RESUMEN
We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.
Asunto(s)
Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Glioma/patología , Proteínas de Choque Térmico HSP27/metabolismo , Osteonectina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adhesión Celular/genética , Ensayos de Migración Celular/métodos , Movimiento Celular/genética , Factor de Crecimiento Epidérmico/genética , Células Epiteliales/metabolismo , Glioma/genética , Glioma/metabolismo , Proteínas de Choque Térmico , Humanos , Sistema de Señalización de MAP Quinasas , Chaperonas Moleculares , Osteonectina/deficiencia , Osteonectina/genética , Fosforilación , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Secreted protein acidic and rich in cysteine (SPARC) regulates cell-extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion.
Asunto(s)
Movimiento Celular/fisiología , Glioma/patología , Proteínas de Choque Térmico/fisiología , Proteínas de Neoplasias/fisiología , Osteonectina/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/genética , Glioma/metabolismo , Proteínas de Choque Térmico HSP27 , Humanos , Chaperonas Moleculares , Invasividad Neoplásica/patología , Osteonectina/genética , Osteonectina/fisiología , Células Tumorales CultivadasRESUMEN
Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and promotes glioma invasion. We have shown by cDNA array analysis that SPARC upregulates membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) transcripts. To confirm these findings at the protein level and determine whether SPARC expression correlates with increased MMP activity, we used Western blot to assess the levels of MT1-MMP, and gelatin zymography to assess MMP-2 levels and activity. We also examined the expression, secretion, and cleavage of galectin-3, a target of MT1-MMP and MMP-2. Our data confirm that SPARC upregulates MT1-MMP levels and MMP-2 activity. There was also an increase in secreted galectin-3, as well as an increase in the proteolytically processed form of galectin-3. Previous studies have demonstrated that MT1-MMP, MMP-2, and galectin-3 are increased in gliomas. Our results suggest that their upregulation and activation may be a consequence of increased SPARC expression. These data provide a provisional mechanism whereby SPARC contributes to brain tumor invasion.