The Bidirectional Roles And Mechanism Study Of Cthrc1 In The Process Of Keloid

Part?Increased Cthrc1 and YAP nuclear location in keloid and keloid fibroblastsObjective:Keloid induces a severe impairment of quality of life for the patients although keloid is a cutaneous benign tumor.Collagen triple helix repeat-containing protein 1(Cthrc1)was identified as a novel gene that was originally found in adventitial fibroblasts after arterial injury.YAP modulates fibroblasts activation and matrix synthesis in the process of wound repair.To address the difference of Cthrc1 and YAP in keloid and normal skin,the expression level of Cthrc1 was assessed in tissue of normal skin and keloid,and fibroblasts derived from normal prepuce(NFs)and keloid(KFs).Methods:The expression level of Cthrc1 and YAP was investigated in keloid and normal skin by using Real Time PCR(RT-PCR),western blotting.Immunohistochemical analysis was used to define the tissue location of Cthrc1 and YAP.Normal fibroblasts and keloid fibroblasts were derived from normal prepuce and keloid tissue and using two-step collagenase digestion method.The fibroblasts were assessed by microscopic observation and immunocytochemistry stain.Cthrc1 and YAP was tested in KFs and NFs by using RT-PCR and western blot.The cellular location of Cthrc1 and YAP in the NFs and KFs was analyzed by immunofluorescence.Resluts:Cthrc1 was enhanced in keloid and KFs,and located in the cytoplasm of hyperplastic fibroblasts.Enhanced YAP protein expression was observed in KFs,but there was no expression difference berween keloid and normal skin.YAP was expressed in the fibrobleasts of keloid's hyperplastc and homogenitic zone.Similarly,YAP was located in the nuclei and cytoplasm of KFs;however,YAP was found only in the cytoplasm in NFs.Conclusion:Increased Cthrc1 and YAP nuclear location may play a role in the process of keloid.Part?Cthrc1 plays bidirectional role in the process of keloid by reversing the TGF-? signaling and modulating YAP subcellular locationObjective:Keloid is characterized pathologically by excessive dermal fibrosis and aberrant wound healing.To date,the patients with keloid and clinical doctors were still puzzled by the high recurrence rate of keloid.In the expriements of part?,we had confirmed that Cthrc1 was increased in the keloid tissue and keloid fibroblasts(KFs),and YAP location was transferred to the nuclei of KFs.To investigate the role and mechanoactivation of enhanced Cthrc1 in keloid formation,in this study we focused on the effect of Cthrc1 on collagen accumulation and the influence for TGF-? and YAP signaling pathway after increasing Cthrc1 expression in keloid fibroblasts and normal fibroblasts.Methods:In NFs and KFs,we enhanced Cthrc1 expression by transfecting lentivirus vectors with Cthrc1.The cellular proliferation and migration were tested by applying Cell Counting Kit 8 and transwell chambers.Real Time PCR(RT-PCR)and western blot were used to detedte the expression of collagen?,TGF-?,and YAP.Meanwhile,YAP and p-Smad2/3 location was verified by immunofluroscence staining.Then,the recombinant TGF-?1 was acted on NFs and KFs to assess the relationship with Cthrc1.Results:The cellular proliferation and migration were increased in KFs compared with NFs.When increased the expression of Cthrc1 in NFs,the NFs proliferation and migration were enhanced,and collagen?expression was still reduced whereas YAP expression was increased by Cthrc1.P-Smad2/3 transfer to nucleus was reversed by Cthrc1 in NFs,but Cthrc1 promoted YAP nuclear location in NFs.Then,when over express Cthrc1 in KFs,KFs' migration was retarded but the cellular proliferation had no significant change.Collagen?and YAP expression was decreased by Cthrc1 in KFs.Meanwhile,Cthrc1 suppressed the p-Smad2/3 and YAP nuclear location.In NFs and KFs,Cthrc1 was increased by TGF-?1.Conclusion:Enhanced Cthrc1 may activate NFs by increasing YAP expression and promoting YAP nuclear location,and inactivate KFs by decreasing YAP expression and inhibiting YAP nuclear location.Cthrc1 as a negative feedback factor of TGF-? signaling reduced the collagen?by reversing p-Smad2/3 nuclear transfer.Cthrc1 may be helpful in the normal wound repair;however,Cthrc1 may inhibit the excessive wound healing.Thus,Cthrc1 may play bidirectional role in the process of keloid.