The Role Of Sufu On Cutaneous Wound Healing And Skin Fibrosis And The Underlying Mechanisms

Part one:The role of Sufu on cutaneous wound healing and the underlying mechanisms.Background:Cutaneous wound healing is a regenerative and interactive pathphysiological process that rapidly and efficiently restores skin integrity.The normal mammalian responses to cutaneous wound involves four phases:(1)a hemostasis phase;(2)an inflammatory phase;(3)a proliferative phase involving the migration and proliferation of keratinocytes,fibroblasts,and endothelial cells,resulting in re-epithelialization and angiogenesis;and(4)a long remodeling phase in which fibroblasts are activated by macrophages and differentiate into myofibroblasts,causing wound contraction.This process is complex and requires dynamic interactions among several cell types,including endothelial cells,blood cells,epidermal keratinocytes,and dermal fibroblasts.Surgical incisions,thermal burns and chronic ulcers are among the conditions in which cutaneous wound healing plays a pivotal role.In addition to acute wounds,there has been a steady rise in chronic skin wounds such as pressure ulcers and diabetic foot ulcers.The prevalence of diabetes,obesity and cardiovascular diseases in aging populations is fostering the growth of chronic skin wounds.The failure of wound healing has become a serious clinical burden not only in surgery,burn or trauma but also in patients with chronic conditions.Dysfunctional healing often imposes socioeconomic problems on the affected individuals and societies.Advances in understanding the molecular and cellular mechanisms of cutaneous wound healing will be critical for improved wound therapy and preclinical prevetion.The Suppressor of fused(Sufu),whose function varies from being a tumor suppressor gene to being a normal tissue development mediator,is a repressor of the Hedgehog(Hh)signaling pathway.Suppressor of fused(Sufu)acts as a negative regulator of the Hedgehog(Hh),Wnt/?-catenin,and other signaling pathways in vertebrates and prevents the development of malignant tumors.Thus,Sufu is essential for skin development and acts as a tumor suppressor.Despite its biological importance,the effects of Sufu on cutaneous wound healing are largely unknown.In this study,we investigated the potential roles of Sufu in cutaneous wound healing.Methods:1.Generation of TG mice overexpressing Sufu in the epidermis.A transgenic(TG)mice overexpressing human Sufu(hSufu)in the epidermis was successfully constructed by including K14 promoter.Sufu morphology in the skin of newborn and adult mice was then examined.2.Examination of in vivo skin repair.Full-thickness incision wounds was created on the dorsal skin of hSufu TG and WT mice.The wound area,epithelial tongues and the granulation tissue was observed and measured by hematoxylin and eosin and masson's trichrome staining.Using Western blot analysis,qRT-PCR and Immunofluorescence to identify the mechanism by which Sufu affect the process.3.The role of sufu in keratinocyte and fibroblast.Using scratch wound assays and CCK8 assay to evaluate the migration and proliferation capability of keratinocytes and fibroblast of hSufu TG and WT mice.Using TGF-? stimulation experiment to assess the differentiation capability of fibroblast into myofibroblast.Results:1.Generation and verification of transgenic mice that overexpress hSufu in the epidermis.Transgenic(TG)mice overexpressing hSufu in the epidermis was generated and were identified by polymerase chain reaction,western blot analysis and immunofluorescence staining.2.Characterization of the K14-hSufu TG mouse model.The dermis of the dorsal skin was significantly reduced in the 7-day-old and 7-week-old TG mice compared with that of the wild-type(WT)controls,whereas the hypodermal adipose tissue was significantly increased in Sufu TG mice at both ages.3.Cutaneous wound healing was delayed in hSufu TG mice.To investigate the role of Sufu in skin homeostasis,the effect of Sufu overexpression in skin repair was examined.Full-thickness incision wounds was created on the dorsal skin of hSufu TG and WT mice.The surface area of the wounds was measured 1,3,7,and 12 days after injury.The wound area was larger in TG mice than in the WT controls from day 7,suggesting that wound healing was delayed significantly in TG mice.Consistent with slower healing,significantly extended epithelial tongues were observed in TG mice at days 7 and 12 after injury.Masson's trichrome staining of excisional wound sections made on day 12 post-injury revealed that the area of granulation tissue in the mid-wound region of the TG mice was significantly larger than that of the WT controls.4.Overexpression of Sufu restrained keratinocyte migration and proliferation.Sufu overexpression can functionally suppress cell migration in vitro.The proliferation capability of keratinocytes was greater in the WT mice than in the Sufu TG mice5.Overexpression of Sufu in the epidermis affects the dermis.Proliferation capability increased in TG fibroblasts compared with WT fibroblasts.Moreover,TGF-?treatment increased the expression of a-smooth muscle actin(?-SMA)in WT fibroblasts compared to TG fibroblasts.6.Overexpression of Sufu retards wound healing via blocking Hh/Gli and Wnt/?-catenin pathway.Conclusion:overexpression of epidermal Sufu regulated the cellular properties of keratinocytes and dermal fibroblasts during skin development and delayed wound healing via inhibition of Hh/Gli1 and Wnt/?-catenin signaling pathways.Part Two:The role of Sufu on skin fibrosis and the underlyin mechanismsBackground:Systemic sclerosis(SSc)is a connective disease of autoimmune origin characterized by the fibrosis of skin and visceral organs.The pathogenesis of SSc involves a triad of autoimmune,vascular and inflammatory alternations resulting in fibrosis.Pathological fibrogenesis is a complex process activated in response to tissue injury.Both the innate and adapative immune system are believed to contribute to pathogenesis of fibrosis as the recruitment of immune cells lead to activation of fibroblasts and conversion of latent transforming growth factor(TGF)-? into the active form and the consequent excessive synthesis of extracellular matrix(ECM).Because there is no effective treatment for patients nowadays,SSc significantly influences patient quality of life and life expectancy,as it eventually causes severe disability and even death.Therefore,in-depth studies on the etiology and possible treatments for SSc are in urgent needs.The mammalian Hh pathway is conserved both molecularly and mechanistically.There are three Hh genes in vertebrates:Sonic,Inidan,and Dessert;among these the Sonic Hedgehog(SHH)has the most prevalent roles.A single homologous Sufu gene has been identified and cloned from the mouse and human genomes,respectively.The human Suppressor of fused(Sufu)gene,which consists of 12 exons and is located in a region often deleted in tumors on 10q24,encodes a protein of 484 amino acids.Human Sufu is expressed in most adult tissues and at various developmental phases with little indication of temporal or spatial regulation.Sufu acts as a negative regulator of Hh signaling and a tumor suppressor.Meanwhile,Sufu acts as a negative regulator of the Wnt/?-catenin and other signaling pathways in vertebrates.The pathogenesis of SSc has been studied in a bleomycin(BLM)-induced scleroderma model.Among fibrotic agents known to contribute to scleroderma,the role of Sufu in skin fibrosis and its underlying mechanisms has not been thoroughly described.In this study,we investigated the potential roles of Sufu in skin fibrosis.Methods:1.Assessing the expression of Sufu in mouse BLM-induced dermal fibroblast.2.Wild type(WT)and transgenic(TG)mice overexpressing Sufu in the epidermis were subjected to the bleomycin model of cutaneous skin scleroderma.BLM or phosphate-buffered saline(PBS)was administered subcutaneously,and skin fibrosis was evaluated by dermal thickness,subcutaneous fat atrophy,myofibroblast count,inflammation and collagen in the dermis.Using western blot and qRT-PCR to explore the possible mechanisms.3.Dermal fibroblasts isolated from WT and TG mice were cultured in vitro,stimulated with BLM,and the expression of profibrotic genes was compared by qRT-PCR and western blotting.Evaluating dermal fibroblast activation and collagen synthesis in response to BLM of WT mice and Sufu TG mice in vitro.Results:1.Sufu was down-regulated in BLM-induced mouse dermal fibroblast.We cultured fibroblast from normal WT mice in vitro and the expression of Sufu was significantly down-regulated after BLM treatment.Western blot results also confirm the above data.2.Sufu TG mice resisted BLM-induced skin fibrosis.BLM administration effectively induced decreased skin thickening in Sufu TG mice versus WT mice(2.02 fold versus 1.56 fold;302±58?m vs.191±41.5?m,p<0.01),while dermal thickness was comparable between PBS-treated WT mice and PBS-treated Sufu TG mice.Similarly,sufu significantly reduced collagen content as expressed by hydroxyproline assy(+376%of control thickness)and TCS blue-stained area(13.6±1.0%vs.30.1±4.2%,p<0.01)in Sufu TG versus WT mice.The reduction in skin thickness and collagen content in Sufu TG mice was accompanied by a significant reduction in the expression of ?-smooth muscle actin(?-SMA)(a hallmark of the myofibroblast phenotype).In addition,we showed the expression of?-SMA in the skin by immune-histochemical staining,and Sufu TG mice attenuated the increase of myofibroblast within the dermis.Similarly,Sufu TG skin injected with BLM had less fibronectin and collagen I expression relative to WT skin injected with BLM.qPCR analysis confirmed up-regulation of collagen transcripts.Also lung fibrosis was evaluated,but the lung histological manifestations were not significantly different between WT and Sufu TG mice with BLM treatment.Together these data demonstrate that Sufu TG mice have reduced dermal fibrosis in the BLM-induced dermal fibrosis model,indicating that Sufu is an important mediator in the development of dermal fibrosis.3.Sufu is associated with dermal fibroblast activation and collagen synthesis in response to BLM.BLM stimulation induced higher levels of ?-SMA,fibronectin,collagen I and cyclinDl protein expression in WT fibroblasts than did Sufu TG fibroblasts.Collectively,sufu TG fibroblasts showed less pro-fibrotic properties in response to BLM than WT.4.Sufu down-regulates the activation of Wnt/?-catenin signaling pathway to inhibit BLM-induced skin fibrosis.We found that Sufu effectively inhibited the activation of ?-catenin in vivo,which,at least in part,likely contributes to preventing BLM-induced dermal fibrosis in this murine model of scleroderma.Protein level and gene expression results of ?-catenin both showed down-regulated by Sufu.Sufu over expression could strongly increase the degradation of ?-catenin.Conclusion:Sufu ameliorates skin involvement in bleomycin-induced fibrosis via down-regulating the activation of Wnt/?-catenin signaling pathway.