Sterol dependent regulation of human TM7SF2 gene expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis

3Beta-hydroxysterol Delta(14)-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3beta-hydroxysterol Delta(14)-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3beta-hydroxysterol Delta(14)-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3beta-hydroxysterol Delta(14)-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Delta(14)-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3beta-hydroxysterol Delta(14)-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3beta-hydroxysterol Delta(14)-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.     

Mutations in the 3beta-hydroxysterol Delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis

Desmosterolosis is a rare autosomal recessive disorder characterized by multiple congenital anomalies. Patients with desmosterolosis have elevated levels of the cholesterol precursor desmosterol, in plasma, tissue, and cultured cells; this abnormality suggests a deficiency of the enzyme 3beta-hydroxysterol Delta24-reductase (DHCR24), which, in cholesterol biosynthesis, catalyzes the reduction of the Delta24 double bond of sterol intermediates. We identified the human DHCR24 cDNA, by the similarity between the encoded protein and a recently characterized plant enzyme--DWF1/DIM, from Arabidopsis thaliana--catalyzing a different but partially similar reaction in steroid/sterol biosynthesis in plants. Heterologous expression, in the yeast Saccharomyces cerevisiae, of the DHCR24 cDNA, followed by enzyme-activity measurements, confirmed that it encodes DHCR24. The encoded DHCR24 protein has a calculated molecular weight of 60.1 kD, contains a potential N-terminal secretory-signal sequence as well as at least one putative transmembrane helix, and is a member of a recently defined family of flavin adenine dinucleotide (FAD)-dependent oxidoreductases. Conversion of desmosterol to cholesterol by DHCR24 in vitro is strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and is increased twofold by the addition of FAD to the assay. The corresponding gene, DHCR24, was identified by database searching, spans approximately 46.4 kb, is localized to chromosome 1p31.1-p33, and comprises nine exons and eight introns. Sequence analysis of DHCR24 in two patients with desmosterolosis revealed four different missense mutations, which were shown, by functional expression, in yeast, of the patient alleles, to be disease causing. Our data demonstrate that desmosterolosis is a cholesterol-biosynthesis disorder caused by mutations in DHCR24.     

Disruption of the oxysterol 7alpha-hydroxylase gene in mice

Mice without oxysterol 7alpha-hydroxylase, an enzyme of the alternate bile acid synthesis pathway with a sexually dimorphic expression pattern, were constructed by the introduction of a null mutation at the Cyp7b1 locus. Animals heterozygous (Cyp7b1(+/-)) and homozygous (Cyp7b1(-/-)) for this mutation were grossly indistinguishable from wild-type mice. Plasma and tissue levels of 25- and 27-hydroxycholesterol, two oxysterol substrates of this enzyme with potent regulatory actions in cultured cells, were markedly elevated in Cyp7b1(-/-) knockout animals. Parameters of bile acid metabolism as well as plasma cholesterol and triglyceride levels in male and female Cyp7b1(-/-) mice were normal. The cholesterol contents of major tissues were not altered. In vivo sterol biosynthetic rates were unaffected in multiple tissues with the exception of the male kidney, which showed a approximately 40% decrease in de novo synthesis versus controls. We conclude that the major physiological role of the CYP7B1 oxysterol 7alpha-hydroxylase is to metabolize 25- and 27-hydroxycholesterol and that loss of this enzyme in the liver is compensated for by increases in the synthesis of bile acids by other pathways. A failure to catabolize oxysterols in the male kidney may lead to a decrease in de novo sterol synthesis.     

PanR1, a dominant negative missense allele of the gene encoding TNF-alpha (Tnf), does not impair lymphoid development

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G(1) mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C-->A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer's patches, suggesting that TNF is not essential for the formation of these lymphoid structures.     

Structure of the rat gene encoding cholesterol 7 alpha-hydroxylase

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P-450 that catalyzes the first and rate-limiting step in bile acid biosynthesis, the major catabolic pathway in cholesterol homeostasis. The gene encoding the rat 7 alpha-hydroxylase has been isolated and characterized. Southern blotting experiments demonstrated that the gene is present in a single copy in the rat genome. DNA sequence analysis showed that the 7 alpha-hydroxylase gene is unique among the characterized cytochrome P-450s in that it contains only six exons. Nuclease S1 and primer-extension mapping experiments positioned the 5'-ends of the 7 alpha-hydroxylase mRNA approximately 20-25 nucleotides downstream of a consensus TATAAA sequence. RNA blotting experiments demonstrated the presence of multiple 7 alpha-hydroxylase mRNAs that differ in the lengths of their 3'-untranslated regions.     

Anosmia does not impair the anxiolytic-like effect of lavender essential oil inhalation in mice

AimThe inhalation of Lavandula angustifolia (lavender) essential oil has anxiolytic-like effects in animal models and humans, but its mechanism of action is still not fully understood. The inhalation of essential oils can induce anxiolytic effects through the central nervous system (e.g., lung absorption and bloodstream transport) or stimulation of the olfactory system and secondary activation of brain regions. Thus, the main objective of the present study was to evaluate whether the perception of lavender essential oil aroma, when inhaled, is necessary to obtain its anxiolytic-like effects in mice tested in the marble-burying test.Main methodsAnosmia was induced by irrigating the nasal cavity with zinc gluconate + zinc acetate so that the mice could not detect odors in the olfactory discrimination test. The marble-burying test was used to evaluate the anxiolytic-like effects of inhaled lavender essential oil.Key findingsAnosmia did not interfere with the anxiolytic-like effect of lavender essential oil inhalation in the marble-burying test at concentrations of 2.5% (number of marbles buried: vehicle, 4.7 ± 1.0; zinc, 6.2 ± 2.2; p > 0.10) and 5% (number of marbles buried: vehicle, 3.4 ± 0.8; zinc, 4.3 ± 0.9; p > 0.10). Lavender essential oil at a concentration of 0.5% was ineffective.SignificanceThese results suggest that olfactory system activation is unlikely to participate in the anxiolytic-like effect of lavender essential oil inhalation.     

Epigallocatechin gallate (EGCG) (TEAVIGO) does not impair nonhaem-iron absorption in man.

A number of studies have shown that tea catechins can inhibit intestinal iron absorption, mostly iron in the nonhaem form. This randomized, double-blind, placebo-controlled, 3-periods cross-over study examined the degree of inhibition of nonhaem iron absorption by pure crystalline epigallocatechin gallate (EGCG). The study was designed to show the maximum inhibitory action of EGCG by selecting 30 healthy women with low iron stores. Treatments were 150 mg, 300 mg EGCG and placebo each for 8 consecutive study days with a wash-out period of 14 days between treatments. Iron incorporation was assessed by supplying 57Fe orally and 58Fe intravenously. Differences in fractional nonhaem iron absorption between the treatments were evaluated by using two-sided ANOVA. Results showed a relative nonhaem iron absorption reduction of 14% with 150mg EGCG and 27% for 300mg EGCG treatment compared to placebo. Differences were statistically significant (p < or = 0.05) between the placebo and the 300mg EGCG treatments and between the 150 and 300 mg EGCG treatments. The inverse relation between EGCG dose and fractional nonhaem iron absorption was linear (p = 0.0002). In this study the magnitude of the inhibitory action of EGCG on nonhaem iron absorption was found to be much lower than that reported in the literature for black tea and similar compounds. The doses of EGCG in supplements, which will be lower than those used in this study, are not expected to have any health relevant effects on iron absorption in subjects with normal iron stores.     

Disruption of the SCS2 ortholog in the alkane-assimilating yeast Yarrowia lipolytica impairs its growth on n-decane, but does not impair inositol prototrophy

Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomyces cerevisiae counterpart.     

Absence of UCP3 in brown adipose tissue does not impair nonshivering thermogenesis

We report on a novel Djungarian hamster mutant lineage that exhibits a loss of uncoupling protein (UCP) 3 mRNA and protein in brown adipose tissue (BAT), whereas UCP3 expression in skeletal muscle is only mildly diminished. In response to 2 d of cold exposure, UCP3 mRNA was 4.5-fold elevated in BAT of wild-type hamsters but remained undetectable in mutant hamsters. Notably, in BAT of warm- and cold-exposed mutant hamsters, UCP1 and UCP2 mRNA levels were increased. The tissue specificity of UCP3 deficiency suggests that the underlying unknown mutation impairs a factor controlling UCP3 gene expression selectively in brown adipocytes. In wild-type but not mutant primary brown adipocytes, UCP3 gene expression was stimulated by treatment with peroxisome proliferator activated receptor (PPAR) ligands. This implies that the underlying mutation causing UCP3 deficiency is expressed within brown adipocytes and disrupts PPAR-dependent transactivation of the UCP3 gene. On the functional level, we found no direct phenotypic consequences of altered UCP expression in BAT. The absence of UCP3 in BAT of cold-acclimated mutant hamsters affected neither maximal nonshivering thermogenesis elicited by noradrenaline nor the uncoupled respiration of isolated mitochondria in the presence of oligomycin and in response to palmitate.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Deficiency in sPLA(2) does not affect HDL levels or atherosclerosis in mice

Secretory non-pancreatic phospholipase A(2) (sPLA(2)) has been implicated in inflammation and has been found in human atherosclerotic lesions. To test the effect of sPLA(2) deficiency on atherosclerosis, C57BL/Ks mice (apoE(+/+) and PLA(2)(++) were bred with C57BL/6 apoE knockout mice which are sPLA(2)(--) due to a spontaneous mutation. Sibling pairs of mice (apoE(--)/sPLA(2)(++) and apoE(--)/sPLA(2)(--)) on high fat Western diets were dissected at 22 weeks. In vitro enzyme assays confirmed higher serum sPLA(2) activity in the sPLA(2)(++) compared to sPLA(2)(--) for both sexes, while sPLA(2)(--) males had slightly higher serum cholesterol and phospholipids. Analysis of lipoprotein profiles by FPLC showed no effect of sPLA(2) genotype on any measured parameters. Atherosclerosis was quantitated by assaying cholesterol in aortic extracts. Male sPLA(2) trended slightly higher than sPLA(2)(++) with no statistical significance. Female sPLA(2)(++) and sPLA(2)(--) mice showed no significant differences in any of the measured parameters. These results suggest that the endogenous mouse sPLA(2) gene does not significantly affect HDL or atherosclerosis in mice.     

Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis

The gene for the secreted acid protease (ACP), a potential virulence factor of Candida species, was inactivated in Candida tropicalis by gene disruption. The disruption was performed by cotransformation of an ade2 C. tropicalis mutant with a linear DNA fragment carrying a deletion in ACP, and the replicative vector pMK16 which carries a selectable ADE2 gene marker. Few of the transformants exhibited lower protease secretion levels and were shown to have one deleted and one unaffected ACP copy, since C. tropicalis is a diploid yeast. These transformants were rendered homozygotic for this deletion by mild UV-treatment. One of the homozygotic acp deletion mutants obtained was completely devoid of extracellular protease activity and grew poorly on bovine serum albumin-containing medium. This mutant could be complemented by an ACP fragment inserted in pMK16, but also by an acid protease gene isolated from C. parapsilosis.     

Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis.

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol.Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.     

Homocysteine thiolactone-induced hyperhomocysteinemia does not alter concentrations of cholesterol and SREBP-2 target gene mRNAS in rats

The present rat study was conducted to test whether hyper-homocysteinemia induced by dietary homocysteine (Hcy) alters the cholesterol concentration in plasma and tissue and the gene expression of genes involved in cholesterol biosynthesis and uptake. Therefore, rats were fed 100 or 200 mg Hcy per kilogram body mass per day (Hcy100 group and Hcy200 group, respectively) as dl-homocysteine thiolactone, or an Hcy-free diet, which served as control, over 14 days. Rats from the Hcy100 group and the Hcy200 group had higher plasma Hcy concentrations (34.4 +/- 4.6 and 69.4 +/- 11.5 microM, respectively) than rats fed an Hcy-free diet (9.5 +/- 1.7 microM). The concentration of Hcy in liver was 2.6 and 3.8 times higher, and in small intestine was 2.6 and 5.1 times higher, in the Hcy100 group and the Hcy200 group, respectively, than in control rats (P < 0.05). The concentrations of cholesterol in plasma, lipoproteins, liver, and small intestine and the relative mRNA concentrations of sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and low-density lipoprotein (LDL) receptor in liver and small intestine were not influenced by dl-homocysteine thiolactone supplementation. In conclusion, in view of the experimental conditions used here, increased plasma and tissue concentrations of Hcy do not alter cholesterol metabolism of liver and intestine.     

Disruption of the thrombospondin-2 gene alters the lamellar morphology but does not permit vascularization of the adult mouse lumbar disc

Introduction

The biological basis for the avascular state of the intervertebral disc is not well understood. Previous work has suggested that the presence of thrombospondin-1 (TSP-1), a matricellular protein, in the outer annulus reflects a role for this protein in conferring an avascular status to the disc. In the present study we have examined thrombospondin-2 (TSP-2), a matricellular protein with recognized anti-angiogenic activity in vivo and in vitro.

Methods

We examined both the location and expression of TSP-2 in the human disc, and its location in the disc and bordering soft tissues of 5-month-old normal wild-type (WT) mice and of mice with a targeted disruption of the TSP-2 gene. Immunohistochemistry and quantitative histology were utilized in this study.

Results

TSP-2 was found to be present in some, but not all, annulus cells of the human annulus and the mouse annulus. Although there was no difference in the number of disc cells in the annulus of TSP-2-null mice compared with that of WT animals, polarized light microscopy revealed a more irregular lamellar collagen structure in null mouse discs compared with WT mouse discs. Additionally, vascular beds at the margins of discs of TSP-2-null mice were substantially more irregular than those of WT animals. Counts of platelet endothelial cell adhesion molecule-1-positive blood vessels in the tissue margin bordering the ventral annulus showed a significantly larger vascular bed in the tissue bordering the disc of TSP-2-null mice compared with that of WT mice (P = 0.0002). There was, however, no vascular ingrowth into discs of the TSP-2-null mice.

Conclusion

These data confirm a role for TSP-2 in the morphology of the disc and suggest the presence of other inhibitors of angiogenesis in the disc. We have shown that although an increase in vasculature was present in the TSP-2-null tissue in the margin of the disc, vascular ingrowth into the body of the disc did not occur. Our results point to the need for future research to understand the transition from the well-vascularized status of the fetal and young discs to the avascular state of the adult human disc or the small mammalian disc.     

Disruption of LRRK2 does not cause specific loss of dopaminergic neurons in zebrafish

Mutations in LRRK2 are genetically linked to Parkinson's disease (PD) but its normal biological function is largely unknown. Sheng et al. recently reported that deletion of the WD40 domain of LRRK2 in zebrafish specifically causes PD-like loss of neurons and behavior defect. However, our similar early study and recent confirming experiments using the same reagents reported by Sheng et al. failed to reproduce the phenotype of the loss of dopaminergic neurons, although the mRNA of LRRK2 was molecularly disrupted. Our study suggests that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation.