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

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that ​Rad54B—a SNF2 helicase-like DNA-repair protein—limits the strength of both the G1/S and G2/M checkpoints. We find that ​Rad54B functions as a scaffold for ​p53 degradation via its direct interaction with the ​MDM2–​MDMX ubiquitin–ligase complex. During the early phases of the DDR, ​Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the ​p53-mediated checkpoint is established, ​Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of ​Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of ​Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.UTokyo Research掲載「がん発生の基盤となる仕組みを探る」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/discovery-of-a-basic-mechanism-of-cancer-development/UTokyo Research "Discovery of a basic mechanism of cancer development" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/discovery-of-a-basic-mechanism-of-cancer-development

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs

Current Overview of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI), or brittle bone disease, is a heterogeneous disorder characterised by bone fragility, multiple fractures, bone deformity, and short stature. OI is a heterogeneous disorder primarily caused by mutations in the genes involved in the production of type 1 collagen. Severe OI is perinatally lethal, while mild OI can sometimes not be recognised until adulthood. Severe or lethal OI can usually be diagnosed using antenatal ultrasound and confirmed by various imaging modalities and genetic testing. The combination of imaging parameters obtained by ultrasound, computed tomography (CT), and magnetic resource imaging (MRI) can not only detect OI accurately but also predict lethality before birth. Moreover, genetic testing, either noninvasive or invasive, can further confirm the diagnosis prenatally. Early and precise diagnoses provide parents with more time to decide on reproductive options. The currently available postnatal treatments for OI are not curative, and individuals with severe OI suffer multiple fractures and bone deformities throughout their lives. In utero mesenchymal stem cell transplantation has been drawing attention as a promising therapy for severe OI, and a clinical trial to assess the safety and efficacy of cell therapy is currently ongoing. In the future, early diagnosis followed by in utero stem cell transplantation should be adopted as a new therapeutic option for severe OI

Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks

Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4−/− compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs

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)

Haploinsufficiency of the Mus81.Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells

The Mus81.Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 orEme1 also causes similar phenotypes.These findings suggest that a complex network of the checkpoint pathways that respond to DNA doublestrand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency