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

Figure 1 Determination of connective tissue growth factor binding to cystine knot motifs of vascular endothelial growth factor-A, platelet-derived growth factor-B, transforming growth factor-β1, and bone morphogenetic protein-4 in yeast two-hybrid LexA system. A: CTGF binding to cystine knot motifs of tested growth factors was assessed using growth assay on selective SD medium lacking leucine (lower panel); B: The liquid β-galactosidase assay was also carried out determining the binding strength of CTGF to cystine knot motifs of tested growth factors. The relative β-galactosidase activity was quantified in graph on the right. ^aP < 0.05; ^bP < 0.01. Values are the average -folds of activation over pB42-AD vector alone (arbitrarily set to 1) from three independent experiments performed in triplicate ± SD. CTGF: Connective tissue growth factor; VEGF: Vascular endothelial growth factor; BMP: Bone morphogenetic protein-4; TGF-β1: Transforming growth factor-β1; PDGF-B: Platelet-derived growth factor-B.

Figure 2 Quantitative measurement of connective tissue growth factor binding to vascular endothelial growth factor 165 and platelet-derived growth factor-BB in surface plasma resonance analysis. The binding kinetics of CTGF to VEGF165 (A) or to PDGF-BB (B) were determined by injecting VEGF165 ranging from 3.879 nmol/L to 52.267 nmol/L or PDGF-BB ranging 19.3 nmol/L to 385 nmol/L over CTGF immobilized channel and reference channel at 30 μL/min for 3 min simultaneously. The reference sensorgram has already deducted to eliminate the signal from a bulk refractive index shift; C: Sensorgrams of SPR analysis showed that the equilibrium dissociation constant for CTGF binding to PDGF-BB or VEGF165 was 43 nmol/L or 1.8 nmol/L respectively. CTGF: Connective tissue growth factor; VEGF: Vascular endothelial growth factor; PDGF-B: Platelet-derived growth factor-B; SPR: Surface plasma resonance.

Figure 3 Connective tissue growth factor is able to enhance the binding of platelet-derived growth factor-BB to platelet-derived growth factor receptorβ. SPR assay was carried out to quantitatively measure the binding of PDGF-BB to PDGFRβ. A: Sensorgrams of SPR analyses showing the binding dissociation equilibrium constant of PDGF-BB and PDGFRβ; B: Measurement of SPR response unit showed that CTGF promoted PDGF-BB/PDGFRβ interaction. A mixture of PDGF-BB (2 nmol/L) with different concentrations of CTGF (0-263.2 nmol/L) was run over PDGFRβ sensor chips. CTGF: Connective tissue growth factor; PDGF-B: Platelet-derived growth factor-B; SPR: Surface plasma resonance; PDGFRβ: Platelet-derived growth factor receptor β.

Figure 4 Connective tissue growth factor enhances platelet-derived growth factor-BB binding to platelet-derived growth factor receptor β in surface plasma resonance analysis. PDGF-BB (2 nmol/L) was mixed with different concentrations of CTGF (0-263.2 nmol/L) and run over PDGFRβ sensor chips. SPR response units were measured and used for quantitative comparisons of interaction between PDGF-BB and PDGFRβ in presence of different concentrations of CTGF proteins in Figure 3B. Representative sensorgrams from two independent trials are shown. CTGF: Connective tissue growth factor; PDGF-B: Platelet-derived growth factor-B; PDGFRβ: Platelet-derived growth factor receptor β.

Figure 5 Connective tissue growth factor is not associated with platelet-derived growth factor receptorβ in surface plasma resonance analysis. CTGF protein (263.2 nmol/L) was run over PDGFRβ sensor chips and the SPR response unit was measured. Sensorgrams showed little binding response between CTGF and PDGFRβ proteins. CTGF: Connective tissue growth factor; PDGF-B: Platelet-derived growth factor-B; SPR: Surface plasma resonance; PDGFRβ: Platelet-derived growth factor receptor β.

Figure 6 Connective tissue growth factor potentiates platelet-derived growth factor-BB stimulated activation of platelet-derived growth factor receptorβ and downstream signal molecule Akt. Western analysis was carried out to determine phosphorylated or total PDGFRβ, ERK1/2 and AKT in rabbit corneal fibroblast cells stimulated by CTGF (6.6 nmol/L), PDGF-B (1.6 nmol/L) or combination. CTGF: Connective tissue growth factor; PDGF-B: Platelet-derived growth factor-B; PDGFRβ: Platelet-derived growth factor receptor β.