MKP-1 is Required for Canonical 1,25(OH)2D3-signaling and Osteoclastogenesis

Vitamin D_3, and its most active form 1,25(OH)₂D₃, are well known to stimulate osteoclastogenesis through stromal cell induction of the receptor activator of nuclear factor kappaB ligand (RANKL). Mitogen activating protein kinase phosphatase-1 (MKP-1) is a phosphatase classically known to negatively regulate the immune response through dephosphorylation of p38, ERK, and JNK activity. Objective: The purpose of this study was to define the role of MKP-1 in genomic 1,25(OH)₂D₃ signaling and downstream osteoclastogenesis through RANKL. Methods: Primary bone marrow stromal cells (BMSCs) from tibias and femurs of MKP-1^-/- and MKP-1^+/+ mice were collected. Gene expression was measured using RT-qPCR, protein expression by immunoblot and ELISA in 1,25(OH)₂D₃ treated cultures. Co-culture osteoclastogenesis assays were conducted as well. Results: RT-qPCR and immunoblot analysis comparing BMSCs revealed that 1,25(OH)₂D₃-induced VDR, CYP24a1 and RANKL mRNA expression and protein were significantly attenuated or absent in MKP-1^-/- BMSCs. Immunoblot analysis from cellular fractions of WT and MKP-1^-/- BMSCs stimulated with 10^-7M 1,25(OH)₂D₃ for 1 hr revealed that RXRa nuclear import was impaired in MKP-1^-/- BMSCs, while VDR import was not affected. Proximal ligation assay experiments addressing VDR-RXRa heterodimer translocation revealed that while baseline levels were unchanged, 1,25(OH)₂D₃-induced nuclear translocation of VDR-RXRa heterodimers was significantly decreased in MKP-1^-/- BMSCs. BMSCs from tibias and femurs of MKP-1^-/- mice treated with 1,25(OH)₂D₃ and co-cultured with RAW 264.7 cells had a 91.1% decrease in osteoclastogenesis and 94.5% decrease in mineralized matrix resorption compared with WT co-cultures (p<0.01). Conclusion: These results reveal an unexpected and novel role for MKP-1 in canonical 1,25(OH)₂D₃ signaling via VDR-RXRa heterodimer nuclear import and downstream osteoclastogenesis through BMSC RANKL expression.