RESUMEN
BACKGROUND: Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in non-cutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis. METHODS: Time- and context-dependent expression of PPL in various mouse models of hepatic and renal disorders were examined by immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions. RESULTS: The hepatic expression of PPL was significantly decreased in Fxr-/- mice. In contrast, the expression was dramatically increased during cholestasis, with massive PPL accumulation observed at the boundaries of hepatocytes in wild-type mice. Interestingly, the hepatic accumulation of PPL resulting from cholestasis was reversible. In addition, similar accumulation of PPL at cellular boundaries was found in epithelial cells around renal tubules upon ureteral obstruction. CONCLUSIONS: PPL may be involved in the temporal accommodation to fluid stasis in different tissues. Further examination of the roles for PPL may lead to the discovery of a novel mechanism for cellular protection by cytolinkers that is applicable to many tissues and in many contexts.
Asunto(s)
Colestasis/metabolismo , Hepatopatías/metabolismo , Hígado/metabolismo , Plaquinas/metabolismo , Animales , Colestasis/inducido químicamente , Colestasis/complicaciones , Concanavalina A , Células Epiteliales , Expresión Génica , Hepatocitos , Túbulos Renales/metabolismo , Hepatopatías/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plaquinas/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Obstrucción Ureteral/metabolismoRESUMEN
The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RAS(V12G) in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses, and increased T helper type 1 (Th1)/Th17 and T cell type 1 (Tc1)/Tc17 skin infiltration. The majority of skin-infiltrating CD8(+) T cells coexpressed IFN-γ and IL-17A. When RAS was expressed on a Rag1-/- background, microabscess formation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation were suppressed. Depletion of CD8(+), but not CD4(+), T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17(+) γδ T cells, and epidermal hyperproliferation to levels similar to a Rag1-/- background. Reconstitution of Rag1-/- inducible RAS mice with purified CD8(+) T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ, but not of IL-17A, in CD8(+) T-cell-reconstituted Rag1-/- mice expressing RAS blocked CD8-mediated skin inflammation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation. These results show that CD8(+) T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.
Asunto(s)
Linfocitos T CD8-positivos/patología , Interferón gamma/inmunología , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Psoriasis/inmunología , Psoriasis/patología , Transducción de Señal/inmunología , Absceso/inmunología , Absceso/patología , Animales , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/patología , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Femenino , Interferón gamma/metabolismo , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Transgénicos , Neutrófilos/inmunología , Neutrófilos/patología , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Psoriasis/genética , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Recent work indicates that PPARalpha is required for perfluorooctanoic acid (PFOA)-induced postnatal lethality resulting from prenatal exposure. The present study tested the hypothesis that relatively modest activation of PPARalpha during prenatal development will cause postnatal lethality, similar to that observed with PFOA, a relatively low affinity PPARalpha agonist. Female wild-type and Pparalpha-null mice were mated overnight with males of the same genotype. The presence of a copulatory plug on the morning after mating was indicative of pregnancy and considered gestation day (GD) 0. Plugged female mice were fed either a control diet or one containing clofibrate (0.5%) or Wy-14,643 (0.005%) until GD18 or until parturition. Mice were examined on GD18 or on postnatal day (PND) 20 following the prenatal exposure period. Dietary administration of clofibrate or Wy-14,643 did not affect maternal weight or weight gain, the average number of implantations, the percentage of litter loss, the average number of live/dead fetuses, average crown-rump length, or the average fetal weight on GD18 in either genotype. An increase in relative maternal liver weight and elevated expression of PPARalpha target genes in maternal and fetal livers on GD18 were observed, indicative of PPARalpha-dependent changes in both the maternal and fetal compartments. However, no defects in postnatal development were observed by either clofibrate or Wy-14,643 in either genotype by PND20. These results demonstrate that relatively low level activation of PPARalpha by clofibrate or Wy-14,643 during prenatal development does not cause postnatal lethality.
Asunto(s)
Clofibrato/farmacología , PPAR alfa/agonistas , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Animales , Femenino , Genotipo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Tamaño de los Órganos/efectos de los fármacos , PPAR alfa/genética , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
The availability of high-affinity agonists for peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) has led to significant advances in our understanding of the functional role of PPARbeta/delta. In this study, a new PPARbeta/delta antagonist, 4-chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), was characterized using in vivo and in vitro models. Orally administered GSK3787 caused antagonism of 4-[2-(3-fluoro-4-trifluoromethyl-phenyl)-4-methyl-thiazol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-acetic acid (GW0742)-induced up-regulation of Angptl4 and Adrp mRNA expression in wild-type mouse colon but not in Pparbeta/delta-null mouse colon. Chromatin immunoprecipitation (ChIP) analysis indicates that this correlated with reduced promoter occupancy of PPARbeta/delta on the Angptl4 and Adrp genes. Reporter assays demonstrated antagonism of PPARbeta/delta activity and weak antagonism and agonism of PPARgamma activity but no effect on PPARalpha activity. Time-resolved fluorescence resonance energy transfer assays confirmed the ability of GSK3787 to modulate the association of both PPARbeta/delta and PPARgamma coregulator peptides in response to ligand activation, consistent with reporter assays. In vivo and in vitro analysis indicates that the efficacy of GSK3787 to modulate PPARgamma activity is markedly lower than the efficacy of GSK3787 to act as a PPARbeta/delta antagonist. GSK3787 antagonized GW0742-induced expression of Angptl4 in mouse fibroblasts, mouse keratinocytes, and human cancer cell lines. Cell proliferation was unchanged in response to either GW0742 or GSK3787 in human cancer cell lines. Results from these studies demonstrate that GSK3787 can antagonize PPARbeta/delta in vivo, thus providing a new strategy to delineate the functional role of a receptor with great potential as a therapeutic target for the treatment and prevention of disease.
Asunto(s)
Receptores Activados del Proliferador del Peroxisoma/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR alfa/farmacología , PPAR gamma/genética , PPAR gamma/metabolismo , PPAR gamma/farmacología , PPAR-beta/genética , PPAR-beta/metabolismo , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/farmacología , Tiazoles , Activación Transcripcional , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Perfluorobutyrate (PFBA) is a short chain perfluoroalkyl carboxylate that is structurally similar to perfluorooctanoate. Administration of PFBA can cause peroxisome proliferation, induction of peroxisomal fatty acid oxidation and hepatomegaly, suggesting that PFBA activates the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In this study, the role of PPAR-alpha in mediating the effects of PFBA was examined using PPAR-alpha null mice and a mouse line expressing the human PPAR-alpha in the absence of mouse PPAR-alpha (PPAR-alpha humanized mice). PFBA caused upregulation of known PPAR-alpha target genes that modulate lipid metabolism in wild-type and PPAR-alpha humanized mice, and this effect was not found in PPAR-alpha null mice. Increased liver weight and hepatocyte hypertrophy were also found in wild-type and humanized PPAR-alpha mice treated with PFBA, but not in PPAR-alpha null mice. Interestingly, hepatocyte focal necrosis with inflammatory cell infiltrate was only found in wild-type mice administered PFBA; this effect was markedly diminished in both PPAR-alpha null and PPAR-alpha humanized mice. Results from these studies demonstrate that PFBA can modulate gene expression and cause mild hepatomegaly and hepatocyte hypertrophy through a mechanism that requires PPAR-alpha and that these effects do not exhibit a species difference. In contrast, the PPAR-alpha-dependent increase in PFBA-induced hepatocyte focal necrosis with inflammatory cell infiltrate was mediated by the mouse PPAR-alpha but not the human PPAR-alpha. Collectively, these findings demonstrate that PFBA can activate both the mouse and human PPAR-alpha, but there is a species difference in the hepatotoxic response to this chemical.