Signaling components of the 1α,25(OH)₂D₃-dependent Pdia3 receptor complex are required for Wnt5a calcium-dependent signaling

Wnt5a and 1α,25(OH)₂D₃ are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)₂D₃ initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A₂ (PLA₂)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA₂, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA₂/PGE₂/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA₂ inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)₂D₃ reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)₂D₃-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)₂D₃-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)₂D₃-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)₂D₃ on PKC and CaMKII. 1α,25(OH)₂D₃ membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)₂D₃ or Wnt5a treatment. This study demonstrates that 1α,25(OH)₂D₃ and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.