RESUMEN
Mast cells are granular immunocytes that reside in the body's barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1-210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.
Asunto(s)
Comunicación Celular/inmunología , Cromogranina A/metabolismo , Cromograninas/metabolismo , Mastocitos/metabolismo , Vesículas Secretoras/metabolismo , Animales , Comunicación Celular/genética , Línea Celular , Línea Celular Tumoral , Cromogranina A/biosíntesis , Cromogranina A/genética , Cromogranina A/fisiología , Cromograninas/biosíntesis , Cromograninas/genética , Cromograninas/fisiología , Humanos , Leucemia de Mastocitos/genética , Leucemia de Mastocitos/inmunología , Leucemia de Mastocitos/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Mastocitoma/genética , Mastocitoma/inmunología , Mastocitoma/metabolismo , Ratones , Células PC12 , Ratas , Vesículas Secretoras/genética , Vesículas Secretoras/inmunología , Vesículas Secretoras/patología , Eliminación de SecuenciaRESUMEN
SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1 inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.
