Biomedical subjects

D Metzger

Publications and source records attributed to D Metzger.

At least 19 recordsLinked to original sources

PubMed2007

Contribution of targeted conditional somatic mutagenesis to deciphering retinoid X receptor functions and to generating mouse models of human diseases.

The last decade has witnessed an enormous rise in the interest for retinoid signalling and its cognate receptors, because of their central role in the coordination of development and homeostasis, through their ability to orchestrate the expression of numerous target genes. These receptors include six nuclear receptor (NR) family members, the retinoic acid receptor (RAR) alpha, beta and gamma, and the retinoid X receptor (RXR) alpha, beta and gamma, which are expressed in many cell types in mammals. Analysis of the development of mouse embryos bearing retinoid receptor null mutations demonstrated that these receptors transduce the effects of retinoic acid (RA, the active derivative of vitamin A) in vivo, and revealed impressive complexity. However, frequent redundancy in receptor functions and lethality of compound RAR-null mutants, as well as of RXRalpha-null mutants, precluded the characterisation of the functions of these receptors during late development and postnatally. We illustrate here how recent developments of conditional targeted somatic mutagenesis have opened new avenues in analysing the physiological functions of retinoid X receptor signalling in a variety of tissues and cell types, as well as in exploring the pathophysiological consequences of their alteration that led to novel mouse models of human diseases.

Adipocytes↗

PubMed2006

Pharmacokinetics and safety profile of ispronicline (TC-1734), a new brain nicotinic receptor partial agonist, in young healthy male volunteers.

Recent research suggests that drugs activating nicotine acetylcholine receptors might be promising therapy in cognitive decline seen in the elderly, including Alzheimer's disease. Ispronicline (TC-1734), a brain-selective alpha4beta2 nicotine acetylcholine receptor partial agonist, has shown memory-enhancing properties in rodents and a good tolerability profile. The safety and the full pharmacokinetic profile of TC-1734 and its N-desalkylated metabolite, TC-1784, were investigated in 2 phase I studies, and results are reported in this article. Study A used a double-blind, placebo-controlled, crossover design with a rising single-dose scheme (2-320 mg). Study B used a double-blind, placebo-controlled, parallel-group design with a rising multiple-dose scheme (doses: 50, 100, and 200 mg, once daily, x 10 days). Cmax of TC-1734 was reached around 1 to 2 hours postdose, and mean terminal half-life (t1/2) ranged from 3 to 5.3 hours (single doses) and from 2.7 to 8.8 hours (repeated doses). No accumulation of TC-1734 was observed after 10 days. Renal clearance appeared to be a minor method of elimination of TC-1734 and TC-1784. A high interindividual variability was noted for all parameters. Across the dose ranges explored, TC-1734 was safe and well tolerated. No changes of clinical significance were seen on laboratory and cardiovascular parameters. Adverse events were generally of mild to moderate intensity, with dizziness and headache being reported most frequently.

Administration, Oral↗

PubMed2005

Functional role of RXRs and PPARgamma in mature adipocytes.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is abundantly expressed in adipocytes, and plays an important role in adipocyte differentiation and fat accretion. It is a heterodimeric partner of the retinoid X receptors alpha, beta and gamma, which are also expressed in the adipose tissue. As lethality of PPARgamma(-/-) and RXRalpha(-/-) mouse fetuses precluded the analysis of PPARgamma and RXRalpha functions in mature adipocytes, we generated RXRalpha(ad-/-) and PPARgamma(ad-/-) mice, in which RXRalpha and PPARgamma are selectively ablated in adult adipocytes, respectively. Even though the adiposity of RXRalpha(ad-/-) mice is similar to that of control mice when fed a regular diet, they are resistant to chemically and dietary-induced obesity. However, mature adipocytes lacking either both RXRalpha and RXRgamma or PPARgamma die, and are replaced by newly formed adipocytes. Thus, in adipocytes, RXRalpha is essential for lipogenesis, but RXRgamma can functionally replace RXRalpha for the adipocyte vital functions exerted by PPARgamma/RXR heterodimers.

Adipocytes↗

PubMed2002

[Genetic dissection of retinoic acid function in epidermis physiology].

The active metabolite of vitamin A (retinoic acid, RA) acts through the nuclear receptors RARalpha, beta and gamma and RXRalpha, beta and gamma. These receptors form RAR/RXR heterodimers, which bind to genetic regulatory DNA sequences and activate transcription of RA target genes. As RXR form heterodimers with a number of other nuclear receptors, such as the vitamin D3 receptor (VDR) and are involved in several signaling pathways. In the skin, RARgamma and RXRalpha predominate, but RARalpha and RXRbeta are also expressed. To elucidate the role of RA in skin physiology, we produced mutant mouse lines null for RAR or RXR. On the one hand, null mutations for RARa or RXRbeta have no effect on the skin, whereas a RARgamma-null mutation induces alterations in the granular cell layer. On the other, genetic inactivation of RXRa leads to embryonic lethality before epidermal development. Consequently, to determine the role of RXRa in adult mice, studies were performed using conditional somatic mutagenesis (permitting inactivation of a given gene in a specific tissue and in a time-dependent manner). Using this novel genetic approach, mutant mice were obtained in which RXRalpha was not expressed in the skin. These mice developed hair follicle degeneration, then alopecia, similar to that observed in VDR-null mutants, suggesting that hair follicle homeostasis depends on RXRalpha/VDR heterodimers. A similar genetic approach applied to the RARgamma locus demonstrated that topical administration of RA on the skin activates RARgamma/RXR heterodimers in suprabasal cells, and induces expression of a paracrine growth factor (HB-EGF) in these cells which, in turn, stimulates the proliferation of basal cells.

Alopecia↗

PubMed2001

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Animals↗

PubMed2001

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

Animals↗

PubMed2001

Substance use among men who have sex with men: comparison with a national household survey.

OBJECTIVE: Compare substance use among men who have sex with men (MSM) at high risk for HIV infection to a nationally representative sample of heterosexual men. METHODS: Compare data from surveys of 3,212 MSM recruited for participation in a Vaccine Preparedness Study (VPS) with an age-standardized group of 2481 single, urban-dwelling men from the 1995 National Household Survey on Drug Abuse (NHSDA). RESULTS: Except for alcohol, relative risk (RR [95% confidence interval (CI)]) for use of any substance was higher in the VPS than the National Household Survey on Drug Abuse (NHSDA) (3.64 [3.01-4.42]). Drugs with the highest relative risks were "poppers" (21.6 [15.2-30.8]), sedatives (6.98 [2.46-19.8]), hallucinogens (6.14 [4.61-8.17]), tranquilizers (4.99 [2.96-8.42]), and stimulants (4.47 [3.58-5.58]). RR was higher for weekly use of poppers (33.5 [12.5-89.6]), stimulants (2.75 [1.79-4.22]), marijuana (2.37 [1.93-2.92]), and cocaine (2.24 [1.32-3.79]); and for daily use of marijuana (1.49 [1.08-2.05]). CONCLUSIONS: Participants in the VPS used more substances than a group of age-standardized, single, urban-dwelling men from the NHSDA. In view of previous data showing that substance use can be associated with unprotected sex, assessing substance use among MSM at high risk for HIV infection is an important component of risk reduction efforts.

Adolescent↗

PubMed2001

Selective ablation of retinoid X receptor alpha in hepatocytes impairs their lifespan and regenerative capacity.

Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRalpha isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRalpha has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRalpha during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436-4444]. However, it is unknown whether RXRalpha plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRalpha was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRalpha is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRalpha plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.

Animals↗

PubMed2001

Are US populations appropriate for trials of human immunodeficiency virus vaccine? The HIVNET Vaccine Preparedness Study.

Questions exist about whether testing of preventive human immunodeficiency virus (HIV)-1 vaccines, which will require rapid recruitment and retention of cohorts with high HIV-1 seroincidence, is feasible in the United States. A prospective cohort study was conducted in 1995-1997 among 4,892 persons at high risk for HIV infection in nine US cities. At 18 months, with an 88% retention rate, 90 incident HIV-1 infections were observed (1.31/100 person-years (PY), 95% confidence interval (CI): 1.06, 1.61). HIV-1 seroincidence rates varied significantly by baseline eligibility criteria--1.55/100 PY among men who had sex with men, 0.38/100 PY among male intravenous drug users, 1.24/100 PY among female intravenous drug users, and 1.13/100 PY among women at heterosexual risk-and by enrollment site, from 0.48/100 PY to 2.18/100 PY. HIV-1 incidence was highest among those men who had sex with men who reported unprotected anal intercourse (2.01/100 PY, 95% CI: 1.54, 2.63), participants who were definitely willing to enroll in an HIV vaccine trial (1.96/100 PY, 95% CI: 1.41, 2.73), and women who used crack cocaine (1.62/100 PY, 95% CI: 0.92, 2.85). Therefore, cohorts with HIV-1 seroincidence rates appropriate for HIV-1 vaccine trials can be recruited, enrolled, and retained.

AIDS Vaccines↗

PubMed2001

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives.

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

Alkaline Phosphatase↗

PubMed2001

The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures.

The active derivative of vitamin A, retinoic acid (RA), is essential for normal embryonic development. The spatio-temporal distribution of embryonic RA results from regulated expression of RA-synthesizing retinaldehyde dehydrogenases and RA-metabolizing cytochrome P450s (CYP26). Excess RA administration or RA deficiency results in a complex spectrum of embryonic abnormalities. As a first step in understanding the developmental function of RA-metabolizing enzymes, we have disrupted the murine Cyp26A1 gene. We report that Cyp26A1-null mutants die during mid-late gestation and show a number of major morphogenetic defects. Spina bifida and truncation of the tail and lumbosacral region (including abnormalities of the kidneys, urogenital tract, and hindgut) are the most conspicuous defects, leading in extreme cases to a sirenomelia ("mermaid tail") phenotype. Cyp26A1 mutants also show posterior transformations of cervical vertebrae and abnormal patterning of the rostral hindbrain, which appears to be partially posteriorly transformed. These defects correlate with two major sites of Cyp26A1 expression in the rostral neural plate and embryonic tail bud. Because all of the Cyp26A1(-/-) abnormalities closely resemble RA teratogenic effects, we postulate that the key function of CYP26A1 is to maintain specific embryonic areas in a RA-depleted state, to protect them against the deleterious effect of ectopic RA signaling.

Abnormalities, Multiple↗

PubMed2001

Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes.

Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.

Adipocytes↗

PubMed2001

Willingness to volunteer in future preventive HIV vaccine trials: issues and perspectives from three U.S. communities.

UNLABELLED: This study examined perceived risks, benefits, and desired information related to willingness to volunteer in preventive HIV vaccine trials. SAMPLE: Purposive sampling was used to select 90 participants among injecting drug users (Philadelphia, PA, U.S.A.); gay men (San Francisco, CA, U.S.A.); and black Americans (Durham, NC, U.S.A.). METHODS: A qualitative interview guide elicited perceived benefits, risks, and desired information relating to trial participation. Themes were developed from the transcribed texts and from freelists. RESULTS: Stated willingness to volunteer in a preventive HIV vaccine trial was similar across the three communities. Eight perceived benefits were reported, including self-benefits, altruism, and stopping the spread of AIDS. Seven perceived risks were reported, including negative side effects and vaccine safety issues, contracting HIV from the vaccine, and social stigmatization. Participants voiced the desire for eight types of information about issues relating to trust and confidentiality in the research process, health complications and later assistance, and vaccine trial methodology. CONCLUSIONS: In this study, many benefits as well as risks of preventive HIV vaccine trial participation were cited. Scientists conducting preventive HIV vaccine trials need to address community perceptions of risks and provide information about the research if trial enrollment is to be diverse and successful.

AIDS Vaccines↗

PubMed2001

Site- and time-specific gene targeting in the mouse.

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter. Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals. However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T). These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

Alleles↗

PubMed2001

Temporally controlled targeted somatic mutagenesis in the mouse brain.

To develop spatio-temporally controlled somatic mutagenesis in the adult mouse nervous system, we established transgenic mice expressing the tamoxifen-inducible Cre-ERT recombinase under the control of the mouse prion protein (PrP) promoter. Cre-ERT was expressed in most regions of the brain and in the retina of one transgenic line, whereas its expression was mostly restricted to the hippocampus and the cerebellum in another line. As tamoxifen efficiently induced Cre-mediated recombination in the various neuronal cell types expressing Cre-ERT in the brain of adult mice, the PrP-Cre-ERT lines should be valuable tools to study the functions of genes involved in neurodegenerative diseases or regeneration, and in complex processes such as behaviour, learning and memory. Some limitations of presently available reporter lines for Cre-mediated recombination in adult mouse CNS are discussed.

Animals↗

PubMed2001

RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations.

RXR-alpha is the most abundant of the three retinoid X receptors (RXRs) in the epidermis. In this study, we have used Cre-mediated recombination to selectively disrupt the mouse gene for RXR-alpha in epidermal and hair follicle keratinocytes. We show that RXR-alpha is apparently dispensable for prenatal epidermal development, while it is involved in postnatal skin maturation. After the first hair pelage, mutant mice develop a progressive alopecia, histologically characterised by the destruction of hair follicle architecture and the formation of utriculi and dermal cysts in adult mice. Our results demonstrate that RXR-alpha plays a key role in anagen initiation during the hair follicle cycle. In addition, RXR-alpha ablation results in epidermal interfollicular hyperplasia with keratinocyte hyperproliferation and aberrant terminal differentiation, accompanied by an inflammatory reaction of the skin. Our data not only provide genetic evidence that RXR-alpha/VDR heterodimers play a major role in controlling hair cycling, but also suggest that additional signalling pathways mediated by RXR-alpha heterodimerised with other nuclear receptors are involved in postnatal hair follicle growth, and homeostasis of proliferation/differentiation of epidermal keratinocytes and of the skin's immune system.

Alopecia↗

PubMed2000

F9 embryonal carcinoma cells engineered for tamoxifen-dependent Cre-mediated site-directed mutagenesis and doxycycline-inducible gene expression.

The study of gene functions in complex genetic environments such as mammalian cells would greatly benefit from systems allowing a tight control of gene expression. The tetracycline-inducible gene expression system and the site-specific Cre/loxP recombination system have gained increasing popularity for conditional expression and gene disruption. To facilitate the analysis of gene functions in a cell autonomous system, we have established an F9 murine embryonal carcinoma cell line, constitutively expressing both the doxycycline-controlled transactivator rtTA and the tamoxifen-dependent Cre recombinase Cre-ER(T). The expression of a reporter gene placed under the control of tetracycline operators was induced about 1000-fold by doxycycline, and tamoxifen-induced excision of a loxP-flanked DNA segment occurred in all cells. This genetically engineered cell line, which allows, upon simple ligand addition, sophisticated genetic manipulations, such as sequential inactivation of loxP-flanked genes, and tightly controlled reexpression of their cDNAs, should be a valuable tool for studying mammalian gene functions.

Carcinoma, Embryonal↗

PubMed2000

Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis.

Nuclear receptors for retinoids (RARs) and vitamin D (VDR), and for some other ligands (TRs, PPARs and LXRs), maybe critical in the development and homeostasis of mammalian epidermis. It is believed that these receptors form heterodimers with retinoid X receptors (RXRs) to act as transcriptional regulators. However, most genetic approaches aimed at establishing their physiological functions in the skin have been inconclusive owing either to pleiotropic effects and redundancies between receptor isotypes in gene knockouts, or to equivocal interpretation of dominant-negative mutant studies in transgenic mice. Moreover, knockout of RXRalpha, the main skin RXR isotype, is lethal in utero before skin formation. Here we have resolved these problems by developing an efficient technique to create spatiotemporally controlled somatic mutations in the mouse. We used tamoxifen-inducible Cre-ER(T) recombinases to ablate RXRalpha selectively in adult mouse keratinocytes. We show that RXRalpha has key roles in hair cycling, probably through RXR/VDR heterodimers, and in epidermal keratinocyte proliferation and differentiation.

Alleles↗