RESUMO
Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Células Epidérmicas/metabolismo , Espermina/metabolismo , Adenosilmetionina Descarboxilase/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epidérmicas/patologia , Técnicas de Silenciamento de Genes , Humanos , Fator 4 Semelhante a Kruppel/metabolismo , Camundongos , Poliaminas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Movimento Celular/genética , Epiderme/fisiologia , Queratinócitos/fisiologia , Cicatrização , Ferimentos e Lesões/metabolismo , Citoesqueleto de Actina/metabolismo , Adenosilmetionina Descarboxilase/genética , Biópsia , Sinalização do Cálcio , Células Cultivadas , Humanos , RNA Interferente Pequeno/genética , Reepitelização/genética , Espermina/metabolismo , Regulação para Cima , Ferimentos e Lesões/genéticaRESUMO
Regulation of gene expression is essential to enable embryonic stem cells (ESCs) to either self-renew or to differentiate. Translational regulation of mRNA plays a major role in regulating gene expression and has been shown to be important for ESC differentiation. Sucrose gradients can be used to separate mRNAs based on the number of associated ribosomes and this can be used as a readout of the rate of translation. Following centrifugation through a sucrose gradient, mRNAs can be recovered, purified, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to determine their ribosomal load in different cell states. Here, we describe how to differentiate mouse ESCs to Neural Precursor Cells (NPCs) and analyze the rate of translation of individual mRNAs by qRT-PCR following polysome fractionation.