Abstract
Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.
Highlights
ClC-3 enhances migration of nasopharyngeal carcinoma cells [11], and pharmacological inhibition with NPPB2 demonstrates a requirement for chloride channels to support glioma invasion [8]
Glioma cells express three members of the ClC family, ClC-2, -3, and -5 [10], and we have found that ClC-3 in particular is a critical regulator of cell volume changes associated with the cell cycle [9]
Using a combination of biochemical and biophysical techniques, we found that calmodulin-dependent protein kinase II (CaMKII) phosphorylates ClC-3 from human glioma cells, leading to an activation of native ClC-3 channels
Summary
Cell Culture—D54 human glioma cells are a World Health Organization grade IV cell line derived from a glioblastoma and gifted to us by Dr D. After incubation in primary antibodies overnight at 4 °C, cells were washed with a 1:3 dilution of the blocking buffer in phosphate-buffered saline and incubated in secondary antibodies for 1 h at room temperature. After further washing with the diluted blocking buffer, cells were incubated with 4Ј,6-diamidino-2-phenylindole (DAPI) at 1:2000 for 5 min at room temperature. 1 l of CaMKII (500,000 units/ml) was diluted in 1ϫ CaMKII reaction buffer, 200 M ATP, 1.2 M calmodulin, and 2 mM CaCl2 This mixture was incubated at 37 °C for 10 min. Primary antibodies, including rabbit anti-ClC-3 (Alpha Diagnostic International; 1:1500), rabbit anti-CaMKII (Abcam; 1:5000), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (Abcam; 1:5000), were incubated for 1 h at room temperature. After four 10-min washes in TBST, blots were incubated with respective horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology; 1:1500) for 1 h at room temperature. One-way analysis of variance was determined, and all data are reported as mean Ϯ S.E. (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001)
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