- 作者: Maja Žugec, Borut Furlani, Maria J. Castañon, Boštjan Rituper, Irmgard Fischer, Giuseppe Broggi, Rosario Caltabiano, Giuseppe M. V. Barbagallo, Michelino Di Rosa, Daniele Tibullo, Rosalba Parenti, Nunzio Vicario, Saša Simčič, Victorio Martin Pozo Devoto, Gorazd B. Stokin, Gerhard Wiche, Jernej Jorgačevski, Robert Zorec & Maja Potokar
- 作者服務機構: 1.Celica Biomedical, Ljubljana, Slovenia 2.Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy 3.Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 4.Department of Medical and Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, University of Catania, Catania, Italy 5.Department of Neurology, Gloucestershire Royal Hospital, Gloucestershire NHS Foundation Trust, Gloucester, UK 6.Institute for Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic 7.Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 8.International Clinical Research Center (ICRC), St. Anne’s University Hospital in Brno, 625 00, Brno, Czech Republic 9.Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 10.Max Perutz Laboratories, Department of Biochemistry and Cell Biology, University of Vienna, Vienna, Austria
- 中文摘要:
- 英文摘要:
Background
The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes.
Methods
In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively.
Results
A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines.
Conclusions
Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs. - 中文關鍵字:
- 英文關鍵字: Astrocyte, Glioblastoma, Plectin, Aquaporin 4, Intermediate flaments, Cytoskeleton, Edema, Cell volume, Cell migration