RESUMEN
BACKGROUND: Transforming growth factor (TGF)-ß1 exerts inhibitory effects on keratinocyte proliferation. OBJECTIVES: To examine whether Smad7, a known inhibitor of TGF-ß1 signalling, is involved in psoriasis-associated keratinocyte hyperproliferation. METHODS: Smad7 was evaluated in skin sections of patients with psoriasis and healthy controls and in mice with Aldara-induced skin pathology by real-time polymerase chain reaction and immunohistochemistry. To assess whether Smad7 positively regulates in vivo keratinocyte growth, mice treated with Aldara received daily cutaneous administration of Smad7 antisense oligonucleotide (AS). Keratin (K)6 and K16, cell-cycle-associated factors, cell-cycle and cell proliferation were evaluated in HaCaT cells either treated with Smad7 AS or transfected with Smad7 plasmid and in mice given Smad7 AS. RESULTS: Smad7 was highly expressed in keratinocytes of patients with psoriasis and of mice treated with Aldara. In HaCaT cells, Smad7 knockdown inhibited cell growth, reduced K6 and K16 expression and promoted accumulation of cells in the S-phase of the cell cycle. Smad7-deficient keratinocytes exhibited reduced levels of CDC25A protein, a phosphatase that facilitates progression of cells through the S-phase, and hyperphosphorylation of eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, Smad7 overexpression in HaCaT cells was followed by induction of K6 and K16 and increased cell proliferation. Topical application of Smad7 AS to Aldara-treated mice reduced epidermal thickness. CONCLUSIONS: Our data show that Smad7 is overexpressed in human and murine psoriasis and suggest a key role of this molecule in the control of keratinocyte proliferation.
Asunto(s)
Proliferación Celular/fisiología , Queratinocitos/patología , Psoriasis/patología , Proteína smad7/fisiología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Animales , Dermatitis/fisiopatología , Relación Dosis-Respuesta a Droga , Epidermis/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Proteína smad7/deficiencia , Regulación hacia Arriba/fisiologíaRESUMEN
P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.
Asunto(s)
Activación de Macrófagos , Macrófagos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Polaridad Celular , Regulación de la Expresión Génica , Imidazoles/farmacología , Interleucina-4/metabolismo , Lipopolisacáridos , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal/inmunología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
T cell differentiation to effector Th cells such as Th1 and Th2 requires the integration of multiple synergic and antagonist signals. Poly(ADP-ribosy)lation is a posttranslational modification of proteins catalyzed by Poly(ADP-ribose) polymerases (PARPs). Recently, many reports showed that PARP-1, the prototypical member of the PARP family, plays a role in immune/inflammatory responses. Consistently, its enzymatic inhibition confers protection in several models of immune-mediated diseases, mainly through an inhibitory effect on NF-κB (and NFAT) activation. PARP-1 regulates cell functions in many types of immune cells, including dendritic cells, macrophages, and T and B lymphocytes. Our results show that PARP-1KO cells displayed a reduced ability to differentiate in Th2 cells. Under both nonskewing and Th2-polarizing conditions, naïve CD4 cells from PARP-1KO mice generated a reduced frequency of IL-4(+) cells, produced less IL-5, and expressed GATA-3 at lower levels compared with cells from wild type mice. Conversely, PARP-1 deficiency did not substantially affect differentiation to Th1 cells. Indeed, the frequency of IFN-γ (+) cells as well as IFN-γ production, in nonskewing and Th1-polarizing conditions, was not affected by PARP-1 gene ablation. These findings demonstrate that PARP-1 plays a relevant role in Th2 cell differentiation and it might be a target to be exploited for the modulation of Th2-dependent immune-mediated diseases.