Different Coactivator Recruitment to Human PPARα/δ/γ Ligand-Binding Domains by Eight PPAR Agonists to Treat Nonalcoholic Fatty Liver Disease.

Three peroxisome proliferator-activated receptor subtypes, PPARα, PPAR(ß/)δ, and PPARγ, exert ligand-dependent transcriptional control in concert with retinoid X receptors (RXRs) on various gene sets harboring PPAR response elements (PPREs) in their promoter regions. Ligand-bound PPAR/RXR complexes do not directly regulate transcription; instead, they recruit multiprotein coactivator complexes to specific genomic regulatory loci to cooperatively activate gene transcription. Several coactivators are expressed in a single cell; however, a ligand-bound PPAR can be associated with only one coactivator through a consensus LXXLL motif. Therefore, altered gene transcription induced by PPAR subtypes/agonists may be attributed to the recruitment of various coactivator species. Using a time-resolved fluorescence resonance energy transfer assay, we analyzed the recruitment of four coactivator peptides (PGC1α, CBP, SRC1, and TRAP220) to human PPARα/δ/γ-ligand-binding domains (LBDs) using eight PPAR dual/pan agonists (bezafibrate, fenofibric acid, pemafibrate, pioglitazone, elafibranor, lanifibranor, saroglitazar, and seladelpar) that are/were anticipated to treat nonalcoholic fatty liver disease. These agonists all recruited four coactivators to PPARα/γ-LBD with varying potencies and efficacy. Only five agonists (bezafibrate, pemafibrate, elafibranor, lanifibranor, and seladelpar) recruited all four coactivators to PPARδ-LBD, and their concentration-dependent responses differed from those of PPARα/γ-LBD. These results indicate that altered gene expression through consensus PPREs by different PPAR subtypes/agonists may be caused, in part, by different coactivators, which may be responsible for the unique pharmacological properties of these PPAR agonists.

Differential Roles of Cystathionine Gamma-Lyase and Mercaptopyruvate Sulfurtransferase in Hapten-Induced Colitis and Contact Dermatitis in Mice.

Hydrogen sulfide (H2S) has been shown to act as both anti-inflammatory and pro-inflammatory mediators. Application of H2S donors generally protects against inflammation; however, experimental results using mice lacking endogenous H2S-producing enzymes, such as cystathionine γ-lyase (CTH) and mercaptopyruvate sulfurtransferase (MPST), are often contradictory. We herein examined two types of model hapten-induced inflammation models, colitis (an inflammatory bowel disease model of mucosal immunity) and contact dermatitis (a type IV allergic model of systemic immunity), in CTH-deficient (Cth-/-) and MPST-deficient (Mpst-/-) mice. Both mice exhibited no significant alteration from wild-type mice in trinitrobenzene sulfonic acid (Th1-type hapten)-induced colitis (a Crohn's disease model) and oxazolone (Th1/Th2 mix-type; Th2 dominant)-induced colitis (an ulcerative colitis model). However, Cth-/- (not Mpst-/-) mice displayed more exacerbated phenotypes in trinitrochlorobenzene (TNCB; Th1-type)-induced contact dermatitis, but not oxazolone, at the delayed phase (24 h post-administration) of inflammation. CTH mRNA expression was upregulated in the TNCB-treated ears of both wild-type and Mpst-/- mice. Although mRNA expression of pro-inflammatory cytokines (IL-1ß and IL-6) was upregulated in both early (2 h) and delayed phases of TNCB-triggered dermatitis in all genotypes, that of Th2 (IL-4) and Treg cytokines (IL-10) was upregulated only in Cth-/- mice, when that of Th1 cytokines (IFNγ and IL-2) was upregulated in wild-type and Mpst-/- mice at the delayed phase. These results suggest that (upregulated) CTH or H2S produced by it helps maintain Th1/Th2 balance to protect against contact dermatitis.

Opposing Roles of Sphingosine 1-Phosphate Receptors 1 and 2 in Fat Deposition and Glucose Tolerance in Obese Male Mice.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.

A High-Methionine Diet for One-Week Induces a High Accumulation of Methionine in the Cerebrospinal Fluid and Confers Bipolar Disorder-like Behavior in Mice.

Methionine (Met) is considered the most toxic amino acid in mammals. Here, we investigated biochemical and behavioral impacts of ad libitum one-week feeding of high-Met diets on mice. Adult male mice were fed the standard rodent diet that contained 0.44% Met (1×) or a diet containing 16 graded Met doses (1.2×-13×). High-Met diets for one-week induced a dose-dependent decrease in body weight and an increase in serum Met levels with a 2.55 mM peak (versus basal 53 µM) on the 12×Met diet. Total homocysteine (Hcy) levels were also upregulated while concentrations of other amino acids were almost maintained in serum. Similarly, levels of Met and Hcy (but not the other amino acids) were highly elevated in the cerebrospinal fluids of mice on the 10×Met diet; the Met levels were much higher than Hcy and the others. In a series of behavioral tests, mice on the 10×Met diet displayed increased anxiety and decreased traveled distances in an open-field test, increased activity to escape from water soaking and tail hanging, and normal learning/memory activity in a Y-maze test, which were reflections of negative/positive symptoms and normal cognitive function, respectively. These results indicate that high-Met ad libitum feeding even for a week can induce bipolar disorder-like disease models in mice.

Homocysteine Hypothesis on the Impaired Peripheral but Not Central Nervous System Oxytocin Responses in Cystathionine γ-Lyase-Deficient Dam Mice.

An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth-/-) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth-/- dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth-/- male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth-/- female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth-/- L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth-/- mice. Therefore, we hypothesize that the blood-brain barrier protects the CNS from high levels of circulatory homocysteine in Cth-/- dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

Cytotoxicity comparison of 35 developmental neurotoxicants in human induced pluripotent stem cells (iPSC), iPSC-derived neural progenitor cells, and transformed cell lines.

The Organization for Economic Co-operation and Development (OECD) test guideline 426 for developmental neurotoxicity (DNT) of industrial/environmental chemicals depends primarily on animal experimentation. This requirement raises various critical issues, such as high cost, long duration, the sacrifice of large numbers of animals, and interspecies differences. This study demonstrates an alternative protocol that is simple, quick, less expensive, and standardized to evaluate DNT of many chemicals using human induced pluripotent stem cells (iPSC) and their differentiation to neural progenitor cells (NPC). Initially, concentration-dependent cytotoxicity of 35 DNT chemicals, including industrial materials, insecticides, and clinical drugs, were compared among iPSC, NPC, and two transformed cells, Cos-7 and HepG2, using tetrazolium dye (MTS)-reducing colorimetric and ATP luciferase assays, and IC50 values were calculated. Next, inhibitory effects of the 14 representative chemicals (mainly insecticides) on iPSC differentiation to NPC were evaluated by measuring altered expression of neural differentiation and undifferentiation marker genes. Results show that both iPSC and NPC were much more sensitive to most DNT chemicals than the transformed cells, and 14 chemicals induced differential patterns of marker gene expression, highlighting the validity and utility of the protocol for evaluation and classification of DNT chemicals and preclinical DNT tests for safety assessment.

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase-An Animal Model of Cystathioninuria.

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia-a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth-/-) pregnant mice display any features of preeclampsia. Cth-/- females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth-/- dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth-/- dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth-/- dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth-/- mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth-/- dams interfere with oxytocin that regulates milk ejection reflex.

Lysophosphatidic acid-induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4.

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased ß-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.

Abnormal Amino Acid Profiles of Blood and Cerebrospinal Fluid from Cystathionine ß-Synthase-Deficient Mice, an Animal Model of Homocystinuria.

Mental retardation is the most common feature among inborn errors of amino acid metabolism. Patients with homocystinuria/homocysteinemia caused by cystathionine ß-synthase (CBS) deficiency suffer from thromboembolism and mental retardation from early ages; therefore, detection by newborn screening is performed. Furthermore, elevated levels of serum homocysteine during pregnancy are associated with the occurrence of neural tube defects (NTDs) in newborns. However, the causes of such central nervous system (CNS) defects are unknown. We found previously impaired learning abilities in Cbs-deficient (Cbs-/-) mice (but not NTD births). Here, we investigated the amino acid profiles of serum and cerebrospinal fluid (CSF) from Cbs-/- mice. Mice deficient in cystathionine γ-lyase (Cth), a downstream enzyme of CBS in transsulfuration, as well as wild-type mice, were analyzed as controls. Cbs-/- and Cth-/- mice were smaller than wild-type mice, and CSF yields in Cbs-/- mice were lower than the others. CSF amino acid levels were generally lower than those in serum, and compared with the dramatic amino acid level alterations in Cbs-/- mouse serum, alterations in CSF were less apparent. However, marked upregulation (versus wild-type) of aspartic acid/asparagine (Asp/Asn), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), methionine (Met), total homocysteine, and citrulline, and downregulation of lysine (Lys) were found in Cbs-/- mouse CSF. Because similar regulation of total homocysteine/citrulline/Lys was observed in the CSF of Cth-/- mice, which are free of CNS dysfunction, the reduced CSF volumes and the level changes of other amino acids could be relevant to Cbs-/--specific CNS defects.

Dietary selenium deficiency or selenomethionine excess drastically alters organ selenium contents without altering the expression of most selenoproteins in mice.

Selenium is an essential trace element, and its deficiency can cause cardiomyopathy, arrhythmias and increased susceptibility to infection. Such clinical symptoms are considered primarily attributed to decreased expression of some of the 25 selenocysteine-containing selenoproteins in humans. Conversely, a selenium-excessive diet can cause acute poisoning and chronic symptoms with unknown mechanisms. To reveal the impact of selenium deficiency and excess on selenoprotein expression in vivo, mice (that possess 24 selenoproteins) were fed with selenium-deficient or selenomethionine-excessive diets for up to 4 weeks, and the expression levels of nine representative selenoproteins [glutathione peroxidase (Gpx) 1/2/3/4, thioredoxin reductase 1/2, deiodinase 1, and selenoprotein P/S] were measured in 10 organs (brain, heart, liver, lung, kidney, pancreas, spleen, testis, skeletal muscle and thymus). We observed a time-dependent decrease in the selenium content of most organs (except testis) of selenium-deficient mice but not in the expression levels of the nine selenoproteins, with the exceptions of Gpx1/2 in the heart/liver/kidney/pancreas/spleen and Gpx3 in the pancreas/spleen. Serum lipid peroxidation levels were up-regulated in response to Se deficiency because of the decreased expression/activity of Gpx3, a plasma-type Gpx. In contrast, a time-dependent increase was observed in the selenium content of all organs but not the expression levels of the nine selenoproteins in most organs of selenomethionine-excessive mice; however, markedly elevated protein-bound selenium levels were observed in the liver/kidney. These results suggest that the systemic response to selenium deficiency and selenomethionine excess involves the down-regulation of some selenoproteins such as Gpx1/Gpx3 and up-regulation of selenium-containing proteins (not selenoproteins), respectively.

The Gα12/13-coupled receptor LPA4 limits proper adipose tissue expansion and remodeling in diet-induced obesity.

White adipose tissue (WAT) can dynamically expand and remodel through adipocyte hypertrophy and hyperplasia. The relative contribution of these 2 mechanisms to WAT expansion is a critical determinant of WAT function and dysfunction in obesity. However, little is known about the signaling systems that determine the mechanisms of WAT expansion. Here, we show that the GPCR LPA4 selectively activates Gα12/13 proteins in adipocytes and limits continuous remodeling and healthy expansion of WAT. LPA4-KO mice showed enhanced expression of mitochondrial and adipogenesis genes and reduced levels of inhibitory phosphorylation of PPARγ in WAT, along with increased production of adiponectin. Furthermore, LPA4-KO mice showed metabolically healthy obese phenotypes in a diet-induced obesity model, with continuous WAT expansion, as well as protection from WAT inflammation, hepatosteatosis, and insulin resistance. These findings unravel a potentially new signaling system that underlies WAT plasticity and expandability, providing a promising therapeutic approach for obesity-related metabolic disorders.

Mutations in Homocysteine Metabolism Genes Increase Keratin N-Homocysteinylation and Damage in Mice.

Genetic or nutritional deficiencies in homocysteine (Hcy) metabolism increase Hcy-thiolactone, which causes protein damage by forming isopetide bonds with lysine residues, generating N-Hcy-protein. In the present work, we studied the prevalence and genetic determinants of keratin damage caused by homocysteinylation. We found that in mammals and birds, 35 to 98% of Hcy was bound to hair keratin via amide or isopeptide bond (Hcy-keratin), while 2 to 65% was S-Hcy-keratin. A major fraction of hair Hcy-keratin (56% to 93%), significantly higher in birds than in mammals, was sodium dodecyl sulfate-insoluble. Genetic hyperhomocysteinemia significantly increased N-Hcy-keratin levels in the mouse pelage. N-Hcy-keratin was elevated 3.5-, 6.3-, and 11.7-fold in hair from Mthfr -/-, Cse -/-, or Cbs -/- mice, respectively. The accumulation of N-Hcy in hair keratin led to a progressive reduction of N-Hcy-keratin solubility in sodium dodecyl sulfate, from 0.39 ± 0.04 in wild-type mice to 0.19 ± 0.03, 0.14 ± 0.01, and 0.07 ± 0.03 in Mthfr -/-, Cse -/-, or Cbs -/-animals, respectively. N-Hcy-keratin accelerated aggregation of unmodified keratin in Cbs -/- mouse hair. Keratin methionine, copper, and iron levels in mouse hair were not affected by hyperhomocysteinemia. These findings provide evidence that pelage keratin is N-homocysteinylated in vivo in mammals and birds, and that this process causes keratin damage, manifested by a reduced solubility.

2D DIGE proteomic analysis reveals fasting-induced protein remodeling through organ-specific transcription factor(s) in mice.

Overnight fasting is a routine procedure before surgery in clinical settings. Intermittent fasting is the most common diet/fitness trend implemented for weight loss and the treatment of lifestyle-related diseases. In either setting, the effects not directly related to parameters of interest, either beneficial or harmful, are often ignored. We previously demonstrated differential activation of cellular adaptive responses in 13 atrophied/nonatrophied organs of fasted mice by quantitative PCR analysis of gene expression. Here, we investigated 2-day fasting-induced protein remodeling in six major mouse organs (liver, kidney, thymus, spleen, brain, and testis) using two-dimensional difference gel electrophoresis (2D DIGE) proteomics as an alternative means to examine systemic adaptive responses. Quantitative analysis of protein expression followed by protein identification using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) revealed that the expression levels of 72, 26, and 14 proteins were significantly up- or downregulated in the highly atrophied liver, thymus, and spleen, respectively, and the expression levels of 32 proteins were up- or downregulated in the mildly atrophied kidney. Conversely, there were no significant protein expression changes in the nonatrophied organs, brain and testis. Upstream regulator analysis highlighted transcriptional regulation by peroxisome proliferator-activated receptor alpha (PPARα) in the liver and kidney and by tumor protein/suppressor p53 (TP53) in the thymus, spleen, and liver. These results imply of the existence of both common and distinct adaptive responses between major mouse organs, which involve transcriptional regulation of specific protein expression upon short-term fasting. Our data may be valuable in understanding systemic transcriptional regulation upon fasting in experimental animals.

Cystathionine γ-Lyase-Produced Hydrogen Sulfide Controls Endothelial NO Bioavailability and Blood Pressure.

Hydrogen sulfide (H2S) and NO are important gasotransmitters, but how endogenous H2S affects the circulatory system has remained incompletely understood. Here, we show that CTH or CSE (cystathionine γ-lyase)-produced H2S scavenges vascular NO and controls its endogenous levels in peripheral arteries, which contribute to blood pressure regulation. Furthermore, eNOS (endothelial NO synthase) and phospho-eNOS protein levels were unaffected, but levels of nitroxyl were low in CTH-deficient arteries, demonstrating reduced direct chemical interaction between H2S and NO. Pretreatment of arterial rings from CTH-deficient mice with exogenous H2S donor rescued the endothelial vasorelaxant response and decreased tissue NO levels. Our discovery that CTH-produced H2S inhibits endogenous endothelial NO bioavailability and vascular tone is novel and fundamentally important for understanding how regulation of vascular tone is tailored for endogenous H2S to contribute to systemic blood pressure function.

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.

Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

Lysophosphatidic acid receptors LPA4 and LPA6 differentially promote lymphocyte transmigration across high endothelial venules in lymph nodes.

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.

2D DIGE proteomic analysis highlights delayed postnatal repression of α-fetoprotein expression in homocystinuria model mice.

Cystathionine ß-synthase-deficient (Cbs (-/-)) mice, an animal model for homocystinuria, exhibit hepatic steatosis and juvenile semilethality via as yet unknown mechanisms. The plasma protein profile of Cbs (-/-) mice was investigated by proteomic analysis using two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. We found hyperaccumulation of α-fetoprotein (AFP) and downregulation of most other plasma proteins. AFP was highly expressed in fetal liver, but its expression declined dramatically via transcriptional repression after birth in both wild-type and Cbs (-/-) mice. However, the repression was delayed in Cbs (-/-) mice, causing high postnatal AFP levels, which may relate to transcriptional repression of most plasma proteins originating from liver and the observed hepatic dysfunction.

The lysophosphatidic acid receptor LPA4 regulates hematopoiesis-supporting activity of bone marrow stromal cells.

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that acts through G protein-coupled receptors (LPA1-6). Although several biological roles of LPA4 are becoming apparent, its role in hematopoiesis has remained unknown. Here, we show a novel regulatory role for LPA4 in hematopoiesis. Lpar4 mRNA was predominantly expressed in mouse bone marrow (BM) PDGFRα(+) stromal cells, known as the components of the hematopoietic stem/progenitor cell (HSPC) niche. Compared with wild-type mice, LPA4-deficient mice had reduced HSPC numbers in the BM and spleen and were hypersusceptible to myelosuppression, most likely due to impairments in HSPC recovery and stem cell factor production in the BM. Analysis of reciprocal BM chimeras (LPA4-deficient BM into wild-type recipients and vice versa) indicated that stromal cells likely account for these phenotypes. Consistently, LPA4-deficient BM stromal cells showed downregulated mRNA expression of stem cell factor and tenascin-c in vitro. Taken together, these results suggest a critical and novel role for the LPA/LPA4 axis in regulating BM stromal cells.