Mechanism Related With Canonical Wnt Signaling Pathway Of SFRP2 Regulating Apoptosis And Function Of Hypertrophic Scar Fibroblast

1. IntroductionThe ideal outcome of human wounding is functional and scarless healing. However, hypertrophic scar(HS) often occurs following cutaneous injury, such as burns and traumas which often result in contractures, severe functional and esthetic defects, and even permanent disability.HS is characterized by abnormal accumulation of a large number of fibroblasts and excessive deposition of extracellular matrix (ECM) components which result from abnormal fibroblast apoptosis and proliferation. Additionally, the differentiation from fibroblasts to myofibroblasts in HS plays an important role in the contracture of scars. Although the mechanism of HS remains unclear, it has been recognized that the inhibition of fibroblast apoptosis and proliferation promotion are key processes in the formation of HS.It is well known that wound healing is regulated by a delicate balance between cell proliferation and cell apoptosis. It was reported that a variety of genes including Bcl-2, Fas, smad3 and p53, were associated with the regulation of fibroblast apoptosis and proliferation. Previously, we screened genes that were differentially expressed in HS and normal skin of burn patients using cDNA microarray and then identified 97 differentially expressed genes. Among these genes, only one anti-apoptotic gene named secreted frizzled-related protein 2 (SFRP2) was conspicuously up-regulated in HS tissue.SFRP2 is a secreted glycoprotein and it has been considered a modulator of Wnt signaling and involved in apoptosis, proliferation and differentiation. Kobayashi et al. confirmed that SFRP2 induced the cardiomyoblast apoptosis and favored collagen deposition and fibrosis. Further, up-regulation of SFRP2 in retinas of patients with retinitis pigmentosa suggested an anti-apoptotic role of SFRP2 in some degree. The role of SFRP2 in the HS formation is not clarified yet. In the present study we inspected the role and mechanim of SFRP2 in HS formation.2. Materials and methods2. 1 Patient specimens and primary cell cultureHSFBs were established as primary cell lines either from normal skin or from HS tissue obtained from patients whom were recovering from severe burn. Individuals knew and signed informed consents. This study was also approved by the Ethnic Committee of Southwest Hospital of the Third Military Medical University (Chongqing, China).2.2 upregulating of SFRP2 by pcDNA3.0/SFRP2Obtaining cDNA of SFRP2 from HSFBs,and then establishing pcDNA3.0/SFRP2. HSFBs were transfected with pcDNA3.0/SFRP2 plasmids. The transfection efficiency was confirmed by immunoflurescence density of SFRP2. The expression levels of SFRP2 were measured by western blot.2.3 Silencing of SFRP2 by shRNAThree pairs of shRNAs targeting different regions of the human SFRP2 transcript (accession number in GenBank: NM₀03013) and one control shRNA were designed and synthesized. shRNA fragments were annealed and cloned into the pSilencer 2.1-U6 neo plasmid (Ambion, USA). HSFBs were transfected with SFRP2 shRNA plasmids. The transfection efficiency was confirmed by immunoflurescence density of SFRP2. The expression levels of SFRP2 were measured by western blot at the second days after the transfection. After the examination, shRNA2 was selected as the most efficient sequence to block the expression of SFRP2.2.4 Cell proliferation assayUsing Cellular growth curve, MTT and BrdU Proliferation assay methods to evaluate the proliferation of HSFBs.2.5 Cell apoptosis assayUsing caspase3 activity and fluorescence-activated cell sorting (FACS) methods to detect the apoptosis of HSFBs.2.6 Cell function assayUsing Fibroblast populated collagen lattice (FPCL) model to evaluate the contractility of HSFbs. Further, real-time PCR and western blot were used to measure the mRNA and protein expression of?-SMA in HSFb. In addition, mRNA levels of types?and?procollagen were assayed by quantitative real-time PCR.2.7 Wnt proteins assayUsing Western blot to measure expression of Wnt1, wnt3a and wat8b of HSFBs. And then apoptosis of HSFBs treated with wnt3a was measured by caspase3 activity and fluorescence-activated cell sorting (FACS).?-catenin and c-myc was detected by western blot.3. Results3.1 upregulating of SFRP2 by pcDNA3.0/SFRP2pcDNA3.0/SFRP2 is the most efficient to upregulate the expression of SFRP2 of HSFBS,and obtain HSFBs transfected with pcDNA3.0/SFRP2(versus HSFBs,p<0.01).3.2 Silencing of SFRP2 by shRNAshRNA2 is the most efficient interference sequence to suppress the expression of SFRP2 of HSFBs, and obtain HSFBs transfected with psilencer 2.1-U6./SFRP2(versus HSFBs and shRNA1, shRNA3,p<0.01).3.3 Cell proliferation assayMTT and BrdU results suggested that Upregulating and downregulating SFRP2 in HSFBs had no significant effect on HSFB proliferation (p>0.05).3.4 Cell apoptosis assayUpregulating SFRP2 in HSFBs had no significant effect on HSFBs apoptosis. But compared with normal HSFBs, SFRP2 shRNA promoted cell caspase3 activity (p<0.01). The results of FACS were same.3.5 Cell function assayCompared with normal HSFBs, the suppression of SFRP2 expression decreasd?-SMA mRNA and protein levels and resulted in loss of contractile ability of HSFBs (p<0.01). SFRP2 shRNA2 downregulated the mRNAs of types?and?procollagen of HSFBs (p<0.01).3.6 wnt proteins assayControlled with NSFBs, the results of WB suggested expression of Wnt3a was upregulated in HSFBs (p<0.01); The level of apoptosis of HSFBs treated with wnt3a was suppressed by SFRP2 and SFRP2 suppressed expression of?-catenin and c-myc of HSFBs (p<0.01).4. Conclusion4.1 Obtaining the HSFBs of overexpressing and downexpressing SFRP2.4.2 In vitro, SFRP2 had no effect on proliferation of HSFBS.4.3 In vitro, SFRP2 shRNA promoted HSFB apoptosis, but upregulating SFRP2 didn't.4.4 SFRP2 suppressed apoptosis of HSFBs by influencing wnt3a/?-catenin.?-catenin and C-myc may be key factors in this progress.4.5 SFRP2 shRNA may be a novel treatment strategy.