Conditional Lpar1 gene targeting identifies cell types mediating neuropathic pain.

LPA1 is one of six known receptors (LPA1-6) for lysophosphatidic acid (LPA). Constitutive Lpar1 null mutant mice have been instrumental in identifying roles for LPA-LPA1 signaling in neurobiological processes, brain development, and behavior, as well as modeling human neurological diseases like neuropathic pain. Constitutive Lpar1 null mutant mice are protected from partial sciatic nerve ligation (PSNL)-induced neuropathic pain, however, the cell types that are functionally responsible for mediating this protective effect are unknown. Here, we report the generation of an Lpar1flox/flox conditional null mutant mouse that allows for cre-mediated conditional deletion, combined with a PSNL pain model. Lpar1flox/flox mice were crossed with cre transgenic lines driven by neural gene promoters for nestin (all neural cells), synapsin (neurons), or P0 (Schwann cells). CD11b-cre transgenic mice were also used to delete Lpar1 in microglia. PSNL-initiated pain responses were reduced following cre-mediated Lpar1 deletion with all three neural promoters as well as the CD11b promoter, supporting involvement of Schwann cells, central and/or peripheral neurons, and microglia in mediating pain. Interestingly, rescue responses were nonidentical, implicating distinct roles for Lpar1-expressing cell types. Our results with a new Lpar1 conditional mouse mutant expand an understanding of LPA1 signaling in the PSNL model of neuropathic pain.

The Role of Lysophosphatidic Acid Receptors in Ovarian Cancer: A Minireview.

Lysophosphatidic acid (LPA) is a bioactive lipid component of ovarian cancer activating factor, which is present at a high concentration in the ascitic fluid and plasma of patients with ovarian cancer. A group of six lysophosphatidic acid receptors (LPARs), LPAR1 through LPAR6, which belong to the G protein-coupled receptor superfamily (GPCR), mediate cellular activities of LPA and activates a series of downstream molecules and cellular responses, including biological and pathological effects. LPARs are widely expressed in normal ovary, benign tumor, and ovarian cancer tissues and cancer cell lines with a broad range of levels. The LPA/LPAR axis is involved in tumorigenesis and development of ovarian cancer through mediating the cellular responses to LPA and influencing the expression and function of oncogenic molecules. In the present review, the roles of LPARs in ovarian cancer, including the expression, function, and downstream molecules, are summarized, and we discuss the implications for ovarian cancer treatment that targets LPARs.

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.

Gintonin, a ginseng-derived ingredient, as a novel therapeutic strategy for Huntington's disease: Activation of the Nrf2 pathway through lysophosphatidic acid receptors.

Gintonin (GT), a ginseng-derived lysophosphatidic acid receptor ligand, regulates various cellular effects and represses inflammation. However, little is known about the potential value of GT regarding inflammation in the neurodegenerative diseases, such as Huntington's disease (HD). In this study, we investigated whether GT could ameliorate the neurological impairment and striatal toxicity in cellular or animal model of HD. Pre-, co-, and onset-treatment with GT (25, 50, or 100 mg/kg/day, p.o.) alleviated the severity of neurological impairment and lethality following 3-nitropropionic acid (3-NPA). Pretreatment with GT also attenuated mitochondrial dysfunction i.e. succinate dehydrogenase and MitoSOX activities, apoptosis, microglial activation, and mRNA expression of inflammatory mediators i.e. IL-1ß, IL-6, TNF-α, COX-2, and iNOS in the striatum after 3-NPA-intoxication. Its action mechanism was associated with lysophosphatidic acid receptors (LPARs) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway activations and the inhibition of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways. These beneficial effects of GT were neutralized by pre-inhibiting LPARs with Ki16425 (a LPAR1/3 antagonist). Interestingly, GT reduced cell death and mutant huntingtin (HTT) aggregates in STHdh cells. It also mitigated neurological impairment in mice with adeno-associated viral (AAV) vector serotype DJ-mediated overexpression of N171-82Q-mutant HTT in the striatum. Taken together, our findings firstly suggested that GT has beneficial effects with a wide therapeutic time-window in 3-NPA-induced striatal toxicity by antioxidant and anti-inflammatory activities through LPA. In addition, GT exerts neuroprotective effects in STHdh cells and AAV vector-infected model of HD. Thus GT might be an innovative therapeutic candidate to treat HD-like syndromes.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model.

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

Lysophosphatidic Acid Receptor Antagonism Protects against Diabetic Nephropathy in a Type 2 Diabetic Model.

Lysophosphatidic acid (LPA) functions through activation of LPA receptors (LPARs). LPA-LPAR signaling has been implicated in development of fibrosis. However, the role of LPA-LPAR signaling in development of diabetic nephropathy (DN) has not been studied. We examined whether BMS002, a novel dual LPAR1 and LPAR3 antagonist, affects development of DN in endothelial nitric oxide synthase-knockout db/db mice. Treatment of these mice with BMS002 from 8 to 20 weeks of age led to a significant reduction in albuminuria, similar to that observed with renin-angiotensin system inhibition (losartan plus enalapril). LPAR inhibition also prevented the decline in GFR observed in vehicle-treated mice, such that GFR at week 20 differed significantly between vehicle and LPAR inhibitor groups (P<0.05). LPAR inhibition also reduced histologic glomerular injury; decreased the expression of profibrotic and fibrotic components, including fibronectin, α-smooth muscle actin, connective tissue growth factor, collagen I, and TGF-ß; and reduced renal macrophage infiltration and oxidative stress. Notably, LPAR inhibition slowed podocyte loss (podocytes per glomerulus ±SEM at 8 weeks: 667±40, n=4; at 20 weeks: 364±18 with vehicle, n=7, and 536±12 with LPAR inhibition, n=7; P<0.001 versus vehicle). Finally, LPAR inhibition minimized the production of 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in podocytes and increased the phosphorylation of AKT2, an indicator of AKT2 activity, in kidneys. Thus, the LPAR antagonist BMS002 protects against GFR decline and attenuates development of DN through multiple mechanisms. LPAR antagonism might provide complementary beneficial effects to renin-angiotensin system inhibition to slow progression of DN.

Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.

KEY POINTS: Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA-induced itch-related behaviours are decreased in TRPA1-knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+ -independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672-673 and KR977-978 (K: lysine, R: arginine). ABSTRACT: Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch-related behaviours but not pain-related behaviours. The LPA-induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA-induced itch is mediated by LPA5 , PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

LPA1 is a key mediator of intracellular signalling and neuroprotection triggered by tetracyclic antidepressants in hippocampal neurons.

Both lysophosphatidic acid (LPA) and antidepressants have been shown to affect neuronal survival and differentiation, but whether LPA signalling participates in the action of antidepressants is still unknown. In this study, we examined the role of LPA receptors in the regulation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and neuronal survival by the tetracyclic antidepressants, mianserin and mirtazapine in hippocampal neurons. In HT22 immortalized hippocampal cells, antidepressants and LPA induced a time- and concentration-dependent stimulation of ERK1/2 phosphorylation. This response was inhibited by either LPA1 and LPA1/3 selective antagonists or siRNA-induced LPA1 down-regulation, and enhanced by LPA1 over-expression. Conversely, the selective LPA2 antagonist H2L5186303 had no effect. Antidepressants induced cyclic AMP response element binding protein phosphorylation and this response was prevented by LPA1 blockade. ERK1/2 stimulation involved pertussis toxin-sensitive G proteins, Src tyrosine kinases and fibroblast growth factor receptor (FGF-R) activity. Tyrosine phosphorylation of FGF-R was enhanced by antidepressants through LPA1 . Serum withdrawal induced apoptotic death, as indicated by increased annexin V staining, caspase activation and cleavage of poly-ADP-ribose polymerase. Antidepressants inhibited the apoptotic cascade and this protective effect was curtailed by blockade of either LPA1 , ERK1/2 or FGF-R activity. Moreover, in primary mouse hippocampal neurons, mianserin acting through LPA1 increased phospho-ERK1/2 and protected from apoptosis induced by removal of growth supplement. These data indicate that in neurons endogenously expressed LPA1 receptors mediate intracellular signalling and neuroprotection by tetracyclic antidepressants.

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication.

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

Preparation of functional human lysophosphatidic acid receptor 2 using a P9∗ expression system and an amphipathic polymer and investigation of its in vitro binding preference to Gα proteins.

Human lysophosphatidic acid receptor 2 (LPA2), a member of the G-protein coupled receptor family, mediates lysophosphatidic acid (LPA)-dependent signaling by recruiting various G proteins. Particularly, it is directly implicated in the progression of colorectal and ovarian cancer through G protein signaling cascades. To investigate the biochemical binding properties of LPA2 against various alpha subunits of G protein (Gα), a functional recombinant LPA2 was overexpressed in E. coli membrane with a P9∗ expression system, and the purified protein was stabilized with an amphipathic polymer that had been synthesized by coupling octylamine, glucosamine, and diethyl aminoproylamine at the carboxylic groups of poly-γ-glutamic acid. The purified LPA2 stabilized with the amphipathic polymer showed selective binding activity to the various Gα proteins as well as agonist-dependent dissociation from Gαi3. Understanding the binding properties of LPA2 against various Gα proteins advances the understanding of downstream signaling cascades of LPA2. The functional LPA2 prepared using a P9∗ expression system and an amphipathic polymer could also facilitate the development of LPA2-targeting drugs.

Autotaxin-Lysophosphatidic Acid: From Inflammation to Cancer Development.

Lysophosphatidic acid (LPA) is a ubiquitous lysophospholipid and one of the main membrane-derived lipid signaling molecules. LPA acts as an autocrine/paracrine messenger through at least six G protein-coupled receptors (GPCRs), known as LPA1-6, to induce various cellular processes including wound healing, differentiation, proliferation, migration, and survival. LPA receptors and autotaxin (ATX), a secreted phosphodiesterase that produces this phospholipid, are overexpressed in many cancers and impact several features of the disease, including cancer-related inflammation, development, and progression. Many ongoing studies aim to understand ATX-LPA axis signaling in cancer and its potential as a therapeutic target. In this review, we discuss the evidence linking LPA signaling to cancer-related inflammation and its impact on cancer progression.

Lysophosphatidic Acid (LPA) Receptor 3-Mediated LPA Signal Transduction Pathways: A Possible Relationship with Early Development of Peri-Implantation Porcine Conceptus.

Lysophosphatidic acid (LPA) is a phospholipid with a variety of fatty acyl groups that mediates diverse biological effects on various types of cells through specific G protein-coupled receptors. LPA appears to play a significant role in many reproductive processes, including luteolysis, implantation, and placentation. Our previous study in pigs demonstrated that LPA and the LPA receptor system are present at the maternal-conceptus interface and that LPA increases uterine endometrial expression of prostaglandin-endoperoxide synthase 2 (PTGS2) through LPA receptor 3 (LPAR3). However, the role of LPA in conceptuses during early pregnancy has not been determined. Therefore, this study examined the effects of LPA in cell proliferation, migration, and activation of the intracellular signaling pathway in porcine conceptuses by using an established porcine trophectoderm (pTr) cell line isolated from Day 12 conceptuses. All examined LPA species with various fatty acid lengths increased proliferation and migration of pTr cells as the dosage increased. Immunoblot analyses found that LPA activated intracellular signaling molecules, extracellular signal-regulated kinase 1/2 (ERK1/2), ribosomal protein S6 kinase 90 kDa (P90RSK), ribosomal protein S6 (RPS6), and P38 in pTr cells. Furthermore, LPA increased expression of PTGS2 and urokinase-type plasminogen activator (PLAU), and the LPA-induced increases in PTGS2 and PLAU expression were inhibited by LPAR3 siRNA. Collectively, these results showed that LPA promotes proliferation, migration, and differentiation of pTr cells by activating the ERK1/2-P90RSK-RPS6 and P38 pathways, indicating that the LPA-LPAR3 system may be involved in the development of trophoblast during early pregnancy in pigs.

Down-regulation of LPA receptor 5 contributes to aberrant LPA signalling in EBV-associated nasopharyngeal carcinoma.

Undifferentiated nasopharyngeal carcinoma (NPC) is a highly metastatic disease that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the contribution of lysophosphatidic acid (LPA) signalling to the pathogenesis of NPC. Here we demonstrate two distinct functional roles for LPA in NPC. First, we show that LPA enhances the migration of NPC cells and second, that it can inhibit the activity of EBV-specific cytotoxic T cells. Focusing on the first of these phenotypes, we show that one of the LPA receptors, LPA receptor 5 (LPAR5), is down-regulated in primary NPC tissues and that this down-regulation promotes the LPA-induced migration of NPC cell lines. Furthermore, we found that EBV infection or ectopic expression of the EBV-encoded LMP2A was sufficient to down-regulate LPAR5 in NPC cell lines. Our data point to a central role for EBV in mediating the oncogenic effects of LPA in NPC and identify LPA signalling as a potential therapeutic target in this disease.

Myelin-related gene silencing mediated by LPA1 - Rho/ROCK signaling is correlated to acetylation of NFκB in S16 Schwann cells.

Lysophosphatidic acid (LPA) initiates demyelination following peripheral nerve injury, which causes neuropathic pain. Our previous in vivo and ex vivo studies using mice have demonstrated that LPA-induced demyelination of spinal dorsal roots is attributed by the LPA1-type receptor-mediated down-regulation of myelin-related molecules, such as MBP and MPZ. In this study using S16 mature-type Schwann cells, we found that LPA-induced down-regulation of myelin-related genes is attributed by the activation of LPA1 receptor, Rho kinase, and p300, leading to an acetylation of NFκB, which down-regulates the transcription of Sox10, MBP and MPZ genes.

Lysophospholipid receptors in drug discovery.

Lysophospholipids (LPs), including lysophosphatidic acid (LPA), sphingosine 1-phospate (S1P), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LysoPS), are bioactive lipids that transduce signals through their specific cell-surface G protein-coupled receptors, LPA1-6, S1P1-5, LPI1, and LysoPS1-3, respectively. These LPs and their receptors have been implicated in both physiological and pathophysiological processes such as autoimmune diseases, neurodegenerative diseases, fibrosis, pain, cancer, inflammation, metabolic syndrome, bone formation, fertility, organismal development, and other effects on most organ systems. Advances in the LP receptor field have enabled the development of novel small molecules targeting LP receptors for several diseases. Most notably, fingolimod (FTY720, Gilenya, Novartis), an S1P receptor modulator, became the first FDA-approved medicine as an orally bioavailable drug for treating relapsing forms of multiple sclerosis. This success is currently being followed by multiple, mechanistically related compounds targeting S1P receptor subtypes, which are in various stages of clinical development. In addition, an LPA1 antagonist, BMS-986020 (Bristol-Myers Squibb), is in Phase 2 clinical development for treating idiopathic pulmonary fibrosis, as a distinct compound, SAR100842 (Sanofi) for the treatment of systemic sclerosis and related fibrotic diseases. This review summarizes the current state of drug discovery in the LP receptor field.

Lysophosphatidic acid receptor type 1 (LPA1) plays a functional role in osteoclast differentiation and bone resorption activity.

Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1-6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1(-/-) mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1(-/-) mice but not in Lpar2(-/-) and Lpar3(-/-) animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1(-/-) osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP(+) osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

Expression and Function of LPA1 in Bladder Cancer.

PURPOSE: LPA is one of several physiologically active lipid mediators that promote cell proliferation and invasion, and are present in serum, ascites and urine. LPA receptor is a G-protein coupled receptor that is considered a potential therapeutic target for some malignant cancers. We evaluated the expression of LPA receptors in bladder cancer and the effect of LPA in bladder cancer invasion. MATERIALS AND METHODS: Using real-time polymerase chain reaction and immunohistochemical staining we determined LPA receptor expression in bladder cancer specimens from patients with bladder cancer, including 12 with Ta or T1 and 15 with T2-T4 disease. ROCK expression, myosin light chain phosphorylation and Matrigel™ invasion assays were done and morphological observations were made to assess LPA effects in T24 cells, which were derived from bladder cancer. RESULTS: Notably LPA1 mRNA expression was significantly higher in muscle invasive bladder cancer specimens than in nonmuscle invasive specimens. Strong LPA1 expression was evident on cell membranes in muscle invasive specimens. T24 cell invasion was increased by LPA treatment and invasiveness was decreased by LPA1 siRNA or LPA1 inhibitor. LPA treatment increased ROCK1 expression and myosin light chain phosphorylation, and induced morphological changes, including lamellipodia formation and cell rounding. CONCLUSIONS: Results indicate that LPA signaling via LPA1 activation promoted bladder cancer invasion. LPA1 might be useful to detect bladder cancer with highly invasive potential and become a new therapeutic target for invasive bladder cancer treatment.

LPA1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferation.

There has been much recent interest in lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, in fibrotic diseases, but the mechanisms by which LPA-LPA1 signaling promotes pathological fibrosis remain to be fully elucidated. Using a mouse peritoneal fibrosis model, we demonstrate central roles for LPA and LPA1 in fibroblast proliferation. Genetic deletion or pharmacological antagonism of LPA1 protected mice from peritoneal fibrosis, blunting the increases in peritoneal collagen by 65.4 and 52.9%, respectively, compared to control animals and demonstrated that peritoneal fibroblast proliferation was highly LPA1 dependent. Activation of LPA1 on mesothelial cells induced these cells to express connective tissue growth factor (CTGF), driving fibroblast proliferation in a paracrine fashion. Activation of mesothelial cell LPA1 induced CTGF expression by inducing cytoskeleton reorganization in these cells, causing nuclear translocation of myocardin-related transcription factor (MRTF)-A and MRTF-B. Pharmacological inhibition of MRTF-induced transcription also diminished CTGF expression and fibrosis in the peritoneal fibrosis model, mitigating the increase in peritoneal collagen content by 57.9% compared to controls. LPA1-induced cytoskeleton reorganization therefore makes a previously unrecognized but critically important contribution to the profibrotic activities of LPA by driving MRTF-dependent CTGF expression, which, in turn, drives fibroblast proliferation.

Involvement of oncogenic K-ras on cell migration stimulated by lysophosphatidic acid receptor-2 in pancreatic cancer cells.

Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA(2) using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA(2) increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA(2) may be enhanced by oncogenic K-ras.

Insights into the biology and prevention of tumor metastasis provided by the Nm23 metastasis suppressor gene.

Metastatic disease is the major cause of death among cancer patients. A class of genes, named metastasis suppressors, has been described to specifically regulate the metastatic process. The metastasis suppressor genes are downregulated in the metastatic lesion compared to the primary tumor. In this review, we describe the body of research surrounding the first metastasis suppressor identified, Nm23. Nm23 overexpression in aggressive cancer cell lines reduced their metastatic potential in vivo with no significant reduction in primary tumor size. A complex mechanism of anti-metastatic action is unfolding involving several known Nm23 enzymatic activities (nucleotide diphosphate kinase, histidine kinase, and 3'-5' exonuclease), protein-protein interactions, and downstream gene regulation properties. Translational approaches involving Nm23 have progressed to the clinic. The upregulation of Nm23 expression by medroxyprogesterone acetate has been tested in a phase II trial. Other approaches with significant preclinical success include gene therapy using traditional or nanoparticle delivery, and cell permeable Nm23 protein. Recently, based on the inverse correlation of Nm23 and LPA1 expression, a LPA1 inhibitor has been shown to both inhibit metastasis and induce metastatic dormancy.