Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.     

Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8 tetrachlorodibenzo-p-dioxin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.     

Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8 tetrachlorodibenzo-p-dioxin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.     

Protein Tyrosine Phosphatase 2 (SHP-2) Moderates Signaling by gp130 but Is Not Required for the Induction of Acute-Phase Plasma Protein Genes in Hepatic Cells

Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for α-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.     

Forkhead Box O1 (FOXO1) Protein,but Not p53, Contributes to Robust Induction of p21 Expression in Fasted Mice

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

The Src Homology and Collagen A (ShcA) Adaptor Protein Is Required for the Spatial Organization of the Costamere/Z-disk Network during Heart Development

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca²⁺/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.     

Restoration of Dlk1 and Rtl1 Is Necessary but Insufficient to Rescue Lethality in Intergenic Differentially Methylated Region (IG-DMR)-deficient Mice

In the Dlk1-Dio3 imprinted domain, an intergenic differentially methylated region (IG-DMR) regulates the parental allele-specific expression of imprinted genes. The maternally inherited deletion of IG-DMR (IG-DMR^(−/+)) results in perinatal lethality because of the overexpression of paternally expressed genes and repression of maternally expressed noncoding RNAs (ncRNAs), including Gtl2. To better understand the possible contribution of paternally expressed genes to the lethality, we attempted to rescue the lethality of IG-DMR^(−/+) mutants by restoring the paternally expressed genes. Because the paternally inherited Gtl2 deletion (Gtl2^(+/−)) induced a decrease in the expression of paternally expressed genes, we crossed female IG-DMR heterozygous mice and male Gtl2 heterozygous mutant mice. The resultant IG-DMR^(−/+)/Gtl2^(+/−) double mutant mice had normal expression levels of paternally expressed genes, and none of them showed perinatal lethality; however, most mice showed postnatal lethality with decreased expression of the maternally expressed ncRNAs. Thus, we inferred that paternally expressed genes are necessary for perinatal survivability and that maternally expressed ncRNAs are involved in postnatal lethality.     

Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; NR1I3), repressing dephosphorylation and sequestering CAR in the cytoplasm

The nuclear constitutive active/androstane receptor (CAR) is inactivated and sequestered in the cytoplasm when Thr-38 is phosphorylated. Here, we have demonstrated that activated ERK1/2 interacts with phosphorylated CAR to repress dephosphorylation of Thr-38. The phosphorylation-dependent interaction between CAR and ERK1/2 was examined by co-immunoprecipitation experiments of ectopically expressed FLAG-tagged CAR T38A and CAR T38D mutants with endogenous phospho-ERK1/2 in Huh-7 cells. Phospho-ERK1/2 coprecipitated only the phosphorylation-mimicking CAR T38D mutant; this coprecipitation was mediated by the interaction with the xenochemical response signal peptide near the C terminus of CAR. This interaction increased after EGF treatment and decreased after treatment with the MEK inhibitor U0126 as well as after knockdown of MEK1/2 by shRNA in Huh-7 cells. The phosphorylation levels of Thr-38 of CAR decreased in U0126-treated Huh-7 cells. Thus, activated ERK1/2 interacts with CAR and represses dephosphorylation of Thr-38, providing a cell signal-regulated mechanism for CAR activation.     

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M₂ muscarinic receptor (M₂R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K⁺ channel I_KACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4^−/−, Rgs6^−/−, and Rgs4^−/−:Rgs6^−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M₂R-I_KACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M₂R-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6^−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6^−/− and Gβ5^−/− mice, suggest that the partial rescue of phenotypes in Rgs4^−/−:Rgs6^−/− mice is attributable to another R7 RGS protein whose influence on M₂R-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

Generation of a 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahrd mouse line harboring the poor-affinity aryl hydrocarbon receptor

Herein, we describe generation of the hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mouse line, which carries human functional CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a1 and Cyp1a2 genes, in a (>99.8%) background of the C57BL/6J genome and harboring the poor-affinity aryl hydrocarbon receptor (AHR) from the DBA/2J mouse. We have characterized this line by comparing it to our previously created hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 line—which carries the same but has the high-affinity AHR of the C57BL/6J mouse. By quantifying CYP1A1 and CYP1A2 mRNA in liver, lung and kidney of dioxin-treated mice, we show that dose-response curves in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mice are shifted to the right of those in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 mice—similar to, but not as robust as, dose-response curves in DBA/2J versus C57BL/6J mice. This new mouse line is perhaps more relevant than the former to human risk assessment vis-à-vis human CYP1A1 and CYP1A2 substrates, because poor-affinity rather than high-affinity AHR occurs in the vast majority of the human population.     

The Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) C Terminus Plays a Key Role in Protein Stability,but Its Farnesylation Is Not Required for Membrane Association in Primary Neurons

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed in neurons. A possible role for UCH-L1 in neurodegeneration has been highlighted because of its presence in Lewy bodies associated with Parkinson disease and neurofibrillary tangles observed in Alzheimer disease. UCH-L1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-L1^C) and a membrane-associated form (UCH-L1^M). Alzheimer brains show reduced levels of soluble UCH-L1^C correlating with the formation of UCH-L1-immunoreactive tau tangles, whereas UCH-L1^M has been implicated in α-synuclein dysfunction. Given these reports of divergent roles, we investigated the properties of UCH-L1 membrane association. Surprisingly, our results indicate that UCH-L1 does not partition to the membrane in the cultured cell lines we tested. Furthermore, in primary cultured neurons, a proportion of UCH-L1^M does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death, and an abnormal intracellular distribution, consistent with protein dysfunction and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared with clonal cell lines and that farnesylation does not account for the membrane association in neurons.