Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Wei Wu,Yi Wei Dong,Peng Cheng Shi,Mei Yu,Da Fu,Chun Yi Zhang,Qian Qian Cai,Qian Lei Zhao,Ming Peng,Li Hui Wu,Xing Zhong Wu

doi:10.1194/jlr.m031450

Abstract

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Highlights

Integrin is important in migration and metastasis of tumor cells
SMMC-7721 cells were treated with exogenous sulfatide, Gal-Cer, or Lacto-Cer for 12, 24, and 36 h (Fig. 1A), and the mRNA level of the integrin ␣V subunit in the cells was analyzed by RT-PCR
Mouse immunoglobulin was used for a negative control as the unrelated primary antibody. (E) The positive rate of the surface integrin ␣V staining by flow cytometry analysis. (F) Integrin ␤3, ␤5, ␤6, ␤8, ␣5, and ␣V subunit mRNA was analyzed by real-time PCR after 24 h treatment with sulfatide. (G) Sulfatide was stained with O4 antibody after SMMC-7721 cells were treated with sulfatide and Lacto-Cer for 24 h, fixed, washed, and permeabilized. (H) SMMC-7721 cells were treated with 2 ␮M sulfatide, Lacto-Cer, ManNpro, and cyclo-ManN-pro for 24 h, respectively

Summary

Introduction

Integrin is important in migration and metastasis of tumor cells. We demonstrated that sulfatide regulated integrin ␣V expression and cell adhesion, which was associated with Erk activation.—Wu, W., Y. Z. Wu. Regulation of integrin ␣V subunit expression by sulfatide in hepatocellular carcinoma cells. The integrin ␣V␤ heterodimers on the cell surface interact with cell adhesive proteins, such as collagen, fibrinogen, fibronectin, and vitronectin. These interactions play an important role in cell adhesion or migration, especially in tumor metastasis. Integrin ␣V␤3 physically associates with phosphorylated and activated insulin-like growth factor receptor, and it may be involved in the HCC cell migration and progression [11]. Inducing the expression of the integrin ␣V [7] or ␤3 [12] subunit in melanoma cells increases their metastatic potential

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