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

Bone. 2016 Mar;84:120-130. doi: 10.1016/j.bone.2015.12.055. Epub 2015 Dec 31.

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 (35)S-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.

Keywords: 7B2; FGF23; Furin; Osteocyte; PC5/6; Posttranslational processing; Proprotein convertase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CHO Cells
  • Cell Differentiation*
  • Cricetinae
  • Cricetulus
  • DNA, Complementary / genetics
  • Fibroblast Growth Factors / metabolism*
  • Glycosylation
  • Humans
  • Immunohistochemistry
  • Mice
  • N-Acetylgalactosaminyltransferases / metabolism
  • Neuroendocrine Secretory Protein 7B2 / metabolism
  • Osteoblasts / cytology*
  • Osteoblasts / metabolism*
  • Osteocytes / cytology*
  • Osteocytes / metabolism*
  • Phosphorylation
  • Proprotein Convertases / metabolism
  • Protein Processing, Post-Translational*
  • Rats
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • DNA, Complementary
  • Neuroendocrine Secretory Protein 7B2
  • Recombinant Proteins
  • Fibroblast Growth Factors
  • fibroblast growth factor 23
  • N-Acetylgalactosaminyltransferases
  • polypeptide N-acetylgalactosaminyltransferase
  • Proprotein Convertases