Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted. Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced, be affected by position-effect variegation, or reside in a chromatin configuration inaccessible for recombination. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites, and the availability of both wild-type (WT) and mutant loxP sites, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.
Using genetic and pharmacological approaches, we demonstrate that both RAR␥/RXR␣ heterodimers involved in repression events, as well as PPAR(␦)/RXR␣ heterodimers involved in activation events, are cell-autonomously required in suprabasal keratinocytes for the generation of lamellar granules (LG), the organelles instrumental to the formation of the skin permeability barrier. In activating PPAR(␦)/RXR␣ heterodimers, RXR␣ is transcriptionally active as its AF-2 activation function is required and can be inhibited by an RXR-selective antagonist. Within repressing RAR␥/RXR␣ heterodimers, induction of the transcriptional activity of RXR␣ is subordinated to the addition of an agonistic ligand for RAR␥. Thus, the ligand that possibly binds and activates RXR␣ heterodimerized with PPAR(␦) cannot be a retinoic acid, as it would also bind RAR␥ and relieve the RAR␥-mediated repression, thereby yielding abnormal LGs. Our data also demonstrate for the first time that subordination of RXR transcriptional activity to that of its RAR partner plays a crucial role in vivo, because it allows RXRs to act concomitantly, within the same cell, as heterodimerization partners for repression, as well as for activation events in which they are transcriptionally active.[Keywords: Conditional somatic mutagenesis; RAR␥; PPAR (␦); skin permeability barrier; transcriptional subordination; ichthyosis] Supplemental material is available at http://www.genesdev.org.
The glucocorticoid receptor (GR) belongs to a superfamily of ligand-regulated nuclear steroid hormone receptors. The steps in the signal transduction pathway leading to the biological effects of glucocorticoids (GCs) include sequentially binding of the steroid to the GR ligand binding domain (LBD), receptor transformation, nuclear translocation and either positive or negative gene transactivation. Rifampicin (RIF) is a macrocyclic antibiotic used as an antituberculosis agents. As the incidence of tuberculosis has been increasing, in part because of the AIDS epidemic, a growing number of patients are being exposed to the adverse effects of this antibiotic. Indeed, this compound, as are the GCs, is often implicated in noxious drug interactions, because of its strong ability to induce drug-metabolizing enzymes. Moreover, in humans, RIF, as are the GCs, has been described as a potential immunodepressor, associated notably with the reduction of mitogenic responsiveness of human peripheral blood lymphocytes. Here, we report that RIF activates the human glucocorticoid receptor (hGR). Transient expression of wild-type, deleted or mutated GRs; sucrose density gradient sedimentation; and the BIAcore technique strongly suggest that RIF binds to the receptor with the physiological consequence that this antibiotic acts as an immunodepressor. Given the wide use of RIF in the treatment of coinfection of tuberculosis and HIV, this report is highly relevant to current medical practice.
-The cytochrome P450 (P450) superfamily represents a group of relevant enzymes in the field of drug metabolism and several exogenous or constitutional factors contribute to regulate its expression. Cattle represent an important source of animal-derived food-products and studies concerning the P450 expression are needed for the extrapolation of pharmacotoxicological data from one species to another and for the evaluation of the consumer's risk associated with the consumption of harmful residues found in foodstuffs. In the present study, possible breed-, genderand species-differences in P4503A (the P450 subfamily more expressed in the human liver) expression were studied in vitro in Piedmontese (PDM) and Limousin (LIM) meat cattle breeds of both sexes and in domestic Ruminants (cattle, sheep and goats). Cytochrome P450 and P4503A contents as well as CYP3A-dependent drug metabolising enzymes (DME) were measured in liver microsomes. Significant lower levels of P450 (P < 0.001) and P4503A (P < 0.05) contents were observed in PDM vs. LIM of both sexes; the P4503A-dependent DME activities were significantly (P values ranging from 0.05 up to 0.001) higher in PDM cattle, particularly in males. A gendereffect in DME activities was noticed (P < 0.05) only in PDM male cattle. With regards to the species, the expression of both P4503A apoprotein and some of the related DME activities were more pronounced in sheep (P < 0.01 vs. cattle) and in goats (P < 0.05 vs. sheep; P < 0.01 vs. cattle) than in cattle. The significant differences in P4503A expression observed in LIM and PDM cattle are consistent with previously published data on strain-and breed-differences pointed out in rats and men. As far as a possible sex-effect is concerned, no clear-cut evidence is likely to be drawn. Finally, P4503A expression was more relevant in small ruminants. ruminants / liver drug metabolism / CYP3A / gender / breed
Nuclear hormone receptors (NR) are important transcriptional regulators of numerous genes involved in diverse pathophysiological and therapeutic functions. Following ligand activation, class II NR share the ability to heterodimerize with the retinoid X receptor (RXR). It is established that RXR activators, rexinoids, transactivate several peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in a PPARalpha-dependent manner. We hypothesized that, once activated, RXR might signal through quiescent NR other than PPARalpha, in an organ-specific manner. To study this putative phenomenon in vivo, we developed an array of 120 genes relevant to the class II NR field. The genes were selected using both published data and high-density screenings performed on RXR or PPARalpha agonist-treated mice. Wild-type C57BL/6J and PPARalpha-deficient mice were treated with fenofibrate (PPARalpha activator) or LGD1069 (RXR activator). Using our customized array, we studied the hepatic, cardiac, and renal expression of this panel of 120 genes and compared them in both murine genotypes. The results obtained from this study confirmed the ability of an RXR agonist to modulate PPARalpha-restricted target genes in the liver and the kidney. Furthermore, we show that various organ-specific regulations occurring in both genotypes (PPARalpha +/+ or -/-) are highly indicative of the ability of RXR to recruit other class II NR pathways. Further development of this molecular tool may lead to a better understanding of the permissiveness of class II nuclear receptor dimers in vivo.
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