RESUMEN
Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 µM. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl D-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transportador 2 de Aminoácidos Excitadores/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Riluzol/farmacología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Transportador 2 de Aminoácidos Excitadores/genética , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Respuesta al Choque Térmico/efectos de los fármacos , Respuesta al Choque Térmico/genética , Humanos , Neuronas/citología , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Elementos de Respuesta/fisiología , Células Madre/citología , Factores de Transcripción/genéticaRESUMEN
Osteopontin (OPN) is both a matrix protein in mineralized tissues and a cytokine, and it has a pivotal role in osteoclast-mediated bone resorption. Here, using a proprietary hydroxyapatite substitute for bone mineral (Osteologic discs), we investigated the requirement for OPN in mineral resorption. Resorption pits formed in the Osteologic discs, revealed by staining with silver nitrite (Von Kossa stain), were analyzed using the NIH Image J program, which can determine the number of pits formed per unit area, their average size, and the fractional area resorbed. After a preincubation of bone marrow cells from OPN -/- and OPN +/+ mice with M-CSF to allow the multiplication of osteoclast precursors on cell culture plastic, osteoclast formation on both Osteologic discs and standard cell culture plates was induced with soluble receptor activator of NFkappaB ligand, sRANKL. We did not detect a dramatic difference in osteoclast formation between OPN +/+ and OPN -/- cells, as judged by staining for tartrate-resistant acid phosphatase in osteoclasts formed on cell culture plastic, nor was there a significant difference in the ability of the osteoclasts to form resorption pits in the Osteologic discs. Additionally, none of six different anti-OPN monoclonal antibodies had a significant and reproducible effect on the formation or subsequent functioning of the OPN+/+ osteoclasts. These studies suggest that, in contrast to what has been found for normal bone, the efficiency of dissolution of a ceramic, protein-free (excepting protein adsorbed from the culture medium) hydroxyapatite/tri-calcium phosphate substrate by osteoclasts is not substantially enhanced by endogenous or exogenous OPN.
Asunto(s)
Resorción Ósea/patología , Durapatita/metabolismo , Osteoclastos/patología , Sialoglicoproteínas/fisiología , Animales , Anticuerpos/farmacología , Células de la Médula Ósea/patología , Células Cultivadas , Glicoproteínas/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Noqueados , Osteoclastos/efectos de los fármacos , Osteopontina , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis Tumoral , Sialoglicoproteínas/genética , Sialoglicoproteínas/inmunologíaRESUMEN
During mouse embryo implantation, trophoblast invasion is controlled in part by a balance of trophoblast-derived proteinases and uterine decidual proteinase inhibitors. Our work has focused on cystatin C, the secreted inhibitor of cathepsins B and L. We have previously shown that cystatin C is synthesized by the uterine decidua and localized to the cells in close contact with the trophoblast during implantation in vivo. In the work reported here we have established that decidualizing cultures show a similar upregulation of cystatin C. Using Northern and Western blotting and immunolocalization techniques both cystatin C mRNA and secreted protein increased with the morphological differentiation of stromal or decidual capsule cultures. In an effort to understand the regulation of cystatin C expression, decidual cells were analyzed under various culture conditions. Cystatin C expression was upregulated by increased cell density and by the presence of serum in the media. The growth factors TGF-beta(1) and EGF were found to induce cystatin C to levels comparable to serum stimulation. Co-culture with ectoplacental cones (EPCs) likewise induced expression and resulted in the localization of cystatin at the decidua:trophoblast interface. This work shows that decidualizing cultures are a good system to study cystatin C expression and that the expression is controlled in part by TGF-beta(1) and EGF signaling.