Figure 5: SIK inhibitors regulate SOST and RANKL expression.
From: SIKs control osteocyte responses to parathyroid hormone
(a) Structure of YKL-04-114 (left), YKL-05-093 (middle) and YKL-05-093 Kd determination curves for SIK2 (right). For the Kd determination curves, the y-axis represents the amount of bound kinase measured by qPCR (see Methods), and the x-axis represents the corresponding compound concentration in nM. (b) Ocy454 cells were treated with YKL-04-114 (10 μM) for the indicated times, followed by immunoblotting of whole cell lysates as indicated. (c) Ocy454 cells were treated with the indicated concentrations of YKL-04-114 for 60 min, followed by immunoblotting of whole cell lysates as indicated. (d) Left: Ocy454 cells were treated with vehicle, PTH (50 nM) or YKL-05-093 (10 μM) for 60 min. Cytosol and nuclear fractions were then generated, followed by immunoblotting as indicated. Right: quantification of nuclear fraction (defined as nuclear/total) of HDAC4 or CRTC2. * indicates P<0.01 comparing treatment versus vehicle. (e) Top: Ocy454 cells were treated with the indicated concentrations of YKL-04-114 for 4 h, followed by RT-qPCR. Bottom: Cells were treated with YKL-04-114 (0.5 μM) for the indicated times. * indicates P<0.05 comparing treatment versus vehicle. (f) Cells lacking SIK2, SIK3 or both were treated with YKL-05-093 (10 μM for 45 min). Quantification of HDAC4 S246 phosphorylation, as assessed by densitometric analysis of immunoblots, is shown. * indicates P<0.01 comparing treatment versus vehicle. # indicates P<0.05 for the same comparison. (g) Control and SIK2/3 deficient cells were treated with YKL-05-093 (0.5 μM) for 4 h and SOST transcript abundance was measured by RT-qPCR. (h) Control and CRTC2 shRNA cells were treated with PTH (1 nM) or YKL-05-093 (0.5 μM) for 4 h and RANKL transcript abundance was measured by RT-qPCR. (i) Control and Gsα-deficient Ocy454 cells were treated with PTH (1 nM), YKL-05-093 (0.5 μM) or forskolin (5 μg ml−1) and SOST and RANKL transcript abundance was determined by RT-qPCR. For (h) and (i), * indicates P<0.05 comparing treatment and vehicle. (j) Control and Gsα-deficient Ocy454 cells were treated with PTH (50 nM), YKL-05-093 (10 μM) or forskolin (5 μg ml−1) for 30 min. Whole cell lysates were generated and immunoblotted as indicated.
