Regulation of bone-derived hormones by post-translational modifications

Bone endocrine functions are mediated by at least two hormones, osteocalcin and fibroblast growth factor 23 (FGF23) which are secreted by the bone cells, osteoblasts and osteocytes. Osteocalcin is an osteoblast-derived hormone regulating glucose and energy metabolism. It improves glucose tolerance and insulin sensitivity, promotes insulin secretion and β-cell proliferation, increases energy expenditure and reduces fat accumulation. Osteocalcin is gamma-carboxylated on three of its glutamic acid residues (Glu), a process that inhibits its endocrine function in mice and humans. It is the acidic pH in the resorption lacuna which decarboxylates osteocalcin releasing the uncarboxylated osteocalcin (ucOCN), the active form of this hormone. Our knowledge on osteocalcin regulation by post-translational modifications is limited to its gamma-carboxylation. Since osteocalcin is secreted by differentiated osteoblasts, a non-classical endocrine cell, we hypothesized that osteocalcin may be subjected to additional post translational modifications (PTMs) in the secretory pathway that regulates its endocrine functions. In the first part of the thesis we showed that osteocalcin’s putative pro-peptide is cleaved in its C-terminus at the basic motif «RLRR», by the proprotein convertase furin. This process occurs independently of osteocalcin gamma-carboxylation. Furin inactivation specifically in osteoblasts in mice totally abolishes osteocalcin processing and impairs its activation and release during bone resorption. Consequently, these mice have decreased serum level of ucOCN resulting in impaired glucose tolerance, reduced insulin secretion and energy expenditure, and increased fat accumulation. Moreover, these mice have a decrease in the appetite independently of osteocalcin. Pair feeding of control mice resulted in more apparent phenotype in furin deficient mice, as it appears at younger age alongside with insulin resistance. In the second part of this thesis, we discovered that mouse osteocalcin is O-glycosylated on serine 8, a process that occurs independently of its gamma-carboxylation and processing. This modification is not conserved in human or any other species and it increases mouse osteocalcin half-life in plasma ex vivo and in vivo. Interestingly, tyrosine 12 in human osteocalcin corresponds to the serine 8 in the mouse sequence. Tyr12Ser mutation was sufficient to O-glycosylate human osteocalcin and to confer this hormone a longer half-life in mouse plasma compared to the native form.  FGF23 is a hormone secreted by osteoblasts and osteocytes which regulates phosphate reabsorption and vitamin D production in the kidney proximal tubule. Its endocrine function is inhibited by endoproteolytic cleavage which releases its N-terminal and C-terminal fragments. Mutations in the «RHTR» motif, a consensus cleavage site for proprotein convertases (PCs), were found in patients with autosomal dominant hypophosphatemic rickets (ADHR). These patients are characterized by an increased intact FGF23 levels, hypophosphatemia and osteomalacia. Despite the importance of FGF23 in the pathology of multiple diseases, the identity of the enzyme(s) involved in FGF23 cleavage is yet unclear, even though furin and the proprotein convertase subtilisin/kexin type 5 (PC5) were shown to cleave FGF23 in vitro. In the third part of the thesis, we addressed this question using mice model deficient in furin and/or PC5 in osteoblasts and osteocytes in mice. Under physiological conditions, furin inactivation resulted in a 25% increase in intact FGF23; however, these mice maintained normal phosphate level and did not shown any sign of osteomalacia. We also showed that under iron restriction, a condition that induce FGF23 expression at the mRNA and protein level, FGF23 processing is totally impaired in furin deficient mice. However, the injection of erythropoietin or interleukin 1-β, two conditions that increase FGF23 production, induce FGF23 serum level while it is still properly processed in mice deficient in furin and/or PC5 in osteoblasts and osteocytes. Interestingly, despite the patterning defects observed in global inactivation of PC5, conditional inactivation of PC5 in osteoblasts and osteocytes in mice did not result in any bone defect. However, furin inactivation in osteoblasts and osteocytes in mice increases trabecular bone parameters and decreases cortical thickness. Moreover, these mice have decreased bone mineral density and bone strength reflecting a poor bone quality. In summary, we described for the first time that furin is a pleotropic regulator of osteoblast and osteocyte function in vivo. It regulates glucose and energy metabolism by mediating pro-osteocalcin processing which is required for osteocalcin maturation and bioactivity, and by regulating appetite independently of osteocalcin. These findings suggest the presence of additional osteokines controlling appetite and which are regulated by furin. Moreover, furin partially regulates FGF23 processing while maintaining normal phosphate homeostasis, suggesting that the regulation of bone mass accrual by furin occurs independently of FGF23. Additionally, we discovered that mouse osteocalcin is subjected to O-glycosylation, a species-specific modification that is not conserved in humans or any other species and increases mouse osteocalcin half-life. Artificial O-glycosylation confer human osteocalcin a longer half-life, thus providing an approach to increase human osteocalcin bioactivity in future therapeutic applications of osteocalcin in human diseases.

Keyphrases: Osteocalcin, FGF23, Furin, PC5, O-glycosylation, Endocrine function, Bone, O-glycosylation, Os