RESUMO
The process by which the periderm transitions to stratified epidermis with the establishment of the skin barrier is unknown. Understanding the cellular and molecular processes involved is crucial for the treatment of human pathologies, where abnormal skin development and barrier dysfunction are associated with hypothermia and perinatal dehydration. For the first time, we demonstrate that retinoic acid (RA) levels are important for periderm desquamation, embryonic skin differentiation and barrier formation. Although excess exogenous RA has been known to have teratogenic effects, little is known about the consequences of elevated endogenous retinoids in skin during embryogenesis. Absence of cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1), a retinoic-acid-degrading enzyme, results in aberrant epidermal differentiation and filaggrin expression, defective cornified envelopes and skin barrier formation, in conjunction with peridermal retention. We show that these alterations are RA dependent because administration of exogenous RA in vivo and to organotypic skin cultures phenocopy Cyp26b1(-/-) skin abnormalities. Furthermore, utilizing the Flaky tail (Ft/Ft) mice, a mouse model for human ichthyosis, characterized by mutations in the filaggrin gene, we establish that proper differentiation and barrier formation is a prerequisite for periderm sloughing. These results are important in understanding pathologies associated with abnormal embryonic skin development and barrier dysfunction.
Assuntos
Sistema Enzimático do Citocromo P-450/deficiência , Pele/embriologia , Pele/metabolismo , Tretinoína/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Proteínas Filagrinas , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Knockout , Ácido Retinoico 4 Hidroxilase , Pele/efeitos dos fármacos , Pele/patologia , Tretinoína/farmacologiaRESUMO
Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.
Assuntos
Fosfatase Alcalina/metabolismo , Coenzima A/metabolismo , Glutationa Transferase/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Fosfatase Alcalina/efeitos dos fármacos , Bioensaio , Coenzima A/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Glutationa Transferase/genética , Humanos , Ligantes , Masculino , Coativador 2 de Receptor Nuclear , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Fatores de Transcrição/genética , Xenobióticos/metabolismo , Xenobióticos/farmacologiaRESUMO
The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.
Assuntos
Padronização Corporal/fisiologia , Encéfalo/embriologia , Sistema Enzimático do Citocromo P-450/fisiologia , Crista Neural/citologia , Animais , Encéfalo/anormalidades , Encéfalo/citologia , Movimento Celular/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Família 26 do Citocromo P450 , Mesencéfalo/citologia , Mesencéfalo/embriologia , Camundongos , Camundongos Knockout , Crista Neural/embriologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Ácido Retinoico 4 Hidroxilase , Transdução de Sinais , Crânio/citologia , Crânio/embriologia , Tretinoína/fisiologiaRESUMO
Germ cells in the mouse embryo can develop as oocytes or spermatogonia, depending on molecular cues that have not been identified. We found that retinoic acid, produced by mesonephroi of both sexes, causes germ cells in the ovary to enter meiosis and initiate oogenesis. Meiosis is retarded in the fetal testis by the action of the retinoid-degrading enzyme CYP26B1, ultimately leading to spermatogenesis. In testes of Cyp26b1-knockout mouse embryos, germ cells enter meiosis precociously, as if in a normal ovary. Thus, precise regulation of retinoid levels during fetal gonad development provides the molecular control mechanism that specifies germ cell fate.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Células Germinativas/fisiologia , Meiose , Oogênese , Transdução de Sinais , Espermatogênese , Tretinoína/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Hibridização In Situ , Óperon Lac , Masculino , Mesonefro/metabolismo , Camundongos , Camundongos Knockout , Naftalenos/farmacologia , Ovário/embriologia , Ovário/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Ácido Retinoico 4 Hidroxilase , Células de Sertoli/enzimologia , Caracteres Sexuais , Testículo/embriologia , Testículo/metabolismo , Técnicas de Cultura de Tecidos , Tretinoína/farmacologiaRESUMO
Nuclear receptors play important roles in the maintenance of the endocrine system, regulation of organ differentiation, and fetal development. Endocrine disruptors exert their adverse effects by disrupting the endocrine system via various mechanisms. To assess the effects of endocrine disruptors on nuclear receptors, we developed a high-throughput method for identifying activators of nuclear receptors. Using this system, we found that triphenyltin and tributyltin were activators of peroxisome proliferator-activated receptor (PPAR) gamma and retinoid X receptor. Because PPARgamma is a master regulator of adipocyte differentiation, we assessed the effect of organotin compounds on preadipocyte 3T3-L1 cells. We found that organotin compounds stimulated differentiation of 3T3-L1 cells as well as expression of adipocyte marker genes.
Assuntos
Adipócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Compostos Orgânicos de Estanho/farmacologia , PPAR gama/fisiologia , Receptores X de Retinoides/fisiologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Clonagem Molecular , Escherichia coli , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Organotin compounds released from antifouling paints, such as tributyltin (TBT) and triphenyltin (TPT), are potent inducers of imposex (a superimposition of male genital tracts, such as penis and vas deferens, on females) in marine gastropods. Little is known about the induction mechanism of gastropod imposex. Here, we show that organotins bind the human retinoid X receptors (hRXRs) with high affinity and that injection of 9-cis retinoic acid (RA), the natural ligand of hRXRs, into females of the rock shell (Thais clavigera) induces the development of imposex. Cloning of the RXR homologue from T. clavigera revealed that the ligand-binding domain of rock shell RXR was very similar to vertebrate RXR and bound to both 9-cis RA and to organotins. These suggest that RXR plays an important role in inducing the development of imposex, namely, the differentiation and growth of male genital tracts in female gastropods.