Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts

AbstractCyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway

Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract

金沢大学大学院自然科学研究科 信頼性システム工学The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication- dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine

Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA).

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2)) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl(2) induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2)-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2). Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1), LPA(2), and LPA(6), which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1) and LPA(2) have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1) and LPA(2) on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1) and LPA(3) antagonist, was adopted to know the LPA(1) function and siRNA was used to knockdown the expression of LPA(2). On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2)-induced hypoxia damage is mediated via LPA(2)

cPA protects against CoCl₂-induced apoptosis in Neuro2A cells.

<p>(A) The effects of cPA and LPA on a number of adhesive Neuro2A cells treated with CoCl₂. Neuro2A cells were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for 24 hours, and the number of cells attached to the surface of dishes was determined (*P<0.05, **P<0.01 vs. the CoCl₂-treated group). (B & C) Analysis of the percent of FITC-Annexin V- and FITC-DEVD-FMK-positive Neuro2A cells after CoCl₂ treatment by flow cytometry. Neuro2A cells were incubated with 300 µM CoCl₂ and various concentrations of either cPA or LPA for 24 hours. Cells were subsequently stained with either FITC-Annexin V (B) or FITC-DEVD-FMK, an activated caspase-3 inhibitor (C) and subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, **P<0.01, ***P<0.001 vs. the CoCl₂-treated group).</p

Treatment with CoCl₂ induces apoptosis in Neuro2A cells.

<p>(A) Effects of CoCl₂ on the viability of Neuro2A cells. Neuro2A cells were incubated with various concentrations of CoCl₂ for 24 hours. Cell viability was estimated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051093#s2" target="_blank">materials and methods</a> section. The data represent the mean ± SE values from triplicate independent experiments. (B) Generation of reactive oxygen species (ROS) induced by CoCl₂. Neuro2A cells were incubated with 300 µM CoCl₂, and ROS generation was measured after 5 and 15 min. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, ***P<0.001 vs. the CoCl₂-treated group). (C) Morphologic changes in the nuclei of CoCl₂-treated Neuro2A cells. Neuro2A cells incubated in the absence (left) or presence (right) of 300 µM CoCl₂ for 24 hours were fixed and stained with DAPI and examined by fluorescence microscopy. (D) Analysis of apoptosis-associated PS exposure on CoCl₂-treated Neuro2A cells by using FITC-Annexin V. Neuro2A cells were treated with (right) or without (left) 300 µM CoCl₂ for 24 hours and stained with FITC-Annexin V. The population of FITC-Annexin V-positive Neuro2A cells was quantified by flow cytometry.</p

Effects of LPA receptors on the neuroprotective functions of cPA and LPA against CoCl₂-induced apoptosis.

<p>(A) Expression of LPA receptors in Neuro2A cells. Total RNA was extracted from Neuro2A cells, and the expression level of each LPA receptor was determined by quantitative real-time PCR. The expression levels were normalized to those of LPA₁ and expressed in terms of the mean ± SE values. (B) The effects of Ki16425 on the neuroprotective functions of cPA and LPA against CoCl₂-induced apoptosis of Neuro2A cells. Neuro2A cells were pretreated with or without 10 µM Ki16425 for 20 min. Subsequently, the cells were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for 24 hours. The cells were then stained with FITC-Annexin V and subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (***P<0.001 vs. the CoCl₂-treated group). (C) Expression of LPA₂ in Neuro2A cells. Neuro2A cells were transfected with siRNA against LPA₂ or non-target siRNA. Total RNA was extracted from each transfected Neuro2A cell, and the expression level of each LPA receptor was determined by quantitative real-time PCR. The expression levels of LPA₂ was normalized to those of Neuro2A cells transfected with non-target siRNA. The resulting data represent the mean ± SE values (***P<0.001 vs. the mock group). (D) The effects of LPA₂ knockdown on the neuroprotective effects of cPA and LPA against CoCl₂-induced apoptosis of Neuro2A cells. Neuro2A cells transfected with either siRNA against LPA₂ or non-target siRNA were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for 24 hours. Cells stained with FITC-Annexin V were subjected to flow cytometric analysis. The data represent the mean ± SE values from triplicate independent experiments (*P<0.05, ***P<0.001 vs. the CoCl₂-treated group; n.s., not significant).</p

Effects of cPA and LPA on the expression of the Bcl-2 protein family.

<p>(A) Neuro2A cells were incubated with 300 µM CoCl₂ in the presence of 10 µM cPA or LPA for the time indicated. Protein levels of Bax, Bcl-2, and β-actin were determined by western blot analysis. (B) The expression of Bax and Bcl-2 was determined using a densitometer, and the Bax/Bcl-2 ratio was calculated and normalized to cells without any treatment. The data represent the mean ± SE values from triplicate independent experiments (***P<0.001 vs. CoCl₂-treated group).</p