Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells

Abstract

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

Fig 1. Upregulation of α₂δ-1 by LPS.

U87 cells were co-transfected with a vector containing the α₂δ-1 subunit promoter and the Renilla luciferase coding sequence (as a reporter gene), and the pRSV-βGal vector, which included the RSV promoter and the β-gal coding sequence to correct for differences related to transfection efficiency. Cells were incubated for 48 h in the absence and presence of the TLR-4 activator, LPS. B) Western blot assay performed on U87 cell lysates in the absence and presence of LPS. The image shows a representative assay of three performed separately. C) Densitometric analysis of protein α₂δ-1 normalized with respect to the expression of β actin and depicted as a fold change from the control. Asterisks denote statistically significant differences (P < 0.05) with respect to the control (Ctl).

Fig 2. Effect of LPS on the proliferation and migration of U87 cells.

A) Comparison of direct and automated cell counting of U87 cells maintained 48 h in culture in the presence and absence of the TLR-4 activator, LPS, as indicated. Cell counts were performed in parallel after treatment with C34, a specific inhibitor of TLR-4, and the vehicle (DMSO) in which it was dissolved. Values are expressed as a percent of control, and each bar represents the mean ± SE of triplicate determinations in 3 separate experiments. B) Cell wound healing assays showing the ability of U87 cells to migrate in culture in the absence and presence of LPS, as indicated. The lower right panel shows a graph of the quantification of the wound area. C) Transwell migration assays of U87 cells in the presence of the activator (LPS) and the inhibitor (C34) of TLR-4, as listed. Typical images from three separate experiments are shown. D, Comparative analysis of transwell migration assay results of U78 cells as in C. Results represent the mean ± SEM of 3 independent experiments. *P < 0.05 compared to untreated cells.

Fig 3. Effect of α₂δ-1 overexpression on the proliferation and migration of U87 cells.

A) Western blot analysis of U87 cell lysates in control and α₂δ-1 transiently transfected cells. The image in the upper panel shows a representative assay of three performed separately. The lower panel shows the densitometric analysis of the protein α₂δ-1 normalized with respect to the expression of β actin and depicted as a fold change from the control. Asterisks denote a statistically significant difference (P < 0.05) with respect to the control (Ctl). B) Comparison of direct and automated cell counting of U87 cells maintained in culture after α₂δ-1 transfection in the presence and absence of the agonist and the antagonist of TLR-4, LPS, and C34, respectively. Values are expressed as the number of cells in each experimental condition, and each bar represents the mean ± SEM of triplicate determinations in 3 separate experiments. *P < 0.05 versus α₂δ-1 overexpressing cells (second bar from the left). ⁋P < 0.05 versus untransfected cells. C) Transwell migration assays of U87 cells in the presence of the TLR-4 activator (LPS). The left panel shows representative images from three separate experiments. The right panel shows the comparative analysis of the mean ± SEM of 3 transwell independent experiments. *P < 0.01 compared to untreated cells.

Fig 4. Effect of gabapentin on U87 cell migration.

A) Representative images from three separate experiments of transwell migration assays in the presence or absence of the TLR-4 activator (LPS) and the antagonist of the α₂δ-1 subunit (gabapentin). B) Comparative analysis of the mean ± SEM of 3 transwell independent experiments. *P < 0.05 compared to untreated cells.

Fig 5. Effect of thrombospondin-1 on proliferation and migration.

A) Comparison of the proliferation of U87 cells in response to various doses of thrombospondin-1 as indicated. B) Representative images from three separate experiments of transwell migration assays in the presence or absence of the thrombospondin-1 (TSP; 1 nM), an activator of the α₂δ-1 subunit, after 48 h of incubation. C) Comparative analysis of three transwell independent experiments as in B. Data show the mean ± SEM of three separate experiments. The asterisk denotes statistically significant differences with respect to the control condition (*P < 0.05 compared to untreated cells).

Fig 6. Effect of α₂δ-1, Sp1, or TLR-4 overexpression on the proliferation and migration of U87 cells.

A) Comparison of cell number after transient transfection of U87 cells with α₂δ-1 subunit, Sp1, and TRL-4 as assessed by direct cell counting. Values are expressed as the percentage of untransfected cells (Ctl), and each bar represents the mean ± SEM of triplicate determinations in 3 separate experiments. *P < 0.05 versus untransfected cells. B) Transwell migration assays of control and cells transiently transfected with α₂δ-1, Sp1, or TLR-4 cDNA clones. Representative images from three independent experiments are shown. C) Comparative analysis of the mean ± SEM of 3 transwell separate experiments as in B. *P < 0.01 compared to untransfected cells.

Fig 7. Effect of α₂δ-1, Sp1, or TLR-4 silencing on the proliferation and migration of U87 cells.

A) Comparison of cell number after α₂δ-1, Sp1, and TRL-4 knockdown with specific siRNAs of U87 cells as assessed by direct cell counting. Values are expressed as the percentage of untransfected cells (Ctl), and each bar represents the mean ± SEM of triplicate determinations in 3 separate experiments. *P < 0.01 versus untransfected cells. B) Transwell migration assays of control and cells transiently transfected with specific α₂δ-1, Sp1, or TLR-4 siRNAs. Representative images from three separate experiments are shown. C) Comparative analysis of the mean ± SEM of 3 transwell independent experiments as in B. *P < 0.05 compared to untransfected cells.

Fig 8. Effect of the knockdown and overexpression of TLR-4 and Sp1 on α₂δ-1 expression.

A) Western blot assays performed on U87 cell lysates in the control condition and after transfection with the Sp1, TLR-4, and α₂δ-1 specific siRNAs. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of α₂δ-1 protein. Data are shown as mean + SEM of three independent experiments in triplicate. B) Western blot assays performed on U87 cells in the control condition and after transfection with the Sp1, TLR-4, and α₂δ-1 cDNA clones. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of α₂δ-1 protein. Data are shown as mean + SEM of 3 independent experiments in triplicate. *P < 0.05 compared to untransfected cells.

Fig 9. Effect of NF-кB inhibition on Sp1 and α₂δ-1 expression.

A) Western blot assays performed on U87 cell lysates in the control condition and after treatment with the TLR-4 agonist LPS and the transcription factor NF-кB inhibitor PDTC. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of α₂δ-1 protein. Data are shown as mean + SEM of 3 independent experiments in triplicate. B) Sp1 Western blot assays performed on U87 cells in the conditions as in A. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of Sp1 protein. Data are shown as mean ± SEM of 3 independent experiments in triplicate. *P < 0.05 compared to untreated cells.