The Effect And Mechanism Of RAMP1 And GPX4 In The Regulation Of Wound Repair

BackgroundWound repair is a dynamic and coordinated process and a variety of functional cells and factors are involved.As one of the most important cells in the process of wound repair,an in-depth study of the function of fibroblasts and related regulatory mechanisms can help to understand the process of normal skin wound repair and provide solutions for abnormal healing of skin wounds.Normal nerve function in the skin is a key factor in the orderly repair of wounds and plays an important role in all stages of wound repair.Abnormal nerve function may lead to excessive scarring or delayed healing resulting in chronic refractory wounds.The calcitonin gene-related peptide(CGRP)has been shown to modulate fibroblast function and promote skin wound healing,but its short half-life and the side effects associated with its direct use in wounds limit its clinical use.The CGRP receptor consists of three subunits,including the guanine nucleotide-binding protein(G protein)-calcitonin receptor like receptor(CLR),the receptor activity modifying protein 1(RAMP1)and the receptor component protein(RCP).RAMP1 can function independently of the CGRP receptor and has previously been shown to influence wound repair by regulating lymphatic vessels and angiogenesis.However,no studies have reported whether RAMP1 acting alone can regulate fibroblast function.Yes-associated protein(YAP)is expressed at high levels in fibroblasts within the wound during the early stages of repair,and G protein-coupled receptor(GPCR)has been shown to regulate YAP activity in different cell types.GPCR activates adenylyl cyclase(AC)through the classical Gas subunit to produce cyclic adenosine monophosphate(cAMP)and stimulate protein kinase A(PKA)and the downstream cAMP response element-binding protein(CREB),while the inhibitory Ga subunit(Gαi)protein inhibits this process.It is not known whether RAMP1 functions through YAP and whether the relevant regulatory mechanisms are through the classical GPCR pathway.In summary,the aim of this study was to investigate the role of RAMP 1 in regulating the proliferation of mouse skin fibroblasts(MSF),the relationship between RAMP1 and YAP regulation,and the underlying mechanisms.Objectives(1)To construct MSF cell lines overexpressing RAMP1 and to examine the effect of RAMP1 on MSF proliferation.(2)To examine the regulatory effect of RAMP 1 on YAP.(3)To investigate the molecular and pathway mechanisms involved in the regulation of YAP by RAMP1.Methods1.Construction of mouse skin wound modelC57BL/6J male mice(8-10 weeks old;weight 30-35 g)were anesthetized,dehaired and disinfected,and the skin wound were prepared on both sides of the back,and the wound tissues were collected after euthanasia on days 1,3,5 and 7 after the wound models were made.2.Total skin protein extractionTotal skin protein was extracted using the Minute Skin Tissue Total Protein Extraction Kit.3.Immunohistochemical stainingImmunohistochemical staining of RAMP 1 was performed on paraffin sections of wound tissues to observe the distribution and expression of RAMP 1 in the tissues.4.Hematoxylin-eosin stainingParaffin sections of wound tissue were stained with hematoxylin-eosin to observe the thickness of the dermis in the wound tissue.5.Construction of MSF cell lines overexpressing RAMP1MSF was transfected with Tet-On-Flag-RAMP1 lentivirus and Tet-On-Flag vector lentivirus for 24 hours and puromycin(3 μg/ml)was screened for 48 hours to obtain stable clones.RAMP1 was overexpressed in MSF cell lines after 48 hours of induction with doxycycline(DOX,5 μg/ml).6.siRNA transfectionMSF cell lines overexpressing RAMP1 were transfected with small interfering RNAs(siGai3-1 and siGai3-2)specifically targeting Gαi3 and a negative control sequence(siNC),respectively.7.Drug interventionThe YAP inhibitor verteporfin,the PKA inhibitor H-89 dihydrochloride,the PKA agonist Bucladesine sodium and the CREB inhibitor KG-501 were used to stimulate the MSF overexpressing RAMP1 at the corresponding concentrations.8.Cell proliferation assayCell proliferation was measured using the IncuCyte S3 Live-Cell Analysis System.9.Protein extractionTotal cellular proteins were extracted by RIPA,and proteins in the nucleus and cytoplasm were extracted by Minute Cytoplasmic and nuclear extraction kit respectively.10.Western Blot assayThe expression level of each target protein in total wound skin protein,total cell protein and nucleus/cytoplasm protein was measured.11.mRNA extractionTrizol was used to extract total cellular mRNA.12.qPCR assayThe levels of the corresponding target mRNAs in the total cellular mRNA were detected by SYBR Green kit.13.Cell immunofluorescence staining analysisImmunofluorescence staining was performed on fixed cells to observe the expression level and localization of the corresponding target protein.14.CUT&RUN assayProtein-DNA interactions were analyzed by CUT&RUN assay kit.15.Dual luciferase reporter assayHEK293T cells were transfected with the reporter plasmid and the dual luciferase reporter kit was used to detect the luciferase activity of each group.16.Statistical analysisStatistical analysis was performed using GraphPad Prism version 7.0.0 software.All experiments were repeated three times and quantitative data were expressed as mean±standard deviation.Differences between groups were compared by using an unpaired t-test or one-way ANOVA,where P<0.05 represents a statistically significant result.Results1.Overexpression of RAMP1 promotes MSF proliferationThe protein level of RAMP 1 in mouse skin wounds changed with the process of wound repair,and the trend of change was consistent with the trend of change in dermal thickness.In vitro MSF cell lines overexpressing RAMP 1 were constructed,and various assays demonstrated that overexpression of RAMP 1 promoted MSF proliferation.2.RAMP1 overexpression increased the expression of Gai3,PKA,CREB and YAPOverexpression of RAMP1 promoted the levels of YAP and pYAP(S127)as well as Gai3,PKA,CREB and its active form pCREB(S133)in MSF.The levels of YAP and CREB were increased in both nucleus and cytoplasm of MSF overexpressing RAMP1,and RAMP1 overexpression also promoted the transcription of YAP.3.RAMP1 overexpression promotes proliferation by increasing YAP protein levelsYAP inhibitors inhibit the proliferation of MSF overexpressing RAMP1 and increase YAP levels in the cytoplasm while decreasing YAP levels in the nucleus and increasing retention of YAP in the cytoplasm,but not YAP transcription.4.Interference with Gai3 down-regulates PKA,CREB and YAP levels and inhibits MSF proliferationInterference with Gai3 in MSF overexpressing RAMP1 inhibited their proliferative capacity and down-regulated the levels of PKA,CREB and YAP total protein and YAP transcription.pCREB(S133),the active form of CREB,and pYAP(S127),the inactive form of YAP,were both down-regulated in the Gai3-interfered group.Gai3 interference also reduced both cytoplasmic and nuclear levels of CREB and YAP.5.PKA inhibitors down-regulate CREB and YAP levels and inhibit MSF proliferationPKA inhibitors inhibited the proliferation of MSF overexpressing RAMP1.PKA inhibitors also reduced the protein levels of PKA,CREB and YAP and the transcript levels of YAP.6.PKA agonists upregulate CREB and YAP levels and promote MSF proliferationPKA agonists promoted the proliferative capacity of MSF overexpressing RAMP1,and increased the protein levels of PKA,CREB and YAP and the transcript levels of YAP.pCREB(S133)and pYAP(S127)in total protein,as well as YAP and CREB levels in the nucleus and cytoplasm were all increased.7.RAMP1-Gαi3-PKA axis regulation of YAP is mediated by CREBCREB inhibitors inhibited the proliferation of MSF overexpressing RAMP1,reducing the levels of YAP and pYAP(S127)and CREB and pCREB(S133),and decreasing the levels of CREB and YAP in both cytoplasm and nucleus,and decreasing the levels of YAP transcripts.Three candidate CREB binding sites in the YAP promoter sequence were predicted using the JASPAR database,and the results of CUT&RUN assay,qPCR and agarose gel electrophoresis confirmed that CREB could bind directly to the YAP promoter at these three sites.By double luciferase reporter assays,it was demonstrated that CREB could activate the wild-type YAP reporter gene and failed to activate the mutant YAP reporter gene after deletion of the above three predicted binding sites,indicating that CREB could act on at least one of the above three sites in the YAP promoter region and regulate the transcription of YAP.Conclusion(1)Protein levels of RAMP 1 are altered during mouse skin wound repair and may be involved in regulating wound repair.(2)Overexpression of RAMP 1 can promote the proliferation of mouse skin fibroblasts by regulating YAP.(3)RAMP1 upregulates YAP activity and functions through the Gαi3-PKA-CREB-YAP axis,ultimately in the form of direct promotion of YAP transcription by CREB.BackgroundWound healing is a continuous and complex biological process;abnormalities in any factors may lead to delayed or no healing,resulting in chronic wounds.In recent years,the increasing prevalence of chronic wounds in the aging population has been a serious challenge for the public health care system.Although chronic wounds are common in clinical work and have been the focus of basic and clinical research,given the complexity of this process,the treatment of chronic wounds remains empirical and inconsistent,and detailed studies to elucidate the mechanisms of chronic would are lacking.Available studies suggest that most common chronic wounds present with exhibit varying degrees of ischemia.Vascular-related lesions that restrict the supply of oxygen,nutrients,and circulating cells are often the primary cause of local ischemia at the wound site,and this ischemia can lead the conversion to chronic wounds.One of the most important steps in classic phases of wound healing(hemostasis,inflammation,proliferation,and remodeling)is wound sealing.Wound closure is usually accomplished through re-epithelialization,cells derived from the interfollicular epidermis and hair follicles(HFs)proliferate,differentiate,and migrate to regenerate the damaged skin.According to reported studies,keratinocytes derived from the interfollicular epidermis and HFs play different but important roles in wound healing,and as the epidermis remodels after wound healing,these keratinocytes of different origins lose their original characteristics and eventually assume an interfollicular epidermal(IFE)-like phenotype.Cell death,including physiological and pathological death,is an important cell fate,and its effects are present throughout wound healing.In 2012,a new form of cell death,ferroptosis,was first identified.The morphological features of ferroptosis mainly include mitochondrial condensation,increased membrane density,and reduced or absent mitochondrial cristae,while its biochemical features mainly include iron accumulation and lipid peroxidation.Related studies have shown that ferroptosis is instrumental in regulating the development of many diseases and has become an essential target;unfortunately,current studies on ferroptosis in skin-related fields,especially in wound healing,are almost nonexistent.To our knowledge,iron is a major factor in maintaining healthy skin,which is also one of the key organs of iron metabolism,and iron is actively excreted from the body through skin desquamation.In parallel,several studies have shown that iron has an important effect on the development of chronic wounds,and although the underlying mechanisms have not been thoroughly investigated,there is consensus that iron affects wound healing by modulating oxidative stress;several iron chelators have been used with some effectiveness as clinical treatments.Based on these reports,we were curious to ask whether ferroptosis induces delayed healing of ischemic wounds and what are the potential regulatory mechanisms of ferroptosis in ischemic wounds.We established animal models combined with agonist/inhibitor use,and cellular models by Oxygen-Glucose Deprivation(OGD),viral transfection and gene silencing,to investigate the effect of ferroptosis on ischemic wound healing in mice,and to further investigate its underlying regulatory mechanisms.Objectives(1)Preliminary exploration of the effect of ferroptosis on healing of ischemic wounds in mouse model combined with topical agonists/inhibitors.(2)Verify ferroptosis in IFE cells under oxygen-glucose deprivation(OGD)conditions by measuring the levels and trends of ferroptosis related indicators.(3)Further exploration of the regulatory mechanism of OGD-induced ferroptosis in IFE cells by means of various molecular biology experiments.Methods1.Construction of mouse skin wound modelC57BL/6J male mice were anesthetized,dehaired and disinfected,and a free flap was created on the right side of the back,followed by a wound model on the flap,while an acute wound was prepared on the left side of the back as a control,and drugs were applied topically to the ischaemic trauma on days 0,2,4 and 6 after the trauma model was created,and trauma healing was assessed on days 1,3,5 and 7 after the trauma model was created.2.Total skin protein extractionUsing the Minute Skin Tissue Total Protein Extraction Kit for extraction of total wound tissue protein.3.Immunofluorescence stainingImmunofluorescence staining of FTH,FTL,GPX4 and Krt14 was performed on paraffin sections of wound tissues to observe the distribution and expression of FTH,FTL,GPX4 and Krt1 4 in the tissues.4.Primary IFE cell extractionExtraction of primary IFE cells from mammary rats by differential adhesion method.5.Flow cytometryThe extracted primary cells were immunolabelled with Krt10,Krt14,Krt15 and p63 and detected by flow cytometry.6.Drug interventions.Ammonium iron(Ⅲ)citrate(AFC),(1S,3R)-RSL3(RSL3),Erastin(Era),Deferoxamine mesylate(DFOM),Ferrostatin-1(Fer-1)Rapamycin(Rap),Chloroquine phosphate(CHQ),Cycloheximide(CHX),MG-132,Nutlin-3a were treated alone or in combination according to the needs of the experiment,followed by the relevant assays.The control group was equipped with the appropriate concentration of DMSO.7.Cell viability assayCell activity was assayed using the IncuCyte S3 Live Cell Analysis System.8.Scanning of transmission electron microscope for mitochondriaThe mitochondria of each group of IFE cells were observed by scanning electron microscopy.9.Mitochondrial membrane potential assayThe mitochondrial membrane potential of each group of IFE cells was measured by flow cytometry using ethyl methylrhodamine.10.Lipid peroxidation assayThe lipid peroxidation levels of each group of IFE cells were measured by flow cytometry using BODIPY 581/591 C11.11.Free Fe2+assayFree Fe2+levels in each group of IFE cells were measured by flow cytometry using FerroOrange.12.GSH/GSSG assayThe levels of GSH and GSSG in each group of IFE cells were measured with the kit.13.Autophagy assayAutophagy levels of each group of IFE cells were measured by flow cytometry using an autophagy probe.14.Adenovirus transfectionUp-regulation of target proteins by transfection with overexpressed adenovirus for subsequent validation.15.Protein ExtractionRIPA standard method for extraction of total cellular proteins.16.Western Blot assayTo detect the expression of each target protein in total trauma protein,total cellular protein and IP protein.17.Cellular immunofluorescence staining analysisImmunofluorescence staining of fixed cells was carried out to observe the expression levels and localization of the corresponding target proteins.18.Co-IP assayOther proteins interacting with the target protein are obtained by the kit and have been subjected to subsequent qualitative analysis.19.Statistical analysisStatistical analysis was performed using GraphPad Prism version 9.0.0 software and all experiments were repeated three times,with quantitative data expressed as mean± standard deviation.Differences between groups were compared by using an unpaired t-test or one-way ANOVA,where P<0.05 means that the results are statistically significant.Results1.Ferroptosis is involved in regulating the repair process of ischemic woundsIn mice,ischemic wound healing was delayed and the levels of FTL,FTH,and GPX4 in the total proteins of the ischemic wound and surrounding tissues were decreased.The levels of these proteins decreased in Krt14-positive IFE cells on day 3 of ischemic wounds.Ferroptosis agonists further inhibited ischemic wound healing,whereas ferroptosis inhibitors significantly promoted ischemic wound healing.2.Elevated iron concentration in the culture environment promotes the upregulation of FTH,FTL and GPX4 in IFE cellsPrimary IFE cells were isolated and characterized.The expression levels of TFRC,FTH,FTL and GPX4 were higher in IFE cells than in mature keratinocytes,and their protein levels increased with the increase of environmental iron concentration.3.Ferroptosis is involved in regulating OGD-induced IFE cell deathFerroptosis agonists promoted OGD-induced IFE cell death,while ferroptosis inhibitors inhibited OGD-induced IFE cell death.The levels of FTH,FTL and GPX4 also changed significantly during OGD treatment,while the ferroptosis marker ACSL4 gradually increased.4.Ferritinophagy is involved in regulating the changes of lipid peroxidation levels in IFE cells induced by OGDFTH and FTL levels in IFE cells under OGD induction were negatively correlated with autophagy levels,while GPX4 levels were not correlated with autophagy.When autophagy was agitated and ferritin levels decreased,free Fe2+ levels increased,whereas the opposite was true for inhibition of autophagy.Increased free Fe2+ levels further promoted OGD-induced upregulation of lipid peroxidation levels in IFE cells.5.Ubiquitination-mediated GPX4 degradation in IFE cells is involved in regulating OGD-induced ferroptosis in IFE cellsGPX4 levels were increased in OGD-induced IFE cells after inhibition of proteasome function,and GPX4 was modified by ubiquitination.Overexpression of GPX4-GFP inhibited OGD-induced IFE cell death,and its effect showed a dependence on the expression level of GPX4-GFP,whose protein level decreased with the duration of OGD induction,and which was also ubiquitinated by GPX4-GFP.6.The E3 ubiquitin ligase MDM2 mediates ubiquitination-proteasome degradation of GPX4 and regulates its homeostasis in IFE cellsThere is an interaction between GPX4-GFP and MDM2,with co-localization of GPX4 and MDM2.Upregulation of MDM2 will result in a decrease in GPX4 levels.MG-132 and Nutlin-3 a both upregulate GPX4 levels in normal culture or in OGD-induced IFE cells overexpressing MDM2.Conclusion(1)Ferroptosis may contribute to the delayed healing of ischaemic wounds in mice.(2)IFE cells are highly expressed in anti-ferroptosis related proteins and are regulated by environmental iron concentrations.Ferroptosis is included in OGD-induced IFE cell death.(3)OGD-induced degradation of FTH and FTL in IFE cells is iron autophagy-dependent and releases iron ions to promote lipid peroxidation.degradation of GPX4 is ubiquitin-proteasome pathway-dependent and mediated by the E3 ubiquitin ligase MDM2.