1257 MKP-1 is Required for Canonical 1,25(OH)2D3-signaling and Osteoclastogenesis

Saturday, March 24, 2012: 9:45 a.m. - 11 a.m.
Presentation Type: Poster Session
A.C. GRIFFIN III, Craniofacial Biology, Medical University of South Carolina, Charleston, SC, and K. KIRKWOOD, Craniofacial Biology and Center for Oral Health Research, Medical University of South Carolina, Charleston, SC
Vitamin D3, 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.
This abstract is based on research that was funded entirely or partially by an outside source: NIH T32DE017551, R01DE018290, and P20 RR017696

Keywords: Cell biology, Cytokine, Mineralization and vitamin D