| 1. |
- Ali, Zeina M F
(author)
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Side-chain oxidized oxysterols as metabolic regulators in liver and brain
- 2014
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Licentiate thesis (other academic/artistic)abstract
- Oxysterols are oxygenated derivatives of cholesterol characterized by a very short half-life and their ability to pass lipophilic membranes easily, thus they are considered as important intermediates in cholesterol excretion pathways and its conversion to bile acids. The two major oxysterols in the circulation of human and mouse are 24S-hydroxycholesterol (24S-OH) and 27-hydroxycholesterol (27-OH), which are formed by the cytochrome P450 enzymes cholesterol 24-hydroxylase (CYP46A1) and sterol 27-hydroxylase (CYP27A1), respectively. The two oxysterols 27-OH and 24S-OH are both strong inhibitors of cholesterol synthesis and activators of LXR in vitro. However, their role as physio- logical regulators under in vivo conditions is controversial. The overall aim of this thesis was to investigate the regulatory role of side chain oxidized oxysterols as metabolic regulators in vivo. In particular we have studied the role of 24S-and 27-hydroxycholesterols (24S- and 27-OH) as regulators of cholesterol synthesis and activators of LXR. We used mouse models with increased levels of 27-OH (CYP27A1) transgenic mice and Cyp7b1 knock-out mice (Cyp7b1-/-) as well as a mouse model with no detectable levels of 27-OH in their circulation, Cyp27a1 knock-out mice (Cyp 27-/-). The latter mice were treated with cholic acid to compensate for the reduced formation of bile acids. In Paper I, we studied a possible regulatory role of 27-OH and 24S-OH in the brain using human CYP27A1 transgenic mice(CYP27A1tg) and Cyp27a1 knock-out (Cyp 27a1-/-) mice. The levels of 27-OH were increased about 12-fold in the brain of CYP27A1tg mice while the levels of 24S-OH was decreased by about 25%, most probably due to increased metabolism by the CYP27A1 enzyme. The mRNA levels of HMG-CoA reductase and HMG-CoA synthase in the brain were increased. In accordance with increased cholesterol synthesis, most of cholesterol precursors were also increased. The increased cholesterol synthesis is likely due to reduced inhibition by 24S-OH. 27-OH is an activator of LXR and in spite of this, there was no upregulation of the LXR-target genes in the brain of the transgenic mice. In contrast, some of the genes were downregulated. In Cyp27a1-/- mouse brain, cholesterol synthesis was slightly increased with increased levels of cholesterol precursors. The increased synthesis is probably the consequence of the absence of an inhibitory effect of the flux of 27-OH into the brain. The results of this study are consistent with the possibility that both 24S-OH and 27-OH have a suppressive effect on cholesterol synthesis in the brain. Since there was no activation of the LXR-target genes in the brain of the transgenic mice, we concluded that 27-OH is not a general activator of LXR in the brain. In Paper II, this study has examined the role of 27-OH in the liver using the above three mouse models. In the liver of CYP27A1tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1, and Abca1. There was no effect on a number of other LXR-regulated genes. There were no significant effects on cholesterol synthesis at the transcriptional level and cholesterol precursors were not affected as well. However, there was a modest decrease in T-MAS levels in the liver of CYP27A1tg mice. In the liver of the Cyp7b1-/-mice, there were also no effects on cholesterol synthesis neither at the transcriptional level nor in the levels of cholesterol precursors, with the exception of increase in desmosterol. In connection to the LXR-target genes in these mice, there were no differences in the expression between the Cyp7b1-/- and the wild type mice. If the high levels of 27-OH are important, the same effects would be expected in the two mouse models. In the liver of the Cyp27a1-/- mice there was a slight activation of some LXR- regulated genes, Abcg5, Abcg8, Fas and Srebp1c. If 27-OH is of importance as a normal activator of the above genes a suppressing effect would be expected. The overall results do not support the contention that 27-OH is an important regulator of cholesterol homeostasis or an activator of LXR-regulated genes under basal conditions in the liver. In conclusion our results suggest that both 24S-OH and 27-OH may be of some regulatory importance for cholesterol synthesis in the brain but not in the liver. Under normal basal conditions 27-OH does not seem to be a general activator of LXR neither in the brain nor in the liver. The different effects on cholesterol synthesis in the two organs may be related to the fact that almost all oxysterols in the brain are in the free form whereas most of them are esterified in the liver.
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| 2. |
- Ali, Zeina M F
(author)
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Side-chain oxidized oxysterols as metabolic regulators in vivo
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Oxysterols are oxygenated derivatives of cholesterol characterized by a very short half- life and their ability to pass lipophilic membranes easily, and they are considered as important intermediates in the excretion pathways of cholesterol and its conversion to bile acids. Evidence has been presented that the production and flux of oxysterols in the brain may also be of some importance for cognitive functions. The two major oxysterols in the circulation of human and mouse are 24S-hydroxycholesterol (24S- OH) and 27-hydroxycholesterol (27-OH), which are formed by the cytochrome P450 enzymes cholesterol 24-hydroxylase (CYP46A1) and sterol 27-hydroxylase (CYP27A1), respectively. The two oxysterols 27-OH and 24S-OH are both strong inhibitors of cholesterol synthesis and activators of LXR in vitro. However, their role as physiological regulators under in vivo conditions is controversial. The overall aim of this thesis was to investigate the regulatory role of side-chain oxidized oxysterols as metabolic regulators in vivo. In particular, we have studied the role of 24S-OH and 27-OH as regulators of cholesterol synthesis and activators of LXR. We used mouse models with increased levels of 27-OH like human CYP27A1 overexpressor mice (CYP27A1tg) and Cyp7b1 knockout mice (Cyp7b1-/-), as well as a mouse model with no detectable levels of 27-OH in the circulation, Cyp27a1 knockout mice (Cyp 27a1-/-). The latter mice were treated with cholic acid to compensate for the reduced formation of bile acids. Using Cyp27a1-/- mice, we have also studied the possibility that 27-OH mediates the negative effects of dietary cholesterol on memory function in mice. In Paper I, we studied a possible regulatory role of 27-OH and 24S-OH in the brain, using CYP27A1tg mice and Cyp27a1-/- mice. The levels of 27-OH were increased by approximately 12-fold in the brain of CYP27A1tg mice while levels of 24S-OH were decreased by about 25%, most probably due to increased metabolism by the CYP27A1 enzyme. Evidence was presented that cholesterol synthesis was increased in the brain of the two mouse models. There was no upregulation of the LXR-target genes in the brain of either of the two models. The increased synthesis in the brain of the Cyp27a1-/- mice is probably the consequence of the absence of an inhibitory effect of the flux of 27-OH into the brain. The increased cholesterol synthesis in the brain of the CYP27A1tg mice is probably due to the reduced levels of 24S-OH, leading to reduced inhibition of its synthesis. The results of this study are consistent with the possibility that both 24S-OH and 27-OH have a suppressive effect on cholesterol synthesis in the brain. We also conclude that 27-OH is not a general activator of LXR in this tissue. In Paper II, we examined the role of 27-OH in the liver using the three mouse models described above. Only very modest effects on cholesterol synthesis and LXR target genes were observed in the three mouse models. The overall results do not support the contention either that 27-OH is an important regulator of cholesterol homeostasis, or that 27-OH is an activator of LXR-regulated genes under basal conditions in the liver. It has been reported that treatment of mice with dietary cholesterol leads to upregulation of some LXR target genes in the liver. In a study by Chen et al, such upregulation of three different LXR target genes was not seen when treating mice lacking 24S-OH, 25-OH and 27-OH with dietary cholesterol (Chen et al., 2007). It was concluded that a 24-, 25- or 27-hydroxylation step mediated the effect of dietary cholesterol. We show here that similar effects were obtained when treating Cyp27a1-/- mice with dietary cholesterol, suggesting that the effects are mediated by 27-OH. Most of these effects could however not be observed at either the protein level, or at the level of enzyme activity. The results suggest that 27-OH is a mediator of the cholesterol-induced effects on some LXR target genes when the mice are challenged with a high load of dietary cholesterol. The physiological importance of this effect is difficult to evaluate. In Paper III we compared the two mouse models CYP27A1tg and Cyp7b1-/- mice, both of which have high levels of 27-OH in the circulation and the brain. In contrast to CYP27A1tg mice, the levels of 24S-OH in the brain were not decreased in the latter model, and cholesterol synthesis was not affected. This supports the proposal that 24S- OH is of regulatory importance for cholesterol synthesis in the brain. It has been reported that cholesterol synthesis is reduced in the kidney of Cyp7b1-/- mice. Cholesterol synthesis in the kidney of CYP27A1tg mice was not affected, however. We conclude that factors other than high levels of 27-OH are of importance for the reduced cholesterol synthesis in the kidney of the Cyp7b1-/- mice. In Paper IV we tested the possibility that the negative effect of dietary cholesterol on cognition in mice is mediated by 27-OH. The negative effect of dietary cholesterol on spatial memory observed in wild-type mice was not observed in Cyp27a1-/- mice. The latter mice were also treated with cholic acid to compensate for the reduced synthesis of bile acids. Treatment with dietary cholesterol was shown to lead to reduced levels of the “memory protein” Arc (Activity-Regulated Cytoskeleton-associated protein) in the hippocampus of the wildtype mice. This effect was not seen in the hippocampus of Cyp27a1-/- mice. The results are consistent with the possibility that 27-OH is a mediator of the negative effects of dietary cholesterol on cognition. In conclusion, our results suggest that both 24S-OH and 27-OH may be of some regulatory importance for cholesterol synthesis in the brain but not in the liver. Under normal basal conditions 27-OH does not seem to be a general activator of LXR, either in the brain or in the liver. We discuss the possibility that the different effects on cholesterol synthesis in the two organs may be related to the fact that almost all oxysterols in the brain are in the free form, whereas in the liver, most of them are esterified. In addition, we demonstrate that the negative effects of dietary cholesterol on cognition is mediated by 27-hydroxylation.
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| 3. |
- Heverin, Maura, et al.
(author)
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On the regulatory importance of 27-hydroxycholesterol in mouse liver
- 2017
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In: Journal of Steroid Biochemistry and Molecular Biology. - : Elsevier. - 0960-0760 .- 1879-1220. ; 169, s. 10-21
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Research review (peer-reviewed)abstract
- 27-Hydroxycholesterol (27OH) is a strong suppressor of cholesterol synthesis and a weak activator of LXR in vitro. The regulatory importance of 27OH in vivo is controversial. Here we utilized male mice with increased levels of 27OH either due to increased production (CYP27A1 transgenic mice) or reduced metabolism (Cyp7b1-/- mice). We also used mice lacking 27OH due to a knockout of Cyp27a1. The latter mice were treated with cholic acid to compensate for reduced bile acid synthesis. The effects of the different levels of 27OH on Srebp- and other LXR-regulated genes in the liver were investigated. In the liver of CYP27tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1 and Abca1. A number of other LXR-regulated genes were not affected. The effect on Abca1 mRNA was not seen in the liver of Cyp7b1-/- mice. There were little or no effects on cholesterol synthesis. In the liver of the Cyp27-/- mice treated with 0.025% cholic acid there was no significant effect of the knockout on the LXR target genes. In a previous work triple-knockout mice deficient in the biosynthesis of 24S-hydroxycholesterol, 25-hydroxycholesterol and 27OH were shown to have impaired response to dietary cholesterol, suggesting side-chain oxidized oxysterols to be mediators in cholesterol-induced effects on LXR target genes at a transcriptional level (Chen W. et al., Cell Metab. 5 (2007) 73-79). The hydroxylated oxysterol responsible for the effect was not defined. We show here that treatment of wildtype mice with dietary cholesterol under the same conditions as in the above study induced the LXR target genes Lpl, Abcg8 and Srebp1c in wild type mice but failed to activate the same genes in mice lacking 27-hydroxycholesterol due to a knockout of Cyp27. We failed to demonstrate the above effects at the protein level (Abcg8) or at the activity level (Lpl). The results suggest that 27OH is not an important regulator of Srebp- or LXR regulated genes under basal conditions in mouse liver. On the other hand 27OH appears to mediate cholesterol-induced effects on some LXR target genes at a transcriptional level under some in vivo conditions.
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| 4. |
- Hleihel, Rita, et al.
(author)
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The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1)
- 2018
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In: Retrovirology. - : BMC. - 1742-4690. ; 15
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Journal article (peer-reviewed)abstract
- Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic human T-cell lymphotropic virus 1 infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling.Results: Adding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm and major increase in the transcription of the NF-ĸB targets Rantes and interleukin-6. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-κB in flies.Conclusions: These data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL.
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