Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells

• 作者: Bryon E Petersen Edward W Scott Gregory S Schultz Liya Pi Masmudur M Rahman Pei-Yu Chung Sriniwas Sriram Wen-Yuan Song
• 来源期刊: 世界生物化学杂志：英文版（电子版）
• 学科: 生物学
• 关键词: CONNECTIVE tissue growth factor Vascular endotheli
• 年，卷（期）: 2015,（4）
• 页码范围: 379-388
• 页数: 10页
• 分类号: Q78

摘要

AIM:To study the binding of connective tissue growth factor(CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor(PDGF)-B signaling. METHODS:The binding strengths of CTGF to cystine knot-containing growth factors including vascular en-dothelial growth factor(VEGF)-A,PDGF-B,bone morphogenetic protein(BMP)-4,and transforming growth factor(TGF)-β1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of Lex A operators and a lac Z reporter plasmid(p80plac Z) containing eight high affinity Lex A binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid β-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance(SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor β(PDGFRβ) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRβ and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. RESULTS:In the LexA-based yeast two-hybrid system,cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein Lex A. Yeast cotransformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine,tryptophan,and uracil. In liquid β-galactosidase assays,CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity,at 29.88 ± 0.91 fold above controls(P < 0.01). Cells containing the cystine knot of BMP-4 expressed