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 cyst...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 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. PDGFBB 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 PDGFBB 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.展开更多
Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in several tissues including skin, liver, kidney and the cornea. In this study, a RNA interference-based approach using siRNAs ...Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in several tissues including skin, liver, kidney and the cornea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and corneal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combination. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein immunolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further increased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb 1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduced SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar formation at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results demonstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rabbit corneas with a triple combination of siRNAs effectively reduced levels of the target genes and SMA, leading to reduced corneal scarring at 14 days, suggesting that this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues.展开更多
基金Supported by NIH RO1 grants,Nos.DK058614 and DK065096 awarded to BEP and EY05587 awarded to GSSChris DiMarco Institutional Research Grant from American Cancer Society award to LP
文摘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 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. PDGFBB 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 PDGFBB 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.
文摘Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in several tissues including skin, liver, kidney and the cornea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and corneal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combination. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein immunolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further increased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb 1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduced SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar formation at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results demonstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rabbit corneas with a triple combination of siRNAs effectively reduced levels of the target genes and SMA, leading to reduced corneal scarring at 14 days, suggesting that this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues.