CaD is specifically up-regulated by GCs and localizes along thick stress fibers. A, effects of DEX on the expression profiles of actin cytoskeletal genes. Real time qPCR of cDNAs made from A549 cells. The mRNA expression levels of actin-interacting proteins (CaD, tropomyosin 1, tropomyosin 2, fascin 1, filamin A, p34Arc, p21Arc, vinculin, zyxin, talin 1, paxillin, α-actinin 1, cortactin 1, β1-catenin, α1-integrin, β1-integrin, gelsolin, profilin 1, cofilin 1, β-actin, and tubulin α1a) are shown. The data were obtained from three independent experiments (mean ± S.D., ^*, p < 0.05; ^***, p < 0.001). B, GCs induced the up-regulation of the CaD protein in A549 cells. The cells were incubated with cortisol (CS) or dexamethasone (DEX) at the indicated concentrations for 48 h. The expression levels of CaD, α-tubulin, and GAPDH were determined by Western blot analysis. The CaD protein levels were quantified using ImageJ and normalized to α-tubulin expression. (mean ± S.D., ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001 compared with vehicle). C, confocal images of DEX-induced reorganization of the actin cytoskeleton. Vehicle- or DEX-treated A549 cells were stained with phalloidin (green) and propidium iodide (PI) (red). Bar, 50 μm. D, DEX-induced reorganization of the actin cytoskeleton. Vehicle- or DEX-treated A549 cells were stained with anti-CaD antibody (green in merged image) and phalloidin (red in merged image). Bar, 50 μm. E, DEX-induced stress fiber formation. Vehicle- or DEX-treated A549 cells were pretreated with 0.1% Triton X-100 in PBS and fixed, followed by staining with anti-CaD (red in merged image) and anti-non-muscle myosin heavy chain (green in merged image) antibodies. Bar, 50 μm. Areas within the insets are shown at higher magnification (right panels, Bar, 10 μm).

GR up-regulates the expression of CaD in A549 cells. A, effect of GR or SRF depletion on the DEX-induced up-regulation of CaD expression. A549 cells transfected with control siRNA, GR siRNA, or SRF siRNA were incubated with vehicle or 1 μm DEX for 48 h. The expression levels of GR, SRF, and CaD were measured by Western blot analysis. B, CaD mRNA expression in the GR- or SRF-depleted cells was measured by real time qPCR (mean ± S.D., ^**, p < 0.01). C, effect of the expression of GRΔC on the CaD mRNA level in A549 cells transiently transfected with pCS2+GRΔC-cFLAG or the empty vector. The cells were incubated for 24 h. The CaD mRNA levels were measured by real time qPCR (mean ± S.D., ^***, p < 0.001). D, expression levels of CaD and GRΔC in GFP- or GRΔC-cFLAG-expressing A549 cells after incubation with vehicle or 1 μm DEX for 48 h. E, cell morphology and CaD localization in GFP- and GRΔC-expressing A549 cells stained with anti-GFP or anti-FLAG (green in merged image), anti-CaD antibodies (red in merged image), and phalloidin (blue in merged image). Bar, 50 μm.

GR binds to the GRE-like sequences in the human CALD1 promoter in vivo and in vitro. A, ChIP assay of A549 cells treated with vehicle or 1 μm DEX for 2 h. The cell extracts were subjected to ChIP using the anti-GR antibody. The DNA fragments of the CALD1 promoter region containing the two GRE-like sequences were amplified by PCR. The SGK1 and GAPDH promoter regions were used as the positive and negative controls, respectively. B, DNA-binding assay of GR to GRE-like sequences. Each biotinylated DNA fragment containing a GRE-like sequence was incubated with in vitro-translated GR protein and collected on streptavidin beads. The DNA fragments with mutated GRE-like sequences were also used to ascertain the binding specificity of GR. DNA-bound GR was detected by Western blot with anti-GR antibody.

Effects of forced expression of GFP-CaD on cell migration and contraction. A, effect of forced expression of GFP-CaD on cell migration. The migration activity of GFP- or GFP-CaD-expressing A549 cells was measured in a Transwell migration chamber with or without 1 μm DEX. (mean ± S.D., ^*, p < 0.05; ^***, p < 0.001). B, effect of forced expression of CaD on serum-induced cell contraction. GFP- or GFP-CaD-expressing A549 cells were treated with vehicle or 1 μm DEX for 48 h. The cells were starved with serum-free DMEM for 18 h and then stimulated with DMEM containing FBS for 80 min. Cell contraction was determined from reduction in cell area after serum stimulation (mean ± S.E.). (GFP-expressing cells: vehicle, n = 54; DEX, n = 39; blebbistatin, n = 39. GFP-CaD expressing cells: vehicle, n = 36). Bar, 50 μm.

Effect of forced expression or depletion of CaD on cell motility. A, cell motility was analyzed by tracing cell movement in time-lapse images, using DIAS software. GFP- or GFP-CaD-expressing A549 cells were incubated with vehicle or 1 μm DEX for 48 h, and representative centroid tracks of the cells are shown (25-min intervals for 5 h). Bar, 100 μm. B, A549 cells transfected with CaD siRNAs (CaD siRNA1 or CaD siRNA2) or control siRNA were incubated with vehicle or 1 μm DEX for 48 h, and representative centroid tracks of the cells are shown (25-min intervals for 5 h). Bar, 100 μm. C, migration speeds of GFP- or GFP-CaD-expressing A549 cells were calculated from the cell tracks (GFP-expressing cells: vehicle, n = 24; DEX, n = 27. GFP-CaD-expressing cells: vehicle, n = 26; DEX: n = 19) (mean ± S.D.). D, migration speeds of CaD-depleted A549 cells calculated from the cell tracks (control siRNA: vehicle, n = 22; DEX, n = 20. CaD siRNA1: vehicle, n = 37; DEX, n = 46. CaD siRNA2: vehicle, n = 21; DEX, n = 27) (mean ± S.D.). E, cumulative percentage plots of the migration speeds of GFP- and GFP-CaD-expressing A549 cells. GFP-expressing cells (•); GFP-CaD-expressing cells (⋄); -DEX (vehicle), solid lines; +DEX, dashed lines. F, cumulative percentage plots of the migration speeds of CaD-depleted and control A549 cells. Control siRNA-transfected cells (•); CaD siRNA1-transfected cells (Δ); CaD siRNA2-transfected cells (□); -DEX (vehicle), solid lines; and +DEX, dashed lines.

Effects of steroid hormones on cell migration and the actin cytoskeleton in A549 cells. A, effects of steroid hormones on cell migration. A549 cells were cultured with the indicated steroid hormones for 48 h and treated with mitomycin C for 2 h preceding the assay. Migration toward FBS in the presence of the steroids was measured in a Transwell migration chamber (mean ± S.D., ^**, p < 0.01 compared with vehicle; n.s., not significant). B, effect of steroid hormones on the actin cytoskeleton. A549 cells were treated with 1 μm of each steroid for 48 h and stained with phalloidin (red in merged image) and anti-vinculin antibody (green in merged image). Bar, 50 μm. C, effect of DEX on Rho activity. The Rho activity was determined by RBD-beads pulldown assay as described under “Experimental Procedures.” The amounts of total and GTP-RhoA protein were detected by Western blot with anti-RhoA antibody. The amounts of Rho downstream effectors, ROCK1, ROCK2, mDia1, cofilin 1, phospho(Ser-3)-cofilin 1, MLC2, phospho(Ser-19)-MLC2, and profilin were also analyzed by Western blot analysis using their corresponding antibodies.

GR antagonist, RU486, blocks DEX-induced up-regulation of CaD expression. A549 cells were incubated with vehicle or 1 μm DEX for the indicated times, and the expression levels of CaD mRNA (from real time qPCR) (A) and CaD protein (from Western blot) (B) are shown (mean ± S.D., ^**, p < 0.01, ^***, p < 0.001; n.s., not significant). C, A549 cells were pretreated with CHX for 2 h and incubated with vehicle or 1 μm DEX for 24 h in the presence of CHX. CaD mRNA was measured by real time qPCR. D and E, A549 cells incubated with 1 μm DEX in the presence of 10 μm RU486 or 10 μm RU28318. The expression of CaD mRNA (after 24 h (D); from real time qPCR) and CaD protein (after 48 h (E); from Western blot) are shown (mean ± S.D., ^***, p < 0.001). F, effect of RU486 or RU28318 on cell migration assayed in Transwell migration chambers. A549 cells were incubated with a combination of 1 μm DEX and 10 μm RU486 or RU28318 (mean ± S.D., ^***, p < 0.001). G, A549 cells were incubated with a combination of 1 μm DEX and 10 μm RU486 or 10 μm RU28318 for 48 h and stained with anti-CaD antibody (green in merged image) and phalloidin (red in merged image). Bar, 50 μm.

Identification of the GC-responsive region in the human CALD1 promoter. A, reporter assay was performed with the human fibroblast-type (-964 to +101) and HeLa-type (-1908 to +207) CALD1 promoters in A549 cells co-transfected with the luciferase reporter plasmid and pGL3β-gal plasmid. The cells were incubated with vehicle or 1 μm DEX for 24 h. The luciferase activities were normalized to the β-galactosidase activity (mean ± S.D., ^***, p < 0.001). B, effect of GR or MR antagonists on the fibroblast-type CALD1 promoter activity (mean ± S.D., ^**, p < 0.01, ^***, p < 0.001). C, effect of GR or SRF depletion on the fibroblast-type CALD1 promoter activity (mean ± S.D., ^**, p < 0.01; ^***, p < 0.001). D, luciferase activities in A549 cells co-transfected with reporter plasmids and the pCS2+GRΔC-cFLAG plasmid were measured after a 24 h incubation (mean ± S.D., ^**, p < 0.01). E, schematic diagram of deletion and mutation constructs of the CArG element or GRE-like sequences in the human fibroblast-type CALD1 promoter. F, GRΔC-responsiveness of the promoter activities of a series of reporter constructs (mean ± S.D., ^**, p < 0.01; ^***, p < 0.001; n.s., not significant).

Effect of forced expression of GFP-CaD on the actin cytoskeleton. A, forced expression of GFP-CaD. The expression levels of GFP-CaD, control GFP, and endogenous CaD in A549 transfectants were analyzed by Western blotting with anti-CaD or anti-GFP antibody. B, effect of DEX on the actin cytoskeleton of A549 cells stably expressing GFP or GFP-CaD. The cells were stained with anti-GFP (green in merged image) and anti-CaD (red in merged image) antibodies and phalloidin (blue in merged image). Bar, 50 μm. C, effect of forced expression of GFP-CaD on stress fiber stability to cytochalasin D (cytD). GFP- or GFP-CaD-expressing A549 cells were incubated with vehicle or 1 μm DEX for 48 h. The cells were treated with or without 0.2 μm cytD for 20 min, and stained with anti-GFP antibody and phalloidin. Bar, 50 μm. D, effect of DEX on focal adhesion assembly of A549 cells stably expressing GFP or GFP-CaD. The cells were stained with anti-GFP (green in merged image) and anti-vinculin (red in merged image) antibodies and phalloidin (blue in merged image). Bar, 50 μm.

Effects of CaD depletion on the actin cytoskeleton and cell migration. A, depletion of CaD expression using siRNA. A549 cells were transfected with CaD siRNAs or control siRNA, and incubated with or without 1 μm DEX for 48 h. The expression levels of CaD protein were detected by Western blot analysis. B, effect of CaD depletion on the formation of stress fibers in response to DEX. The cells were stained with anti-CaD antibody (green in merged image) and phalloidin (red in merged image). Bar, 50 μm. C, cell migration of CaD-depleted cells was measured in a Transwell migration chamber with or without 1 μm DEX (mean ± S.D., ^***, p < 0.001). D, GFP-CaD expression counteracts the effects of endogenous CaD depletion. GFP- and GFP-CaD-expressing A549 cells transfected with CaD siRNA2 were incubated with or without 1 μm DEX. CaD siRNA2 repressed the expression of endogenous CaD, but not of exogenous GFP-CaD, which lacked the siRNA target, the 3′-untranslated region. E, cell migration of CaD siRNA2-transfected GFP-CaD- or GFP-expressing cells in a Transwell migration chamber with or without 1 μm DEX. (mean ± S.D., ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; n.s., not significant).