Differential Expression Of Connective Tissue Growth Factor In Keloid Derived Fibroblasts Versus Normal Dermal Fibroblasts And The Regulatory Mechanism

Objective Keloids are pathological scars representing a pathological wound-healing response. There is no ideal treatment for keloid. Connective Tissue Growth Factor (CTGF) is an important pro-fibrotic growth factor. In this study, we aimed to compare CTGF expression between keloid derived fibroblasts (KFs) and normal dermal fibroblast (NFs). Serum stimulation was employed in this study as an in vitro model to mimic a component of the wound microenvironment for the examination of CTGF gene expression in KFs versus NFs. CTGF RNA interference was employed to inhibit CTGF expression in KFs. The expression of type I collagen and the proliferation of KFs were studied. We further explored the signal transduction pathways that trigged the over expression of CTGF after serum stimulation in KFs and the transcription regulation mechanism of CTGF promoter. By investigating the expression and regulation of CTGF, we wish to understand the molecular mechanism of keloid pathogenesis. Furthermore, we wish to identify a new therapeutic target for the treatment of keloids.Experiment 1 Differential expression and RNA interference study of CTGF in KFs and NFsMethods The expression of CTGF and its downstream target gene type I collagen in KFs and NFs was analyzed with q RT-PCR. Three CTGF siRNA sequences were cloned into pRNAT-6.1/Neo vector and transfected into KFs or NFs respectively. The expression of CTGF and type I collagen was analyzed with q RT-PCR 24h after transfection. The growth curve of KFs and NFs was draw after transfection.Results The expression of CTGF and type I collagen was elevated significantly in KFs versus NFs(p<0.05). CTGF mRNA level up-regulated 1h after serum stimulation in KFs, up to 8-17 folds before stimulation. While CTGF mRNA level only slightly increased in NFs after serum stimulation, which was about 2 fold before stimulation. All of the three CTGF siRNA vectors showed inhibitory effects on CTGF mRNA expression in KFs, among those, the CTGF siRNA3 vector was the most potent one(.p<0.05)Transfection of CTGF siRNA3 vector inhibited the type I collagen expression both in KFs and NFs(p<0.05). CTGF siRNA3 vector also decreased the proliferation rate of KFs and NFs(p<0.05).Experiment 2 The study of signal transduction pathways related to the over-expression of CTGF in KFsMethods Cycloheximide was added to the medium 30min before serum stimulation to block the new protein synthesis. CTGF expression was analyzed with q RT-PCR. Small molecule synthesized inhibitors of various signal pathways were added to culture medium 30 min before serum stimulation to block specific signal transduction pathways, including PI3K inhibitor wortmannin (100 nM);ERK inhibitor PD98059 (50 mM);p38 MAPK inhibitor SB203580 (10 mM);JNK inhibitor SP600125 (25 mM), and TGF-βtype I receptor inhibitor SB431542 (0.1μM, 0.5μM, 1μM and 10μM). RNA was collected 0, 1, 6, 12 and 24 hours after serum stimulation and the expression of CTGF was analyzed with q RT-PCR. The protein of KFs and NFs was collected 0h, 15min, 30min, 1h, 2h, 3h, 6h and 24h after serum stimulation. The expression and phorsphoralation of signal molecules of MAPK, PI3K and TGF-βpathways were assayed with Western blot.Results Pretreatment of cycloheximide did not inhibit the expression of CTGF in either KFs or NFs, demonstrated the transcriptional response of CTGF was in an immediate early fashion as previously described. Among all the inhibitors that were studied, only JNK inhibitor SP600125 and TGF-βtype I receptor inhibitor SB431542 decreased the CTGF up-regulation in KFs after serum stimulation, indicated that both JNK and TGF-βsignaling pathways took part in the over-expression of CTGF in KFs. Western blot showed that the expression of ERK, P38 and JNK did not change before or after serum stimulation, while the phosphorylation of P38 and JNK was significant higher and more prolonged in KFs versus in NFs. The down stream transcription factors ATF-2, Elk-1 and c-JUN were quickly phosphorylated within 15 min after serum stimulation in KFs, and maintained at high level up to 24 h in KFs. While in NFs, the phosphorylation was transient. The activation of other signal molecules such as P90 and AKT showed no difference between KFs and NFs. The type I receptor of TGF-βand SMAD3 were phosphorylated within 15 min after serum stimulation and maintained at high level up to 24 h in both KFs and NFs. Our results suggested that after serum stimulation, multiple signal pathways were activated in both KFs and NFs, including ERK, P38, JNK, PI3K and TGF-β, while only P38 and JNK showed significant differences between KFs and NFs.Experiment 3 The promoter analysis of CTGF gene and the transcriptional regulation studyMethods CTGF promoter reporter plasmids were constructed with various length of CTGF promoter sequences into PGL3-basic, namely pCTGF-1999,pCTGF-736,pCTGF-625,pCTGF-140,pCTGF-72. Each CTGF promoter/Luc fusion plasmids were transient transfected into KFs and NFs, and the luciferase activity was assessed. The site-directed point mutagenesis of SMAD binding site or AP-1 binding site was performed on pCTGF-625 construct, and the subsequent luciferase activity was assessed. Nuclear extracts were prepared from KFs and NFs before and 1 hour after serum stimulation, and was analyzed with electrophoretic mobility shift assay (EMSA). Supershift experiments were carried out with antibodies against c-jun, SMAD2 and SMAD3.Results Promoter analysis demonstrated a higher promoter activity of CTGF in KFs versus NFs. The fragment from -140/-72 conferred increased basal expression and the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and Smad binding site were both necessary for serum responsiveness. EMSA showed specific bands indicating proteins binding to the AP-1 site and Smad binding site in KFs that increases in intensity 1h after serum stimulation. This increase binding activity with serum stimulation was not detected in NFs. The addition of antibodies specific for c-Jun, SMAD2 and SMAD3 was able to supershift both the dominant band and the induced band indicating that c-Jun, SMAD2 and SMAD3 presented in that complex that binding the AP-1 site and the SMAD binding site. Conclusion These data suggest the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding. KFs express elevated level of CTGF, and have a stronger response to serum stimulation in terms of over-expression of CTGF. The over expression of CTGF following serum stimulation in KFs is mediated by JNK and TGF-βsignaling pathways, and the activation of JNK pathway is believed to be unique in KFs. The CTGF promoter fragment from -140/-72 conferres increased basal expression. The fragment from -625/-140 conferres increased serum responsiveness, which could be the AP-1 site and the SMAD binding site. The phosphorylated c-Jun, SMAD2 and SMAD3 formed the transcription factor complex and coordinatively binds to the AP-1 site and the SMAD binding site, up-regulated the expression of CTGF after serum stimulation. CTGF RNA interference could inhibit the expression of CTGF in KFs, and down-regulated the production of type I collagen and decreased the proliferation of KFs. Therefore the RNA interference of CTGF may have the therapeutic potential in the treatment of keloids.