Repression of Smoothened by Patched-Dependent (Pro-)Vitamin D3 Secretion (original) (raw)
Figure 6
Analysis of Vitamin D3 as a Specific Smo Antagonist
(A) Shown are the Gli reporter inhibitions by 7-DHC, AY-9944, a Dhcr7 inhibitor, vitamin D3, and a combination of the latter two. Also shown is the inhibition that can be conferred by the well-known Smo antagonist cyclopamine and the previously observed effects for Ptch1 transfectant conditioned medium as well as Ptch1 cotransfection (data taken from Figure 4B and Figure 2B, respectively). Cells were stimulated with the various compounds for 6 h (16 h past transfection), except for the cotransfection, which was lysed after 24 h ( n ≥ 4).
(B) A range of concentrations of vitamin D3 was tested for inhibition of Gli reporter activity (also in SmoM2-transfected cells), MTT viability assay, and N-terminal repressor form Gli3 levels. Cells were stimulated with vitamin D3 for 6 h before lysis. For the reporter assays, RLU values were corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells ( n = 4). The MTT assays shown were raw absorption data expressed as percent difference to control stimulated ( n = 8); MTT agent was added for 3 h. N-terminal repressor form Gli3 was determined by quantifying ECL signal from Western blot and corrected for β-actin ( n = 3). Depicted is the mean ± SEM.
(C) Gli specificity of the inhibitory effect of 10 μM vitamin D3 was assayed by using a panel of luciferase reporter constructs, one of which was a mGli reporter (mGli-LUC). Cells were stimulated for 6 h with vitamin D3, and the luciferase activity was assayed; values were calculated from RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells ( n ≥ 4). Depicted is the mean ± SEM.
(D) Different cell types share the inhibitory response to vitamin D3 on Gli activity. C3H/10T1/2 and MDA-MD-231 cells were used earlier in the mix-and-match and medium transfer experiments. Ptch1–/– MEFs served as a control for any possible effects of vitamin D3 on Ptch1 function. Cells were stimulated for 6 h, and the luciferase activity was assayed,; values are RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells ( n = 4). Depicted is the mean ± SEM.
(E) Proposed mechanism of vitamin D3 action. First panel: in the presence of Hh, Ptch1 is inactive and does not inhibit Smo. The Hh pathway is active, and Gli activity can be measured in a reporter assay. Second panel: in the absence of Hh, Ptch1 is active and uses vitamin D3 to inhibit Smo. The pathway is inactive, and low Gli activity is measured. Third panel: in the presence of Hh as well as exogenous D3, Smo is inhibited independently of Ptch1, and Hh can no longer elicit a Gli response.
(F) Confluent Shh-LIGHT II stable Gli reporter transfectants were stimulated with 10 μM vitamin D3 and/or 200 ng/ml Shh overnight in the presence of 0.5% FCS. Luciferase activity was assayed; values are RLU values corrected for an internal CMV Renilla standard and expressed as percent difference to control stimulated cells (0 μM vitamin D3; 0 ng/ml Shh). In the presence of vitamin D3, Shh is no longer able to induce reporter activity. n = 4; depicted is the mean ± SEM.