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

Abstract

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.

A review of 1α,25(OH)2D3 dependent Pdia3 receptor complex components in Wnt5a non-canonical pathway signaling

“…To elucidate whether Wnt5a works via a similar mechanism to that of 1a,25(OH) 2 D 3 , we studied the rapid effects of Wnt5a treatment on MC3T3-E1 osteoblasts. Wnt5a increased CaMKII and PKC activities at 9 min after treatment in osteoblasts [28] (Fig. 1).…”

“…2) [28]. To elucidate the roles of Pdia3, PLAA, VDR, Cav-1, and CaM, we took the advantage of already established models of silenced Pdia3 (ShPdia3), silenced Plaa (ShPlaa), silenced Vdr (ShVdr) and silenced Cav-1 (ShCav-1) MC-3T3-E1 cells and the CaM inhibitor, W-7 [28].…”

“…Additionally, binding of Wnt5a to its receptor complex can activate the planar cell polarity pathway, which works via RhoA, Rasrelated C3 botulinum toxin substrate (Rac), and c-Jun N-terminal kinase (JNK)-dependent signaling pathways [26]. Results of a recent study have suggested that components of the 1a,25(OH) 2 D 3 membrane-associated receptor complex play a role in Wnt5a calcium-dependent signaling [28]. This review will focus on the roles of Pdia3 receptor complex components in the Wnt5a pathway.…”

Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation

“…Recent work suggests that 1α,25-dihydroxyvitamin D3, or 1α,25(OH) 2 D 3 , which also synergistically affects osteoblast response in combination with surface roughness, may compete with Wnt5a to regulate proliferation and differentiation in osteoblasts. This may have implications in patients receiving vitamin D treatment (Boyan et al 1998;Doroudi et al 2014).…”

ESWT Enhances Expression of Pdia-3 which is a Key Factor of 1α,25-Dihydroxyvitamin D3 Rapid Membrane Signaling Pathway in Treatment of Early Osteoarthritis Knee

“…These findings indicated altered protein expression in the pathogenesis of OA and illuminated a novel therapeutic avenue for treatment in OA disease. Among these proteins, protein Pdia-3 in the knee joint, which was reported to osteoblast and chondrocyte cells integrity and function [19][20][21].…”