RESUMEN
The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.
Asunto(s)
Ciclo Celular/genética , Diferenciación Celular/genética , Melaninas/genética , Melanoma Experimental/genética , Proteínas del Tejido Nervioso/genética , Adenoviridae/genética , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular , Diferenciación Celular/fisiología , Línea Celular Tumoral , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente Directa , Colorantes Fluorescentes/metabolismo , Vectores Genéticos , Inmunohistoquímica , Hibridación in Situ , Indoles/metabolismo , Melaninas/fisiología , Melanoma Experimental/fisiopatología , Ratones , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso/fisiología , Fenotipo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Transducción GenéticaRESUMEN
The hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a highly conserved protein that is expressed in developing and regenerating tissues. In this study, Hn1 expression was evaluated in human and murine malignant gliomas. Hn1 mRNA and protein were detected in the murine GL261 glioma cell line and in GL261 brain tumors in vivo. HN1 is also expressed in human U118MG and U87MG cell lines. Evaluation of human brain tumors using an anti-Hn1 polyclonal antibody detected strong immunoreactivity in high-grade (WHO III and IV) malignant gliomas. The rate of GL261 cell proliferation in vitro was unaltered by Hn1 depletion using an anti-Hn1 siRNA. However, tumors established from Hn1-depleted GL261 cells formed significantly smaller volumes than those established from control-treated cells. These data suggest a role for Hn1 in the biology of malignant brain tumors.