Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid-liquid phase separation.

Myofibroblasts cause tissue fibrosis by producing extracellular matrix proteins, such as collagens. Humoral factors like TGF-ß, and matrix stiffness are important for collagen production by myofibroblasts. However, the molecular mechanisms regulating their ability to produce collagen remain poorly characterised. Here, we show that vestigial-like family member 3 (VGLL3) is specifically expressed in myofibroblasts from mouse and human fibrotic hearts and promotes collagen production. Further, substrate stiffness triggers VGLL3 translocation into the nucleus through the integrin ß1-Rho-actin pathway. In the nucleus, VGLL3 undergoes liquid-liquid phase separation via its low-complexity domain and is incorporated into non-paraspeckle NONO condensates containing EWS RNA-binding protein 1 (EWSR1). VGLL3 binds EWSR1 and suppresses miR-29b, which targets collagen mRNA. Consistently, cardiac fibrosis after myocardial infarction is significantly attenuated in Vgll3-deficient mice, with increased miR-29b expression. Overall, our results reveal an unrecognised VGLL3-mediated pathway that controls myofibroblasts' collagen production, representing a novel therapeutic target for tissue fibrosis.

Prostaglandin E2 receptor Ptger4b regulates female-specific peptidergic neurons and female sexual receptivity in medaka.

In vertebrates, female receptivity to male courtship is highly dependent on ovarian secretion of estrogens and prostaglandins. We recently identified female-specific neurons in the medaka (Oryzias latipes) preoptic area that express Npba, a neuropeptide mediating female sexual receptivity, in response to ovarian estrogens. Here we show by transcriptomic analysis that these neurons express a multitude of neuropeptides, in addition to Npba, in an ovarian-dependent manner, and we thus termed them female-specific, sex steroid-responsive peptidergic (FeSP) neurons. Our results further revealed that FeSP neurons express a prostaglandin E2 receptor gene, ptger4b, in an ovarian estrogen-dependent manner. Behavioral and physiological examination of ptger4b-deficient female medaka found that they exhibit increased sexual receptivity while retaining normal ovarian function and that their FeSP neurons have reduced firing activity and impaired neuropeptide release. Collectively, this work provides evidence that prostaglandin E2/Ptger4b signaling mediates the estrogenic regulation of FeSP neuron activity and female sexual receptivity.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Effects of an ω3 fatty acid-biased diet on luteolysis, parturition, and uterine prostanoid synthesis in pregnant mice.

The ω3 polyunsaturated fatty acids (PUFAs) are known to have beneficial effects on health and diseases, and hence their intake is encouraged. However, it remains unknown as to how ω3 PUFAs affect female reproduction processes, in which ω6 PUFA-derived prostaglandin (PG) E2 and PGF2α play crucial roles. We therefore compared female reproductive performance between ω3 PUFA-biased linseed oil diet-fed (Lin) mice and ω6 PUFA-biased soybean oil diet-fed (Soy) mice. In Lin mice, the uterine levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were 0.42 fold and 16 fold of those in Soy mice, respectively, with the EPA/AA ratio being 0.7 (vs 0.02 in Soy mice). Lin mice showed no alterations in any of the fertility indexes, including luteolysis and parturition. The uterine PG synthesis profiles of Lin mice were similar to those of Soy mice, but the levels of PGF2α and PGE2 were 50% of those in Soy mice, as a result of the increased EPA/AA ratio. PGF3α and PGE3 were undetectable in the uterine tissues of Soy and Lin mice. Interestingly, in Lin mice, 'luteolytic' PGF2α synthesis was considerably maintained even in the ω6 PUFA-reduced condition. These results suggest the existence of an elaborate mechanism securing PGF2α synthesis to a level that is sufficient for triggering luteolysis and parturition, even under ω6 PUFA-reduced conditions.

Expression of leukotriene B4 receptor 1 defines functionally distinct DCs that control allergic skin inflammation.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.

Lipocalin-type prostaglandin D synthase regulates light-induced phase advance of the central circadian rhythm in mice.

We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

Prostaglandin E2-EP4 Axis Promotes Lipolysis and Fibrosis in Adipose Tissue Leading to Ectopic Fat Deposition and Insulin Resistance.

Lipolysis, the breakdown of triglyceride storage in white adipose tissue, supplies fatty acids to other tissues as a fuel under fasting conditions. In morbid obesity, fibrosis limits adipocyte expandability, resulting in enforced lipolysis, ectopic fat distribution, and ultimately insulin resistance. Although basal levels of lipolysis persist even after feeding, the regulatory mechanisms of basal lipolysis remain unclear. Here, we show the important role of adipocyte prostaglandin (PG) E2-EP4 receptor signaling in controlling basal lipolysis, fat distribution, and collagen deposition during feeding-fasting cycles. The PGE2-synthesis pathway in adipocytes, which is coupled with lipolysis, is activated by insulin during feeding. By regulating the lipolytic key players, the PGE2-EP4 pathway sustains basal lipolysis as a negative feedback loop of insulin action, and perturbation of this process leads to "metabolically healthy obesity." The potential role of the human EP4 receptor in lipid regulation was also suggested through genotype-phenotype association analyses.

Advanced Oxidation Protein Products Contribute to Renal Tubulopathy via Perturbation of Renal Fatty Acids.

Background: Renal proximal tubulopathy plays a crucial role in kidney disease, but its molecular mechanism is incompletely understood. Because proximal tubular cells consume a lot of energy during reabsorption, the relationship between fatty acids (FAs) and proximal tubulopathy has been attracting attention. The purpose of this study is to investigate the association between change in renal FA composition and tubulopathy. Methods: Mice with cisplatin-induced nephrotoxicity were used as a model of AKI and 5/6-nephrectomized mice were used as a model of CKD. Renal FA composition in mice was measured by GC-MS. Human tubular epithelial cells (HK-2 cells) were used for in vitro studies. Results: In kidneys of AKI mice, increased stearic acid (C18:0) and decreased palmitic acid (C16:0) were observed, accompanied by increased expression of the long-chain FA elongase Elovl6. Similar results were also obtained in CKD mice. We show that C18:0 has higher tubular toxicity than C16:0 via induction of ER stress. Using adenovirus-expressing Elovl6 or siRNA for Elovl6 in HK-2 cells, we demonstrated that increased Elovl6 expression contributes to tubulopathy via increasing C18:0. Elovl6 knockout suppressed the increased serum creatinine levels, renal ER stress, and inflammation that would usually result after 5/6 nephrectomy. Advanced oxidation protein products (AOPPs), specifically an oxidized albumin, was found to induce Elovl6 via the mTORC1/SREBP1 pathway. Conclusions: AOPPs may contribute to renal tubulopathy via perturbation of renal FAs through induction of Elovl6. The perturbation of renal FAs induced by the AOPPs-Elovl6 system could be a potential target for the treatment of tubulopathy.

Cutaneous p38 mitogen-activated protein kinase activation triggers psoriatic dermatitis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by IL-17-mediated immune responses. p38 is known to be highly activated in the psoriatic epidermis; however, whether p38 is involved in the development of psoriasis is unclear. OBJECTIVE: We sought to demonstrate that activation of p38 mitogen-activated protein kinase is sufficient to induce psoriatic inflammation in mice and that cutaneous p38 activities are the topical therapeutic targets for psoriasis. METHODS: A p38 activator, anisomycin, was applied daily to murine skin. Transcriptomic analyses were performed to evaluate the similarities of the skin responses to those in human psoriasis and the existing animal model. BIRB796, a small-molecule inhibitor targeting p38 activities, was applied to the murine psoriatic models topically or to human psoriatic skin specimens ex vivo. RESULTS: Topical treatment with anisomycin induced key signatures in psoriasis, such as epidermal thickening, neutrophil infiltration, and gene expression of Il1a, Il1b, Il6, Il24, Cxcl1, Il23a, and Il17a, in treated murine skin. These responses were fully abrogated by topical treatment with BIRB796, and were reduced in IL-17A-deficient mice. Transcriptomic analyses demonstrated the similarities of anisomycin-induced dermatitis to human psoriasis and imiquimod-induced murine psoriatic dermatitis. Furthermore, BIRB796 targeting of p38 activities reduced expression of psoriasis-related genes in both human keratinocytes stimulated with recombinant IL-17A in vitro and psoriatic skin specimens ex vivo. CONCLUSION: Therefore our findings suggest that cutaneous p38 activation can be a key event in patients with psoriasis and a potential topical therapeutic target of a small molecule.

Essential role of prostaglandin E2 and the EP3 receptor in lymphatic vessel development during zebrafish embryogenesis.

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

Epithelial TRAF6 drives IL-17-mediated psoriatic inflammation.

Epithelial cells are the first line of defense against external dangers, and contribute to induction of adaptive immunity including Th17 responses. However, it is unclear whether specific epithelial signaling pathways are essential for the development of robust IL-17-mediated immune responses. In mice, the development of psoriatic inflammation induced by imiquimod required keratinocyte TRAF6. Conditional deletion of TRAF6 in keratinocytes abrogated dendritic cell activation, IL-23 production, and IL-17 production by γδ T cells at the imiquimod-treated sites. In contrast, hapten-induced contact hypersensitivity and papain-induced IgE production were not affected by loss of TRAF6. Loss of psoriatic inflammation was not solely due to defective imiquimod sensing, as subcutaneous administration of IL-23 restored IL-17 production but did not reconstitute psoriatic pathology in the mutant animals. Thus, TRAF6 was required for the full development of IL-17-mediated inflammation. Therefore, epithelial TRAF6 signaling plays an essential role in both triggering and propagating IL-17-mediated psoriatic inflammation.

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

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

Olfactory receptor for prostaglandin F2α mediates male fish courtship behavior.

Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2α acts not only as a female reproductive hormone, facilitating ovulation and spawning, but also as a sex pheromone inducing male reproductive behaviors. Here, we unravel the molecular and neural circuit mechanisms underlying the pheromonal action of prostaglandin F2α in zebrafish. Prostaglandin F2α specifically activates two olfactory receptors with different sensitivities and expression in distinct populations of ciliated olfactory sensory neurons. Pheromone information is then transmitted to two ventromedial glomeruli in the olfactory bulb and further to four regions in higher olfactory centers. Mutant male zebrafish deficient in the high-affinity receptor exhibit loss of attractive response to prostaglandin F2α and impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2α and its cognate olfactory receptor in fish reproductive behavior.

p13 overexpression in pancreatic ß-cells ameliorates type 2 diabetes in high-fat-fed mice.

We examined the pancreatic function of p13 encoded by 1110001J03Rik, whose expression is decreased in pancreatic islets in high-fat-fed diabetic mice, by generating transgenic mice overexpressing p13 (p13-Tg) in pancreatic ß-cells. p13-Tg mice showed normal basal glucose metabolism; however, under high-fat feeding, these animals showed augmented glucose-induced first-phase and total insulin secretion, improved glucose disposal, greater islet area and increased mitotic insulin-positive cells. In addition, high-fat diet-induced 4-hydroxynonenal immunoreactivity, a reliable marker and causative agent of lipid peroxidative stress, was significantly decreased in p13-Tg mouse islets. These results indicate that p13 is a novel pancreatic factor exerting multiple beneficial effects against type 2 diabetes.

Comparative gene expression profiles in pancreatic islets associated with agouti yellow mutation and PACAP overexpression in mice.

In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic ß-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F1 offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with >16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.

Prostaglandin E2-induced inflammation: Relevance of prostaglandin E receptors.

Prostaglandin E2 (PGE2) is one of the most typical lipid mediators produced from arachidonic acid (AA) by cyclooxygenase (COX) as the rate-limiting enzyme, and acts on four kinds of receptor subtypes (EP1-EP4) to elicit its diverse actions including pyrexia, pain sensation, and inflammation. Recently, the molecular mechanisms underlying the PGE2 actions mediated by each EP subtype have been elucidated by studies using mice deficient in each EP subtype as well as several compounds highly selective to each EP subtype, and their findings now enable us to discuss how PGE2 initiates and exacerbates inflammation at the molecular level. Here, we review the recent advances in PGE2 receptor research by focusing on the activation of mast cells via the EP3 receptor and the control of helper T cells via the EP2/4 receptor, which are the molecular mechanisms involved in PGE2-induced inflammation that had been unknown for many years. We also discuss the roles of PGE2 in acute inflammation and inflammatory disorders, and the usefulness of anti-inflammatory therapies that target EP receptors. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Dermal Vγ4(+) γδ T cells possess a migratory potency to the draining lymph nodes and modulate CD8(+) T-cell activity through TNF-α production.

A large number of gamma delta T cells (γδ T cells) are located within epithelial tissues including the skin. In mice, epidermal and dermal γδ T cells consist of distinct subsets and have specific roles in cutaneous immune responses. A recent study demonstrated that γδ T cells and cutaneous dendritic cells migrate from the skin to the draining lymph nodes (LNs). However, it remains unclear whether they regulate the antigen-specific immune response within the LNs. Herein, we investigated their properties and role in the LNs using the Mycobacterium bovis bacille Calmette-Guérin (BCG) infection model. In vivo cell labeling analysis revealed that most of the migratory subset comprised dermal Vγ4(+) cells. This population transmigrated from the skin to the LNs in a Gi-coupled chemokine receptor-independent manner. By depleting Vγ4(+) cells, the intranodal expansion of CD8(+) T cell against BCG was significantly attenuated. In addition, in vitro analysis revealed that Vγ4(+) cells produced TNF-α and enhanced IL-12 production by dendritic cells. Taken together, these findings suggest that dermal Vγ4(+) cells are a unique subset that possesses a migratory potency to the skin-draining LNs and enhances the dendritic cell function therein.

Roles of prostaglandin receptors in female reproduction.

Prostaglandins (PGs) have long been known to play roles in various processes of female reproduction; however, the molecular mechanisms therein remained unsolved until recently. This review summarizes the recent progress towards understanding the molecular mechanisms underlying PG actions in fertilization and parturition. A series of studies using EP2-deficient mice demonstrated that after ovulation chemokine signalling in the cumulus cells stimulates integrin activation and cumulus extracellular matrix (ECM) assembly through the RhoA/ROCK/actomyosin pathway, although excessive chemokine signalling disturbs sperm penetration. PGE2-EP2 signalling suppresses such a chemokine signalling and stimulates cumulus ECM disassembly, which contributes to successful fertilization. A series of studies using FP-deficient mice revealed that PGF(2α)-FP signalling induces parturition at least by terminating progesterone production; however, some other EP signals are likely to be involved in parturition by inducing myometrial contraction. Therefore, it should be clarified as to which EP and/or FP receptor signals are physiologically essential for myometrial contraction and successful parturition.