-SPIKE complex revealed promising candidates, including lupron, neflamapimod, and nilotinib, stating the importance of drug repurposing in the drug discovery process. These drugs exhibited the ability to bind and inhibit the LPAR receptor activity and the SPIKE protein and interfere with LPAR-SPIKE protein interaction. Through a combined network and targeted-based therapeutic intervention approach, this study has identified several drugs that could be repurposed for treating COVID-19 due to their expected interference with LPAR(1, 3, and 6) and spike protein complexes. In addition, it can also be hypothesized that the co-administration of these identified drugs during COVID-19 infection may not only help mitigate the impact of the virus but also potentially contribute to the prevention or management of post-COVID complications related to AD and DM.

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.

Lysophosphatidic acid (LPA) receptor-mediated signaling and cellular responses to anticancer drugs and radiation of cancer cells.

Lysophosphatidic acid (LPA) is a simple physiological lipid and structurally consists of a fatty, a phosphate and a glycerol. LPA binds to G protein-coupled LPA receptors (LPA1 to LPA6). LPA receptor-mediated signaling mediates a variety of biological responses, such as cell growth, migration, morphogenesis, differentiation and protection from apoptosis. It is considered that LPA receptor-mediated signaling plays an important role in the pathogenesis of human malignancies. So far, genetic and epigenetic alterations of LPA receptors have been found in several cancer cells as well as abnormal LPA production. In addition, LPA receptor-mediated signaling regulates the promotion of malignant behaviors, including chemo- and/or radiation-resistance. Chemotherapy and radiotherapy are the common approaches to the treatments of cancers. However, resistance to anticancer drugs and irradiation is the most critical limitation for chemotherapy and radiotherapy. In this review, we provide the roles of LPA receptor-mediated signaling in the regulation of cellular responses induced by chemotherapeutic agents and irradiation and its biological utility as a possible molecular target for improving cancer cell responses to chemotherapy and radiotherapy.

A dual role of lysophosphatidic acid type 2 receptor (LPAR2) in nonsteroidal anti-inflammatory drug-induced mouse enteropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.

Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts.

BACKGROUND: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer-associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation-related molecules. METHODS: Cluster analysis, immunoblotting, real-time quantitative-PCR, CRISPR-Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA-LPAR1 on B7-H3 in tumor promotion of CAFs. RESULTS: In this study, we found that LPAR1 and CD276 (B7-H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7-H3 up-expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7-H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non-small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7-H3. CONCLUSION: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7-H3 targeting therapies.

Expression of lysophosphatidic acid receptors in the rat uterus: cellular distribution of protein and gestation-associated changes in gene expression.

Though lysophosphatidic acid (LPA) shows a variety of regulatory roles in reproduction, its action mechanisms in the gestational organs are still largely unknown. We here characterized cellular distribution of its six kinds of specific receptors (LPA1-6) in rat uteri by immunohistochemistry and quantitatively analyzed changes in Lpar1-6 mRNAs expression throughout pregnancy. Among LPA1-6, evident expression of LPA3, LPA4, and LPA6 was immunologically detected and less expression of immunoreactive LPA1 and LPA2 was also found. Luminal and glandular epithelial cells, stromal cells, and myometrial cells are sites of positive immunoreactions, and they are all likely to express three or more subtypes. All of Lpar1-6 mRNAs were expressed, and their alterations were variable depending on subtypes and gestational age. The present information suggests that diverse actions of LPA in the uterus involve varied expression of LPA receptors dependent on tissue/cell types, receptor subtype(s), and organ reproductive states and helps to understand uterine biology of LPA.

Social avoidance and altered hypothalamic-pituitary-adrenal axis in a mouse model of anxious depression: The role of LPA1 receptor.

Anxious depression is a prevalent disease with devastating consequences. Despite the lack of knowledge about the neurobiological basis of this subtype of depression, recently our group has identified a relationship between the LPA1 receptor, one of the six characterized G protein-coupled receptors (LPA1-6) for lysophosphatidic acid, with a mixed depressive-anxiety phenotype. Dysfunctional social behaviors, which have been related to increased activation of the hypothalamus-pituitary-adrenal (HPA) axis, are key symptoms of depression and are even more prominent in patients with comorbid anxiety and depressive disorders. Social behavior and HPA functioning were assessed in animals lacking the LPA1 receptor. For these purposes, we first examined social behaviors in wild-type and LPA1 receptor-null mice. In addition, a dexamethasone (DEX) suppression test was carried out. maLPA1-null mice exhibited social avoidance, a blunted response to DEX administration and an impaired circadian rhythm of corticosterone levels, which are features that are consistently dysregulated in many mental illnesses including anxious depression. Here, we have strengthened the previous experimental evidence for maLPA1-null mice to represent a good animal model of anxious depression, providing an opportunity to explore new therapeutic targets for the treatment of mood disorders, particularly this subtype of depression.

Induction of lysophosphatidic acid (LPA) receptor-mediated signaling regulates cell motility and survival to anticancer drugs in cancer cells treated with hydrogen peroxide.

Hydrogen peroxide (H2O2) is one of reactive oxygen species (ROS) and promotes malignant properties of cancer cells. Lysophosphatidic acid (LPA) signaling via LPA receptor (LPA1 to LPA6) regulates a variety of cellular functions, such as cell growth, migration and differentiation. This study aimed to evaluate the effects of LPA receptors on the cell motility and survival to anticancer drugs by H2O2 in colon cancer DLD-1 cells. To obtain H2O2 treated (DLD- H2O2) cells, cells were maintained in culture medium containing H2O2 (60 µM) for 2 months. LPAR2 and LPAR4 gene expressions were markedly elevated in DLD-H2O2 cells. The cell motility of DLD-H2O2 cells was significantly lower than that of DLD-1 cells. DLD-H2O2 cell motility was suppressed by LPA2 knockdown and stimulated by LPA4 knockdown. The cell survival rates to fluorouracil (5-FU), irinotecan (CPT-11) and oxaliplatin (L-OHP) of DLD-H2O2 cells were significantly higher than those of DLD-1 cells. The cell survival rate to 5-FU of DLD-H2O2 cells was decreased by LPA2 knockdown. Conversely, LPA4 knockdown enhanced the cell survival rate to 5-FU of DLD-H2O2 cells. In the tumor microenvironment, high levels of H2O2 production are observed under hypoxic conditions. The cell survival rate to 5-FU of DLD-H2O2 cells cultured at 1% O2 was significantly higher than that of DLD-1 cells cultured at 1% O2, correlating with LPAR2 gene expression. The present results suggest that the induction of LPA receptor-mediated signaling plays an important role in regulating cellular functions of DLD-1 cells treated with H2O2.

PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity.

Chromatin remodeling and N6-methyladenosine (m6A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m6A methylation of poly(A)+ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m6A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m6A methylation was located at A1881. The level of m6A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m6A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity.

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity.