RESUMO
High estradiol levels in late puberty induce growth plate closure and thereby cessation of growth in humans. In mice, the growth plates do not fuse after sexual maturation, but old mice display reduced longitudinal bone growth and high-dose estradiol treatment induces growth plate closure. Estrogen receptor (ER)-α stimulates gene transcription via two activation functions (AFs), AF-1 and AF-2. To evaluate the role of ERα and its AF-1 for age-dependent reduction in longitudinal bone growth and growth plate closure, female mice with inactivation of ERα (ERα(-/-)) or ERαAF-1 (ERαAF-1(0)) were evaluated. Old (16- to 19-mo-old) female ERα(-/-) mice showed continued substantial longitudinal bone growth, resulting in longer bones (tibia: +8.3%, P < 0.01) associated with increased growth plate height (+18%, P < 0.05) compared with wild-type (WT) mice. In contrast, the longitudinal bone growth ceased in old ERαAF-1(0) mice (tibia: -4.9%, P < 0.01). Importantly, the proximal tibial growth plates were closed in all old ERαAF-1(0) mice while they were open in all WT mice. Growth plate closure was associated with a significantly altered balance between chondrocyte proliferation and apoptosis in the growth plate. In conclusion, old female ERα(-/-) mice display a prolonged and enhanced longitudinal bone growth associated with increased growth plate height, resembling the growth phenotype of patients with inactivating mutations in ERα or aromatase. In contrast, ERαAF-1 deletion results in a hyperactive ERα, altering the chondrocyte proliferation/apoptosis balance, leading to growth plate closure. This suggests that growth plate closure is induced by functions of ERα that do not require AF-1 and that ERαAF-1 opposes growth plate closure.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Lâmina de Crescimento/fisiologia , Transativadores/fisiologia , Absorciometria de Fóton , Envelhecimento/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Desenvolvimento Ósseo/efeitos dos fármacos , Proliferação de Células , Condrócitos/fisiologia , Primers do DNA , Estradiol/sangue , Receptor alfa de Estrogênio/genética , Feminino , Lâmina de Crescimento/anatomia & histologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígeno Nuclear de Célula em Proliferação/metabolismo , Maturidade Sexual/fisiologia , Tíbia/crescimento & desenvolvimento , Transativadores/genéticaRESUMO
The bone-sparing effect of estrogen is primarily mediated via estrogen receptor-α (ERα), which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand binding domain. To evaluate the role of ERα AF-1 and ERα AF-2 for the effects of estrogen in bone in vivo, we analyzed mouse models lacking the entire ERα protein (ERα(-/-)), ERα AF-1 (ERαAF-1(0)), or ERα AF-2 (ERαAF-2(0)). Estradiol (E2) treatment increased the amount of both trabecular and cortical bone in ovariectomized (OVX) WT mice. Neither the trabecular nor the cortical bone responded to E2 treatment in OVX ERα(-/-) or OVX ERαAF-2(0) mice. OVX ERαAF-1(0) mice displayed a normal E2 response in cortical bone but no E2 response in trabecular bone. Although E2 treatment increased the uterine and liver weights and reduced the thymus weight in OVX WT mice, no effect was seen on these parameters in OVX ERα(-/-) or OVX ERαAF-2(0) mice. The effect of E2 in OVX ERαAF-1(0) mice was tissue-dependent, with no or weak E2 response on thymus and uterine weights but a normal response on liver weight. In conclusion, ERα AF-2 is required for the estrogenic effects on all parameters evaluated, whereas the role of ERα AF-1 is tissue-specific, with a crucial role in trabecular bone and uterus but not cortical bone. Selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial actions in cortical bone, constituting 80% of the skeleton, while minimizing effects on reproductive organs.
Assuntos
Osso e Ossos/fisiologia , Receptor alfa de Estrogênio/fisiologia , Estrogênios/fisiologia , Animais , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Feminino , Camundongos , Camundongos Mutantes , Tamanho do Órgão , Radiografia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Timo/anatomia & histologia , Timo/efeitos dos fármacos , Timo/fisiologia , Ativação Transcricional , Útero/anatomia & histologia , Útero/efeitos dos fármacos , Útero/fisiologiaRESUMO
Glucocorticoid-induced apoptosis of thymocytes is one of the first recognized forms of programmed cell death. It was shown to require gene activation induced by the glucocorticoid receptor (GR) translocated into the nucleus following ligand binding. In addition, the necessity of the glucocorticoid-induced, but transcription-independent phosphorylation of phosphatidylinositol-specific phospholipase C (PI-PLC) has also been shown. Here we report that retinoic acids, physiological ligands for the nuclear retinoid receptors, enhance glucocorticoid-induced death of mouse thymocytes both in vitro and in vivo. The effect is mediated by retinoic acid receptor (RAR) alpha/retinoid X receptor (RXR) heterodimers, and occurs when both RARα and RXR are ligated by retinoic acids. We show that the ligated RARα/RXR interacts with the ligated GR, resulting in an enhanced transcriptional activity of the GR. The mechanism through which this interaction promotes GR-mediated transcription does not require DNA binding of the retinoid receptors and does not alter the phosphorylation status of Ser232, known to regulate the transcriptional activity of GR. Phosphorylation of PI-PLC was not affected. Besides thymocytes, retinoids also promoted glucocorticoid-induced apoptosis of various T-cell lines, suggesting that they could be used in the therapy of glucocorticoid-sensitive T-cell malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Glucocorticoides/farmacologia , Receptores de Glucocorticoides/metabolismo , Retinoides/farmacologia , Linfócitos T/efeitos dos fármacos , Alitretinoína , Animais , Células Cultivadas , Fragmentação do DNA , Dexametasona/efeitos adversos , Deleção de Genes , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Compostos Orgânicos/farmacologia , Fosfoinositídeo Fosfolipase C/metabolismo , Fosforilação , Multimerização Proteica/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/agonistas , Receptores X de Retinoides/metabolismo , Linfócitos T/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Tretinoína/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.
Assuntos
Transformação Celular Neoplásica , Receptores ErbB/metabolismo , Proteínas F-Box/metabolismo , Queratinócitos/metabolismo , Presenilinas/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Proteína 7 com Repetições F-Box-WD , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Hiperplasia , Técnicas Imunoenzimáticas , Integrases/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Knockout , Ubiquitina/metabolismoRESUMO
Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose-response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-alpha-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.
Assuntos
Estrogênios/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Peptídeos/genética , Adrenomedulina , Animais , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Receptor beta de Estrogênio/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteína 3 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Útero/metabolismo , Útero/fisiologiaRESUMO
Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)-dependent and nonclassical membrane ER-mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane-associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence-activated cell sorting (FACS) studies indicated the attachment of ER-alpha but not ER-beta to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER-alpha. In FACS studies on ER-alpha knockout astrocytes, only a few membrane ER-positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP-kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER-alpha. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen-mediated protective effects on midbrain dopamine neurons.
Assuntos
Astrócitos/metabolismo , Membrana Celular/metabolismo , Sistema Nervoso Central/metabolismo , Receptor alfa de Estrogênio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases da Família src/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Citoproteção/fisiologia , Receptor alfa de Estrogênio/genética , Citometria de Fluxo , Citometria por Imagem , Mesencéfalo/citologia , Mesencéfalo/crescimento & desenvolvimento , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Confocal , Neurônios/metabolismoRESUMO
Estrogen regulates proliferation and differentiation of epithelial cells in the mammalian oviduct, but pathways for cell-specific differentiation are not well understood. In the epithelial cells of the developing rat oviduct, we found estrogen receptor (ER) alpha is expressed at birth and persists in all cells through neonatal day (ND) 7 when ciliated cells appear. To determine a specific function of ER and foxj1, a transcription factor known to have fundamental roles in ciliogenesis in the lung, in differentiation of the ciliated epithelial cells, we treated newborn rats from ND 0 to 5 with estradiol-17beta (E2) with and without a selective ER antagonist. E2 enhanced the number of proliferating cells and accelerated the process of epithelial cell differentiation resulting in ciliogenesis by ND 5, and co-treatment with an ER antagonist inhibited these changes. Foxj1 was expressed only in the infundibulum and ampulla (INF/AMP). That expression preceded the appearance of cilia and was induced by E2. Cilia were absent in oviducts of foxj1-deficient mice, indicating that foxj1 plays a critical role in oviductal ciliogenesis. However, we found the presence of cilia in the ERalpha-deficient mouse oviduct. The widespread expression of ERalpha in oviductal epithelium, but restriction of cilia to the INF/AMP regions, and importantly, the presence of cilia in the ERalpha-deficient mice, suggested ER signaling is not essential for ciliated epithelial cell differentiation. These observations demonstrate that, although E2 stimulates the differentiation process of ciliated epithelial cells, foxj1 is directly required for epithelial cell ciliogenesis of the neonatal oviduct.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Oviductos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Fatores de Transcrição Forkhead , Humanos , Masculino , Camundongos , Oviductos/citologia , Oviductos/efeitos dos fármacos , RatosRESUMO
Peroxisome proliferator-activated receptor (PPAR) are nuclear hormone receptors that are activated by endogenous lipid metabolites. Previous studies have demonstrated that PPAR-alpha activation stimulates keratinocyte differentiation in vitro and in vivo, is anti-inflammatory, and improves barrier homeostasis. Recent studies have shown that PPAR-beta/delta activation induces keratinocyte differentiation in vitro. This study demonstrated that topical treatment of mice with a selective PPAR-beta/delta agonist (GW1514) in vivo had pro-differentiating effects, was anti-inflammatory, improved barrier homeostasis, and stimulated differentiation in a disease model of epidermal hyperproliferation [corrected]. In contrast to PPAR-alpha activation, PPAR-beta/deltain vivo did not display anti-proliferative or pro-apoptotic effects. The pro-differentiating effects persisted in mice lacking PPAR-alpha, but were decreased in mice deficient in retinoid X receptor-alpha, the major heterodimerization partner of PPAR. Furthermore, in vitro PPAR-beta/delta activation, aside from stimulating differentiation-related genes, additionally induced adipose differentiation-related protein (ADRP) and fasting induced adipose factor (FIAF) mRNA in cultures keratinocytes, which was paralleled by increased oil red O staining indicative of lipid accumulation, the bulk of which were triglycerides (TG). Comparison of differentially expressed genes between PPAR-beta/delta and PPAR-alpha activation revealed distinct profiles. Together, these studies indicate that PPAR-beta/delta activation stimulates keratinocyte differentiation, is anti-inflammatory, improves barrier homeostasis, and stimulates TG accumulation in keratinocytes.
Assuntos
Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Administração Tópica , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Citocinas/farmacologia , Dermatite/prevenção & controle , Células Epidérmicas , Epiderme/metabolismo , Regulação da Expressão Gênica , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Masculino , Camundongos , Camundongos Pelados , Permeabilidade , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/administração & dosagem , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Raios UltravioletaRESUMO
Although estradiol (E(2)) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E(2) (80 microg/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E(2) increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E(2), whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E(2). To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ERalpha or ERbeta has been disrupted were ovariectomized and treated, or not, with E(2). Basal NO production was increased and the sensitivity to acetylcholine decreased in ERbeta knockout mice in response to E(2), whereas this effect was abolished in ERalpha knockout mice. Finally, these effects of E(2) on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E(2) in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ERalpha, but not ERbeta, mediates the beneficial effect of E(2) on basal NO production.
Assuntos
Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Estradiol/administração & dosagem , Óxido Nítrico/metabolismo , Receptores de Estrogênio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Fatores Biológicos/metabolismo , Peso Corporal/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Implantes de Medicamento , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Técnicas In Vitro , Ionóforos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Ovariectomia , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Útero/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Two isoforms of estrogen receptor (ER) have been described: ERalpha and ERbeta. The initial gene targeting of ERalpha, consisting in the introduction of a Neo cassette in exon 1 [alphaERKO, hereafter called ERalpha-Neo KO (knockout)], was reported in 1993. More recently, another mouse deficient in ERalpha because of the deletion of exon 2 (ERalphaKO, hereafter called ERalpha-delta2 KO) was generated. In ovariectomized ERalpha-wild-type mice, estradiol (E(2)) increases uterine weight and basal production of endothelial nitric oxide (NO). Both of these effects are abolished in ERalpha-delta2 KO mice. In contrast, we show here that both of these effects of E(2) are partially (uterine weight) or totally (endothelial NO production) preserved in ERalpha-Neo KO. We also confirm the presence of two ERalpha mRNA splice variants in uterus and aorta from ERalpha-Neo KO mice. One of them encodes a chimeric ERalpha protein (ERalpha55), partially deleted in the A/B domain, that was detected in both uterus and aorta by Western blot analysis. The other ERalpha mRNA splice variant codes for an isoform deleted for the A/B domain (ERalpha46), which was detected in uterus of ERalpha-Neo KO, and wild-type mice. This protein isoform was not detected in aorta. The identification of these two N-terminal modified isoforms in uterus, and at least one of them in aorta, probably explains the persistence of the E(2) effects in ERalpha-Neo KO mice. Furthermore, ERalpha-Neo KO mice may help in the elucidation of the specific functions of full-length ERalpha (ERalpha66) and ERalpha46, both shown to be physiologically generated in vivo.
Assuntos
Estradiol/farmacologia , Óxido Nítrico/biossíntese , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Processamento Alternativo , Animais , Aorta/metabolismo , Western Blotting , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Receptor alfa de Estrogênio , Éxons , Feminino , Hipertrofia , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Modelos Genéticos , Mutagênese Insercional , Tamanho do Órgão , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/patologiaRESUMO
Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ERalpha or ERbeta in mice. Behavior consistent with increased anxiety was observed principally in ERbeta mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression in medial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERbeta-mediated estrogen signaling in the processing of emotional behavior.
Assuntos
Ansiedade/fisiopatologia , Plasticidade Neuronal/fisiologia , Receptores de Estrogênio/fisiologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Tonsila do Cerebelo/fisiologia , Animais , Ansiedade/metabolismo , Comportamento Animal , Eletrofisiologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Estrogênio/genética , Serotonina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.
Assuntos
Carcinoma Embrionário/patologia , Carcinoma Embrionário/fisiopatologia , Receptores do Ácido Retinoico/fisiologia , Retinoides/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Embrionário/tratamento farmacológico , Carcinoma Embrionário/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/genética , Engenharia Genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Receptores do Ácido Retinoico/deficiência , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RAR gamma (A alpha/A gamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 A alpha/A gamma embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 A alpha/A gamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RAR alpha and RAR beta (A alpha/A beta mutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an A alpha/A beta-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an A alpha/A gamma-like phenotype. Altogether, our data suggest that RAR alpha and/or RAR gamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RAR beta is subsequently involved in setting up the caudal boundary of this territory.
Assuntos
Proteínas Aviárias , Padronização Corporal , Proteínas Oncogênicas , Receptores do Ácido Retinoico/fisiologia , Rombencéfalo/embriologia , Animais , Proteínas de Ligação a DNA/genética , Proteína 2 de Resposta de Crescimento Precoce , Desenvolvimento Embrionário e Fetal , Proteínas de Homeodomínio/genética , Fator de Transcrição MafB , Camundongos , Morfogênese , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/genética , Receptor gama de Ácido RetinoicoRESUMO
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.
Assuntos
Epiderme/fisiologia , Queratinócitos/fisiologia , Peroxissomos/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Cicatrização/genética , Animais , Adesão Celular , Divisão Celular , Movimento Celular , Colágeno/metabolismo , Elastina/metabolismo , Células Epidérmicas , Folículo Piloso/lesões , Queratinócitos/citologia , Macrófagos/citologia , Camundongos , Camundongos Mutantes , Neutrófilos/citologia , Pele/lesões , Acetato de Tetradecanoilforbol/farmacologia , Regulação para CimaRESUMO
Lethal toxicity levels of two inorganic water pollutants, chromium (Cr6+) and arsenic (As3+), were determined toward six freshwater macroinvertebrate species collected from a single field site. Crustaceans were represented by two amphipod species, an epigean one (Gammarus fossarum) and a hypogean one (Niphargus rhenorhodanensis), and by an isopod species (Asellus aquaticus). There were two insect larvae, Heptagenia sulphurea (Ephemeroptera) and Hydropsiche pellucidula (Trichoptera) and a snail, Physa fontinalis. Median lethal concentrations (LC50s) were determined over 96-h and 240-h periods for chromium and over a 240-h period for arsenic. Arsenic bioaccumulation was studied, too. The macroinvertebrates tested showed a wide range of sensitivity and bioaccumulation. A comparison between 96-h and 240-h experiments demonstrated that there was an increase in toxicity values following a longer time exposure for chromium. Also chromium was more toxic toward crustaceans than arsenic; conversely, arsenic was more toxic for the insect larvae and snail tested here. The lethal concentrations determined for the two metals were discussed and compared to results from other toxicity studies. The use of such macroinvertebrates, collected in the field and tested for longer exposure periods than within the standardized 96-h tests, should provide more suitable results for monitoring the general environmental quality of freshwater systems.
Assuntos
Arsênio/toxicidade , Carcinógenos Ambientais/toxicidade , Cromo/toxicidade , Crustáceos/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Arsênio/farmacocinética , Disponibilidade Biológica , Carcinógenos Ambientais/farmacocinética , Cromo/farmacocinética , Ecossistema , Monitoramento Ambiental , Dose Letal Mediana , Dinâmica Populacional , Distribuição Tecidual , Poluentes Químicos da Água/farmacocinéticaRESUMO
Recent advances in the field of in vitro chromatin assembly have led to in vitro transcription systems which reproduce in the test tube, in vivo characteristics of ligand-dependent transcriptional activation by nuclear receptors. Dissection of these systems has begun to provide us with information concerning the underlying molecular mechanisms. Through recruitment of coactivator proteins, nuclear receptors act first to remodel chromatin within the promoter region and then to recruit the transcriptional machinery to the promoter region in order to initiate transcription. Here we present a possible sequential mechanism for ligand-dependent transcriptional activation by nuclear receptors and discuss the in vitro and in vivo data that support this model.
Assuntos
Cromatina/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Transativadores/fisiologia , Transcrição Gênica/genética , Animais , Cromatina/metabolismo , Humanos , Modelos BiológicosRESUMO
Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Drosophila , Células-Tronco de Carcinoma Embrionário , Epitopos/genética , Heterocromatina/genética , Histonas/química , Histonas/genética , Técnicas In Vitro , Mamíferos , Camundongos , Dados de Sequência Molecular , Mutagênese/fisiologia , Células-Tronco Neoplásicas , Nucleossomos/genética , Nucleossomos/metabolismo , Oligopeptídeos , Peptídeos/genética , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Técnicas de Transferência de Genes , Proteínas Virais , Alelos , Animais , Antineoplásicos Hormonais/farmacologia , Núcleo Celular/metabolismo , Cruzamentos Genéticos , Citoplasma/metabolismo , Epiderme/metabolismo , Escherichia coli/enzimologia , Humanos , Integrases/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Recombinação Genética , Tamoxifeno/farmacologia , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor alpha. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf-Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.
Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Cromossomos Humanos Par 8/genética , Proteínas Nucleares/genética , Processamento Alternativo , Sequência de Aminoácidos , Sítios de Ligação , Northern Blotting , Neoplasias da Mama/patologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Feminino , Amplificação de Genes , Expressão Gênica , Genes/genética , Histona-Lisina N-Metiltransferase , Humanos , Hibridização in Situ Fluorescente , Íntrons , Dados de Sequência Molecular , Poli A/genética , Isoformas de Proteínas/genética , RNA/genética , RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.