Ko Fujimori

Interferon-γ Interferes with Transforming Growth Factor-β Signaling through Direct Interaction of YB-1 with Smad3

Transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-γ on α2(I) procollagen gene (COL1A2) transcription through the IFN-γ response element located between –161 and –150. Here we report that YB-1 counter-represses TGF-β-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around –265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-γ-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-γ in the absence and presence of TGF-β. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-β and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-γ/YB-1 counteracts TGF-β/Smad3. They also indicate that IFN-γ/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-γ response element and through a cross-talk with the TGF-β/Smad signaling pathway.

Identification of mu-class glutathione transferases M2-2 and M3-3 as cytosolic prostaglandin E synthases in the human brain.

Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH2 to PGE2 at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH2 were 141 μM and 10.8 min−1 for GSTM2-2 and 1.5 mM and 130 min−1 for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE2 in the brain.

Identification of a Novel Prostaglandin F2α Synthase in Trypanosoma brucei

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Protective effects of quercetin against hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells

Hydrogen peroxide (H(2)O(2)) is a major reactive oxygen species that has been implicated in various neurodegenerative diseases. Quercetin, one of the plant flavonoids, has been reported to harbor various physiological properties including antioxidant activity. In this study, we investigated the neuroprotective effects of quercetin against H(2)O(2)-induced apoptosis in human neuronal SH-SY5Y cells. H(2)O(2)-mediated cytotoxicity and lactate dehydrogenase release were suppressed in a quercetin concentration-dependent manner. In addition, quercetin repressed the expression of the pro-apoptotic Bax gene and enhanced that of the anti-apoptotic Bcl-2 gene in SH-SY5Y cells. Moreover, quercetin effectively inhibited the activation of the caspase cascade that leads to DNA fragmentation, a key feature of apoptosis, and subsequent cell death. These results indicate the importance of quercetin in protecting against H(2)O(2)-mediated neuronal cell death. Thus, quercetin might potentially serve as an agent for prevention of neurodegenerative diseases caused by oxidative stress and apoptosis.

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2α Synthase

Prostaglandin (PG) F2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF2α suppressed adipocyte differentiation by acting through FP receptors.

NMR Solution Structure of Lipocalin-type Prostaglandin D Synthase EVIDENCE FOR PARTIAL OVERLAPPING OF CATALYTIC POCKET AND RETINOIC ACID-BINDING POCKET WITHIN THE CENTRAL CAVITY

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2, an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD2-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel β-barrel and a long α-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH2 analog, revealed that PGH2 almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH2 binding mode. These results indicated that the two binding sites for PGH2 and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.

A Novel Pathway to Enhance Adipocyte Differentiation of 3T3-L1 Cells by Up-regulation of Lipocalin-type Prostaglandin D Synthase Mediated by Liver X Receptor-activated Sterol Regulatory Element-binding Protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.

Pronounced adipogenesis and increased insulin sensitivity caused by overproduction of prostaglandin D2in vivo

Lipocalin‐type prostaglandin (PG) D synthase is expressed in adipose tissues and involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes. However, the physiological roles of PGD2 in adipogenesis in vivo are not clear, as lipocalin‐type prostaglandin D synthase can also act as a transporter for lipophilic molecules, such as retinoids. We generated transgenic (TG) mice overexpressing human hematopoietic PGDS (H‐PGDS) and investigated the in vivo functions of PGD2 in adipogenesis. PGD2 production in white adipose tissue of H‐PGDS TG mice was increased approximately seven‐fold as compared with that in wild‐type (WT) mice. With a high‐fat diet, H‐PGDS TG mice gained more body weight than WT mice. Serum leptin and insulin levels were increased in H‐PGDS TG mice, and the triglyceride level was decreased by about 50% as compared with WT mice. Furthermore, in the white adipose tissue of H‐PGDS TG mice, transcription levels of peroxisome proliferator‐activated receptor γ, fatty acid binding protein 4 and lipoprotein lipase were increased approximately two‐fold to five‐fold as compared with those of WT mice. Finally, H‐PGDS TG mice showed clear hypoglycemia after insulin clamp. These results indicate that TG mice overexpressing H‐PGDS abundantly produced PGD2 in adipose tissues, resulting in pronounced adipogenesis and increased insulin sensitivity. The present study provides the first evidence that PGD2 participates in the differentiation of adipocytes and in insulin sensitivity in vivo, and the H‐PGDS TG mice could constitute a novel model mouse for diabetes studies.

Drug transporters on arachnoid barrier cells contribute to the blood-cerebrospinal fluid barrier.

The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.

De novo synthesis, uptake and proteolytic processing of lipocalin-type prostaglandin D synthase, β-trace, in the kidneys

Lipocalin‐type prostaglandin D synthase (L‐PGDS) is a multifunctional protein that produces prostaglandin D2 and binds and transports various lipophilic substances after secretion into various body fluids as β‐trace. L‐PGDS has been proposed to be a useful diagnostic marker for renal injury associated with diabetes or hypertension, because the urinary and plasma concentrations are increased in patients with these diseases. However, it remains unclear whether urinary L‐PGDS is synthesized de novo in the kidney or taken up from the blood circulation. In crude extracts of monkey kidney and human urine, we found L‐PGDS with its original N‐terminal sequence starting from Ala23 after the signal sequence, and also its N‐terminal‐truncated products starting from Gln31 and Phe34. In situ hybridization and immunohistochemical staining with monoclonal antibody 5C11, which recognized the amino‐terminal Ala23–Val28 loop of L‐PGDS, revealed that both the mRNA and the intact form of L‐PGDS were localized in the cells of Henle’s loop and the glomeruli of the kidney, indicating that L‐PGDS is synthesized de novo in these tissues. However, truncated forms of L‐PGDS were found in the lysosomes of tubular cells, as visualized by immunostaining with 10A5, another monoclonal antibody, which recognized the three‐turn α‐helix between Arg156 and Thr173. These results suggest that L‐PGDS is taken up by tubular cells and actively degraded within their lysosomes to produce the N‐terminal‐truncated form.