EGR1/NOX4 Pathway Regulates Oxidative Stress And Further Facilitates Fibrosis Progression In Keloids Responses To TGF-?1

Background:Keloid disorder,a paradigm of fibrotic skin diseases,is characterized by continuous activation of fibroblasts and unremitting accumulation of the extracellular matrix(ECM),primarily collagen.The biosynthetic pathways,which lead to ECM accumulation,are activated by several cytokines,but particularly by transforming growth beta 1(TGF-?1)signalling.The sustained activation of the canonical TGF-?/Smad signaling is a key factor in the framework of scarring and fibrosis,which leads to aberrant collagen synthesis and deposition in keloids.Recently,it was suggested that reactive oxygen species(ROS)are emerging as key players in the ECM remodeling process of cutaneous scar formation.Oxidative stress,which results in the generation of ROS,mainly in the form of superoxide and hydrogen peroxide,plays a significant role in the initiation and progression of liver fibrosis,renal fibrosis,and pulmonary fibrosis.Just a few research projects have investigated the ROS mechanisms that account for keloids.Although multiple research projects have demonstrated that NOX4-derived ROS facilitate TGF-?-mediated fibrosis,the underlying molecular mechanisms remain unclear.The early growth response factor-1(EGR1),is related to tissue fibrosis,including lung,liver fibrosis,and scleroderma.Also,it was discovered that EGR1 might play a critical role in keloids and hypertrophic scar development.Currently,how EGR1 plays a master regulatory role in the process of keloids fibrosis remains poorly understood.Some research projects have revealed that TGF-?1 up-regulates the expression of EGR1 through the activation of Smad3-dependent signaling pathways in the fibroblasts of organ fibrosis.Some research projects have revealed that the transcription factor,EGR1,can directly bind to promoter regions to guide the expression of NOX4.Therefore,we hypothesized that TGF-?1 up-regulates the expression of EGR1 through the Smad pathway,and EGR1 targets and binds to the NOX4 promoter region to regulate the level of ROS,thereby promoting the process of keloid fibrosis.The elucidation of this mechanism will provide new ideas for the treatment of keloids.Research purposes:In this experiment,we aim to analyze the expression of TGF-?1,EGR1,NOX4 and the level of ROS in keloids,clarify the molecular mechanism of TGF-?1 regulating EGR1 through Smad3 pathway and EGR1 regulating NOX4 to affect the level of ROS,and by further clarify the effect of TGF-?1/EGR1/NOX4 axis on the process of keloid fibrosis by regulating oxidative stress,thus provide a new concept and new basis for the targeted therapy of keloids.Methods:1.Using keloid tissue and normal skin tissue samples to culture keloid primary fibroblasts(KF)and normal skin fibroblasts(NF)by tissue explant and collagenase digestion,and identify primary cell phenotype by immunofluorescence(IF)and RTPCR.2.The m RNA and protein expression levels of differential gene in keloid tissue and normal skin tissue samples were analyzed by RNA sequencing,Western blotting(WB)and immunohistochemistry(IHC).The m RNA and protein expression levels and cellular localization of differential genes in KF cells and NF cells were analyzed by RTPCR and IF.The intracellular ROS levels of KF,NF and HSF were detected by DCFHDA staining or flow cytometry.3.By adding exogenous TGF-?1 cytokine or TGF-?1/Smad pathway inhibitor SB431542 to HSF cells or KF to construct a cell model of TGF-?1/Smad pathway activation or blocking.The expression of the Smad pathway corresponding molecules and fibrosis-related genes(Col1,Col3,Vimentin and ?-SMA)in cell models were detected RT-PCR and Western Bolt to confirm the molecular mechanism of TGF-?1regulating EGR1 to affect cell fibrosis phenotype.The changes of ROS level and NOX4 protein expression were used to clarify the effect of TGF-?1/EGR1 axis on oxidative stress in the process of keloid fibrosis.4.Constructing EGR1/NOX4 overexpressing HSF cell model or EGR1/NOX4 knockdown KF cell model by transfecting overexpression plasmid or si RNA interference fragment.After verifying that the cell model is successfully constructed,we detected the expression of fibrosis-related genes and NOX4 in the cell model and the intracellular ROS levels to confirm the effect of EGR1 differential expression on NOX4-catalyzed oxidative stress and cell fibrosis phenotype and confirm the effect of NOX4-derived ROS on fibroblast fibrosis phenotype.We used dual-luciferase reporter experiments to confirm the targeted regulatory relationship between EGR1 and NOX4.5.Three rescue experiments were performed by knocking down the EGR1/NOX4 gene in the TGF-?1/Smad activated cell model or knocking down the NOX4 gene in the EGR1-overexpressing cell model,and simultaneously detected the expression of fibrosis-related genes in the cell model and the changes of intracellular ROS levels to further clarify the effect of TGF-?1/EGR1/NOX4 axis regulating oxidative stress on keloid fibrosis process.Results:1.Normal skin tissue is thinner,and it is suitable for dispase dispase method to remove epidermis to reduce the mixing of epidermal cells.In the process of tissue block culture,compared with NF,KF is released from the tissue block earlier,and the growth rate is faster.It can occupy all the spaces between the tissues when cultured for 12-15 days.During the collagenase digestion method,the number of viable primary fibroblasts is the largest when normal skin tissue and keloid tissue are digested for about1 h and 3 h,respectively.Culturing of tissue samples after 4 hours in vitro increases the chance of bacterial contamination and oil droplet contamination.IF test showed that the proportion of Vimentin-positive cells in P3 primary cells was close to 100%,indicating that the purity of fibroblasts was high.RT-PCR test results showed that compared with NF cells,m RNA expression levels Col1(p < 0.001)and ?-SMA(p < 0.01)were significantly increased in KF cells,and KF cells presented a fibrotic-like phenotype.X2.Differential gene expression profiles of keloid tissue and normal skin tissue were detected by RNA-seq technology.The results showed that compared with normal skin tissue,the m RNA expression levels of COL1A1,COL1A2 and COL3A1 genes encoding ECM proteins in keloid tissue were significantly increased(p < 0.001),the m RNA expression level of the pro-fibrotic cytokine TGF-?1 was also significantly increased(p < 0.01),and the m RNA expression level of NOX4 was significantly upregulated(p < 0.001).KEGG enrichment analysis of differential gene showed that the differential genes were mainly involved in ECM-receptor interaction,focal adhesion and other signaling pathways,and the fibrosis-related TGF-? signaling pathway was also significantly activated(p <0.05).We then confirmed by WB detection that the protein levels of TGF-?1,Col1,Col3,Vimetnin,?-SMA,N-cad,E-cad and NOX4 were significantly increased in keloid tissue compared with normal skin tissue.3.We detected the expression of EGR1 in multiple tissue samples by WB,and the results showed that the protein expression of EGR1 was significantly up-regulated in keloids compared with normal skin tissue(p < 0.001)and the same results were obtained by IHC analysis.Subsequently,we detected the m RNA expression levels of EGR1 and TGF-?1 in 9 pairs of primary fibroblasts by RT-PCR,and the results showed that the m RNA level of EGR1 was significantly increased in KF cells compared with NF cells(p < 0.01),and the m RNA level of TGF-?1 was up-regulated(p < 0.05),and Pearson correlation analysis showed that the two changes were positively correlated.By IF detection,we found that compared with human normal skin fibroblasts(HSF)or NF cells,EGR1 was mainly increased in the nucleus of KF cells,?-SMA was mainly upregulated in the cytoplasm of KF cells,and the upregulation of N-cad in KF cells and the downregulation of E-cad were observed in both the nucleus and cytoplasm.4.In the cell model with TGF-?1/Smad pathway activation or blockade,we observed that 10 ng/m L TGF-?1 significantly increased the m RNA levels of EGR1 and Col1(p < 0.001)compared to control(0 ng/m L),and up-regulated the protein levels of EGR1,Col1 and Smad3 phosphorylation(p < 0.001).In the time course effect,we found that the m RNA and protein levels of EGR1 were significantly up-regulated at 6h,while the m RNA and protein levels of Col1,Col3 and ?-SMA were significantly upregulated at 48 h,and Smad3 phosphorylation reached the highest level at 12 h.0.1 ?M of SB431542 inhibited the upregulation of EGR1 in response to TGF-?1(p < 0.001)and decreased the phosphorylation level of Smad3(p < 0.001).SB431542 at a concentration of 20 ?M significantly inhibited the protein expression of EGR1 in KF cells(p < 0.001)and down-regulated the phosphorylation level of Smad3(p < 0.001).The above results suggest that TGF-?1 upregulates the expression of EGR1 in keloids by activating the Smad pathway and promotes the fibrotic phenotype of KF cells.5.By DCFH-DA staining and flow cytometry,we found that the fluorescence intensity of KF cells was significantly higher than that of NF cells and HSF cells,that is,KF cells produced higher levels of ROS and were in a state of high oxidative stress.TGF-?1(10 ng/m L)significantly up-regulated intracellular ROS levels in HSF(p <0.001),and this stimulatory effect of TGF-?1 on ROS was significantly blocked by SB431542(0.1 ?M,1 ?M and 10 ?M)(p < 0.001,p < 0.001,p < 0.01).In addition,SB431542(0.1 ?M and 10 ?M)also significantly inhibited the high levels of ROS in KF cells(p < 0.001,p < 0.01).The above results indicated that TGF-?1 increased the level of ROS in keloids through the Smad pathway.In addition,we confirmed by WB that changes in EGR1 and ROS levels were also accompanied by changes in protein expression of fibrosis-related genes(Col1,Col3,Vimentin,?-SMA),and these expression changes were consistent with the trend of NOX4 gene changes.These results confirm our previous speculation that TGF-?1 promotes ROS formation mainly by inducing NOX4 expression in keloid fibrosis progression,and EGR1 plays a key regulatory role in this process.6.After EGR1 overexpression(p < 0.001),ROS levels in HSF cells were significantly increased(p < 0.001),and the protein expressions of NOX4 and fibrosisrelated genes were also significantly increased with EGR1 overexpression.In turn,the expression of NOX4 and fibrosis-related genes were significantly down-regulated with knockdown of EGR1 in KF cells(p < 0.001).In addition,the levels of ROS were also significantly decreased(p < 0.001).The above results suggest that EGR1 can directly XII regulate NOX4-dependent ROS levels and fibrotic phenotype in KF cells.In addition,we confirmed the targeted regulatory relationship between EGR1 and NOX4 by dual luciferase experiments.The results showed that the relative firefly luciferase activity of NOX4 promoter gradually increased with the increase of EGR1 overexpression plasmid concentration.Taken together,EGR1 targets NOX4 to regulate oxidative stress and fibrosis in keloids.7.By overexpressing NOX4(p < 0.001),we observed a significant upregulation of ROS levels in HSF cells(p < 0.05).Moreover,these fibrosis-related genes were significantly increased with NOX4 overexpression.That is,NOX4-derived ROS can promote the fibrosis-like phenotype of HSF cells.Additionally,in NOX4 knockdown KF cells(p < 0.05),we observed that both intracellular ROS levels(p < 0.05)and the expression of these fibrosis-related genes were significantly down-regulated.The above results further confirmed that NOX4-derived ROS promoted keloid fibrosis.8.Rescue experiments showed that when the expression of EGR1 and NOX4 was significantly increased in response to TGF-?1,intracellular ROS levels were also significantly increased.However,this effect could be inhibited by EGR1 or NOX4 knockdown.In addition,NOX4 knockdown also rescued the elevated ROS levels caused by EGR1 overexpression in HSF cells.The expression of fibrosis-related genes was consistent with the change trend of ROS level.Conclusions:1.The TGF-?1/Smad pathway is activated in keloids to promote keloid fibrosis.Meanwhile,it affects the level of ROS in KF cells.2.The up-regulation of EGR1 in keloid tissue and KF cells is positively correlated with the expression of TGF-?1.TGF-?1 upregulates the expression of EGR1 in keloids by activating the Smad pathway,and promotes the fibrotic phenotype of KF cells.3.EGR1 can target and regulate the expression of NOX4.The EGR1/NOX4 axis regulates the level of ROS in KF cells and affects the expressions of Col1,Col3,?-SMA and Vimentin.4.The activation of TGF-?1/Smad pathway regulates the level of ROS through the EGR1/NOX4 axis,thereby promoting the process of keloid fibrosis.