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Abstract: FR-PO519

Effects of Fetuin-A-Containing Calciprotein Particle (CPP) on Posttranslational Modifications of Fetuin-A in HepG2 Cells

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Uedono, Hideki, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Ochi, Akinobu, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Nakatani, Shinya, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Tsuda, Akihiro, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Mori, Katsuhito, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Emoto, Masanori, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Inaba, Masaaki, Osaka City University Graduate School of Medicine, Osaka, Japan
  • Shoji, Tetsuo, Osaka City University Graduate School, Osaka, Japan
Background

Fetuin-A is a liver-derived circulating glycoprotein that has potent calcification-inhibitory capacity. Under calcification stress such as CKD, fetuin-A prevent ectopic calcification by forming colloidal complexes that comprise calcium, phosphate and fetuin-A, termed fetuin-A-containing calciprotein particle (CPP). Recent reports suggest that CPP may have physiological and pathological functions including induction of inflammation. The objective of this study was to investigate the effects of CPP on fetuin-A expression in hepatocytes.

Methods

Synthetic CPP was prepared as previously reported. Because fetuin-A is posttranslationally modified, the molecular weight (MW) of fetuin-A is known to be approximately 60 kDa (fully modified fetuin-A: FM-Fet), which is much higher than deduced MW based on amino acid sequence. We focused on the effects of synthetic CPP on FM-Fet expression using the human hepatoma HepG2 cell line.

Results

CPP increased protein expression of FM-Fet in dose- (462% increase by 100 μg/mL CPP for 24hr) and time-dependent manner. However,CPP did not affect the mRNA expression of fetuin-A . Although we focused on the effects of CPP on degradation pathways of fetuin-A such as the lysosome and the ubiquitin-proteasome system, these pathways were not involved in CPP-induced FM-Fet expression. Since FM-Fet contains N-linked and O-linked glycosylation sites, cell lysates containing CPP-induced FM-Fet were incubated with N-glycosidase PNGase F (peptide-N-glycosidase F) and/or O-glycosidase. Treatment with N-glycosidase and/or O-glycosidase increased the considerable mobility of 60 kDa form of FM-Fet, suggesting that CPP modulate N- and O- glycosidations of fetuin-A. After HepG2 cells were pretreated for 30 min with or without 1 μg/mL brefeldin A, which blocks the transport of proteins from the endoplasmic reticulum to the Golgi complex, cells were incubated in 100 μg/mL CPP for 24 hr. Treatment with brefeldin-A inhibited CPP-induced FM-Fet expression concomitant appearance of approximately 50-55 kDa form.

Conclusion

CPP could upregulate posttranslational modifications of fetuin-A in HepG2 cells. These findings suggest a positive feedback loop between increased CPP which may reflect fetuin-A deficiency in advanced CKD and up-regulation of secreted fetuin-A.