Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1

Abstract

Background and purpose: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation.

Experimental approach: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng.mL(-1)) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators.

Key results: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC(50)= 520 +/- 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 +/- 8%) and asthmatic subjects (57 +/- 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21(Waf/Cip-1), p27(Kip1), cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC(50)= 450 +/- 100 pM) PDGF-induced cell cycle progression.

Conclusions and implications: Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.

Figure 1

Airway smooth muscle (ASM) DNA synthesis and vitamin D receptor (VDR) activation. (A) [³H]-thymidine incorporation was measured in quiescent ASM cells derived from normal subjects and those with asthma. Platelet-derived growth factor (PDGF), thrombin and the combination markedly enhanced [³H]-thymidine incorporation in both normal and asthma ASM. There was a dramatic enhancement in thymidine incorporation in asthma ASM as compared with normal ASM stimulated by PDGF or thrombin. The data represent mean ± standard error of the mean from four separate cell lines with each condition representing a minimum of six replicates. Statistics were performed using analysis of variance with Bonferroni–Dunn correction; P < 0.05 considered significant. (B) Quiescent ASM cells from normal subjects were stimulated with varying concentrations of calcitriol, and expression of CYP24A1, VDR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) total mRNA was determined by reverse transcription–polymerase chain reaction. Data shown are representative of three separate experiments from four different cell lines derived from normal subjects or those with asthma.

Figure 2

Calcitriol, but not dexamethasone, inhibits DNA synthesis in airway smooth muscle (ASM) cells. ASM were grown to near confluence, serum-deprived and then stimulated with growth factors. Thymidine incorporation was determined as described in Methods. Calcitriol inhibited platelet-derived growth factor (PDGF)-induced (A) or thrombin-induced (B) DNA synthesis in ASM from normals and subjects with asthma. (C) Dexamethasone augmented PDGF-induced DNA synthesis in normal ASM, while modestly inhibiting DNA synthesis in asthma cells. (D) Dexamethasone augmented thrombin-induced DNA synthesis in ASM from normal subjects, but had no effect on asthma ASM. All data were normalized to maximum stimulation by a growth factor after the basal thymidine incorporation counts were subtracted. All data in (A) and (B) represent P values > 0.05 when compared to maximum PDGF-induced DNA synthesis (100%). In (C) and (D), growth augmentation by dexamethasone on PDGF- and thrombin-induced DNA synthesis was significant at concentrations of 0.1 and 1 µM for PDGF, and at all tested concentrations for thrombin-induced DNA synthesis (P < 0.05). Dexamethasone effects in asthma ASM cells were not different in comparison to thrombin-induced DNA synthesis, and significant at concentrations of 1 and 10 µM dexamethasone plus PDGF when compared with responses stimulated by PDGF alone (P > 0.05). All data represent means ± SEM. Significance was assessed using analysis of variance with Bonferroni–Dunn correction, with significance demonstrated at P < 0.05.

Figure 3

Calcitriol inhibited cell cycle progression but had no effect on airway smooth muscle (ASM) apoptosis. (A) Quiescent ASM cells were stimulated with platelet-derived growth factor (PDGF), calcitriol, dexamethasone or the combination, and after 24 h, flow cytometry and characterization of cells entering the S phase of the cell cycle were performed as described in Methods. (B) Calcitriol had no effect on ASM apoptosis. ASM cells were stimulated with PDGF, calcitriol, the combination or ceramide for 48 h, and caspase-3 activity was measured as described in Methods. Ceramide served as a positive control demonstrating induction of apoptosis as measured by caspase-3 activity in ASM cells. All data represent means ± SEM. Each data point represents a minimum of four replicates from four separate cell lines derived from normal subjects and those with asthma.

Figure 4

(A) Platelet-derived growth factor (PDGF) induced p42/44 mitogen-activated protein kinase (MAPK) activation, but calcitriol had no little effect. Quiescent airway smooth muscle (ASM) cells from normal subjects were pretreated for 30 min with calcitriol at varying concentrations and then with PDGF (10 ng·mL⁻¹) for 10 min. Activation of p42/44 MAPK was characterized by immunoblot analysis as described in Methods. The bar graphs represent quantitation of the displayed immunoblots. Parallel experiments performed in ASM derived from subjects with asthma were identical to these results. Equal protein loadings were confirmed in immunoblots stripped of primary antibodies and re-probed using a β-actin antibody (data not shown). These data represent means ± standard deviations from four separate cell lines derived from normal subjects. (B) Calcitriol had no effect on S6K1 activation by PDGF. Ribosomal S6K1 activation was determined in quiescent ASM cells pretreated with calcitriol (1 µM) for 30 min or 18 h, and then stimulated with PDGF (10 ng·mL⁻¹). PDGF induced S6K1 activation; however, calcitriol had no effect. There was no difference in S6K1 activation induced by PDGF in ASM derived from normals and from subjects with asthma. These data are representative of three separate experiments from four different ASM cell lines. (C) Calcitriol had no effect on cyclin D1 or E2F protein expression in ASM. ASM monolayers from normal subjects were treated with 10 ng·mL⁻¹ PDGF, 1 µM calcitriol or the combination for 18 h. Cells were then lysed, and immunoblot analysis was performed as described in Methods. Antibodies specific for cyclin D1, E2F1–E2F4 and β-actin were used to characterize immunoblot analysis. All blots were stripped and then probed with antibodies to β-actin that serve as a control for total protein loading per well. There was no difference in protein expression profile among normal ASM and those derived from subjects with asthma. The data are representative of a single experiment repeated in triplicate in four separate cell lines.

Figure 5

(A) Calcitriol inhibited platelet-derived growth factor (PDGF)-induced hyperphosphorylation of retinoblastoma (Rb) protein. Quiescent airway smooth muscle (ASM) cells derived from normal subjects were stimulated for 12 h with PDGF (10 ng·mL⁻¹) in the presence and absence of a 30 min pretreatment with calcitriol (1 µM). Cells were lysed, and the supernatant was analysed by gel electrophoresis and immunoblot analysis using specific antibodies to phospho-Rb protein or actin. The immunoblot analysis is representative of triplicate experiments performed in four different cell lines derived from normal and subjects with asthma. The data are plotted as Rb phosphorylation as % control. Statistical analysis was performed examining the PDGF effects versus basal in multiple gels in multiple cell lineages. (B) Lack of effects of calcitriol on inhibitors of cell cycle progression. Confluent ASM cells were stimulated with PDGF (10 ng·mL⁻¹), calcitriol (1 µM) or the combination. Cells were then lysed and the protein extracted. Immunoblot analysis was performed using specific antibodies to the cell signalling molecules as described in the Methods section. In each case, the immunoblots were stripped and reprobed with a β-actin antibody to demonstrate equal loading. Calcitriol had no effect on PDGF-induced effects on inhibitors of cell cycle progression.

Figure 6

(A) Chk1 activity was essential for platelet-derived growth factor (PDGF)-induced cell cycle progression. Quiescent airway smooth muscle (ASM) cells from normal subjects were treated with PDGF (10 ng·mL⁻¹) for 24 h following pretreatment with varying concentrations (0.1–100 nM) of Chk1 inhibitor (SB218078). Following the 24 h time period, the S phase cell cycle progression was analysed by flow cytometry as described in the Methods section. The data are representative of a single experiment repeated in triplicate in four separate cell lines derived from normal and subjects with asthma. (B) Calcitriol inhibited Chk1 phosphorylation in PDGF-stimulated ASM cells. Quiescent ASM cells were pretreated with calcitriol (1 µM) or SB218078 for 2 h before stimulating with PDGF (10 ng·mL⁻¹) for the indicated times. Immunoblot analysis used specific antibodies to phospho-Chk1. Immunoblots were stripped and re-probed with a β-actin antibody to demonstrate equal loading. PDGF-induced Chk1 phosphorylation was inhibited by calcitriol at 1 µM in a time-dependent manner. (C) Densitometric analysis of Chk1 and Chk2 phosphorylation after 24 h of PDGF treatment in calcitriol-treated and untreated ASM cells. Data are expressed as % basal phospho-Chk1 after normalizing for actin. Pretreatment of ASM cells with calcitriol (1 µM) markedly inhibited PDGF-induced Chk1 phosphorylation with no effect on Chk2 phosphorylation. The data are representative of repetitive densitometric measurements of triplicate immunoblots from four separate ASM cell lines derived from normal and subjects with asthma.