Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Supplemental effects of dietary lysophospholipids in lactation diets on sow performance, milk composition, gut health, and gut-associated microbiome of offspring.

Dietary lysophospholipids (LPL) would influence milk composition of sows, thus positively affect intestinal health of offspring. The objective of this study was to determine effects of dietary LPL fed to lactating sows on performance, milk characteristics, gut health, and gut-associated microbiome of offspring. Sixty pregnant sows were allotted to 2 treatments in a randomized complete block design with parity and BW as blocks on day 110 of gestation. Treatments were CON (no added LPL) and LPL (0.05% LPL; Lipidol-Ultra, Pathway Intermediates, Shrewsbury, UK). Sows were fed 2 kg/d from day 110 of gestation until farrowing and ad libitum after farrowing. Diets were formulated to meet NRC requirement for lactating sows. Colostrum and milk samples from 12 sows per treatment were collected to measure nutrients and immunoglobulins on days 1 and 18 of lactation, respectively. Twelve piglets per treatment (1 piglet per litter) were euthanized on day 18 to collect tissues to measure tumor necrosis factor-α, interleukin-8 (IL-8), malondialdehyde, protein carbonyl, IgA, histomorphology, crypt cell proliferation rate, and microbiota in the jejunum and colon. Data were analyzed using the MIXED procedure of SAS, and the mortality was analyzed using the GLIMMIX procedure of SAS. There was no difference in sow BW, parity, and litter size between treatments on day 0 of lactation. Sows fed LPL had increased (P < 0.05) litter BW gain (53.9 vs. 59.4 kg) and decreased piglet mortality (13.9% vs. 10.6%) on day 18 of lactation. Sows fed LPL had increased (P < 0.05) omega-6:omega-3 (22.1 vs. 23.7) and unsaturated:saturated (1.4 vs. 1.6) fatty acids ratios with increased oleic acid (29.1% vs. 31.4%) and tended to have increased (P = 0.092) IgG (1.14 vs. 1.94 g/L) and linoleic acid (17.7% vs. 18.7%) in the milk on day 18 of lactation. Piglets from sows fed LPL had increased (P < 0.05) IL-8 (184 vs. 245 pg/mg) and crypt cell proliferation rate (39.4% vs. 40.9%) and tended to have increased (P = 0.095) Firmicutes:Bacteroidetes ratio (1.0 vs. 3.5) in the jejunum. In conclusion, sows fed with LPL had milk with increased IgG, oleic acids, and linoleic acids without changes in BW and backfat during lactation. These changes could contribute to improved survivability and intestinal health of piglets by increasing IL-8 concentration, enhancing balance among gut-associated microbiome, and increasing enterocyte proliferation in the jejunum.

Declining BRCA-Mediated DNA Repair in Sperm Aging and its Prevention by Sphingosine-1-Phosphate.

Recent data suggest that paternal age can have major impact on reproductive outcomes, and with increased age, there is increased likelihood of chromosomal abnormalities in the sperm. Here, we studied DNA damage and repair as a function of male aging and assessed whether sphingosine-1-phosphate (S1P), a ceramide-induced death inhibitor, can prevent sperm aging by enhancing DNA double-strand breaks (DSB) repair. We observed a significant increase in DNA damage with age and this increase was associated with a decline in the expression of key DNA DSB repair genes in mouse sperm. The haploinsufficiency of BRCA1 male mice sperm showed significantly increased DNA damage and apoptosis, along with decreased chromatin integrity when compared to similar age wild type (WT) mice. Furthermore, haploinsufficiency of BRCA1 male mice had lower sperm count and smaller litter size when crossed with WT females. The resulting embryos had a higher probability of growth arrest and reduced implantation. S1P treatment decreased genotoxic-stress-induced DNA damage in sperm and enhanced the expressions of key DNA repair genes such as BRCA1. Co-treatment with an ATM inhibitor reversed the effects of S1P, implying that the impact of S1P on DNA repair is via the ATM-mediated pathway. Our findings indicate a key role for DNA damage repair mechanism in the maintenance of sperm integrity and suggest that S1P can improve DNA repair in sperm. Further translational studies are warranted to determine the clinical significance of these findings and whether S1P can delay male reproductive aging. There is mounting evidence that sperm quality declines with age, similar to that of the oocyte. However, the reasons behind this decline are poorly understood and there is no medical intervention to improve sperm quality. Our study suggests a strong role for DNA damage repair in maintenance of sperm quality, and for the first time, a potential pharmaceutical approach to prevent sperm aging.

Sequential Bone-Marrow Cell Delivery of VEGFA/S1P Improves Vascularization and Limits Adverse Cardiac Remodeling After Myocardial Infarction in Mice.

Microvascular dysfunction and resulting tissue hypoxia is a major contributor to the pathogenesis and evolution of cardiovascular diseases (CVD). Diverse gene and cell therapies have been proposed to preserve the microvasculature or boost angiogenesis in CVD, with moderate benefit. This study tested in vivo the impact of sequential delivery by bone-marrow (BM) cells of the pro-angiogenic factors vascular endothelial growth factor (VEGFA) and sphingosine-1-phosphate (S1P) in a myocardial infarction model. For that, mouse BM cells were transduced with lentiviral vectors coding for VEGFA or sphingosine kinase (SPHK1), which catalyzes S1P production, and injected them intravenously 4 and 7 days after cardiac ischemia-reperfusion in mice. Sequential delivery by transduced BM cells of VEGFA and S1P led to increased endothelial cell numbers and shorter extravascular distances in the infarct zone, which support better oxygen diffusion 28 days post myocardial infarction, as shown by automated 3D image analysis of the microvasculature. Milder effects were observed in the remote zone, together with increased proportion of capillaries. BM cells delivering VEGFA and S1P also decreased myofibroblast abundance and restricted adverse cardiac remodeling without major impact on cardiac contractility. The results indicate that BM cells engineered to deliver VEGFA/S1P angiogenic factors sequentially may constitute a promising strategy to improve micro-vascularization and oxygen diffusion, thus limiting the adverse consequences of cardiac ischemia.

Sphingosine 1-phosphate promotes mesenchymal stem cell-mediated cardioprotection against myocardial infarction via ERK1/2-MMP-9 and Akt signaling axis.

AIMS: The sphingolipid metabolite sphingosine 1­phosphate (S1P) has emerged as a potential cardioprotective molecule against ischemic heart disease. Moreover, S1P triggers mobilization and homing of bone marrow-derived stem/progenitor cells into the damaged heart. However, it remains elusive whether S1P promotes mesenchymal stem cells (MSCs)-mediated cardioprotection against ischemic heart diseases. MAIN METHODS: Adipose tissue-derived MSCs (AT-MSCs) were obtained from GFP transgenic mice or C57BL/6J. Myocardial infarction (MI) was induced in C57BL/6J mice by ligation of the left anterior descending coronary artery (LAD). Subsequently, S1P-treated AT-MSCs or vehicle-treated AT-MSCs were intravenously administered for 24 h after induction of MI or sham procedure. KEY FINDINGS: Pre-conditioning with S1P significantly enhanced the migratory and anti-apoptotic efficacies of AT-MSCs. In MI-induced mice, intravenous administration of S1P-treated AT-MSCs significantly augmented their homing and engraftment in ischemic area. Besides, AT-MSCs with S1P pre-treatment exhibited enhanced potencies to inhibit cardiomyocyte apoptosis and fibrosis, and stimulate angiogenesis and preserve cardiac function. Mechanistic studies revealed that S1P promoted AT-MSCs migration through activation of ERK1/2-MMP-9, and protected AT-MSCs against apoptosis via Akt activation. Further, S1P activated the ERK1/2 and Akt via S1P receptor 2 (S1PR2), but not through S1PR1. S1PR2 knockdown by siRNA, however, significantly attenuated S1P-mediated AT-MSCs migration and anti-apoptosis. SIGNIFICANCE: The findings of the present study revealed the protective efficacies of S1P pretreatment on the survival/retention and cardioprotection of engrafted MSCs. Pre-conditioning of donor MSCs with S1P is an effective strategy to promote the therapeutic potential of MSCs for ischemic heart diseases.

Effects of dietary supplemental lysophospholipids and vitamin C on performance, antioxidant enzymes, lipid peroxidation, thyroid hormones and serum metabolites of broiler chickens reared under thermoneutral and high ambient temperature.

The effects of dietary supplemental lysophospholipids (LPLs) and vitamin C (VC) on performance, activity of antioxidant enzymes, and thyroid hormones of broiler chickens reared under thermoneutral and high ambient temperatures were evaluated. A total of 1,680 broiler chicks (Cobb 500) in finishing rearing period (days 21-38 of age) were allotted to two groups: thermoneutral (TN) and heat stress (HS). In the TN group, 480 chicks were subjected to four treatments with four replicates (n = 30) and maintained in usual ambient temperature (24 ± 1°C). In HS group, the remaining 1,200 chicks were subjected to four treatments with 10 replicates (n = 30) and exposed to high ambient temperature (34 ± 1°C for 8 hr daily). In both groups, four iso-caloric and iso-nitrogenous experimental diets based on a 2 × 2 factorial arrangements including supplemental LPLs (0 or 1,000 mg/kg) and VC (0 or 500 mg/kg) were formulated and used. Supplemental LPLs decreased (p < 0.05) body weight gain and increased FCR in the TN and HS groups. In the TN group, increased (p < 0.05) serum glucose was observed in chickens fed with dietary supplemental VC. In the HS group, decreased (p < 0.05) total protein concentration was detected in birds fed with supplemental LPLs. In both TN and HS groups, decreased (p < 0.05) uric acid concentration was detected in chicks fed with the VC-supplemented diets. A significant (p < 0.05) interaction between LPLs and VC on lactate concentration in the TN group was observed. In the HS group, decreased breast malondialdehyde concentration was detected in birds fed with the VC-supplemented diet. In the TN group, increased serum total antioxidant status was detected in birds fed with the LPLs-supplemented diet. In conclusion, LPLs improved oxidative stability and increased the antioxidant capacity of the serum. In addition, vitamin C modified heat stress and reduced lipid peroxidation.

Lysophosphatidic acid induces neuronal cell death via activation of asparagine endopeptidase in cerebral ischemia-reperfusion injury.

Lysophosphatidic acid (LPA), an extracellular signaling molecule, influences diverse biological events, including the pathophysiological process induced after ischemic brain injury. However, the molecular mechanisms mediating the pathological change after ischemic stroke remain elusive. Here we report that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is regulated by LPA during stroke. AEP proteolytically cleaves tau and generates tauN368 fragments, triggering neuronal death. Inhibiting the generation of LPA reduces the expression of AEP and tauN368, and alleviates neuronal cell death. Together, this evidence indicates that the LPA-AEP pathway plays a key role in the pathophysiological process induced after ischemic stroke. Inhibition of LPA could be a useful therapeutic for treating neuronal injury after stroke.

2-O-Carba-oleoyl cyclic phosphatidic acid induces glial proliferation through the activation of lysophosphatidic acid receptor.

Lysophosphatidic acid (LPA) and cyclic phosphatidic acid (cPA) are one of the lipid mediators regulating cell proliferation and differentiation through the activation of LPA receptors. An LPA receptor-mediated signal is important for the development of the central nervous system, while it has been demonstrated that LPA caused microglial activation and astroglial dysfunction. Previously, we have reported that cPA and carba analog of cPA, 2-O-carba-cPA (2ccPA), protected neural damage caused by transient ischemia. However, little is known about the target cell of cPA/2ccPA in the central nervous systems. Here, we examined the effect of 2ccPA on glial proliferation and differentiation using the primary astrocytes and oligodendrocyte precursor cells (OPCs) cultures. 2ccPA increased the DNA synthesis of astrocytes and OPCs, but it did not reduce the formazan production in the mitochondria. Further, 2ccPA increased the cell number and cell survival against oxidative stress. The inhibition of LPA receptors by ki16425 abolished 2ccPA-induced DNA synthesis. Extracellular signal-regulated kinase (ERK) was activated by 2ccPA, which contributed to the astroglial DNA synthesis. These results suggest that 2ccPA is a beneficial regulator of glial population through the activation of LPA receptor without reduction of mitochondrial activity.

Lysophosphatidic acid protects against acetaminophen-induced acute liver injury.

We investigated the effect of lysophosphatidic acid (LPA) in experimental acetaminophen (APAP)-induced acute liver injury. LPA administration significantly reduced APAP-challenged acute liver injury, showing attenuated liver damage, liver cell death and aspartate aminotransferase and alanine aminotransferase levels. APAP overdose-induced mortality was also significantly decreased by LPA administration. Regarding the mechanism involved in LPA-induced protection against acute liver injury, LPA administration significantly increased the glutathione level, which was markedly decreased in APAP challenge-induced acute liver injury. LPA administration also strongly blocked the APAP challenge-elicited phosphorylation of JNK, ERK and GSK3ß, which are involved in the pathogenesis of acute liver injury. Furthermore, LPA administration decreased the production of TNF-α and IL-1ß in an experimental drug-induced liver injury animal model. Mouse primary hepatocytes express LPA1,3-6, and injection of the LPA receptor antagonist KI16425 (an LPA1,3-selective inhibitor) or H2L 5765834 (an LPA1,3,5-selective inhibitor) did not reverse the LPA-induced protective effects against acute liver injury. The therapeutic administration of LPA also blocked APAP-induced liver damage, leading to an increased survival rate. Collectively, these results indicate that the well-known bioactive lipid LPA can block the pathogenesis of APAP-induced acute liver injury by increasing the glutathione level but decreasing inflammatory cytokines in an LPA1,3,5-independent manner. Our results suggest that LPA might be an important therapeutic agent for drug-induced liver injury.

Analgesic effects of novel lysophosphatidic acid receptor 5 antagonist AS2717638 in rodents.

Lysophosphatidic acid (LPA) is a bioactive lipid that acts via at least six G protein-coupled receptors, LPA receptors 1-6 (LPA1-6), for various physiological functions. We examined (1) whether LPA5 is involved in pain signaling in the spinal cord; and (2) the pharmacological effects of a novel LPA5 antagonist on intrathecal prostaglandin (PG)- and (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced allodynia, and neuropathic and inflammatory pain in rodents. Intrathecal injection of a selective LPA5 agonist, geranylgeranyl diphosphate, and a non-selective agonist, LPA, induced allodynia in wild type, but not in LPA5 knockout mice. These novel results suggest that LPA5 is important for pain signal transmission in the spinal cord. AS2717638 (6,7-dimethoxy-2-(5-methyl-1,2-benzoxazol-3-yl)-4-(piperidin-1-ylcarbonyl)isoquinolin-1(2H)-one) bound to the LPA-binding site on LPA5 and selectively inhibited LPA-induced cyclic adenosine monophosphate accumulation in human LPA5-but not LPA1-, 2-, or 3-expressing cells. Further, oral administration of AS2717638 inhibited LPA5 agonist-induced allodynia in mice. AS2717638 also significantly improved PGE2-, PGF2α-, and AMPA-induced allodynia, while both pregabalin and duloxetine alleviated only PGE2-induced allodynia in mice. Similarly, AS2717638 significantly ameliorated static mechanical allodynia and thermal hyperalgesia in rat models of chronic constriction injury (CCI)-induced neuropathic pain. AS2717638 also showed analgesic effects in a rat model of inflammatory pain. These findings suggest that LPA5 antagonists elicit broad analgesic effects against both neuropathic and inflammatory pain. Accordingly, pharmacological LPA5 antagonists are attractive development candidates for potential novel pain therapies.

Sphingosine-1-phosphate reduces adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel walls by protecting endothelial surface glycocalyx.

Sphingosine-1-phosphate (S1P) is a sphingolipid in plasma that plays a critical role in cardiovascular and immune systems. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. To test the hypothesis that S1P can reduce tumor cell adhesion to microvessel walls by protecting the ESG, we quantified the ESG and MDA-MB-231 tumor cell adhesion in the presence and absence of 1µM S1P, and in the presence of the matrix metalloproteinase (MMP) inhibitor in post-capillary venules of rat mesentery. We also measured the microvessel permeability to albumin as an indicator for the microvessel wall integrity. In the absence of S1P, ESG was ~10% of that in the presence of S1P, whereas adherent tumor cells and the permeability to albumin and were ~3.5-fold (after 30 min adhesion) and ~7.7-fold that in the presence of S1P, respectively. In the presence of the MMP inhibitor, the results are similar to those in the presence of S1P. Our results conform to the hypothesis that protecting ESG by S1P inhibits MDA-MB-231 tumor cell adhesion to the microvessel wall.

Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression.

AIM: Sphingosine 1-phosphate (S1P) has been suggested to be a positive regulator of plasminogen activator inhibitor 1 (PAI-1) in adipocytes, while some studies are not consistent with this prothrombotic property of S1P. Since S1P is bound to apolipoprotein M (apoM) on HDL or to albumin in plasma, we compared the properties of these two forms on the PAI-1 induction. METHODS: We investigated the associations of S1P, apoM, and PAI-1 concentrations in the plasma of normal coronary artery (NCA), stable angina pectoris (SAP), and acute coronary syndrome (ACS) subjects (n=32, 71, and 38, respectively). Then, we compared the effects of S1P with various vehicles on the PAI-1 expression in 3T3L1 adipocytes. We also investigated the modulation of the PAI-1 levels in mice infected with adenovirus coding apoM. RESULTS: Among ACS subjects, the PAI-1 level was positively correlated with the S1P level, but not the apoM level. In adipocytes, S1P bound to an apoM-rich vehicle induced PAI-1 expression to a lesser extent than the control vehicle, while S1P bound to an apoM-depleted vehicle induced PAI-1 expression to a greater extent than the control vehicle in 3T3L1 adipocytes. Additionally, apoM overexpression in mice failed to modulate the plasma PAI-1 level and the adipose PAI-1 expression level. S1P bound to albumin increased PAI-1 expression through the S1P receptor 2-Rho/ROCK-NFκB pathway. CONCLUSION: S1P bound to albumin, but not to apoM, induces PAI-1 expression in adipocytes, indicating that S1P can exert different properties on the pathogenesis of vascular diseases, depending on its vehicle.

Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients.

The pathophysiology of human keratoconus (KC), a bilateral progressive corneal disease leading to protrusion of the cornea, stromal thinning, and scarring, is not well-understood. In this study, we investigated a novel sphingolipid (SPL) signaling pathway through which KC may be regulated. Using human corneal fibroblasts (HCFs) and human KC cells (HKCs), we examined the SPL pathway modulation. Both cell types were stimulated by the three transforming growth factor (TGF)-ß isoforms: TGF-ß1 (T1), TGF-ß2 (T2), and TGF-ß3 (T3). All samples were analyzed using lipidomics and real-time PCR. Our data showed that HKCs have increased levels of signaling SPLs, ceramide (Cer), and sphingosine 1-phosphate (S1P). Treatment with T1 reversed the increase in Cer in HKCs and treatment with T3 reversed the increase in S1P. S1P3 receptor mRNA levels were also significantly upregulated in HKCs, but were reduced to normal levels following T3 treatment. Furthermore, stimulation with Cer and S1P led to significant upregulation of fibrotic markers in HCFs, but not in HKCs. Additionally, stimulation with a Cer synthesis inhibitor (FTY720) led to significant downregulation of specific fibrotic markers in HKCs (TGF-ß1, collagen type III, and α smooth muscle actin) without an effect on healthy HCFs, suggesting a causative role of Cer and S1P in fibrogenesis. Overall, this study suggests an association of the SPL signaling pathway in KC disease and its relation with the TGF-ß pathway.

The effect of lysophosphatidic acid together with interferon tau on the global transcriptomic profile in bovine endometrial cells.

In cows, lysophosphatidic acid (LPA), which acts in an auto/paracrine manner, serves as a luteotropic factor during early pregnancy by stimulating progesterone and prostaglandin E2 secretion, thus protecting the bovine corpus luteum and early embryo development. Our hypothesis was that LPA exerted some local effects on the bovine endometrium prior to early embryo-maternal interactions and that interferon tau (IFNτ), the pregnancy recognition signal, modulated this action. In the present study, we applied an in vitro model involving whole-transcriptomic profiling to examine the effects of LPA on gene expression in bovine endometrial cells. Microarray analyses revealed 36, 269 and 284 differentially expressed transcripts in bovine endometrial cells in the control vs. LPA, control vs. LPA + IFNτ and LPA vs. LPA + IFNτ groups, respectively. The expression of matrix metalloproteinase 13 (MMP13) and radical S-adenosyl methionine domain containing 2 (RSAD2) was increased in the LPA-treated endometrial cells. Among the transcripts differentially regulated by LPA together with IFNτ, many of the genes were classical- or novel-type I IFN-stimulated genes (ISGs). The results indicated that 10 of the 16 analyzed genes showed a positive correlation with their corresponding microarray data upon real-time PCR validation, indicating a considerable consistency between both techniques. In summary, these transcriptional profiling studies identified a number of genes that were regulated by LPA alone and LPA together with IFNτ in endometrial cells from the bovine uterus. Available studies support the idea that LPA, which acts in an auto/paracrine manner on the endometrium, alters the expression of genes that are probably important for uterine receptivity, maternal immune tolerance to the embryo and conceptus growth and development during early pregnancy. Moreover, the differentially expressed genes (DEGs) that increased in the LPA + IFNτ-treated endometrial cells are largely in response to IFNτ actions and are possibly associated with crucial biological processes during the peri-implantation period of pregnancy.

Biodistribution of negatively charged iron oxide nanoparticles (IONPs) in mice and enhanced brain delivery using lysophosphatidic acid (LPA).

Effective treatment of brain disorders requires a focus on improving drug permeability across the blood-brain barrier (BBB). Herein, we examined the pharmacokinetic properties of negatively charged iron oxide nanoparticles (IONPs) and the capability of using lysophosphatidic acid (LPA) to transiently disrupt the tight junctions and allow IONPs to enter the brain. Under normal conditions, IONPs had a plasma half-life of six minutes, with the liver and spleen being the major organs of deposition. Treatment with LPA enhanced accumulation of IONPs in the brain and spleen (approximately 4-fold vs. control). LPA and IONP treated mice revealed no sign of peripheral immune cell infiltration in the brain and no significant activation of microglia or astrocytes. These studies show improved delivery efficiency of IONPs following LPA administration. Our findings suggest transient disruption of the BBB may be a safe and effective method for increasing IONP delivery to the brain.

Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid-Mediated Autocrine Mechanism.

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat-stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non-CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere-forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC-associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1-specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA-stimulated CSC properties. Autotaxin, an LPA-producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA-producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA-dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin-LPA-LPA receptor 1-AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs.

LPA signaling is required for dopaminergic neuron development and is reduced through low expression of the LPA1 receptor in a 6-OHDA lesion model of Parkinson's disease.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that activates at least five known G-protein-coupled receptors (GPCRs): LPA1-LPA5. The nervous system is a major locus for LPA1 expression. LPA has been shown to regulate neuronal proliferation, migration, and differentiation during central nervous system development as well as neuronal survival. Furthermore, deficient LPA signaling has been implicated in several neurological disorders including neuropathic pain and schizophrenia. Parkinson's disease (PD) is a neurodegenerative movement disorder that results from the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). The specific molecular pathways that lead to DA neuron degeneration, however, are poorly understood. The influence of LPA in the differentiation of mesenchymal stem cells (MSCs) into DA neurons in vitro and LPA1 expression in a 6-hydroxydopamine (6-OHDA) lesion model of PD in vivo were examined in the present study. LPA induced neuronal differentiation in 80.2 % of the MSC population. These MSCs developed characteristic neuronal morphology and expressed the neuronal marker, neuron-specific enolase (NSE), while expression of the glial marker, glial fibrillary acidic protein (GFAP), was absent. Moreover, 27.6 % of differentiated MSCs were positive for tyrosine hydroxylase (TH), a marker for DA neurons. In the 6-OHDA PD rat model, LPA1 expression in the substantia nigra was significantly reduced compared to control. These results suggest LPA signaling via activation of LPA1 may be necessary for DA neuron development and survival. Furthermore, reduced LPA/LPA1 signaling may be involved in DA neuron degeneration thus contributing to the pathogenesis of PD.

Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson's disease and in MPP+ -treated MN9D cells in vitro.

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson's disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP(+))-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP(+) exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP(+)-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP(+)-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Transforming growth factor-ß inhibits IQ motif containing guanosine triphosphatase activating protein 1 expression in lung fibroblasts via the nuclear factor-κB signaling pathway.

IQ motif containing guanosine triphosphatase activating protein 1 (IQGAP1) is associated with idiopathic pulmonary fibrogenesis (IPF); however, characterization of the expression of IQGAP1 in lung fibroblasts has remained elusive. The present study therefore evaluated IQGAP1 expression in mouse and human lung fibroblasts under fibrotic conditions via western blot analysis. It was revealed that IQGAP1 expression levels were significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice than in those of control mice. Transforming growth factor-ß (TGF-ß) induced differentiation, as well as decreased expression of IQGAP1 in WI-38 cells human lung fibroblasts. Furthermore, inhibition of nuclear factor (NF)-κB activation restored the TGF-ß-induced inhibition of IQGAP1 expression in WI-38 cells. In lysophosphatidic acid (LPA)-challenged WI-38 cells, the expression of IQGAP1 was also decreased, while neutralized anti-TGF-ß antibody treatment restored the LPA-induced inhibition of IQGAP1 expression. These data indicated that TGF-ß inhibited IQGAP1 expression in lung fibroblasts via the NF-κB signaling pathway, presenting a potential novel therapeutic target for the treatment of IPF.