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EGR1/NOX4 pathway regulates oxidative stress and further facilitates fibrosis progression in keloids responses to TGF-β1

Open AccessPublished:December 25, 2022DOI:https://doi.org/10.1016/j.jdermsci.2022.12.009

      Highlights

      • TGF-β1 facilitates the expression of EGR1 through the SMAD pathway in keloids.
      • NOX4-derived ROS may play important biological role in keloid fibrosis.
      • EGR1 could regulate the levels of ROS by targeting NOX4.

      Abstract

      Background

      As classic benign fibroproliferative tumors, keloids remain a major therapeutic challenge due to their complex pathological mechanisms.

      Objective

      To determine the functional role of transforming growth factor β1 (TGF-β1)/early growth response factor-1 (EGR1)/NADPH oxidases 4 (NOX4) axis in the pathogenesis of keloid fibrosis.

      Methods

      Differentially expressed genes in keloid tissues and normal skins were analyzed by RNA sequencing. Then, the human skin fibroblast cell line was treated with TGF-β1 at a dose of 10 ng/mL in order to stimulate the TGF-β1/SMAD pathway and the pathway was blocked using the SB431542. Furthermore, EGR1/NOX4 was over-expressed and inhibited by transfecting overexpression plasmids and small interfering RNAs, respectively. The levels of intracellular reactive oxygen species were measured using the DCFH-DA assay, and the expression levels of fibrosis‐related genes were assessed by Western blot analysis. Alternately, dual-luciferase reporter analysis verified the targeting relationship between EGR1 and NOX4.

      Results

      The TGF-β1/SMAD signaling pathway was significantly activated in keloid tissues to promote dermal fibrosis. The level of ROS was increased in keloid fibroblasts. Moreover, TGF-β1 could facilitate the expression of EGR1 through regulating the SMAD pathway in keloids and promoting the fibrotic phenotype of keloid fibroblasts. EGR1 could regulate the production of ROS by targeting NOX4. Furthermore, NOX4-derived ROS could promote fibrotic-like phenotype of keloid fibroblasts and play an important role in keloid fibrosis.

      Conclusion

      Our findings provide new insights into the mechanisms of the TGF-β1/EGR1/NOX4 pathway in keloid fibrosis, and the TGF-β1/EGR1/NOX4 axis may serve as a potential therapeutic target for keloids.

      Keywords

      Abbreviations:

      NOX4 (Nicotinamide adenine dinucleotide phosphate oxidase4), EGR1 (early growth response factor-1)

      1. Introduction

      Keloids represent benign fibroproliferative tumors, which originate in response to trauma to the skin. They are esthetically disfiguring and can cause pain, itching, discomfort, and psychological stress, which often affect the quality of life of an individual [
      • Ojeh N.
      • Bharatha A.
      • Gaur U.
      • Forde A.L.
      Keloids: current and emerging therapies.
      ]. Currently, several treatment methods for keloids exist, including the combination therapy of surgical incision and intralesional steroid therapy; however, keloids have high recurrence rates regardless of these current treatment methods [
      • Macarak E.J.
      • Wermuth P.J.
      • Rosenbloom J.
      • Uitto J.
      Keloid disorder: fibroblast differentiation and gene expression profile in fibrotic skin diseases.
      ].
      Keloid disorder, a paradigm of fibrotic skin diseases, is characterized by 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-β1 (TGF-β1) [
      • Andrews J.P.
      • Marttala J.
      • Macarak E.
      • Rosenbloom J.
      • Uitto J.
      Keloids: the paradigm of skin fibrosis – pathomechanisms and treatment.
      ]. It is currently accepted that the activation of the canonical and non-canonical signaling pathways can be induced by TGF-β. In the canonical signaling pathways, upon ligand binding, TGFβ-receptor 1 recruits and phosphorylates SMAD2 and SMAD3: phosphorylated SMAD2 or SMAD3 then associates with SMAD4 to form heterodimeric complexes, which translocate to the nucleus, where they can trigger downstream transcriptional responses [
      • Zhang T.
      • Wang X.F.
      • Wang Z.C.
      • Lou D.
      • Fang Q.Q.
      • Hu Y.Y.
      • et al.
      Current potential therapeutic strategies targeting the TGF-beta/Smad signaling pathway to attenuate keloid and hypertrophic scar formation.
      ]. The sustained activation of the canonical TGF-β/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 [
      • Andre-Levigne D.
      • Modarressi A.
      • Pepper M.S.
      • Pittet-Cuenod B.
      Reactive oxygen species and NOX enzymes are emerging as key players in cutaneous wound repair.
      ].
      ROS are necessary at low concentrations for cellular signaling and are usually generated in physiological cell processes; however, excessive ROS production has been associated with oxidative stress-related pathological conditions, especially organ fibrosis [
      • Cheresh P.
      • Kim S.J.
      • Tulasiram S.
      • Kamp D.W.
      Oxidative stress and pulmonary fibrosis.
      ,
      • Touyz R.M.
      • Rios F.J.
      • Alves-Lopes R.
      • Neves K.B.
      • Camargo L.L.
      • Montezano A.C.
      Oxidative stress: a unifying paradigm in hypertension.
      ,
      • Vona R.
      • Giovannetti A.
      • Gambardella L.
      • Malorni W.
      • Pietraforte D.
      • Straface E.
      Oxidative stress in the pathogenesis of systemic scleroderma: an overview.
      ,
      • Rodriguez M.J.
      • Sabaj M.
      • Tolosa G.
      • Herrera Vielma F.
      • Zuniga M.J.
      • Gonzalez D.R.
      • et al.
      Maresin-1 prevents liver fibrosis by targeting Nrf2 and NF-kappaB, reducing oxidative stress and inflammation.
      ]. 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. The NADPH oxidases (NOXs) are a major source of ROS in the parenchymal organ and mediate fibrogenic responses. Accumulative evidence shows the involvement of NOXs-dependent redox signaling in the profibrotic responses mediated by TGF-β. Some research projects have demonstrated that the TGF-β/SMAD3 pathway increases NOX4 expression in hematopoietic stem cells and lung mesenchymal cells, correlating with the degree of fibrosis [
      • Roehlen N.
      • Crouchet E.
      • Baumert T.F.
      Liver fibrosis: mechanistic concepts and therapeutic perspectives.
      ,
      • Hecker L.
      • Vittal R.
      • Jones T.
      • Jagirdar R.
      • Luckhardt T.R.
      • Horowitz J.C.
      • et al.
      NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury.
      ]. The silencing of NOX4 decreased the ROS production and expression of type I collagen and α-smooth muscle actin (α-SMA, a marker of myofibroblast), which are involved in TGF-β-induced differentiation of cardiac fibroblasts to myofibroblasts [
      • Jiang F.
      • Liu G.S.
      • Dusting G.J.
      • Chan E.C.
      NADPH oxidase-dependent redox signaling in TGF-beta-mediated fibrotic responses.
      ]. Although multiple research projects have demonstrated that NOX4-derived ROS facilitate TGF-β-mediated fibrosis, the underlying molecular mechanisms remain unclear.
      The zinc-finger transcription factor, early growth response factor-1 (EGR1), is an immediate-early gene product that shows inducible expression in response to diverse stimuli. The recent research projects show that EGR1 is related to tissue fibrosis, including lung, liver fibrosis, and scleroderma [
      • Guan M.
      • Li W.
      • Xu L.
      • Zeng Y.
      • Wang D.
      • Zheng Z.
      • et al.
      Metformin improves epithelial-to-mesenchymal transition induced by TGF-beta1 in renal tubular epithelial NRK-52E cells via inhibiting Egr-1.
      ]. Also, it was discovered that EGR1 might play a critical role in keloids and hypertrophic scar development: the analysis of the protein-protein interaction network maps gotten from microarray data helped with this discovery [
      • Zhang L.
      • Qin H.
      • Wu Z.
      • Chen W.
      • Zhang G.
      Identification of the potential targets for keloid and hypertrophic scar prevention.
      ]. Currently, how EGR1 plays a master regulatory role in the process of keloids fibrosis remains poorly understood. Chen et.al. identified EGR1 as a novel intracellular TGF-β target, which is necessary for the maximal stimulation of collagen gene expression in fibroblasts [
      • Chen S.J.
      • Ning H.Y.
      • Ishida W.
      • Sodin-Semrl S.
      • Takagawa S.
      • Mori Y.
      • et al.
      The early-immediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression.
      ]. The up-regulation of TGF-β signaling (upon bleomycin treatment) was attenuated in EGR1-null mice, and this ameliorated scleroderma-like dermal fibrosis [
      • Wu M.
      • Varga J.
      In perspective: murine models of scleroderma.
      ,
      • Bhattacharyya S.
      • Wu M.
      • Fang F.
      • Tourtellotte W.
      • Feghali-Bostwick C.
      • Varga J.
      Early growth response transcription factors: key mediators of fibrosis and novel targets for anti-fibrotic therapy.
      ]. Collectively, TGF-β exerts its function by modulating the expression of the transcription factor, EGR1, in fibrosis progression. Interestingly, Gabrielli et al. discovered that the combination of TGF-β and hypoxia might be responsible for sustained EGR1 expression that results in increased NOX4 activity and oxidative stress, further exacerbating fibrogenesis in systemic sclerosis patients [
      • Gabrielli A.
      • Avvedimento E.V.
      • Krieg T.
      Scleroderma.
      ]. Some research projects have revealed that the transcription factor, EGR1, can directly bind to promoter regions to guide the expression of NOX4 [
      • Hu F.
      • Xue M.
      • Li Y.
      • Jia Y.J.
      • Zheng Z.J.
      • Yang Y.L.
      • et al.
      Early growth response 1 (Egr1) is a transcriptional activator of NOX4 in oxidative stress of diabetic kidney disease.
      ].
      In this research, it is demonstrated that TGF-β1 up-regulates the expression of EGR1: the activation of SMAD3-dependent signaling pathways in the fibroblasts of keloids is how this is carried out. Experimental pieces of evidence that EGR1 binds to the promoters of NOX4, which induces its expression and increases ROS production, which is critical for the progression of keloids fibrosis, are provided. The aim is to provide new insight into the treatment of keloids.

      2. Materials and methods

      The authors declare that all supporting data are available within the article and its Online supplementary files. A detailed Methods section can be found in the Online Data Supplement.

      3. Results

      3.1 Differential gene expression in keloids

      To identify novel mediators of keloid fibrosis, RNA sequencing of tissue samples was carried out. The results showed that a total of 1345 significantly dysregulated mRNA transcripts were identified in keloid tissue (KT) compared with the normal skins. The cluster analysis of differentially expressed mRNAs is shown in Fig. 1A. Of these differential genes, the expression levels of genes encoding ECM proteins, such as type I collagen α 1 (COL1A1) and α 2 (COL1A2), and type III collagen α 1 (COL3A1) were significantly increased in the keloids (p < 0.001). The profibrotic cytokine, TGF-β1, was also significantly elevated in the keloid samples (p < 0.01). Given the strong link of ROS with fibrosis, the next focus was on the expression of several genes associated with oxidative stress. It was observed that the expression levels of NOX4 were dramatically upregulated in the keloids compared with normal skins (p < 0.001) (Supplementary Table S1). Furthermore, gene ontology (GO) terms and KEGG pathway analyses were carried out to evaluate the potential mechanism underlying the identified differential gene. GO term analysis shows that these genes are mainly associated with ECM and ECM organization (p < 0.05, Fig. 1B). Moreover, KEGG pathway analysis demonstrates that these differential genes are involved in ECM-receptor interaction, focal adhesion, protein digestion and absorption, and so on. Among these, the TGF-beta signaling pathway was most strongly associated with fibrosis and ECM remodeling (p < 0.05, Fig. 1C).
      Fig. 1
      Fig. 1Differential gene expression in keloids. (A) Cluster analysis of differentially expressed mRNAs. Red indicates increased expression, and blue denotes decreased expression. (B) Gene Ontology (GO) analysis of differentially expressed mRNAs, *p < 0.05. (C) KEGG analysis of differentially expressed mRNAs, *p < 0.05. (D)Representative pictures of Western blot (WB) analysis of TGF-β1, COL1, COL3, NOX4, α-SMA, and vimentin in KT and normal skin sections. (E) Immunohistochemistry (IHC) for TGF-β1 and α-SMA in keloid tissue (KT) and normal skin sections. Positive staining for TGF-β1 and a-SMA is brown, and counterstaining for nuclei is blue. Most of the positive staining for TGF-β1 was observed in KT and the highest amount of α-SMA + staining was observed in the cytoplasm. Scale bar, 200 µm. IHC was carried out on up to three tissue sections from three patients. Representative images are shown. (F) Representative pictures of immunofluorescence staining of α-SMA in keloid fibroblasts (KF), normal fibroblasts (NF), and human normal skin fibroblasts line (HSF) cells. Red = α-SMA, blue = DAPI. KF and HSF, Scale bar,40 µm; NF, Scale bar, 20 µm.
      To validate the findings identified by mRNA sequence, western blot (WB) was carried out on skin tissue from normal skin and keloids. Similarly, WB results showed the significantly elevated protein levels of TGF-β1, COL1, COL3, and NOX4 in the KT (Fig. 1D). Moreover, to quantify the level of myofibroblasts activation, the expression of α-SMA and vimentin was examined. The results revealed a significant upregulation of α-SMA and vimentin in KT (Fig. 1D and E). In addition, the keloid fibroblasts (KFs) significantly increased α-SMA expression compared with the normal skin fibroblasts (NFs) and the human skin fibroblast cell line (HSF) (Fig. 1F).
      Taken together, the observed upregulation of TGF-β1 and NOX4 expression and the higher expression levels of fibrotic-related genes (COL1, COL3, vimentin, α-SMA) in keloids suggest a potential mechanism underlying TGF-β1 and NOX4 interactions in the pathogenesis of keloid fibrosis.

      3.2 EGR1 is upregulated in keloids and positively correlated with the TGF-β1 levels in keloid fibroblasts

      It has been shown that TGF-β1 may exert its function by modulating the expression of transcription factors, such as EGR1, in fibrosis progression. To confirm the relationship between TGF-β1 and EGR1, the expression levels of EGR1 in KT were examined, firstly. The outcomes showed that the expression of EGR1 protein was upregulated in KT compared with normal skins tissues (p < 0.001, Fig. 2A and B). Moreover, immunohistochemistry (IHC) analyses showed that EGR1 expression was significantly elevated in keloids compared to normal skins. The overabundance of ECM, resulting from activated KFs, represents the vital pathophysiology underlying keloids. To further evaluate the role of KFs in the progression of keloid fibrosis, the expression levels of EGR1 in KFs and NFs were examined next. RT-PCR results demonstrated that the mRNA levels of EGR1 were significantly increased in KFs (p < 0.01, Fig. 2C), so were the mRNA levels of TGF-β1 (p < 0.05, Fig. 2D). It was also observed that EGR1 was increased in KFs and was mainly found within the cell nucleus compared with HSF or NFs (Fig. 2F). To test if the increase of EGR1 in the nucleus in KFs is linked to the upregulation of TGF-β1, the Pearson correlation analysis between the mRNA level of EGR1 and the mRNA level of TGF-β1 in KFs and NFs was carried out. The outcome showed that EGR1 mRNA expression was positively correlated with TGF-β1 mRNA expression (r = 0.511; p = 0.03) (Fig. 2E).
      Fig. 2
      Fig. 2Early growth response 1 (EGR1) is upregulated in keloids and positively correlated with the TGF-β1 levels in KFs. (A) WB analysis of EGR1 in KT (n = 25) and normal skins sections (n = 24), ***p < 0.001, keloids vs. normal skins. (B) IHC for EGR1 in KT and normal skins sections. Positive staining for EGR1 is brown, and counterstaining for nuclei is blue. Scale bar, 200 µm. Most of the positive staining for EGR1 was observed in KT. (C) The real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of the mRNA expression of EGR1 in KFs (n = 9) and NFs (n = 9), ## p < 0.01, KF vs. NF. (D) The RT-PCR analysis of the mRNA expression of TGF-β1 in KFs (n = 9) and NFs (n = 9), # p < 0.05, KF vs. NF. (E) Pearson correlation analysis between the EGR1 and TGF-β1 mRNA levels in KFs and NFs, r = 0.511, p = 0.03. (F) Representative pictures of immunofluorescence staining of EGR1 in KF, NF, and HSF cells. Red = EGR1, blue = DAPI. Scale bar, 20 µm.

      3.3 TGF-β1 up-regulates the expression of EGR1 through the SMAD pathway in keloids

      To test whether TGF-β1 impacts EGR1 expression in the progression of keloid fibrosis via the SMAD pathway, HSF was exposed to the cytokine TGF-β1 for varying concentrations for six hours. It was discovered that TGF-β1 caused a significant increase of EGR1 mRNA and protein at a concentration of 10 ng/mL (p < 0.001, Fig. 3A and C). The highest protein expression of p-SMAD3 also came at 10 ng/mL of TGF-β1 (Fig. 3C), even the expression levels of SMAD3 mRNA and protein did not change significantly with the increase of TGF-β1 concentration (Fig. 3B and C). Moreover, this effect was markedly blocked by treatment with SB431542, a TGF-β1/SMAD3 signaling-specific inhibitor. Low concentrations of SB431542 could inhibit the elevation of EGR1 responses to TGF-β1, and decrease the level of p-SMAD3 (Fig. 3D, 0.1 μM SB431542 +10 ng/mL TGF-β1 vs. 10 ng/mL TGF-β1). These results suggest that TGF-β1 stimulates HSF cells to synthesize EGR1 mainly through the activation of the TGF-β1/SMAD3 pathway.
      Fig. 3
      Fig. 3TGF-β1 up-regulates the expression of EGR1 through the SMAD pathway in HSF and KFs. (A and B) The RT-PCR analysis of the mRNA expression of EGR1 and SMAD3 in HSF treated with varying concentrations of TGF-β1 for six hours. (C) The WB analysis of the protein expression of EGR1 in HSF treated with varying concentrations of TGF-β1 for six hours. (D) The WB analysis of the protein expression of EGR1 and p-SMAD3 in HSF cells treated with 10 ng/mL TGF-β1 and varying concentrations of SB431542. (E) The WB analysis of the protein expression of EGR1 and p-SMAD3 in KFs treated with varying concentrations of SB431542. (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control).
      To further assess whether the activation of SMAD3 pathway responses to TGF-β1 is needed for the upregulation of EGR1 in KFs, the activation of TGF-β1/SMAD3 pathway was blocked by treatment with varying concentrations of SB431542. It was observed that the protein expression of EGR1 was significantly inhibited by SB431542 (0.1, 10, 20, 50 μM), along with the decrease of p-SMAD3 in KFs (0.1, 10, 50 μM) (Fig. 3E). Collectively, these results further confirmed that TGF-β1 could up-regulate the expression of EGR1 through the SMAD pathway in keloids.

      3.4 TGF-β1/EGR1 regulates the levels of ROS and fibrotic phenotype

      As suggested by numerous research projects, ROS play a significant role in the initiation and progression of organ fibrosis and the involvement of NOXs-dependent redox signaling in the profibrotic responses mediated by TGF-β [
      • Jiang F.
      • Liu G.S.
      • Dusting G.J.
      • Chan E.C.
      NADPH oxidase-dependent redox signaling in TGF-beta-mediated fibrotic responses.
      ]. Interestingly, a significant upregulation of NOX4 in KTs was observed, and it was speculated that oxidative stress takes on a special role in the progression of keloid fibrosis.
      To demonstrate this, the level of intracellular ROS was first assessed by the DCFH-DA assay. The results obtained showed that the fluorescence intensity of KFs was significantly higher than that of NFs and HSF cells (Fig. 4A). That is, KF cells are under high oxidative stress and generate higher levels of ROS. It was suggested that the high level of ROS is mediated by TGF-β1/EGR1, thus, impairing the fibrogenic phenotype of KFs. To prove this point, intracellular ROS levels were studied further after treatment for six hours with TGF-β1 in HSF cells. As expected, it was discovered that the levels of intracellular ROS were significantly elevated after stimulation by TGF-β1(10 ng/mL) (Fig. 4B; p < 0.001, Fig. 4D). As with the results of EGR1 (Fig. 3D), this stimulatory effect on ROS was markedly blocked by treatment with SB431542 (0.1 μM, 1 μM and 10 μM) (Fig. 4B and D). Similarly, as with the results of EGR1 (Fig. 3E), the high level of ROS in KFs could be significantly inhibited by SB431542 (0.1 μM and 10 μM) (Fig. 4C and E).
      Fig. 4
      Fig. 4TGF-β1/EGR1 regulates the levels of reactive oxygen species (ROS) and fibrotic phenotypes in HSF and KFs. (A–C) The levels of intracellular ROS were detected by DCFH-DA (10 μM), scale bar, 200 µm. (D and E) ROS contents were detected by DCFH-DA and analyzed by flow cytometer (FCM), **p < 0.01, ***p < 0.001 vs. control; ## p < 0.01, ### p < 0.001 vs. 10 ng/mL TGF-β1. (F) The WB analysis of the expression of fibrosis‐related genes (COL1, COL3, Vimentin, and α-SMA) and NOX4 in HSF cells treated with 10 ng/mL TGF-β1 and varying concentrations of SB431542. (G) The WB analysis of the protein expression of fibrosis‐related genes and NOX4 in KFs treated with varying concentrations of SB431542.
      The results above establish that TGF-β1 raises the levels of ROS through the SMAD pathway in keloids. Moreover, there is a close relationship between the up-regulation of EGR1 and the elevated ROS level response to TGF-β1. In addition, it was confirmed that the variation of EGR1 and the levels of ROS were also accompanied by the changes in protein expression of fibrosis‐related genes by WB (Fig. 4F and G). Moreover, these expression changes agree with NOX4′s change of gene expression (Fig. 4F and G). These results corroborate our previous speculation that TGF-β1 can promote ROS formation mainly through the induction of NOX4 expression in the progression of keloid fibrosis. In this process, EGR1 plays a key role.

      3.5 EGR1 targeting of NOX4 promotes oxidative stress and fibrosis in keloids

      TGF-β1/EGR1 has been confirmed to possess the ability to regulate the levels of ROS and fibrotic phenotype in KFs and HSF cells. In this process, the expression of NOX4 was significantly altered. As a transcription factor, EGR1 regulates the expression of target genes by binding to promoter regions of these genes. Given this, it was speculated that EGR1 could promote oxidative stress and fibrosis by targeting NOX4 in keloids. To test this hypothesis: an investigation was first carried out to know if EGR1 siRNA knockdown and over-expression could also modulate intracellular ROS levels. It was discovered that upon overexpression of EGR1, the levels of ROS were significantly elevated in HSF cells (p < 0.001 vs. vector, Fig. 5A and B). Alternatively, it was observed that the protein expression of NOX4 and fibrosis‐related genes were significantly increased with the overexpression of EGR1 (Fig. 5C).
      Fig. 5
      Fig. 5EGR1 targeting of NOX4 promotes oxidative stress and fibrosis in keloids. (A and D) The levels of cellular ROS were detected by DCFH-DA (10 μM), scale bar, 200 µm. (B and E) ROS contents were detected by DCFH-DA and analyzed by FCM. (C and F) The WB analysis of the expression of fibrosis‐related genes, EGR1 and NOX4. (G) Luciferase assay of NOX4 promoter activity with different concentrations of EGR1 overexpressing plasmid, n = 4. The relative activity was calculated by firefly luciferase activity from NOX4 reporter co-transfected with EGR1 overexpressing plasmid (normalized to renilla luciferase activity) divided by that of firefly luciferase activity from NOX4 reporter co-transfected with negative control (empty plasmid) (normalized to renilla luciferase activity). (***p < 0.001, EGR1OE vs. Vector, EGR1si vs. siNC).
      Conversely, the expression of NOX4 and fibrosis‐related genes was markedly down-regulated with the EGR1 knockdown in KFs (Fig. 5F). Also, the levels of ROS were significantly down-regulated (p < 0.001 vs. siNC, Fig. 5D and E). The results above establish that EGR1 could directly regulate the NOX4-dependent ROS level and fibrotic phenotype in KFs. If so, whether EGR1 directly binds to promoter regions to guide the expression of NOX4 remained unclear at that moment. Next, the NOX4 promoter (− 2000 to + 100) region was cloned into a luciferase reporter construct: then co-transfection of the EGR1 overexpression plasmid or negative control (empty plasmid) in 293 T cells was carried out: to see the effect of overexpression of EGR1 on the promoter activity of NOX4. It was further observed that: the relative firefly luciferase activity gradually increased with elevating EGR1 overexpressing plasmid concentration (Fig. 5G). The results above establish that EGR1 targeting of NOX4 promotes oxidative stress and fibrosis in keloids.

      3.6 EGR1/NOX4 axis regulates oxidative stress and further facilitates fibrosis progression in keloids responses to TGF-β1

      To further demonstrate that the high levels of ROS are primarily derived from NOX4 and further promote a fibrotic-like phenotype in KFs, some experiments were performed. First, it was observed that the intracellular ROS levels were significantly elevated in HSF cells by overexpressing NOX4 genes (p < 0.05 vs. vector, Fig. 6A and B). Also, these fibrosis‐related genes were significantly increased with the overexpression of NOX4 (Fig. 6C). That is, NOX4-derived ROS could promote a fibrotic-like phenotype in HSF. Alternatively, the same molecular mechanism in KFs was observed by siRNA-mediated NOX4 knockdown. Results show that the intracellular ROS levels and the expression of these fibrosis‐related genes were all significantly down-regulated in KFs with NOX4 knockdown (p < 0.05 vs. siNC, Fig. 6D–F). Here, it was further confirmed that NOX4-derived ROS promote fibrosis in keloids.
      Fig. 6
      Fig. 6NOX4-derived ROS promote fibrosis in keloids. (A and D) The levels of cellular ROS were detected by DCFH-DA (10 μM), scale bar, 200 µm. (B and E) ROS contents were detected by DCFH-DA and analyzed by FCM. (C and F) The WB analysis of the expression of fibrosis‐related gene sand NOX4 (*p < 0.05, NOX4OE vs. Vector, NOX4si vs. siNC).
      Overall, the present results suggest that EGR1/NOX4 axis regulates oxidative stress and further facilitates fibrosis progression in keloids responses to TGF-β1. To provide further evidence of their regulatory role, some rescue experiments were performed. It was discovered that intracellular ROS levels were increased (Supplementary Fig. S1 A and B) when the expression of EGR1 and NOX4 were significantly elevated in response to TGF-β1 (Supplementary Fig. S1 D and E), but it was inhibited upon EGR1 or NOX4 knockdown (p < 0.05, Supplementary Fig. S1A and B; Supplementary Fig. S1 D and E). Of note, NOX4 knockdown also rescued the ROS elevation seen with EGR1 overexpression in HSF cells (Supplemental Fig. S1 C and F). Moreover, the expression of these fibrosis‐related genes also changed like ROS levels (Supplementary Fig. S1 D–F). Therefore, it was reported that the high level of ROS was responsible for keloid fibrosis progression mediated by EGR1/NOX4 pathway responses to TGF-β1.

      4. Discussion

      As classic benign fibroproliferative tumors, keloids remain a major therapeutic challenge due to their complex pathological mechanisms. The current research regarding the pathogenesis of keloids mainly focuses on genetic predisposition, wound repair mechanisms, key regulator molecules of fibrosis, and fibrotic signaling cascades [
      • Andrews J.P.
      • Marttala J.
      • Macarak E.
      • Rosenbloom J.
      • Uitto J.
      Keloids: the paradigm of skin fibrosis – pathomechanisms and treatment.
      ]. With the growing accumulation of expression data about fibrotic signaling cascades in keloids, some research projects are exploring the possibilities of targeted therapies. For example, antisense oligonucleotide (ASO) treatment directed against TGF-β mRNA transcripts demonstrated down-regulation of matrix metallopeptidase 9, SMAD2, SMAD4, and reduced secretion from fibroblasts in vitro [
      • Sadick H.
      • Herberger A.
      • Riedel K.
      • Bran G.
      • Goessler U.
      • Hoermann K.
      • et al.
      TGF-beta1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts.
      ,
      • Bran G.M.
      • Sommer U.J.
      • Goessler U.R.
      • Hormann K.
      • Riedel F.
      • Sadick H.
      TGF-ss1 antisense impacts the SMAD signalling system in fibroblasts from keloid scars.
      ,
      • Zhang Q.
      • Wu Y.
      • Ann D.K.
      • Messadi D.V.
      • Tuan T.L.
      • Kelly A.P.
      • et al.
      Mechanisms of hypoxic regulation of plasminogen activator inhibitor-1 gene expression in keloid fibroblasts.
      ]. However, it is still a puzzle to translate their use to the clinical setting due to the multiple physiological functions of the TGF-β/SMAD pathway.
      The TGF-β/SMAD signaling pathway is the canonical pathway involved in the formation of collagen. The formation is done by targeting COL1A1 and COL3A1 and the activation of EMT. Additionally, TGF-β/SMAD could also induce the generation of ROS that mediate myofibroblast transdifferentiation during organ fibrosis progress [
      • Richter K.
      • Konzack A.
      • Pihlajaniemi T.
      • Heljasvaara R.
      • Kietzmann T.
      Redox-fibrosis: impact of TGFbeta1 on ROS generators, mediators and functional consequences.
      ,
      • Crosas-Molist E.
      • Fabregat I.
      Role of NADPH oxidases in the redox biology of liver fibrosis.
      ]. Based on the above, focus was placed on the role of the TGF-β1/SMAD pathway and oxidative stress, and exploration of the underlying mechanisms in the pathogenesis of keloid fibrosis was done. According to the findings of this research: the TGF-beta signaling pathway was aberrantly activated, with the significant upregulation of fibrosis‐related genes in keloids. Simultaneously, for the first time, significant upregulation of NOX4 (a major source of ROS) was observed in KTs. In addition, we also observed that KF cells are under high oxidative stress and generate higher levels of ROS, consistent with earlier works [
      • Lee Y.J.
      • Kwon S.B.
      • Kim C.H.
      • Cho H.D.
      • Nam H.S.
      • Lee S.H.
      • et al.
      Oxidative damage and nuclear factor erythroid 2-related factor 2 protein expression in normal skin and keloid tissue.
      ,
      • De Felice B.
      • Garbi C.
      • Santoriello M.
      • Santillo A.
      • Wilson R.R.
      Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts.
      ]. Although the role of NADPH oxidase-dependent redox signaling in the progression of multiple organ fibrosis has been extensively studied, its function in keloids is far from clear [
      • Samarakoon R.
      • Overstreet J.M.
      • Higgins P.J.
      TGF-beta signaling in tissue fibrosis: redox controls, target genes and therapeutic opportunities.
      ]. In this research, we discovered that TGF-β1 could markedly induce intracellular ROS generation accompanied by up-regulation of fibrosis‐related genes, and this effect can be abolished by NOX4 siRNA in HSF cells. Alternatively, we directly blocked both the ROS production as well as the expression of fibrosis‐related genes by the inhibition of NOX4 with siRNA in KFs. From this, we conclude that oxidative stress plays an important biological role in keloid fibrosis. However, the link between endogenously produced ROS and the activation of TGF-β/SMAD signaling has remained unclear in keloids.
      EGR1 is a key profibrotic factor for matrix remodeling in skin, lung, and kidney [
      • Ghosh A.K.
      • Quaggin S.E.
      • Vaughan D.E.
      Molecular basis of organ fibrosis: potential therapeutic approaches.
      ,
      • Shen J.
      • Xing W.H.
      • Gong F.Q.
      • Wang W.
      • Yan Y.F.
      • Zhang Y.Y.
      • et al.
      MiR-150-5p retards the progression of myocardial fibrosis by targeting EGR1.
      ,
      • Liu F.
      • Zhang Z.P.
      • Xin G.D.
      • Guo L.H.
      • Jiang Q.
      • Wang Z.X.
      miR-192 prevents renal tubulointerstitial fibrosis in diabetic nephropathy by targeting Egr1.
      ]. Currently, some research projects demonstrate that EGR1 plays a relevant role in the process of keloid formation [
      • Zhang L.
      • Qin H.
      • Wu Z.
      • Chen W.
      • Zhang G.
      Identification of the potential targets for keloid and hypertrophic scar prevention.
      ,
      • Shi K.
      • Qiu X.
      • Zheng W.
      • Yan D.
      • Peng W.
      MiR-203 regulates keloid fibroblast proliferation, invasion, and extracellular matrix expression by targeting EGR1 and FGF2.
      ,
      • Zhu H.Y.
      • Bai W.D.
      • Wang H.T.
      • Xie S.T.
      • Tao K.
      • Su L.L.
      • et al.
      Peroxisome proliferator-activated receptor-gamma agonist inhibits collagen synthesis in human keloid fibroblasts by suppression of early growth response-1 expression through upregulation of miR-543 expression.
      ]. Alternatively, the summary of numerous research projects indicates an important regulatory relationship between TGF-β1 and EGR1, which then promotes fibrosis [
      • Hu F.
      • Xue M.
      • Li Y.
      • Jia Y.J.
      • Zheng Z.J.
      • Yang Y.L.
      • et al.
      Early growth response 1 (Egr1) is a transcriptional activator of NOX4 in oxidative stress of diabetic kidney disease.
      ,
      • Yang L.
      • Tang J.
      • Chen H.T.
      • Ge D.W.
      • Sui T.
      • Que J.
      • et al.
      Taurine reduced epidural fibrosis in rat models after laminectomy via downregulating EGR1.
      ]. Our results revealed that the mRNA and protein of EGR1 were all upregulated to varying degrees with various concentrations of TGF-β1-treated in HSF cells. Moreover, the effects of TGF-β1 on EGR1 expression were abolished by the TGF-β1 inhibitor SB431542. Among the KF cells, SB431542 could similarly prevent the activation of the TGF-β1/SMAD pathway, which, in turn, down-regulates EGR1 expression. Although we identified EGR1 upregulation in KTs through IHC and WB, and its upregulations were mainly detected within cell nucleus in KFs, consistent with findings by others [
      • Shi K.
      • Qiu X.
      • Zheng W.
      • Yan D.
      • Peng W.
      MiR-203 regulates keloid fibroblast proliferation, invasion, and extracellular matrix expression by targeting EGR1 and FGF2.
      ,
      • Zhu H.Y.
      • Bai W.D.
      • Wang H.T.
      • Xie S.T.
      • Tao K.
      • Su L.L.
      • et al.
      Peroxisome proliferator-activated receptor-gamma agonist inhibits collagen synthesis in human keloid fibroblasts by suppression of early growth response-1 expression through upregulation of miR-543 expression.
      ], the important question remains as to why EGR1 is overexpressing and how EGR1 exerts its biological effects in keloids.
      The present research represents a comprehensive investigation of the role of EGR1 in keloids and KFs. Firstly, we observed that the protein levels of fibrosis‐related genes (COL1, COL3, Vimentin, and a-SMA) were significantly upregulated by overexpressing EGR1 in HSF cells. Conversely, we revealed that EGR1 knockdown suppressed the effects of EGR1 on fibrosis‐related genes expression in KF cells. Chen et al. have reported that the upregulated endogenous EGR1 could increase the promoter activity of COL1A2 through interacting with guanine-cytosine-rich DNA sequences of the human COL1A2 promoter in response to TGF-β1 [
      • Chen S.J.
      • Ning H.Y.
      • Ishida W.
      • Sodin-Semrl S.
      • Takagawa S.
      • Mori Y.
      • et al.
      The early-immediate gene EGR-1 is induced by transforming growth factor-beta and mediates stimulation of collagen gene expression.
      ], which may promote the expression of COL1. Interestingly, we also observed that EGR1 overexpression significantly increased intracellular ROS levels accompanied by up-regulation of NOX4 in HSF, and this effect was significantly blocked by NOX4 knockdown. Alternatively, NOX4-derived ROS in KFs and the elevated ROS levels in TGF-β1-treated HSF could both be directly downregulated by EGR1 knockdown. Combining the above results together, we carried out further experiments to confirm that NOX4 is a vital downstream target of EGR1 in keloids. Using a dual-luciferase reporter system assay, we discovered that the relative firefly luciferase activity of NOX4 promoter gradually increased with elevating EGR1 overexpressing plasmid concentration. Altogether, our research project is the first to confirm such a targeted regulatory relationship between EGR1 and NOX4 during keloid fibrosis progress.
      In conclusion, we revealed that TGF-β1 up-regulates the expression of EGR1 through the SMAD pathway in keloids. Furthermore, we indicated that NOX4-derived ROS play important biological roles in keloid fibrosis. Finally, we confirm that EGR1 can regulate the levels of ROS by targeting NOX4. Overall, our findings provide new insights into the mechanism of the TGF-β1/EGR1/NOX4 pathway in keloid fibrosis, and the TGF-β1/EGR1/NOX4 axis might be a potential therapeutic target for the treatment of keloids.

      CRediT authorship contribution statement

      Conceptualization, H.Q., S.S. and Z.G.; Methodology, H.Q., S.S. and M.L.; Software, L.B, W.N. and H.Q.; Validation, Z.G., M.L. and L.B.; Formal analysis, Z.G., H.Q., M.L. and B.B.; Data curation, Z.G., H.Q., G.L, M.L. and B.B.; Writing – original draft, Z.G., H.Q., G.L, M.L., S.S. and L.B.; Writing – review & editing, Z.G., H.Q., G.L, M.L., S.S. and L.B.; Visualization, Z.G., H.Q., G.L, M.L., Y.L. and L.B.; Supervision, H.Q. and Z.G.; Project administration, H.Q. and Z.G.; Funding acquisition, H.Q. and Z.G., Y.L. All authors have read and agreed to the published version of the manuscript.

      Funding

      This research was funded by the National Natural Science Foundation of China (Nos. 82072184 and 81971842), the Jilin Scientific and Technological Development Program (Nos. 20200201371JC and 20200201580JC), and the Key R & D Project of Science and Technology Department of Jilin Province (Nos. 20210204109YY and 20200404179YY).

      Declaration of Competing Interest

      The authors have no conflict of interest to declare.

      Appendix A. Supplementary material

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