MKP-1 is essential for canonical vitamin D-induced signaling through nuclear import and regulates RANKL expression and function

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-B ligand (RANKL). MAPK phosphatase-1 (MKP-1) is a phosphatase classically known to negatively regulate the innate immune response through dephosphorylation of p38, ERK, and c-Jun N-terminal kinase activity. This paper describes a new function of MKP-1 in permitting genomic 1,25(OH)₂D₃ signaling and downstream osteoclastogenesis through RANKL. Initially, quantitative RT-PCR (qRT-PCR) and immunoblot analysis comparing bone marrow stromal cells (BMSC) revealed that 1,25(OH)₂D₃-induced vitamin D receptor (VDR), cytochrome P 45024a1, and RANKL mRNA expression and protein were significantly attenuated or absent in MKP-1/ BMSC. Immunoblot analysis from cellular fractions of wild type and MKP-1 ^/ BMSC stimulated with 10 7 M 1,25(OH)₂D₃ revealed retinoid X receptor (RXR)÷ nuclear import was impaired in MKP-1^/ BMSC, whereas VDR import was not. Proximity ligation assays revealed that baseline VDR-RXR÷ heterodimer translocation was unchanged, yet 1,25(OH)₂D₃-induced nuclear translocation of VDR-RXR÷ heterodimers was reduced in MKP-1^/ BMSC. A functional consequence was observed as BMSC from MKP-1^/ mice treated with 1,25(OH)₂D₃ and cocultured with RAW 264.7 cells had a 91% decrease in osteoclastogenesis and a 94.5% decrease in mineralized matrix resorption compared with wild-type cocultures (P 0.01). These results reveal an unexpected, permissive role for MKP-1 in canonical 1,25(OH)₂D₃ signaling via VDR-RXR÷ heterodimer nuclear import and downstream osteoclastogenesis through stromal cell RANKL expression.