p38MAPK-MSK1 activation by 1,25(OH)₂D₃ depends on RhoA–ROCK and is necessary for the interference of the Wnt–β-catenin pathway. (A) SW480-ADH cells were incubated with 1,25(OH)₂D₃ in the presence of 2 μM Ro318220 or vehicle. The [Ca²⁺]_cyt was determined after 1,25(OH)₂D₃ addition. Mean ± SEM of 28–30 cells (three independent experiments). (B) Mock and N19-RhoA cells were incubated with 1,25(OH)₂D₃ or vehicle (2 h) and the levels of total and phospho-p38MAPK, -MSK1, -CREB, and -ATF1 were determined by WB. Mean ± SD (n = 3). (C) Mock and N19-RhoA cells were pretreated with vehicle, 30 μM Ro318220, or 30 μM SB203580 (2 h) and then incubated with 1,25(OH)₂D₃ for additional 4 h, and the level of E-cadherin RNA was determined by qRT-PCR. Mean ± SD (three independent experiments performed in triplicate). (D) Mock and N19-RhoA cells were incubated (4 h) with 1,25(OH)₂D₃ in the presence of vehicle, Ro318220, or SB203580, and the level of CYP24 RNA and quantification was determined as in C. (E) SW480-ADH cells were transfected with wild-type (TOP) or mutant (FOP) reporter plasmids for β-catenin–TCF activity. After overnight incubation, the cells were treated (48 h) with either vehicle or 1,25(OH)₂D₃ in the presence or absence of 1 μM Ro318220. Mean ± SD (n = 3). (F) Scheme of the mechanism of action of 1,25(OH)₂D₃ in human SW480-ADH cells. A rapid, VDR-dependent nongenomic signaling pathway that starts with Ca²⁺ influx and continues with the sequential activation of RhoA GTPase and the kinases ROCK, p38MAPK, and MSK1 converges in the nucleus with ligand-activated VDR to regulate gene expression and interfere with the Wnt–β-catenin pathway leading to proliferation arrest and epithelial differentiation. MSK1 may target VDR and/or its coregulators and/or downstream transcription factors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.