Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin, Especially Decreased Sialylation

Kotone Sano,Yasunori Miyamoto,Nana Kawasaki,Noritaka Hashii,Satsuki Itoh,Misaki Murase,Kimie Date,Miki Yokoyama,Chihiro Sato,Ken Kitajima,Haruko Ogawa

doi:10.1074/jbc.m109.077016

Abstract

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn(167). Highly sialylated O-glycans were found to be present in the Thr(110)-Thr(124) region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

Highlights

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes
The effect of partially hepatectomized (PH)-VN on rat hepatic stellate cell (HSC) (rHSCs) spreading was found to be decreased to one-half of that of NO- or SH-VN (Fig. 1A). rHSC spreading was decreased on neuraminidase-treated VN compared with untreated VN (Fig. 1B), whereas it was decreased less on de-Nglycosylated NO-VN (Fig. 1C)
These results indicate the importance of glycosylation, sialylation, of VN in rHSC spreading

Summary

Survival Signals of Hepatic Stellate Cells in Liver

Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin, Especially Decreased Sialylation*□S. The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Vitronectin Glycan Controls Hepatic Stellate Cell Survival with type-1 plasminogen activator inhibitor, urokinase-type plasminogen activator, and urokinase receptor [3, 7,8,9] Besides this function, VN plays a key role in cell adhesion and cellular motility during tissue remodeling through binding to major ECM receptors, integrins such as ␣v␤1, ␣v␤3, ␣v␤5, ␣v␤6, and ␣v␤8, and other ECM components like collagen and proteoglycans [3]. The present study attempted to determine the structure and changes of rat VN glycans during liver regeneration with a particular focus on the relationship between hepatic stellate cell (HSC) adhesion and the glycosylation of VN. The present findings provide novel insight into the significance of VN glycosylation in regulating HSC survival during tissue remodeling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION