Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor

Adrien Bernard,Jacqueline Gao-Li,Claudio-Areias Franco,Tahar Bouceba,Alexis Huet,Zhenlin Li

doi:10.1074/jbc.m809259200

Abstract

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A(2)/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44-77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120-210). A 25-mer peptide named P46 (aa 46-70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

Highlights

A ligand/receptor interaction at the cell membrane seemed likely, in addition to interactions within extracellular matrices, because of the diverse effects and ubiquitous expression of Pigment epithelium-derived factor (PEDF) and the fact that most PEDF deposits remain within extracellular matrices [20]
Solid tumors need new vessels to grow, so selectively preventing angiogenesis has become a promising approach in anti-cancer therapy
Our results provide a molecular basis for this anti-angiogenic activity

Summary

EXPERIMENTAL PROCEDURES

Y2H Screening of PEDF Partners—We used a Matchmaker GAL4 two-hybrid system of Saccharomyces cerevisiae AH109 strain (Clontech) to screen a human skeletal muscle Matchmaker cDNA library (Clontech), with their 5Ј ends proximal to the activation domain (AD) of the GAL4 transcription factor in a pACT2 vector. Y2H Method to Identify Laminin Receptor and PEDF Interaction—We PCR-amplified different human PEDF fragments (encoding amino acids 2– 418, 140 – 418, 206 – 418, 374 – 418, 2–326, 2–140, 2– 86, 2– 44, 44 –121, and 44 –77; see Fig. 1) and cloned them into the EcoRI and BamHI sites of the pGBKT7 vector. PEDF Expression in Insect Cells and Laminin Receptor Expression in Escherichia coli—We used the primers PEDF2 (5Ј-AATGAATTCCAGGCCCTGGTGCTACTCCTC-3Ј) and PEDFR (5Ј-CCTCTAGACTGGGGCCCCTGGGGTCCAG3Ј) to PCR amplify human PEDF cDNA [2– 418]. We cloned this fragment into the EcoRI and XbaI sites of the pIB/V5-His/ CAT vector (Invitrogen). We analyzed results with BIA evaluation software version 4.1

F46 Binding to Cells—We grew

Findings

DISCUSSION