Prevention of age-associated neuronal hyperexcitability with improved learning and attention upon knockout or antagonism of LPAR2.

Recent studies suggest that synaptic lysophosphatidic acids (LPAs) augment glutamate-dependent cortical excitability and sensory information processing in mice and humans via presynaptic LPAR2 activation. Here, we studied the consequences of LPAR2 deletion or antagonism on various aspects of cognition using a set of behavioral and electrophysiological analyses. Hippocampal neuronal network activity was decreased in middle-aged LPAR2-/- mice, whereas hippocampal long-term potentiation (LTP) was increased suggesting cognitive advantages of LPAR2-/- mice. In line with the lower excitability, RNAseq studies revealed reduced transcription of neuronal activity markers in the dentate gyrus of the hippocampus in naïve LPAR2-/- mice, including ARC, FOS, FOSB, NR4A, NPAS4 and EGR2. LPAR2-/- mice behaved similarly to wild-type controls in maze tests of spatial or social learning and memory but showed faster and accurate responses in a 5-choice serial reaction touchscreen task requiring high attention and fast spatial discrimination. In IntelliCage learning experiments, LPAR2-/- were less active during daytime but normally active at night, and showed higher accuracy and attention to LED cues during active times. Overall, they maintained equal or superior licking success with fewer trials. Pharmacological block of the LPAR2 receptor recapitulated the LPAR2-/- phenotype, which was characterized by economic corner usage, stronger daytime resting behavior and higher proportions of correct trials. We conclude that LPAR2 stabilizes neuronal network excitability upon aging and allows for more efficient use of resting periods, better memory consolidation and better  performance in tasks requiring high selective attention. Therapeutic LPAR2 antagonism may alleviate aging-associated cognitive dysfunctions.

Expression of the type 1 lysophosphatidic acid receptor in osteoblastic cell lineage controls both bone mineralization and osteocyte specification.

Lysphosphatidic acid (LPA) is a major natural bioactive lipid mediator whose biological functions affect multiple organs. These include bone as demonstrated by global Lpar1-knockout mice (Lpar1-/-) which present a bone growth defect. LPA acts on all bone cells including osteoblasts, that are responsible for bone formation, and osteoclasts, which are specialized cells that resorb bone. LPA appears as a potential new coupling molecule during bone remodeling. LPA1 is the most ubiquitous LPA receptor among the six LPA receptor family members (LPA1-6). To better understand the specific role of LPA via its receptor LPA1 in osteoblastic cell lineage we generated osteoblast-specific Lpar1 knockout mice (Lpar1-∆Ob) by crossing Lpar1flox/flox and Osx:Cre+ mouse lines. Lpar1-∆Ob mice do not recapitulate the bone defects of Lpar1-/- mice but revealed reduced bone mineralization and decreased cortical thickness, as well as increased bone porosity associated with an augmentation in the lacunae areas of osteocyte and their apoptotic yield. In vitro, primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts revealed a remarkable premature expression of alkaline phosphatase, reduced cell proliferation associated with decreased YAP-P nuclear accumulation, and reduced mineralization activity. Osteocyte specification is markedly impaired as demonstrated by reduced expression of early (E11) and late (DMP1, DKK1, SOST) osteocyte markers ex vivo in enriched osteocytic fractions of Lpar1-∆Ob mouse bone explants. In addition, E11 expression and dendrite formation induced by FGF2 are markedly impaired in both primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts. Taken together these results suggest a new role for LPA in bone mass control via bone mineralization and osteocyte function.

Lysophosphatidic Acid Receptor 1 Is Important for Intestinal Epithelial Barrier Function and Susceptibility to Colitis.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

Autotaxin-lysophosphatidic acid-LPA3 signaling at the embryo-epithelial boundary controls decidualization pathways.

During pregnancy, up-regulation of heparin-binding (HB-) EGF and cyclooxygenase-2 (COX-2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G-protein-coupled receptor LPA3 in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA-producing enzyme autotaxin (ATX) in pregnant mice leads to HB-EGF and COX-2 down-regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA3 induces decidualization via up-regulation of HB-EGF and COX-2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre-implantation-stage embryos, respectively. Our results indicate that ATX-LPA-LPA3 signaling at the embryo-epithelial boundary induces decidualization via the canonical HB-EGF and COX-2 pathways.

maLPA1-null mice as an endophenotype of anxious depression.

Anxious depression is a prevalent disease with devastating consequences and a poor prognosis. Nevertheless, the neurobiological mechanisms underlying this mood disorder remain poorly characterized. The LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intracellular signalling molecule. The loss of this receptor induces anxiety and several behavioural and neurobiological changes that have been strongly associated with depression. In this study, we sought to investigate the involvement of the LPA1 receptor in mood. We first examined hedonic and despair-like behaviours in wild-type and maLPA1 receptor null mice. Owing to the behavioural response exhibited by the maLPA1-null mice, the panic-like reaction was assessed. In addition, c-Fos expression was evaluated as a measure of the functional activity, followed by interregional correlation matrices to establish the brain map of functional activation. maLPA1-null mice exhibited anhedonia, agitation and increased stress reactivity, behaviours that are strongly associated with the psychopathological endophenotype of depression with anxiety features. Furthermore, the functional brain maps differed between the genotypes. The maLPA1-null mice showed increased limbic-system activation, similar to that observed in depressive patients. Antidepressant treatment induced behavioural improvements and functional brain normalisation. Finally, based on validity criteria, maLPA1-null mice are proposed as an animal model of anxious depression. Here, for we believe the first time, we have identified a possible relationship between the LPA1 receptor and anxious depression, shedding light on the unknown neurobiological basis of this subtype of depression and providing an opportunity to explore new therapeutic targets for the treatment of mood disorders, especially for the anxious subtype of depression.

Lysophosphatidic acid signaling through its receptor initiates profibrotic epithelial cell fibroblast communication mediated by epithelial cell derived connective tissue growth factor.

The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA1-deficient (LPA1-/-) or -sufficient (LPA1+/+) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA1-/-Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA1-/-Col-GFP mice. LPA-LPA1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis.

LPA1 Mediates Antidepressant-Induced ERK1/2 Signaling and Protection from Oxidative Stress in Glial Cells.

Antidepressants have been shown to affect glial cell functions and intracellular signaling through mechanisms that are still not completely understood. In the present study, we provide evidence that in glial cells the lysophosphatidic acid (LPA) receptor LPA1 mediates antidepressant-induced growth factor receptor transactivation, ERK1/2 signaling, and protection from oxidative stress. Thus, in C6 glioma cells and rat cortical astrocytes, ERK1/2 activation induced by either amitriptyline or mianserin was antagonized by Ki16425 and VPC 12249 (S), which block LPA1 and LPA3 receptors, and by AM966, which selectively blocks LPA1 Cell depletion of LPA1 with siRNA treatment markedly reduced antidepressant- and LPA-induced ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant-induced phosphorylation of the transcription factors CREB and Elk-1. Antidepressants and LPA signaling to ERK1/2 was abrogated by cell treatment with pertussis toxin and by the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases. Both Ki16425 and AM966 suppressed antidepressant-induced phosphorylation of FGF-R. Moreover, blockade of LPA1 or inhibition of FGF-R and PDGF-R activities prevented antidepressant-stimulated Akt and GSK-3ß phosphorylations. Mianserin protected C6 glioma cells and astrocytes from apoptotic cell death induced by H2O2, as indicated by increased cell viability, decreased expression of cleaved caspase 3, reduced cleavage of poly-ADP ribose polymerase and inhibition of DNA fragmentation. The protective effects of mianserin were antagonized by AM966. These data indicate that LPA1 constitutes a novel molecular target of the regulatory actions of tricyclic and tetracyclic antidepressants in glial cells.

Positive and Negative Cross-Talk between Lysophosphatidic Acid Receptor 1, Free Fatty Acid Receptor 4, and Epidermal Growth Factor Receptor in Human Prostate Cancer Cells.

Lysophosphatidic acid (LPA) is a lipid mediator that mediates cellular effects via G protein-coupled receptors (GPCRs). Epidermal growth factor (EGF) is a peptide that acts via a receptor tyrosine kinase. LPA and EGF both induce proliferation of prostate cancer cells and can transactivate each other's receptors. The LPA receptor LPA1 is particularly important for LPA response in human prostate cancer cells. Previous work in our laboratory has demonstrated that free fatty acid 4 (FFA4), a GPCR activated by ω-3 fatty acids, inhibits responses to both LPA and EGF in these cells. One potential mechanism for the inhibition involves negative interactions between FFA4 and LPA1, thereby suppressing responses to EGF that require LPA1 In the current study, we examined the role of LPA1 in mediating EGF and FFA4 agonist responses in two human prostate cancer cell lines, DU145 and PC-3. The results show that an LPA1-selective antagonist inhibits proliferation and migration to both LPA and EGF. Knockdown of LPA1 expression, using silencing RNA, blocks responses to LPA and significantly inhibits responses to EGF. The partial response to EGF that is observed after LPA1 knockdown is not inhibited by FFA4 agonists. Finally, the role of arrestin-3, a GPCR-binding protein that mediates many actions of activated GPCRs, was tested. Knockdown of arrestin-3 completely inhibits responses to both LPA and EGF in prostate cancer cells. Taken together, these results suggest that LPA1 plays a critical role in EGF responses and that FFA4 agonists inhibit proliferation by suppressing positive cross-talk between LPA1 and the EGF receptor.

Deficiency or inhibition of lysophosphatidic acid receptor 1 protects against hyperoxia-induced lung injury in neonatal rats.

AIM: Blocking of lysophosphatidic acid (LPA) receptor (LPAR) 1 may be a novel therapeutic option for bronchopulmonary dysplasia (BPD) by preventing the LPAR1-mediated adverse effects of its ligand (LPA), consisting of lung inflammation, pulmonary arterial hypertension (PAH) and fibrosis. METHODS: In Wistar rats with experimental BPD, induced by continuous exposure to 100% oxygen for 10 days, we determined the beneficial effects of LPAR1 deficiency in neonatal rats with a missense mutation in cytoplasmic helix 8 of LPAR1 and of LPAR1 and -3 blocking with Ki16425. Parameters investigated included survival, lung and heart histopathology, fibrin and collagen deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in LPA signalling and BPD pathogenesis. RESULTS: LPAR1-mutant rats were protected against experimental BPD and mortality with reduced alveolar septal thickness, lung inflammation (reduced influx of macrophages and neutrophils, and CINC1 expression) and collagen III deposition. However, LPAR1-mutant rats were not protected against alveolar enlargement, increased medial wall thickness of small arterioles, fibrin deposition and vascular alveolar leakage. Treatment of experimental BPD with Ki16425 confirmed the data observed in LPAR1-mutant rats, but did not reduce the pulmonary influx of neutrophils, CINC1 expression and mortality in rats with experimental BPD. In addition, Ki16425 treatment protected against PAH and right ventricular hypertrophy. CONCLUSION: LPAR1 deficiency attenuates pulmonary injury by reducing pulmonary inflammation and fibrosis, thereby reducing mortality, but does not affect alveolar and vascular development and, unlike Ki16425 treatment, does not prevent PAH in neonatal rats with experimental BPD.

Activation of Lysophosphatidic Acid Receptor Type 1 Contributes to Pathophysiology of Spinal Cord Injury.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator involved in many physiological functions that signals through six known G-protein-coupled receptors (LPA1-LPA6). A wide range of LPA effects have been identified in the CNS, including neural progenitor cell physiology, astrocyte and microglia activation, neuronal cell death, axonal retraction, and development of neuropathic pain. However, little is known about the involvement of LPA in CNS pathologies. Herein, we demonstrate for the first time that LPA signaling via LPA1 contributes to secondary damage after spinal cord injury. LPA levels increase in the contused spinal cord parenchyma during the first 14 d. To model this potential contribution of LPA in the spinal cord, we injected LPA into the normal spinal cord, revealing that LPA induces microglia/macrophage activation and demyelination. Use of a selective LPA1 antagonist or mice lacking LPA1 linked receptor-mediated signaling to demyelination, which was in part mediated by microglia. Finally, we demonstrate that selective blockade of LPA1 after spinal cord injury results in reduced demyelination and improvement in locomotor recovery. Overall, these results support LPA-LPA1 signaling as a novel pathway that contributes to secondary damage after spinal cord contusion in mice and suggest that LPA1 antagonism might be useful for the treatment of acute spinal cord injury. SIGNIFICANCE STATEMENT: This study reveals that LPA signaling via LPA receptor type 1 activation causes demyelination and functional deficits after spinal cord injury.

Regulation of T cell motility in vitro and in vivo by LPA and LPA2.

Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1-6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2-/- CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.

GPR87 mediates lysophosphatidic acid-induced colony dispersal in A431 cells.

We have previously reported that an orphan G protein-coupled receptor GPR87 was activated by lysophosphatidic acid (LPA) and that it induced an increase in the intracellular Ca(2+) levels in the CHO cells genetically engineered to express GPR87-Gα16 fusion protein. Because the Ca(2+) response was blocked by the LPA receptor antagonist Ki16425, GPR87 was suggested to be a putative LPA receptor. However, further studies are required to confirm whether GPR87 is an LPA receptor. A previous study showed that colonies of A431 cells treated with LPA showed rapid and synchronized dissociation. Because A431 cells have been shown to express GPR87, we used these cells to examine whether GPR87 acted as an LPA receptor. When A431 cells were treated with gpr87-specific siRNA, the expression of GPR87 was decreased and LPA-induced colony dispersal was significantly reduced. Treatment of the cells with lpa1 siRNA had an additive effect in decrease in the colony dispersal. Studies on the LPA-mediated signaling pathway in A431 cells indicated that transactivation of the epidermal growth factor receptor (EGFR) by LPA led to cell scattering. PD153035, an inhibitor of tyrosine-kinase of EGFR, and BB94, an inhibitor of metalloprotease which produces a ligand for EGFR, significantly prevented the LPA-induced scattering of A431 cells pretreated with lpa1 or gpr87-siRNA. These results strongly suggested that GPR87 acts as an LPA receptor and induces colony dispersal via the transactivation of EGFR in A431 cells.

Lysophosphatidic acid receptor-2 deficiency confers protection against bleomycin-induced lung injury and fibrosis in mice.

Idiopathic pulmonary fibrosis is a devastating disease characterized by alveolar epithelial cell injury, the accumulation of fibroblasts/myofibroblasts, and the deposition of extracellular matrix proteins. Lysophosphatidic acid (LPA) signaling through its G protein-coupled receptors is critical for its various biological functions. Recently, LPA and LPA receptor 1 were implicated in lung fibrogenesis. However, the role of other LPA receptors in fibrosis remains unclear. Here, we use a bleomycin-induced pulmonary fibrosis model to investigate the roles of LPA2 in pulmonary fibrogenesis. In the present study, we found that LPA2 knockout (Lpar2(-/-)) mice were protected against bleomycin-induced lung injury, fibrosis, and mortality, compared with wild-type control mice. Furthermore, LPA2 deficiency attenuated the bleomycin-induced expression of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagen in lung tissue, as well as levels of IL-6, transforming growth factor-ß (TGF-ß), and total protein in bronchoalveolar lavage fluid. In human lung fibroblasts, the knockdown of LPA2 attenuated the LPA-induced expression of TGF-ß1 and the differentiation of lung fibroblasts to myofibroblasts, resulting in the decreased expression of FN, α-SMA, and collagen, as well as decreased activation of extracellular regulated kinase 1/2, Akt, Smad3, and p38 mitogen-activated protein kinase. Moreover, the knockdown of LPA2 with small interfering RNA also mitigated the TGF-ß1-induced differentiation of lung fibroblasts. In addition, LPA2 deficiency significantly attenuated the bleomycin-induced apoptosis of alveolar and bronchial epithelial cells in the mouse lung. Together, our data indicate that the knockdown of LPA2 attenuated bleomycin-induced lung injury and pulmonary fibrosis, and this may be related to an inhibition of the LPA-induced expression of TGF-ß and the activation and differentiation of fibroblasts.

Necessity of lysophosphatidic acid receptor 1 for development of arthritis.

OBJECTIVE: Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA receptors 1-6 [LPA1-6 ]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA1 on the development of arthritis. METHODS: Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The effects of abrogation of LPA1 on collagen-induced arthritis (CIA) were evaluated using LPA1 -deficient mice or LPA1 antagonist. Migrating fluorescence-labeled CD11b+ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4+ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined. RESULTS: LPA1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA1 -deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b+ macrophages from LPA1 -deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA1 deficiency or LPA1 inhibition in vitro. CONCLUSION: Collectively, these results indicate that LPA/LPA1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA1 may be a promising target molecule for RA therapy.

Lpa2 is a negative regulator of both dendritic cell activation and murine models of allergic lung inflammation.

Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2(-/-) mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB-driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2(-/-) DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2(-/-) mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.

Lysophosphatidic acid receptor 1 suppression sensitizes rheumatoid fibroblast-like synoviocytes to tumor necrosis factor-induced apoptosis.

OBJECTIVE: To investigate the role of lysophosphatidic acid (LPA) receptors in the proliferation and apoptosis of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Expression of LPA receptors 1-3 was analyzed by real-time polymerase chain reaction (PCR). LPAR1 and LPAR2 were suppressed in RA FLS by small interfering RNA (siRNA) transfection. Proliferation of RA FLS after tumor necrosis factor (TNF) and LPA stimulation was determined with a luminescent cell viability assay. Apoptosis was analyzed by quantification of nucleosome release and measurement of activated caspase 3/7. Genes involved in the apoptotic response were identified with a human apoptosis PCR array and validated with Western blot assays. The requirement of these genes for apoptosis sensitization was assessed by siRNA transfection. Secretion of mediators of inflammation was analyzed by enzyme-linked immunosorbent assay. RESULTS: Only LPAR1 and LPAR2 were expressed by RA FLS, and their levels were higher than those in osteoarthritis (OA) FLS. Suppression of LPAR1 abrogated TNF-induced proliferation and sensitized the RA FLS, but not the OA FLS, to TNF-induced apoptosis. These changes occurred despite an increased early inflammatory response to TNF. Sensitization to apoptosis was associated with changes in expression of multiple apoptosis-related genes. Three of the up-regulated proapoptotic genes were further studied to confirm their involvement. In contrast, suppression of LPAR2 showed no effect in any of these analyses. CONCLUSION: LPA(1) is an important receptor in RA FLS. Its suppression is accompanied by a global increase in the response to TNF that is ultimately dominated by sensitization to apoptosis.

11-deoxy prostaglandin F(2α), a thromboxane A2 receptor agonist, partially alleviates embryo crowding in Lpar3((-/-)) females.

OBJECTIVE: To determine cyclooxygenase-derived prostanoid signaling in alleviating embryo crowding in the Lpar3((-/-)) females. DESIGN: Experimental mouse model. SETTING: Research laboratories. ANIMAL(S): Wild-type, Lpar3((+/-)), and Lpar3((-/-)) mice. INTERVENTION(S): Intraperitoneal (IP) administration of prostaglandin E(2) (PGE(2)), cPGI (a stable analogue of PGI(2)), and 11-deoxy prostaglandin F(2α) (11-deoxy PGF(2α), a thromboxane A(2) receptor agonist) to preimplantation gestation day 3.5 Lpar3((-/-)) females. MAIN OUTCOME MEASURE(S): Implantation sites were detected by blue dye reaction and embryo spacing was determined by the distribution of the implantation sites along the uterine horns on gestation day 4.5; pregnancy outcome was measured by litter size at birth. RESULT(S): Administration of PGE(2) + cPGI on gestation day 3.5 Lpar3((-/-)) females restored on-time implantation but did not affect embryo spacing or the number of implantation sites detected on gestation day 4.5; PGE(2) + cPGI treatment increased litter size at birth. Administration of PGE(2) + cPGI + 11-deoxy PGF(2α) on gestation day 3.5 Lpar3((-/-)) females rescued on-time implantation, partially dispersed the clustered implantation sites normally observed in the Lpar3((-/-)) females, increased the number of implantation sites detected on gestation day 4.5, and increased litter size at birth. CONCLUSION(S): The thromboxane A(2) receptor agonist 11-deoxy PGF(2α) can partially alleviate embryo crowding in the Lpar3((-/-)) females and embryo crowding likely contributes to reduced litter size in the Lpar3((-/-)) females.

Lysophosphatidic acid signaling protects pulmonary vasculature from hypoxia-induced remodeling.

OBJECTIVE: Lysophosphatidic acid (LPA) is a bioactive lipid molecule produced by the plasma lysophospholipase D enzyme autotaxin that is present at ≥100 nmol/L in plasma. Local administration of LPA promotes systemic arterial remodeling in rodents. To determine whether LPA contributes to remodeling of the pulmonary vasculature, we examined responses in mice with alterations in LPA signaling and metabolism. METHODS AND RESULTS: Enpp2(+/-) mice, which are heterozygous for the autotaxin-encoding gene and which have reduced expression of autotaxin/lysophospholipase D and approximately half normal plasma LPA, were hyperresponsive to hypoxia-induced vasoconstriction and remodeling, as evidenced by the development of higher right ventricular (RV) systolic pressure, greater decline in peak flow velocity across the pulmonary valve, and a higher percentage of muscularized arterioles. Mice lacking LPA(1) and LPA(2), 2 LPA receptors abundantly expressed in the vasculature, also had enhanced hypoxia-induced pulmonary remodeling. With age, Lpar1(-/-)2(-/-) mice spontaneously developed elevated RV systolic pressure and RV hypertrophy that was not observed in Lpar1(-/-) mice or Lpar2(-/-) mice. Expression of endothelin-1, a potent vasoconstrictor, was elevated in lungs of Lpar1(-/-)2(-/-) mice, and expression of endothelin(B) receptor, which promotes vasodilation and clears endothelin, was reduced in Enpp2(+/-) and Lpar1(-/-)2(-/-) mice. CONCLUSIONS: Our findings indicate that LPA may negatively regulate pulmonary vascular pressure through LPA(1) and LPA(2) receptors and that in the absence of LPA signaling, upregulation in the endothelin system favors remodeling.

The lysophosphatidic acid receptor LPA1 promotes epithelial cell apoptosis after lung injury.

Increased epithelial cell apoptosis in response to lung injury has been implicated in the development of idiopathic pulmonary fibrosis (IPF), but the molecular pathways promoting epithelial cell apoptosis in this disease have yet to be fully identified. Lysophosphatidic acid (LPA), which we have previously demonstrated to mediate bleomycin lung injury-induced fibroblast recruitment and vascular leak in mice and fibroblast recruitment in patients with IPF, is an important regulator of survival and apoptosis in many cell types. We now show that LPA signaling through its receptor LPA(1) promotes epithelial cell apoptosis induced by bleomycin injury. The number of apoptotic cells present in the alveolar and bronchial epithelia of LPA(1)-deficient mice was significantly reduced compared with wild-type mice at Day 3 after bleomycin challenge, as was lung caspase-3 activity. Consistent with these in vivo results, we found that LPA signaling through LPA(1) induced apoptosis in normal human bronchial epithelial cells in culture. LPA-LPA(1) signaling appeared to specifically mediate anoikis, the apoptosis of anchorage-dependent cells induced by their detachment. Similarly, LPA negatively regulated attachment of R3/1 rat alveolar epithelial cell line cells. In contrast, LPA signaling through LPA(1) promoted the resistance of lung fibroblasts to apoptosis, which has also been implicated in IPF. The ability of LPA-LPA(1) signaling to promote epithelial cell apoptosis and fibroblast resistance to apoptosis may therefore contribute to the capacity of this signaling pathway to regulate the development of pulmonary fibrosis after lung injury.

Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA1 receptor knockout mice.

BACKGROUND: The lysophosphatidic acid LPA1 receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA1 receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. METHODOLOGY/PRINCIPAL FINDINGS: Male LPA1-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice. CONCLUSIONS/SIGNIFICANCE: These results reveal that the absence of the LPA1 receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA1 receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.