DMP1 Rescues FGF23 and Bone Mineralization Defects in CKD Mice

Fibroblast growth factor (FGF)-23 is a bone derived hormone that regulates phosphate homeostasis. Serum intact FGF23 levels result from a balance between FGF23 transcription and cleavage: phosphorylation of the serine 180 by FAM20C promotes FGF23 cleavage by Furin, while glycosylation by GALNT3 at the same site protects FGF23 from cleavage. During chronic kidney disease (CKD) progression, alterations in mineral and bone metabolism lead to elevations in serum intact FGF23 that contribute to increased risk of cardiovascular disease and mortality. Dentin matrix protein (DMP)-1 is a bone matrix protein that stimulates mineralization and regulates FGF23. We hypothesized that DMP1 overexpression will rescue alterations of bone mineralization and FGF23 elevations in Col4a3KO mice, a well-characterized CKD model. We analyzed the osseous and osteocyte phenotype in femurs collected from 20 week-old wild-type (WT), mice overexpressing DMP1 in bone (DMP1TG), Col4a3KO and compound (Col4a3KO/DMP1TG) littermates. We also measured serum levels of intact and total (cleaved + intact) FGF23. We assessed in vitro matrix mineralization by alizarin red S (ARS) staining and osteoblast differentiation by alkaline phosphatase staining of cultured bone marrow stromal cells (BMSCs). Then, we stimulated FGF23 expression in WT and DMP1TG BMSCs for 48h using calcium (12mM) and assessed the effects of DMP1 on mRNA expression of FGF23 and regulatory enzymes of FGF23 cleavage (GALNT3, FAM20C and furin) by qRT-PCR. Col4a3KO mice displayed a 5% decrease in bone mineral density and a rounder, less polarized osteocyte morphology (osteocyte roundness index: 0.43 ± 0.17 vs. 0.34 ± 0.12) (p<0.05 vs. WT for each). Col4a3KO mice also displayed increased serum FGF23 levels (intact FGF23: +7-fold; total FGF23: +4-fold; p<0.05 vs. WT). In vitro, Col4a3KO BMSCs showed impaired activity and matrix mineralization (-40% ARS; p<0.05 vs. WT). Overexpression of DMP1 in Col4a3KO prevented all alterations in cell morphology, activity and mineralization in vivo and in vitro. In Col4a3KO/DMP1TG mice, serum intact FGF23 levels were reduced by 50% while total FGF23 levels remained unchanged compared to Col4a3KO mice. As expected, Ca2+ treatment in WT BMSCs stimulated FGF23 mRNA expression (+1150-fold; p<0.05 vs. untreated WT). This was associated with reduced GALNT3 (-1.6-fold), increased FAM20C (+2.2-fold) and increased furin (+3.4-fold) expressions (p<0.05 vs untreated WT for each). Overexpression of DMP1 in DMP1TG BMSCs partially prevented Ca2+-induced stimulation of FGF23 mRNA expression (+650-fold), further decreased GALNT3 (-3.7-fold) and further increased FAM20C (+3-fold) (p<0.05 vs untreated WT for each). Thus, DMP1 prevented intrinsic bone cell alterations in CKD. DMP1 also prevented elevations of serum FGF23 by regulating mechanisms of FGF23 transcription and cleavage. These data suggest that DMP1 could serve as a novel therapeutic target to improve bone health and reduce FGF23 levels in CKD.