1alpha,25-Dihydroxyvitamin D(3) antiproliferative actions involve vitamin D receptor-mediated activation of MAPK pathways and AP-1/p21(waf1) upregulation in human osteosarcoma

Abstract

The molecular mechanisms underlying antiproliferative actions of the steroid 1alpha,25-dihydroxy vitamin D(3) (1,25D) in human osteosarcoma cells are known only partially. To better understand the signaling involved in 1,25D anti-tumorigenic properties in bone, we stably silenced vitamin D receptor (VDR) expression in the human osteosarcoma SaOS-2 cell line. We found that 1,25D treatment reduced cell proliferation by approximately 25% after 3 days only in SaOS-2 cells expressing native levels of VDR protein, and involved activation of MAPK/AP-1/p21(waf1) pathways. Both sustained (3 days) and transient (15min) 1,25D treatment activated JNK and ERK1/2 MAPK signaling in a nongenomic VDR-dependent manner. However, only sustained exposure to hormone led to upregulation of p21 and subsequent genomic control of the cell cycle. Specific blockade of MEK1/MEK2 cascade upstream from ERK1/2 abrogated 1,25D activation of AP-1 and p21, and subsequent antiproliferative effects, even in the presence of a nuclear VDR. We conclude that 1,25D-induced inhibition of human osteosarcoma cell proliferation occurs via sustained activation of JNK and MEK1/MEK2 pathways downstream of nongenomic VDR signaling that leads to upregulation of a c-Jun/c-Fos (AP-1) complex, which in turn modulates p21(waf1) gene expression. Our results demonstrate a cross-talk between 1,25D/VDR nongenomic and genomic signaling at the level of MAP kinase activation that leads to reduction of cell proliferation in human osteosarcoma cells.

Fig. 1

1,25D induces activation of MAPK cascades in human SaOS-2 and rat ROS 17/2.8 cells. Osteosarcoma cells were grown in the absence (-) or presence (+) of 10 nM 1,25D for 1 and 3 days. Western blots from whole cell lysates show increasing levels of phosphorylated (p-) JNK and c-Jun (A and B), and higher expression of c-Fos protein (A) in hormone-treated cell cultures. Beta-actin and vinculin were used for normalization of gel loading and transfer. Increasing levels of VDR protein were also detected over time under sustained treatment with hormone.

Fig. 2

Stable VDR silencing in SaOS-2 cells. (A) VDR protein expression levels in native non-transfected (Non-T), control (vector transfected, V-T), and siRNA VDR transfected (siRNA-T) Saos-2 cells. VDR levels are quantitatively shown in (B) as a measure of blot densities. (C) Immunofluorescence detection of VDR in control V-T (left), and VDR silenced (siRNA-T, right). Cells treated with a goat antibody against human VDR were visualized with a secondary anti-goat Cy3-conjugate antibody. VDR was profusely localized in the cell cytoplasm and nucleus of control SaOS-2 cells. Note significantly decreased fluorescence intensity levels corresponding with lower VDR protein levels in VDR silenced versus control V-T cells. Digital images were obtained with identical exposure settings for comparison. (D) 1,25D induction of alkaline phosphatase (ALP) activity requires a VDR. Endogenous ALP enzyme activity was measured in the absence (open bars) and presence (filled bars) of 10 nM 1,25D added to the culture medium for 3 days in native Non-T, control V-T, and siRNA VDR transfected SaOS-2 cells. *, p<0.001, n=3.

Fig. 3

(A,B) Anti-proliferative effects of 1,25D occur in the presence of a VDR. Cell density values, as measured with the CellTiter96 AQueous non-radioactive cell proliferation assay (Promega), were obtained every 24 h over a period of 5 days from SaOS-2 cell cultures treated either with 10 nM 1,25D for 15 min on day 0 (A, transient), or grown in continuous presence of hormone (B, sustained). Squares represent data obtained from control Non-T cells in the absence of 1,25D. Inverse triangles, circles, and upright triangles represent data obtained from siRNA VDR knock-down, control Non-T (3) and control V-T (4) cells in the presence of 10 nM 1,25D; n=5, *, p<0.001. (C,D) Transient and sustained 1,25D treatment induces JNK activation and c-Fos upregulation only in cells expressing a VDR. Western blots obtained for activated (p-) JNK, p-c-Jun, and c-Fos in native Non-T, control V-T, and VDR knock-down (siRNA-T) SaOS-2 cells treated with 10 nM 1,25D for 15 min (transient, C) or 3 days (sustained, D). Activated JNK and c-Jun proteins were detected with commercial antibodies raised against phosphorylated residues. VDR protein expression levels are also shown for the same cell lysates.

Fig. 4

1,25D-induced upregulation of AP-1 and p21 luciferase reporters occurs only in the presence of a classic VDR. Native (Non-T), control vector transfected (V-T), and siRNA VDR transfected (siRNA-T) SaOS-2cells co-transfected with AP-1 (x7) enhancer (A) or p21-promoter luciferase constructs (B) were cultured in the presence of 10 nM 1,25D for 15 min (open bars) or 3 days (filled bars). Relative light units (RLU) from luciferase activity were measured as a function of AP-1 enhancer and p21 promoter activities. *, p<0.001, n=3. RLU values were normalized for transfection efficiency with β-galactosidase activity.

Fig. 5

Blockade of MEK1/MEK2 activity abolishes 1,25D-induction of AP-1 and p21, and prevents hormone anti-proliferative effects. Native, non-transfected (Non-T) and control vector transfected (V-T) SaOS-2 cells, co-transfected with AP-1 (x7) enhancer (A) or p21 promoter (B) luciferase constructs, were cultured for 3 days in the presence of 10 nM 1,25D, with (filled bars) or without (open bars) specific MEK1/MEK2 blocker U0126 (25 μM). Relative light units (RLU) were measured as a function of AP-1 enhancer and p21 promoter activities. Results show that inhibition of MEK1/MEK2 signaling prevented AP-1 and p21 upregulation, confirming that p21 gene transactivation occurs downstream 1,25D-induced MAPK activity. *, p<0.001, n=4. C: Measurements for cell densities obtained for native (non-transfected) SaOS-2 cell cultures treated with 10 nM 1,25D in the presence or absence of U0126 (25 μM) for 3 days. *, p<0.01, n=3.

Fig. 6

Proposed model for the integration of anti-proliferative genomic and nongenomic 1,25D pathways via MAPK cross-talk through a classic VDR in human osteosarcoma cells. Shortly after arrival at the cell surface, 1,25D hormone interacts with a VDR protein localized at the cytoplasm in close proximity to the plasma membrane that triggers nongenomic effects of the steroid. Cytoplasmic 1,25D/VDR complex rapidly (transient pathway) activates a JNK MAP kinase, on one hand, and upregulates c-Fos expression via a MEK1/MEK2/ERK pathway, on the other. Only under sustained presence of hormone, phosphorylated c-Jun, a downstream product of JNK activation, dimerizes with c-Fos at the cell nucleus, and the complex binds to an AP-1 site upstream the p21^waf1 gene involved in the control of cell cycle progression. In addition, under continuous presence of hormone and according to traditional genomic pathways, 1,25D/VDR complex translocates to the cell nucleus and binds to a vitamin D response element (VDRE) upstream of the p21 gene. As a result of this genomic and nongenomic cross-talk, the cdk inhibitor p21 is expressed leading to cell cycle arrest and decreased cell proliferation in a time frame of days. As seen from our results, a classic VDR is required for nongenomic activation of MAPK signaling.