Dexamethasone Treatment Increases the Intracellular Calcium Level Through TRPV6 in A549 Cells

Abstract

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.

Keywords: A549 cells; TRPV6; calcium; dexamethasone.

Figure 1

The intracellular calcium levels under dexamethasone treatment for 6, 12, and 24 h. A549 cells were seeded at 3 × 10⁵ in coverglass-bottom dish for microscopy and co-transfected with 0.5 μg of pGP-CMV-GCaMP6f and CMV-R-GECO1.2 then 1.5 μL of Lipofectamine in 50 μL of Opti-MEM medium at room temperature for 5 min. Intracellular calcium levels were increased by dexamethasone (DEX) treatment for 6, 12, and 24 h after pGP-CMV-GCaMP6f and CMV-R-GECO1.2 transfection. Increased intracellular calcium levels by DEX were determined by using lionheart microscopy. (A) Expression of pGP-CMV-GCaMP6f (green) and CMV-R-GECO1.2 (red) detected after 6, 12, and 24 h co-transfection. Nuclei were stained with Hoechst (blue). (B) The green fluorescent protein (GFP) intensities by pGP-CMV-GCaMP6f (green) were plotted for each of the 6, 12, and 24 h groups. (C) The red fluorescent protein (RFP) intensities by CMV-R-GECO1.2 (red) were plotted for each of the 6, 12, and 24 h groups. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus DEX. Scale bar, 25 μm.

Figure 2

Intracellular calcium response affected by dexamethasone. Intracellular calcium response was increased by DEX at 24 h after attachment. DEX and a glucocorticoid receptor antagonist RU486 affected intracellular calcium response. Comparison of intracellular calcium response determined by confocal microscopy. DEX, 10⁻⁸ M of dexamethasone; DEX + RU, 10⁻⁸ M of dexamethasone treated with 10⁻⁶ M of RU486; RU, 10⁻⁶ M of RU486.

Figure 3

Regulation of calcium-processing gene expression by dexamethasone in A549 cells. Effect of DEX and its antagonist (RU486) on a transcriptional level of (A) transient receptor potential cation channel subfamily V member 6 (TRPV6) by real-time PCR, (B) TRPV6 by Western blotting, (C) sodium-calcium exchanger (NCX1) by real-time PCR, (D) NCX1 by Western blotting, (E) plasma membrane calcium ATPase 1 (PMCA1) by real-time PCR, and (F) PMCA1 by Western blotting. The mRNA level was measured by performing real-time PCR and was normalized by GAPDH. Quantification of protein levels determined by using NIH ImageJ software. Protein level was normalized by β-actin. * p < 0.05 versus Control; ** p < 0.01 versus Control; *** p < 0.001 versus control; ^# p < 0.05 versus DEX; ^## p < 0.01 versus DEX; ^### p < 0.001 versus DEX.

Figure 4

Effect of EGTA on calcium-processing genes in A549 cells. Effect of EGTA and DEX on the transcriptional level of (A) transient receptor potential cation channel subfamily V member 6 (TRPV6), (B) sodium-calcium exchanger (NCX1), and (C) plasma membrane calcium ATPase 1 (PMCA1) by real-time PCR. The mRNA level was measured by performing real-time PCR and was normalized by GAPDH. *** p < 0.001 versus control; ^## p < 0.01 versus EGTA; ^### p < 0.001 versus EGTA.

Figure 5

Regulation of surfactant protein and mucin genes expressions by dexamethasone in A549 cells. Effect of DEX and its antagonist (RU486) on the transcriptional level of (A) surfactant protein A (SFTPA1), (B) surfactant protein B (SFTPB), (C) surfactant protein C (SFTPC), (D) surfactant protein D (SFTPD), (E) mucin 1 (MUC1), and (F) mucin 5AC (MUC5AC) by real-time PCR. The mRNA level was measured by performing real-time PCR and was normalized by GAPDH. ** p < 0.01 versus Control; *** p < 0.001 versus control; ^# p < 0.05 versus DEX; ^### p < 0.001 versus DEX.

Figure 6

Effect of EGTA on surfactant protein and mucin genes in A549 cells. Effect of EGTA and DEX on the transcriptional level of (A) surfactant protein A (SFTPA), (B) surfactant protein B (SFTPB), (C) surfactant protein C (SFTPC), (D) surfactant protein D (SFTPD), (E) mucin 1 (MUC1), and (F) mucin 5AC (MUC5AC) by real-time PCR. The mRNA level was measured by performing real-time PCR and was normalized by GAPDH. ** p < 0.01 versus Control; *** p < 0.001 versus control; ^# p < 0.05 versus EGTA; ^## p < 0.01 versus EGTA; ^### p < 0.001 versus EGTA.

Figure 7

A simplified schematic diagram of the DEX effect on the intracellular calcium level through TRPV6 in A549 cells. DEX influences the intracellular calcium level through TRPV6 and affects the pulmonary surfactant genes (SFTPA, -B, -C, and -D) and the secreted airway mucin genes (MUC1 and MUC5AC) in A549 cells. ↑, increase; ↓, decrease.