Vasohibin induces prolyl hydroxylase-mediated degradation of hypoxia-inducible factor-1α in human umbilical vein endothelial cells.

Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF-1α expression under oxidative stress induced by hydrogen peroxide (H2O2) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H2O2-pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF-1α expression during H2O2-pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF-1α degradation via PHD.

Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity.

Drug induced allergies are believed to be induced by conjugates consisting of biological macromolecules and active metabolites. The present study investigated whether guinea pig glutathione S-transferase (gpGST), a protein that binds with sulfanilamide (SA) and sulfamethoxazole (SMX), could be detected in the liver cytosol fraction of guinea pigs that intraperitoneally received SA or SMX, and whether gpGST is a carrier protein. We synthesized three nitroso compounds, i.e., 4-nitroso-sulfanilamide (SA-NO), 4-nitrososulfamethoxazole (SMX-NO) and fluorescent-labeled nitroso compound (DNSBA-NO), and examined binding quantities of nitroso compounds to gpGST purified from untreated female guinea pigs. Furthermore, the concentrations of IgG in serum antibody for nitroso compounds were estimated using ELISA. When guinea pigs were sensitized using the three nitroso compounds, the dose dependent skin reactions were confirmed with each compound. In addition, sensitized guinea pigs using each nitroso compound showed positive skin reactions at an elicitation test performed using gpGST alone. The results confirmed synthesis of antibody against gpGST due to hapten sensitization. Therefore, when a nitroso compound binds with gpGST in the body of guinea pigs, nitroso-gpGST acts as a neoantigen, which induces synthesis of autoantibody. Thus, gpGST appears to be one of the carrier proteins that induce sulfa drug-induced allergies. Immunization of guinea pigs with active metabolite of drugs may give information for predicting the occurrence of delayed type hypersensitivity in human.

Proxisome proliferator-activated receptor-α mediates high-fat, diet-enhanced daily oscillation of plasminogen activator inhibitor-1 activity in mice.

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitation showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCK/BMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARα/retinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.

Bezafibrate induces plasminogen activator inhibitor-1 gene expression in a CLOCK-dependent circadian manner.

A functional interaction between peroxisome proliferator-activated receptor alpha (PPARalpha) and components of the circadian clock has been suggested, but whether these transcriptional factors interact to regulate the expression of their target genes remains obscure. Here we used a PPARalpha ligand, bezafibrate, to search for PPARalpha-regulated genes that are expressed in a CLOCK-dependent circadian manner. Microarray analyses using hepatic RNA isolated from bezafibrate treated-wild type, Clock mutant (Clk/Clk), and PPARalpha-null mice revealed that 136 genes are transcriptionally regulated by PPARalpha in a CLOCK-dependent manner. Among them, we focused on the plasminogen activator inhibitor-1 (PAI-1) gene, because its expression typically shows circadian variation, and it has transcriptional response elements for both PPAR and CLOCK. The bezafibrate-induced expression of PAI-1 mRNA was attenuated in Clk/Clk mice and in PPARalpha-null mice. The protein levels of PPARalpha were reduced in Clk/Clk hepatocytes. However, the overexpression of PPARalpha could not rescue bezafibrate-induced PAI-1 expression in Clk/Clk hepatocytes, suggesting that impaired bezafibrate-induced PAI-1 expression in Clk/Clk mice is not due to reduced PPARalpha expression. Luciferase reporter and chromatin immunoprecipitation analyses using primary hepatocytes demonstrated that DNA binding of both PPARalpha and CLOCK is essential for bezafibrate-induced PAI-1 gene expression. Pull-down assays in vitro showed that both PPARalpha and its heterodimerized partner retinoic acid receptor-alpha can serve as potential interaction targets of CLOCK. The present findings revealed that molecular interaction between the circadian clock and the lipid metabolism regulator affects the bezafibrate-induced gene expression.

Fasting promotes the expression of SIRT1, an NAD+ -dependent protein deacetylase, via activation of PPARalpha in mice.

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD(+)/NADH ratio in cells and results in activation of SIRT1, an NAD(+)-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARalpha, PPARgamma, and PPARbeta/delta, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARgamma by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARalpha correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARalpha in the livers, where the NAD(+) levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD(+) salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD(+) or NADH showed that the mRNA expression of both SIRT1 and PPARalpha can be enhanced by addition of NAD(+), and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARalpha agonist suggested that PPARalpha is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.

Biochemistry and neurobiology of prosaposin: a potential therapeutic neuro-effector.

Prosaposin, a 66 kDa glycoprotein, was identified initially as the precursor of the sphingolipid activator proteins, saposins A-D, which are required for the enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. While mature saposins are distributed to lysosomes, prosaposin exists in secretory body fluids and plasma membranes. In addition to its role as the precursor, prosaposin shows a variety of neurotrophic and myelinotrophic activities through a receptor-mediated mechanism. In studies in vivo, prosaposin was demonstrated to exert a variety of neuro-efficacies capable of preventing neuro-degeneration following neuro-injury and promoting the amelioration of allodynia and hyperalgesia in pain models. Collective findings indicate that prosaposin is not a simple house-keeping precursor protein; instead, it is a protein essentially required for the development and maintenance of the central and peripheral nervous systems. Accumulating evidence over the last decade has attracted interests in exploring and developing new therapeutic approaches using prosaposin for human disorders associated with neuro-degeneration. In this review we detail the structure characteristics, cell biological feature, in vivo efficacy, and neuro-therapeutic potential of prosaposin, thereby providing future prospective in clinical application of this multifunctional protein.

Modulation of circadian rhythm of DNA synthesis in tumor cells by inhibiting platelet-derived growth factor signaling.

Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 microg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: its regulation of dimeric transcription factor formation.

Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of prosaposin on the oxidative stress-induced cell death. Furthermore, prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.

Anti-apoptotic roles of plasminogen activator inhibitor-1 as a neurotrophic factor in the central nervous system.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin gene family, is the primary inhibitor of urokinase-type and tissue-type PAs. PAI-1 plays an important role in the process of peripheral tissue remodeling and fibrinolysis through the regulation of PA activity. This serpin is also produced in brain tissues and may regulate the neural protease sequence in the central nervous system (CNS), as it does in peripheral tissues. In fact, PAI-1 mRNA is up-regulated in mouse brain after stroke. The serpin activity of PAI-1 helps to prevent tissue-type PA-induced neuron death. However, we have previously found that PAI-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. In neuronally differentiated rat pheochromocytoma (PC-12) cells, a deficiency of PAI-1 in vitro caused a significant reduction in Bcl-2 and Bcl-X(L) mRNAs and an increase in Bcl-X(S) and Bax mRNAs. The change in the balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins promoted the apoptotic sequence: caspase-3 activation, cytochrome c release from mitochondria and DNA fragmentation. Our results indicate that PAI-1 has an anti-apoptotic role in neurons. PAI-1 prevented the disintegration of the formed neuronal networks by maintaining or promoting neuroprotective signaling through the MAPK/ERK pathway, suggesting that the neuroprotective effect of PAI-1 is independent of its action as a protease inhibitor. This review discusses the neuroprotective effects of PAI-1 in vitro, together with the relevant data from other laboratories. Special emphasis is placed on its action on PC-12 cells.

Glucocorticoid regulation of 24-hour oscillation in interferon receptor gene expression in mouse liver.

Although the antiviral effect of interferon (IFN) varies depending on 24-h oscillation in the expression of its specific receptor, the mechanism of oscillation remains to be clarified. Here we report that oscillation in the expression of the IFN receptor gene (IFN-alpha/beta R1) in mouse liver is caused by the endogenous rhythm of glucocorticoid secretion. Brief exposure of mouse hepatic cells (Hepa 1-6) to corticosterone (CORT) resulted in a significant decrease in mRNA levels of IFN-alpha/beta R1. The CORT-induced decrease in IFN-alpha/beta R1 mRNA levels was reversed by pretreating the cells with RU486, a glucocorticoid receptor antagonist. The mRNA levels of IFN-alpha/beta R1 gene in the liver of adrenalectomized mice were consistently increased throughout the day. However, a single administration of CORT to adrenalectomized mice significantly decreased the mRNA levels of IFN-alpha/beta R1 in the liver. Furthermore, the rhythmic phase of IFN-alpha/beta R1 expression was modulated after the alteration of rhythmicity in glucocorticoid secretion, which was induced by restricted daily feeding. As a consequence, under manipulation of the feeding schedule, 2'-5' oligoadenylate synthase activities, as an index of antiviral effect, in plasma and liver at 24 h after IFN-alpha injection also varied depending on the alteration of glucocorticoid secretion rhythm. These results suggest that the endogenous rhythm of glucocorticoid secretion is involved in the circadian regulation of IFN-alpha/beta R1 expression in mouse liver. Our findings also support the notion that monitoring the 24-h variation in IFN receptor function is useful for selecting the most appropriate time of day to administer IFN.

Circadian regulation of mouse topoisomerase I gene expression by glucocorticoid hormones.

Because glucocorticoid hormones modulate various biological processes, the endogenous rhythm of their secretion is thought to be an important factor affecting the efficacy and/or toxicity of many drugs. Topoisomerase I (Topo I) is a nuclear target of the anticancer drug camptothecin (CPT). In this study, we demonstrate that Topo I expression in tumor-bearing mice and the efficacy of CPT on the tumor are affected by the 24-h variation in circulating glucocorticoid levels. A single administration of corticosterone (CORT) to the tumor-bearing mice resulted in a significant increase in Topo I mRNA levels not only in the tumor masses but also in other healthy tissues such as liver and skeletal muscle. The CORT-induced increase in Topo I mRNA was suppressed by pretreating the mice with RU486, a glucocorticoid receptor antagonist. Significant 24-h oscillations in the Topo I mRNA levels were observed in the tumor and healthy liver without exogenous CORT, and were eliminated by adrenalectomy of the mice. This result suggests that endogenous glucocorticoid hormones are involved in the circadian regulation of Topo I gene expression. Furthermore, the anti-tumor efficacy of the Topo I inhibitor CPT-11 on the tumor-bearing mice was enhanced by administering the drug at the time when the Topo I activity was increased. Our present results demonstrate that glucocorticoid is involved in the 24-h oscillation mechanism of Topo I gene expression and suggest that monitoring the circadian rhythm in Topo I activity is useful for choosing the most appropriate time of day to administer of Topo I inhibitors.

Chronic treatment with prednisolone represses the circadian oscillation of clock gene expression in mouse peripheral tissues.

Although altered homeostatic regulation, including disturbance of 24-h rhythms, is often observed in the patients undergoing glucocorticoid therapy, the mechanisms underlying the disturbance remains poorly understood. We report here that chronic treatment with a synthetic glucocorticoid, prednisolone (PSL), can cause alteration of circadian clock function at molecular level. Treatment of cultured hepatic cells (HepG2) with PSL induced expression of Period1 (Per1), and the PSL treatment also attenuated the serum-induced oscillations in the expression of Period2 (Per2), Rev-erbalpha, and Bmal1 mRNA in HepG2 cells. Because the attenuation of clock gene oscillations was blocked by pretreating the cells with a Per1 antisense phosphothioate oligodeoxynucleotide, the extensive expression of Per1 induced by PSL may have resulted in the reduced amplitude of other clock gene oscillations. Continuous administration of PSL into mice constitutively increased the Per1 mRNA levels in liver and skeletal muscle, which seems to attenuate the oscillation in the expressions of Per2, Rev-erbalpha, and Bmal1. However, a single daily administration of PSL at the time of day corresponding to acrophase of endogenous glucocorticoid levels had little effect on the rhythmic expression of clock genes. These results suggest a possible pharmacological action by PSL on the core circadian oscillation mechanism and indicate the possibility that the alteration of clock function induced by PSL can be avoided by optimizing the dosing schedule.

24-hour oscillation of mouse methionine aminopeptidase2, a regulator of tumor progression, is regulated by clock gene proteins.

Methionine aminopeptidase2 (MetAP2) plays an important role in the growth of endothelial cells during the tumor angiogenesis stage. Recently, we have clarified that mouse methionine aminopeptidases (mMetAPs) show a 24-hour rhythm in implanted tumor masses. In the present study, we investigated the mechanism underlying the 24-hour rhythm of mMetAP2 activity in tumor-bearing mice under a light-dark (lights on from 7 a.m. to 7 p.m.) cycle. The 5' flanking region of mMetAP2 included eight E-boxes. The transcription of the mMetAP2 promoter was enhanced by the mCLOCK:mBMAL1 heterodimer, and its activation was inhibited by mPER2 or mCRY1. Deletion and mutation of the E-boxes in the region indicated that the E-box nearest to the initiation start site played an important role in the transcriptional regulation by clock genes. In sarcoma180-bearing mice, the pattern of binding of mCLOCK and mBMAL1 to the E-box and transcription of the mMetAP2 promoter showed a 24-hour rhythm with higher levels from the mid-light to early dark phase. The pattern of mMetAP2 transcription was closely associated with that of mMetAP2 mRNA expression in three types of tumor-bearing mice. mMetAP2 protein expression varied with higher levels from the late-dark to early light phase. The rhythmicity of the protein expression was synchronous with that of the activity of mMetAPs but out of phase with that of the mMetAP2 mRNA expression. These results suggest that the 24-hour rhythm of mMetAP2 activity is regulated by the transcription of clock genes within the clock feedback loops.

A molecular mechanism regulating circadian expression of vascular endothelial growth factor in tumor cells.

Because angiogenesis is essential for tumor growth and metastasis, inhibition of angiogenesis has emerged as a new therapy to treat cancers. Hypoxia-induced expression of vascular endothelial growth factor (VEGF) plays a central role in tumor-induced angiogenesis. In this study, we found that expression of VEGF in hypoxic tumor cells was affected by the circadian organization of molecular clockwork. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop in which CLOCK and BMAL1 are positive regulators, and Period and Cryptochrome genes act as negative ones. The levels of VEGF mRNA in tumor cells implanted in mice rose substantially in response to hypoxia, but the levels fluctuated rhythmically in a circadian fashion. Luciferase reporter gene analysis revealed that Period2 and Cryptochrome1, whose expression in the implanted tumor cells showed a circadian oscillation, inhibited the hypoxia-induced VEGF promoter activity. These results suggest that the negative limbs of the molecular loop periodically inhibit the hypoxic induction of VEGF transcription, resulting in the circadian fluctuation of its mRNA expression. We also showed that the antitumor efficacy of antiangiogenic agents could be enhanced by administering the drugs at the time when VEGF production increased. These findings support the notion that monitoring of the circadian rhythm in VEGF production is useful for choosing the most appropriate time of day for administration of antiangiogenic agents.

Optimizing the dosing schedule of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol] enhances its antitumor and antiangiogenic efficacies.

Many drugs vary in potency and/or toxicity according to the time of day when they are administered. In this study, we investigated whether antitumor efficacy of angiogenesis inhibitor, TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], could be improved by optimizing the dosing schedule. Tumor-bearing mice were housed under standardized light/dark cycle conditions (lights on at 7:00 AM, off at 7:00 PM) with food and water ad libitum. The antitumor effect of TNP-470 (30 mg/kg s.c.) was more potent in mice injected with the drug at the early light phase than it was when administered at the early dark phase. The diurnal change in the antitumor effect of TNP-470 was parallel to that in its antiangiogenic activity. The variation in the effects of TNP-470 was closely related to the diurnal variations in its inhibitory action on methionine aminopeptidase activity in tumor masses. There was a significant dosing time-dependent change in the concentration of TNP-470 in plasma. The higher concentration of TNP-470 in plasma was observed when its antitumor and antiangiogenic activities were increased. These results suggest that therapeutic efficacy of TNP-470 can be enhanced by choosing the most appropriate time of day to administer the drug.