Optimal bone fracture repair requires 24,25-dihydroxyvitamin D and its effector molecule, FAM57B2 - There is an emerging effort to develop novel strategies to stimulate bone fracture repair. A certain percentage
of fractures display impaired healing, and any improvement in fracture treatment would be of considerable benefit
to patients. When studying bone healing in Cyp24a1-deficient mice, which cannot synthesize the vitamin D
metabolite, 24,25(OH)2D, we have measured a significant, reproducible impairment in callus formation during
fracture repair. The callus formation defect can be corrected by exogenous administration of 24,25(OH)2D. We
subsequently cloned Fam57b2, encoding a transmembrane protein that specifically interacts with 24,25(OH)2D.
Our preliminary results show that mice deficient for FAM57B2 in chondrocytes exhibit the same impaired callus
formation during fracture repair than Cyp24a1-deficient mice.
FAM57B2 contains a domain that suggests a potential enzymatic activity within the sphingolipid synthetic
pathway. We have measured 24,25(OH)2D-dependent production of lactosylceramide (LacCer) by FAM57B2,
supporting allosteric regulation of the enzymatic activity of FAM57B2 by the vitamin D metabolite.
Our results strongly suggest that FAM57B2 is an effector of 24,25(OH)2D-mediated signaling during fracture
repair. We hypothesize that FAM57B2 is a lactosylceramide synthase whose enzymatic activity is
stimulated by 24,25(OH)2D binding and that LacCer acts as a second messenger to optimize
endochondral ossification during fracture repair. We have identified the following specific aims to test the
proposed hypothesis using a combination of molecular, biochemical, and genetic approaches:
1. Identify the ligand-binding pocket and the enzymatic moiety of FAM57B2.
2. Examine the response of chondrocytes to treatment with LacCer.
3. Ascertain the source of 24,25(OH)2D in fracture repair using cell-type specific inactivation of Cyp24a1.
4. Characterize and rescue the fracture repair phenotype of chondrocyte-specific Fam57b-deficient mice.
We will examine structure-function relationships of the FAM57B2 protein and confirm the allosteric regulation
of its enzymatic activity. Chondrocytes will be treated with LacCer to examine impact on cellular responses.
Cyp24a1 will be inactivated in chondrocytes, osteoblasts, or macrophages to determine the relevant site of
synthesis of 24,25(OH)2D for optimal fracture healing. Rescue of the callus formation defect phenotype of
chondrocyte-specific Fam57b-deficient mice by administration of LacCer will confirm that it acts as a second
messenger to transduce the 24,25(OH)2D signal. Bone healing during fracture repair will be assessed using an
array of techniques including histomorphometry, gene expression monitoring, and biomechanical testing.
The research that we propose will demonstrate the physiological role of 24,25(OH)2D and its effector
molecule FAM57B2 in fracture repair. Our work could lead to the use of 24,25(OH)2D, suitable analogs, or
lactosylceramide in the therapeutic management of fracture and surgical osteotomy healing.
