Posttranslational processing of FGF23 in osteocytes during the osteoblast to osteocyte transition

Abstract

FGF23 is an O-glycosylated circulating peptide hormone with a critical role in phosphate homeostasis; it is inactivated by cellular proprotein convertases in a pre-release degradative pathway. We have here examined the metabolism of FGF23 in a model bone cell line, IDG-SW3, prior to and following differentiation, as well as in regulated secretory cells. Labeling experiments showed that the majority of 35S-labeled FGF23 was cleaved to smaller fragments which were constitutively secreted by all cell types. Intact FGF23 was much more efficiently stored in differentiated than in undifferentiated IDG-SW3 cells. The prohormone convertase PC2 has recently been implicated in FGF23 degradation; however, FGF23 was not targeted to forskolin-stimulatable secretory vesicles in a regulated cell line, suggesting that it lacks a targeting signal to PC2-containing compartments. In vitro, PC1/3 and PC2, but not furin, efficiently cleaved glycosylated FGF23; surprisingly, PC5/6 accomplished a small amount of conversion. FGF23 has recently been shown to be phosphorylated by the kinase FAM20C, a process which was shown to reduce FGF23 glycosylation and promote its cleavage; our in vitro data, however, show that phosphorylation does not directly impact cleavage, as both PC5/6 and furin were able to efficiently cleave unglycosylated, phosphorylated FGF23. Using qPCR, we found that the expression of FGF23 and PC5/6, but not PC2 or furin, increased substantially following osteoblast to osteocyte differentiation. Western blotting confirmed the large increase in PC5/6 expression upon differentiation. FGF23 has been linked to a variety of bone disorders ranging from autosomal dominant hypophosphatemic rickets to chronic kidney disease. A better understanding of the biosynthetic pathway of this hormone may lead to new treatments for these diseases.

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein

“…Our new findings suggest osteocytes use the constitutive secretory pathway for PTHrP. A similar mechanism is used for FGF23 secretion by IDG‐SW3 osteocytes . As we observed for PTHrP, FGF23 used the constitutive secretory pathway, rather than the regulated secretory pathway .…”

“…A similar mechanism is used for FGF23 secretion by IDG-SW3 osteocytes. (60) As we observed for PTHrP, FGF23 used the constitutive secretory pathway, rather than the regulated secretory pathway. (60) PTHrP biological activity through PTHR1 is destroyed by kexin-2 cleavage carboxyl to Arg 19 Arg 20 Arg 21 .…”

Proprotein convertase furin regulates osteocalcin and bone endocrine function

“…The insulin receptor precursor (pro-INSR), another protein cleaved by furin and PACE4 in cell culture (32) and involved in the regulation of energy metabolism by osteoblasts (33), was still processed in the bones of Furin osb-/-mice and in Furin -/-osteoblasts (Supplemental Figure 1, C and D). In addition, OCN was still processed when PC5, another PC known to be expressed in osteoblasts (34) and implicated in skeletal development (35), was inactivated in this cell type (Supplemental Figure 1E). …”

“…PC5 is another PC that is required for normal patterning of the axial skeleton through the processing of progrowth differentiation factor 11 (pro-GDF11) (35). PC5 has been reported to be expressed in osteoblasts and osteocytes, where it may process FGF23, another bone-derived hormone regulating phosphate homeostasis (34). However, our data show that, although PC5A is expressed in primary osteoblasts and cleaves pro-OCN in vitro, the inactivation in this cell type of Pcsk5, encoding PC5A and PC5B isoforms, does not affect pro-OCN processing in cell culture or in vivo.…”

Multiple faces of fibroblast growth factor-23

“…In contrast, loss-of-function mutations in polypeptide N -acetylgalactos-aminyltransferase 3 (GalNAc-T3) that prevents O-glycosylation of Thr 178 within the RXXR cleavage site causes tumoral calcinosis by increasing FGF-23 proteolysis. In vitro , PC1/3, PC2 and PC5/6, but not furin, cleaves FGF-23 [29]. Understanding the role of posttranslational regulation of FGF-23 in health and disease requires additional study.…”