Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Biol Chem. Nov 26, 2015; 6(4): 379-388
Published online Nov 26, 2015. doi: 10.4331/wjbc.v6.i4.379
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
Liya Pi, Pei-Yu Chung, Sriniwas Sriram, Masmudur M Rahman, Wen-Yuan Song, Edward W Scott, Bryon E Petersen, Gregory S Schultz
Liya Pi, Bryon E Petersen, Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States
Pei-Yu Chung, Johnson and Johnson Vision Care, Jacksonville, FL 32256, United States
Sriniwas Sriram, Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114, United States
Masmudur M Rahman, Edward W Scott, Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, United States
Wen-Yuan Song, Department of Plant Pathology, University of Florida, Gainesville, FL 32610, United States
Gregory S Schultz, Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL 32610, United States
Author contributions: Pi L and Chung PY are co-first authors and share equal contribution; Pi L performed yeast two-hybrid analysis; Chung PY carried SPR experiments; Rahman MM contributed to SPR analysis; Sriram S established rabbit corneal fibroblast cell culture; Pi L, Chung PY, Scott EW, Petersen BE, and Schultz GS substantially contributed to the design of the study, acquisition, analysis and interpretation of data; Pi L and Chung PY wrote the paper; all authors made critical comments related to the intellectual content of the manuscript, and approved the final version of the article to be published.
Supported by NIH RO1 grants, Nos. DK058614 and DK065096 awarded to BEP and EY05587 awarded to GSS; Chris DiMarco Institutional Research Grant from American Cancer Society award to LP.
Conflict-of-interest statement: No conflict of interest exists.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at lpi@peds.ufl.edu.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Liya Pi, PhD, Research Assistant Professor, Department of Pediatrics, University of Florida, Gainesville, FL 32610, United States. lpi@peds.ufl.edu
Telephone: +1-352-2945679 Fax: +1-352-2945734
Received: May 29, 2015
Peer-review started: May 30, 2015
First decision: June 18, 2015
Revised: July 29, 2015
Accepted: September 29, 2015
Article in press: September 30, 2015
Published online: November 26, 2015
Processing time: 177 Days and 11.4 Hours
Abstract

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 endothelial 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 LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA 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 LexA. Yeast co-transformants 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 the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-β1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of β-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRβ receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRβ and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2.

CONCLUSION: CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

Keywords: Connective tissue growth factor; Vascular endothelial growth factor A; Platelet derived growth factor B; Transforming growth factor-β; Bone morphogenetic protein-4; Yeast two-hybrid analysis; Surface plasma resonance; Rabbit corneal fibroblast cells; Platelet-derived growth factor receptor β; Platelet-derived growth factor-B signaling

Core tip: The relative binding strength of connective tissue growth factor (CTGF) was vascular endothelial growth factor-A > bone morphogenetic protein-4 > transforming growth factor-β1 > platelet-derived growth factor (PDGF)-B. CTGF binding could potentiate PDGF-B signaling as evidenced by enhanced phosphorylation of PDGF receptor β and downstream AKT molecules in rabbit corneal fibroblast cells. Our findings provide deep insight into CTGF action in fine-tuned regulation of extracellular signaling mediated by different cysteine knot containing growth factors.