DUOX1 in mammalian disease pathophysiology

Abstract

Dual oxidase 1 (DUOX1) is a member of the protein family of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidases. DUOX1 has several normal physiological, immunological, and biochemical functions in different parts of the body. Dysregulated oxidative metabolism interferes with various disease pathologies and numerous therapeutic options are based on targeting cellular redox pathways. DUOX1 forms an important enzymatic source of biological oxidants, and DUOX1 expression is frequently dysregulated in various diseases. While this review shortly addresses the biochemical and cellular properties and proposed physiological roles of DUOX1, its main purpose is to summarize the current knowledge with respect to the potential role of DUOX1 enzyme in disease pathology, especially in mammalian organisms. Although DUOX1 is normally prominently expressed in epithelial lineages, it is frequently silenced in epithelial-derived cancers by epigenetic mechanisms. While an abundance of information is available on DUOX1 transcription in different diseases, increasing number of mechanistic studies indicate a causative relationship between DUOX1 function and disease pathophysiology. Additionally, specific functions of the DUOX1 maturation factor, DUOXA1, will also be addressed. Lastly, urgent and outstanding questions on the field of DUOX1 will be discussed that could provide valuable new diagnostic tools and novel therapeutic options.

Introduction

The family of NADPH oxidases consists of seven members, NOX 1–5 and Dual oxidases 1 and 2 (DUOX1, DUOX2), and represent enzymes that mediate regulated cellular production of reactive oxygen species (ROS). These enzymes play various functional roles in biological processes that include innate immunity, oxidative signal transduction, and biochemical reactions, e.g., biosynthesis of the thyroid hormones (1). ROS can, however, damage various molecules including nucleic acids, proteins, carbohydrates and lipids. Excessive exposure to ROS results in oxidative stress and could lead, for instance, to genetic mutations. ROS has been correlated with the pathophysiology of many diseases that tend to occur late in life, including atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer and Alzheimer’s disease. Insufficient DUOX2 activity in rodent models leads to hypothyroidism and DUOX2 mutations have been associated with this disease in humans (1). Human DUOX1 is highly expressed in the lung, pancreas, placenta, prostate, testis, and salivary gland (2). It was suggested that DUOX1 has a crucial role in innate host defense (3). Previous studies over the past years have, however, highlighted associations of DUOX1 gene expression with numerous diseases. While several, excellent, prior reviews focused on both DUOX enzymes, on their physiological roles in all living organisms, the specific goal of this review is to summarize scientific literature linking DUOX1 gene expression to human and murine pathophysiologies.

Structural features and discovery of DUOX1

DUOX enzymes were originally described as thyroid oxidases because they were first identified in the thyroid gland and linked to calcium-sensitive H₂O₂ generation (4, 5). Edens et al. explained the cloning of homologous sequences from Caenorhabditis elegans and suggested the DUOX nomenclature, according to the structural features of the proteins (2). The location of DUOX1 and DUOX2 is on human chromosome 15 and both genes are arranged in a head-to-head configuration (2). The two DUOX genes are separated by a 16 kb region (6). The length of the DUOX1 gene is 36 kb containing 35 exons (2). The human DUOX1 protein is composed of 1,551 amino acids, and shows 83% sequence similarity with the human DUOX2 amino acid sequence (2). In addition to its NADPH oxidase portion, the unique N-terminal peroxidase homology domain shows high sequence similarity to peroxidases (2, 7). While peroxidases contain heme molecules (2, 7), the peroxidase homology domains of DUOX proteins do not bind heme, because they lack key amino acid residues (7) that are crucial for heme binding and are present in highly conserved positions in all peroxidases (8). Calcium ions regulate the enzymatic activity of DUOX proteins (H₂O₂ production) due to the presence of two EF hand motifs between the peroxidase homology domain and the NADPH oxidase portion (8). The DUOXA (maturation factor) proteins are essential for full maturation of the DUOX proteins and coordinate their localization to the plasma membrane, although immature DUOX proteins within the endoplasmic reticulum (ER) have also been shown to possess catalytic activity (9–11). DUOXA1 is arranged in a head-to-head configuration and is co-expressed with DUOX1 (11). DUOXA1 co-expression enables DUOX1 transition from ER to the Golgi complex, maturation, and translocation to the plasma membrane (11). DUOXA1 mRNA was first shown to be expressed in the thyroid gland and, at lower level, in the esophagus (11). Two transcripts of ~2.9 and ~3.5 kb sizes were detected, compatible with alternative splicing of the 5’-untranslated exons and the use of alternative 3’-polyadenylation signals (11). The DUOXA1 open reading frame was confirmed by sequencing from human thyroid cDNA (11). The splicing sites are conserved at the single codon level (11). Transcriptional modes of regulation of DUOX1 and its corresponding maturation factor is not completely characterized yet (12). Multiple studies detected the expression of DUOX1 in a variety of cell types: keratinocytes (13), urothelial cells (14), primary respiratory epithelial cells, and non-epithelial cells such as T cells (14), innate lymphoid cells (15) and alveolar macrophages (16, 17). This reported expression pattern determines the proposed physiological roles of DUOX1.

Physiological role of DUOX1

DUOX1 has a crucial role in ROS generation. ROS have an important role in various physiological processes including host defense, mitogenesis, hormone biosynthesis, apoptosis and fertilization (18). ROS-producing systems, for instance, play an important function in phagocytic cells where ROS generated during phagocytosis in a partnership by the NOX2-based NADPH oxidase and myeloperoxidase contributes to microbial killing and host defense (19). A ROS-based, antimicrobial system analogous to this phagocytic system has been proposed in the airway epithelium. Respiratory epithelial cells were proposed to orchestrate a fast and potent, oxidative, extracellular, antimicrobial system consisting of the protein lactoperoxidase (LPO), the thiocyanate anion (SCN⁻) and H₂O₂ (20–23). Antimicrobial LPO is present in large amounts in mammalian airways and its inhibition leads to weakened microbial clearance in vivo (3, 20, 21, 23). LPO is mainly secreted by submucosal glands in the respiratory mucosa (3, 20). SCN⁻ is abundant in the airway surface liquid to support LPO function (20, 22). SCN⁻ is not produced by the airway epithelium itself, it is transported from the blood into tissues through the epithelium and concentrated in the airway surface liquid by several epithelial transporters (24, 25). LPO oxidizes SCN⁻ using H₂O₂ into antimicrobial hypothiocyanite (OSCN⁻) in the airway surface liquid (26). It has been suggested that DUOX1, a Ca²⁺-regulated NADPH oxidase highly expressed in the apical membrane of the respiratory epithelium producing large amounts of extracellular H₂O₂, works in partnership with LPO to produce antimicrobial OSCN⁻ (5, 7, 27). As discussed earlier, the main in vivo function of DUOX2 is the production of H₂O₂ for thyroid hormone biosynthesis since both, DUOX2-deficient human patients and DUOX2-deficient mice suffer only from hypothyroidism, and not other, reported conditions (28–30). The in vivo role of DUOX1 remains largely unknown. DUOX1-deficient human patients have not been identified yet. DUOX1 is the dominant NADPH oxidase expressed and the main H₂O₂ source in the respiratory epithelium (7, 31–34). DUOX1 is expressed in well-differentiated, polarized, ciliated, air-liquid interface cultures of primary respiratory epithelial cells of several mammalian species (human, rat, mouse) but not in most cell lines used as in vitro models of the respiratory epithelium (7, 31, 32).

In addition, DUOX1-generated H₂O₂ plays an essential role as a chemoattractant for the immune system. In zebrafish, the tissue-scale gradient of H₂O₂ induces recruitment of leukocytes to the location of injury, and DUOX1 knockdown decreased immune cell recruitment (35). The murine respiratory epithelium and cultured tracheal epithelial cells isolated from mice express DUOX1 (15, 36). Ongoing studies are focusing on the DUOX1/H₂O₂/LPO/SCN⁻ system as an effective, broad spectrum antimicrobial system exploring the antimicrobial role of LPO in mammalian experimental models (3). Moreover, DUOX1 has been proposed to have an important function in the normal wound healing process due to its ability to activate the transition of fibroblasts to myofibroblasts and subsequent deposition of the collagen matrix (37). There are several, recent reviews that summarize the proposed physiological roles of DUOX1 (3, 38–40). The goal of this particular review article is to provide an updated summary on the recent knowledge associated with DUOX1-generated H₂O₂ and various diseases such as allergic conditions, pulmonary diseases and cancer (15, 37, 41, 42).

DUOX1 and its maturation factor DUOXA1

The DUOX1 gene is normally expressed in a coordinated manner with its maturation factor or activator, DUOXA1. The DUOXA1 protein consists of five alpha helical transmembrane regions, with extracellular loops and an intracellular tail, and associates with immature DUOX1 within the endoplasmic reticulum to promote its proper folding, glycosylation and transport to the plasma membrane to generate a mature, fully functional enzyme (11, 43, 44). Though, immature DUOX1 still shows intrinsic catalytic activity, as well (10). Several studies show that DUOX1-DUOXA1 protein interactions also potentially include intramolecular disulfide bonds between their extracellular parts (45, 46). Moreover, it was recently observed that the specificity of produced ROS, O2⁻ as an intermediate or H₂O₂ as its end product, is determined by sequences in the extracellular A loop of the DUOX proteins, which bind to the N-termini of DUOXA proteins (47). DUOXA1 has numerous splice variants whose physiological roles remain still unclear (43). It has been previously shown that specific splice variants of DUOXA1 can also combine with DUOX2, which allows H₂O₂ generation at almost the exact levels as that of the matching DUOX2-DUOXA2 heterodimers, that was, however, not true for mispairing DUOX1 with DUOXA2 (43, 47). Beyond being associated with corresponding DUOX proteins, the DUOXA1 protein might also have a role independent of DUOX1. DUOXA1 was first recognized in Drosophila melanogaster, as a complimentary part to a master regulator of the Notch cell differentiation pathway, Numb, and was then named Numb Interacting Protein (NIP) (11, 48, 49). The characteristics of sequence in the NIP proteins indicated that they are membrane-associated proteins, and it was suggested that NIP regulates the subcellular localization of Numb by sequestering Numb to the plasma membrane during asymmetric cell division (48). DUOX-dependent ROS production is regulated by mammalian NIP proteins, and hence were known as DUOX activators, DUOXA1 and DUOXA2 (11, 48). Knowing the essential function of the Notch signaling pathway as a main controller of cellular differentiation, changing the expression of DUOXA1 gene could have obvious downstream impacts on the Notch pathway, thus causing abnormalities in cell differentiation and/or tumor progression. Additionally, aberrant Notch signaling was reported in various types of cancers, with its signaling or expression either being pro- or anti-tumorigenic that highly relies on the tissue and cellular context (50–52). Surprisingly, Notch signaling and oncogenic EGFR signaling have been considered correlative (6), which could obviously be linked to a main function for DUOX1/DUOXA1 on these processes. Further than its function as a maturation factor for DUOX1, or in controlling the notch pathway, DUOXA1 might also have other independent functional roles. For example, recent studies indicated that DUOXA1 has a role in cellular differentiation in muscle satellite cells (53) and neuronal cells (50, 54), which include interactions with p53, promoting its functions in tumor suppressor, cell cycle stages and differentiation (50). Additionally, DUOXA1 was proposed to possess other, DUOX1-independent roles in different tissues including the brain and breast (50, 54, 55). Overall, DUOX1 expression requires a functional DUOXA1 protein and was reported to be associated with numerous diseases in several tissues.

DUOX1 in the skin

In a previous study, using a DNA microarray it was shown that of the seven NOX/DUOX family members expressed in normal human epidermal keratinocytes (NHEK), IL-4/IL-13 treatment only induced gene expression of DUOX1 (13). Moreover, this induced DUOX1 expression was demonstrated to be associated with high H₂O₂ production that was significantly inhibited both by diphenyleneiodonium, an inhibitor of NADPH oxidases, and by small interfering RNA against DUOX1 (13). Lastly, the elevated level of DUOX1 in IL-4/IL-13-treated NHEKs induced STAT6 phosphorylation through oxidative inactivation of the protein tyrosine phosphatase 1B. This study discovered a new function of IL-4/IL-13-induced DUOX1 gene expression in creating a positive feedback loop for IL-4/IL-13 signaling in keratinocytes (13). Previous studies in nonmammalian organisms indicated that DUOX1 is involved in innate cutaneous host defense to infectious triggers and in epidermal wound responses (35, 56, 57). In another study assessing the effects of cytokines on DUOX1 gene expression in cultured NHEK cells, DUOX1 levels were found to be 50-fold higher compared to DUOX2 levels. DUOX1 gene expression was augmented by IL-4 and IFN-γ (58). On the other hand, in the human bronchial epithelial cell line HBE1, IL-4 upregulated only DUOX1, while IFN-γ enhanced only DUOX2, not DUOX1, gene expression (58). These studies demonstrate that epithelial DUOX1 gene expression in different tissues can vary in their cytokine regulation (58). DUOX1 has been associated with Ca²⁺-dependent keratinocyte differentiation (59). DUOX1 represents the predominant DUOX isoform in human keratinocytes, the major epidermal cell type in the skin (>90%) (35). DUOX1 is also present in nonepithelial cells such as macrophages and innate lymphoid cells that also reside in the skin. DUOX1 plays a proposed role in innate lymphoid cells and macrophage polarization (17, 35). Atopic dermatitis and allergic contact dermatitis are caused by exaggerated immune responses to allergic stimuli characterized by high level of IgE antibodies (60). Atopic dermatitis can be followed by food allergy, allergic rhinitis and/or allergic asthma, in a process named as the atopic march (60). DUOX1 can also play a role in the pathogenesis of atopic dermatitis as it is induced by type 2 cytokines and it is present in epidermal keratinocytes (13, 61, 62). The local generation of DUOX1-derived H₂O₂ by TNF-deficient keratinocytes in the skin activates the transcription factor NF-κB, which then enhances the induction of genes encoding pro-inflammatory molecules (63). Moreover, complete pharmacological inhibition of DUOX1 eliminated skin inflammation. DUOX1 is highly expressed in the skin tissues of psoriasis and lichen planus patients (63). These studies suggest that pharmacologic and genetic approaches targeting DUOX1 could provide innovative methods to the treatment of atopic dermatitis, psoriasis, lichen planus, and other chronic inflammatory diseases. (13, 63).

DUOX1 in the respiratory system

1. DUOX1 in allergy and asthma

DUOX1 gene is expressed in airway epithelial cells and plays a proposed role in innate airway epithelial responses to infection or injury (64). DUOX1 is highly expressed in the nasal mucosa of patients with chronic sinusitis (65). Elevated DUOX1 expression was reported in cultured nasal or bronchial epithelial cells from patients with allergic asthma (15). The in vivo role of DUOX1 is best understood in allergic inflammation (15). Using Duox1-deficient mice and siRNA-mediated DUOX1 down-regulation, Habibovic et al. showed the essential role of DUOX1 in the induction of type 2 inflammatory responses, mucus metaplasia, subepithelial remodeling and airway hyperresponsiveness to methacholine (15). In previous studies, DUOX1 was shown to interfere with neutrophilic rather than eosinophilic inflammation in severe exacerbations of asthma (66). Severe exacerbation of asthma is usually dominated by neutrophilic inflammation, and it is more resistant to current treatments than other types of asthma (66). DUOX1 inhibition might offer a novel therapeutic option for patients with allergic asthma. DUOX1 enhances the activation of Src and EGFR that can cause mucosal hyperplasia and allergic airway inflammation (15). IL-4 or IL-13 induces DUOX1 gene expression in the epithelium of the respiratory tract. These cytokines enhance the Th2 response and antibody production, thus stimulate various types of allergies (67). In a previous study, DUOX1 was shown to regulate the expression of 50% of the genes induced upon IL-4/IL-13 stimulation by using a combination of DNA microarray analysis and DUOX1 siRNA knockdown. These studies suggested that DUOX1 might regulate the function of some transcription factors, and induces STAT1, 3 and 6 phosphorylation (12). Elevated DUOX1 gene expression is associated with multiple allergic conditions. Thus, selective inhibition of DUOX1 might be an attractive alternative strategy for treating severe allergic asthma. DUOX1-induced H₂O₂ production could also induce cellular proliferation and mucosal hyperplasia due to its ability to recruit inflammatory cells and activate Src and EGFR signaling (15). This might contribute to the pathogenesis of chronic rhinosinusitis in patients with nasal polyps (68). Until recently, the regulation of DUOX1 by proinflammatory cytokines has remained ambiguous. In a previous study, DUOX1 expression was enhanced in primary respiratory tracheobronchial epithelial cells grown on air-liquid interface cultures for one week and treated for 24 hours with various types of cytokines. DUOX1 expression was induced by Th2 cytokines such as IL-4 (4.5-fold elevation) and IL-13 (4.4-fold elevation). DUOX1 gene expression increased in a time- and dose-dependent manner (73). DUOX1 is the main DUOX isoform in the respiratory epithelium (66). Maximal DUOX1 expression can be reached at low levels of Th2 cytokines. DUOX1 expression is persistent and takes long time to disappear. DUOX1 may be an essential component of IL-4, IL-13 signaling leading to mucus secretion (73), bronchial smooth muscle hyperplasia and hyperatrophy. Abnormal expression of IL-13 can augment DUOX1 gene expression, subsequently lead to mucus hypersecretion (73) and muscular hyperplasia which exacerbate the disease and associate with poor outcomes (74, 75). High DUOX1 expression in asthmatic patients is correlated with severe symptoms and poor prognosis (64, 66).

2. DUOX1 in nasal polyposis

Nasal polyps are painless, smooth, noncancerous projections on the interior surface of the nasal canals or sinuses. Polyps project like teardrops or grapes. Polyps are caused by chronic inflammation and are linked to asthma, persistent infection, allergies, drug sensitivity and some immune diseases (69). In a previous study, DUOX1 expression in nasal mucosal tissues from patients with chronic rhinosinusitis with or without polyps was compared to that of non-inflamed control nasal tissues. DUOX1 was upregulated in nasal tissues from patients with chronic rhinosinusitis with polyps, in comparison to the controls. The level of DUOX1 in specimens from patients with chronic nasal sinusitis without polyps was similar to the controls (70). DUOX1 generates H₂O₂ into the extracellular space (71). H₂O₂ levels were elevated in nasal secretions from patients with chronic sinusitis without polyps. Furthermore, in patients with chronic rhinosinusitis with polyps, H₂O₂ levels were three-fold higher compared to the control levels. These results suggest the link between the presence of H₂O₂ in nasal secretions and DUOX1 expression in nasal tissues (70). H₂O₂ is produced in the airway mucosa and is normally exhaled during expiration. In various experiments, it was noted that individuals with inflammatory lung diseases exhale higher levels of H₂O₂ than healthy, control individuals (71). The extent of inflammation and the white blood cell count linked positively with exhaled H₂O₂, which is proved to be generated both by the epithelium and by activated white blood cells during airway inflammation (72). Measurements of H₂O₂ in nasal fluids cannot differentiate between the H₂O₂ generated by the epithelium or by mucosal white blood cells, thus, it is complicated to quantify the fraction of DUOX1- vs. phagocyte (NOX2)-mediated H₂O₂ production. In previous studies, it has been proven that DUOX-generated H₂O₂ attracts leukocytes (65). In one study, it was observed that the level of inflammatory cytokines such as eotaxin, monokine induced by gamma interferon, and TNF-α were prominently escalated in patients with chronic rhinosinusitis with polyps and their amount in nasal fluids was closely linked with DUOX1 gene expression (65). DUOX1 is upregulated in patients with chronic rhinosinusitis. Close correlations between DUOX1 expression and H₂O₂ release, and correlation between key inflammatory cytokines and DUOX1 expression, indicate a role of DUOX1 in the inflammatory response in chronic rhinosinusitis (65).

3. DUOX1 in chronic obstructive pulmonary disease (COPD)

Cigarette smoking is considered as a predisposing factor for various chronic respiratory illnesses involving chronic obstructive pulmonary diseases (COPD) and asthma (76). COPD involves emphysema and chronic bronchitis. COPD is the fourth leading cause of mortality in the United States (40) and it comes in the third place as a cause of death in 2020 (41). COPD symptoms are caused by constant airflow constriction that is often progressive and linked to various predisposing factors, leading to the exacerbation of the disease. COPD is characterized by constant inflammation and damage of the airways and the lung parenchyma. Exposure to smoking has been linked to enhanced oxidative stress and interferes with multiple biological mechanisms, including carcinogenesis and inflammation (77, 78). The COPD pathophysiology is characterized by chronic inflammation and damage to the small airways and the lung parenchyma (37). It is believed that oxidative stress is increased in patients with COPD due to chronic exposure to cigarette smoke, a main risk factor, which contains a high concentration of oxidants (39). Exposure to cigarette smoke augments cell calcium trafficking, induces mRNA and protein expression of DUOX1 in murine lungs and human cells. Furthermore, DUOX1 generates H₂O₂ that activates protein kinase C, leads to secretion of proinflammatory cytokines and blocks A2A adenosine receptor-stimulated wound repair (79). A previous study has used the human bronchial epithelial cell line Nuli-1 and C57BL/6 mice, and has shown that silencing the expression of DUOX1 partially enhanced the resistance of epithelial cells to the negative effects of cigarette smoke on wound healing after injury (79). Cigarette exposure regulates DUOX1 expression in the lower respiratory tract. These studies indicate that airway expression of DUOX1 is diversely associated with smoking and COPD (69). Acute cigarette smoke exposure promotes intracellular calcium mobilization to activate DUOX1. It is still not well-understood how DUOX1 is regulated in smoking patients with COPD. In one study, epithelial DUOX1 expression was investigated in the airways of the smokers, and the association between this expression and COPD in the early stages of the disease. First, bronchoscopy was used to brush the tracheal and bronchial epithelium from patients who have never smoked and current smokers (69). DUOX1 gene expression in harvested tracheal and bronchial epithelium was significantly downregulated in current smokers as compared to cases with no history of smoking. Furthermore, laser capture microdissection and microscope-assisted manual dissection were used in surgically resected lung tissues to harvest bronchiolar epithelium and alveolar septa (69). Smoker patients with mild/moderate COPD had lower bronchiolar DUOX1 expression than subjects who have never smoked, while a difference between former smokers, with and without COPD, was observed only for DUOX1. The expression of DUOX1 in the alveoli was low and was similar between the groups. These results imply that the airway expression of DUOX1 and DUOX2 is diversely associated with smoking and COPD. (69). A recent study reported that downregulation of epithelial DUOX1 gene in COPD contributes to disease pathogenesis (80). DUOX1 levels in the small airways were decreased in advanced COPD, and correlated with the loss of lung function and markers of emphysema and remodeling. DUOX1 gene downregulation was also observed in correlation with extracellular matrix remodeling observed in a genetic model of COPD (80). DUOX1 deficiency exacerbated both, the development of subepithelial airway fibrosis in acrolein-exposed mice, and alveolar emphysema in elastase-administered animals. These studies further highlight that DUOX1 downregulation is a contributing feature of COPD pathogenesis.

4. DUOX1 in lung fibrosis

Several factors and diseases can cause lung fibrosis including cytotoxic drugs, inflammation, allergic diseases, connective tissue diseases such as sarcoidosis, autoimmune diseases including rheumatoid arthritis and systemic lupus erythamtosis, and inhalation of organic materials such as asbestos (81). Excessive production and deposition of extracellular matrix in pulmonary fibrosis cause lung tissue damage, which leads to the impairment of pulmonary function (81). DUOX1 is expressed in fibroblasts in response to lung tissue damage. DUOX1-generated H₂O₂ enhances the TGF-β1 signaling pathway in fibroblasts by inhibiting phospho-Smad3 degradation (37). DUOX1 is highly expressed in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF). This elevation was accompanied with an escalation in the expression of α-SMA (smooth muscle actin), a marker of myofibroblasts (37). Induction of lung damage by bleomycin or radiation enhanced the expression of DUOX1 and DUOXA1. This elevation was linked to high mRNA expression of fibrosis markers including collagen (Col1a1), α smooth muscle actin (ACTA2) and the profibrotic factor Tgf-β (37). Duox1-deficient mice were used to examine tissue damage due to bleomycin and radiation, and it was found that these mice had less radiation-induced lung damage indicating that Duox1 is involved in the process of fibrosis formation (37). After bleomycin treatment, Duox1-deficient mice showed elevation in TGF-β1 in the lung tissue. In addition, elastase, which is released from activated neutrophils, was significantly lower in Duox1-deficient mice compared to control animals (37). The expressions of both DUOX1 and DUOXA1 mRNAs were stimulated by TGF-β1 in a dose- and time-dependent manner in wild-type fibroblasts. DUOX1 also inhibited the expression of α-SMA and collagen I mRNAs (37). Extracellular H₂O₂ enhances phosphorylation of Smad2/3 and elimination of DUOX1 enhanced the reduction of phosphorylated Smad2/3 levels (37). Phosphorylation of Smad2/3 was inhibited by catalase indicating that this step was H₂O₂-sensitive (37). Knockdown of DUOX1 in IPF-derived cells using RNAi caused a significant decrease in extracellular H₂O₂ generation, which lead to decreased TGF-β1-dependent accumulation of phospho-Smad3 (37). This study mechanistically proves that DUOX1-derived H₂O₂ determines the strength and duration of Smad signalling in the TGF-β1 pathway (37). NEDD4L is known as the main ubiquitin ligase that selectively interacts with activated Smad2/3 for destruction. The effect of NEED4L depletion on TGF-β1-induced Smad3 phosphorylation was evaluated by RNAi-mediated knockdown (37). NEDD4L knockdown inhibited the decline in phospho-Smad3 accumulation in DUOX1-deficient fibroblasts, indicating that the absence of DUOX1 enhances the action of NEDD4L to enhance TGF-β1 signaling (37). The NEDD4L oligomeric state is crucial for polyubiquitin chain assembly. Moreover, the enzyme catalytic site has a cysteine portion, which is essential for its activity (82). Both the oligomeric state of NEDD4L and its activity could be affected by H₂O₂-mediated oxidation (37). Selective DUOX1 inhibitors could, therefore, represent a novel therapeutic option against pulmonary fibrosis (37). IPF was shown to be linked to a high risk of pulmonary malignancy due to the appearance of unusual or dysplastic epithelial changes in fibrosis which develop into invasive malignancy (83).

DUOX1 in cancer

DUOX1 can promote or attenuate cancer growth according to its tissue expression and the type of malignancy. Although, in some types of cancers DUOX1-generated H₂O₂ can induce cellular proliferation, migration and angiogenesis, DUOX1-mediated immune cell recruitment can restrict tumor growth and metastasis (84) and promotes tumor cell clearance (63). The fraction of innate immune cells, including NK cells, monocytes, dendritic cells, and mast cells, was elevated in patients with high DUOX1 levels (61). The mechanism by which ROS are produced by tumor cells remained incompletely understood until the discovery over the last 15 years of the family of NADPH oxidases (85). DUOX1 was reported to act as a selective tumor-suppressor gene during tumor initiation and progression (4). The G2/M phase of the cell cycle acts as a cycle checkpoint and inhibits the entry into mitosis (86). It plays an important role in the control and maintenance of tissue homeostasis, when tumor growth is induced by DNA damage (60). In various types of malignancies, restoring DUOX1 expression in cancer cells with low DUOX1 expression significantly inhibited the growth of cancer cells by arresting the G2/M phase of the cell cycle and enhancing ROS production (4). A study found an association between intracellular ROS accumulation and tumor suppression (53). ROS can cause a specific type of cell death, termed anoikis, in normal cells. Although anoikis is a barrier to abnormal growth and metastasis, cancer cells have often acquired increased threshold for anoikis and thus elevated metastatic potential. Moreover, elevated DUOX1 expression was associated with enhanced immune pathways related to interferon (IFN)-alpha, IFN-gamma, and natural killer (NK) cell signaling. Malignancies such as lung cancer, hepatocellular carcinoma, follicular thyroid carcinoma, and breast cancer have low DUOX1 expression (42, 87–90), others including pancreatic ductal adenocarcinoma, gastric carcinoma and cervical carcinoma have high reported DUOX1 expression (91–94). Subsequently, ROS might enhance anti-apoptotic, proliferative, metastatic and angiogenic signaling, on the other hand, ROS may also contribute to cytotoxic and proapoptotic effects that constrain malignant progression and carcinogenesis according to the tissue location and type of cancer (84). DUOX1 gene expression is silenced in both lung cancer and hepatocellular carcinomas compared to surrounding normal tissues. It was proposed to be silenced due to DUOX1 promoter hypermethylation since both directions of the promotor regions of DUOX1 and DUOX1A are rich in CpG islands (87). Similarly to DUOX1, DUOXA1 was downregulated in different cancers as shown by TCGA data analysis of DUOXA1 mRNA expression (84). Surprisingly, recent research in lung malignant cells proved that the overexpression of DUOX1, but not DUOXA1, could reestablish epithelial characteristics and decrease invasive and migratory properties (41). A previous study has shown that DUOX1 expression was significantly lower in most cell lines of liver cancer and primary hepatocellular carcinoma tissues compared to non-tumor tissues (42). The prognosis of hepatocellular carcinoma after hepatectomy can be predicted by the level of DUOX1 expression (42). Individuals with elevated DUOX1 mRNA levels have prolonged disease-free and overall survival compared to those with low DUOX1 mRNA expression. The level of DUOX1 mRNA expression was an independent prognostic factor for overall survival in hepatocellular carcinoma patients (55). DUOX1 could work as a tumor suppressor gene in the initiation of hepatic carcinoma and could be considered as a novel biomarker for prognosis and therapy in liver cancer (55). In follicular thyroid carcinoma with poor differentiation, elevated DUOX1 expression is linked to low mortality rate (89), however, data from TCGA analysis indicate DUOXA1 mRNA expression is enhanced in other thyroid cancers (84). Thus, DUOX1 and DUOX1A might work independently in thyroid malignancies. Additionally, DUOX1 gene expression was proven to be decreased in breast cancer cell lines and in breast cancers when compared to their non-tumor counterparts (65). This led to increased cell proliferation rates and adhesion properties, which are typical features of transformed cells (65). Moreover, some studies in breast cancer cells examined the functional significance of DUOXA1 downregulation and showed that decreased DUOXA1 expression in metastatic cell lines, altered cell adhesion, and susceptibility to doxorubicin after transient overexpression of DUOXA1 (55). Pancreatic inflammation due to ROS production and cytokine release have been linked to repeated inflammation in pancreatic tissues, and to the development and progression of pancreatic ductal adenocarcinoma (91, 95). DUOX1 mRNA and protein levels were 57.9-fold higher in patients with cervical carcinoma than in normal controls (92). DUOX1 mRNA levels were prominently increased in patients diagnosed with human papilloma virus HPV 16 compared to patients diagnosed with human papilloma virus HPV 18 and human papilloma virus HPV 45 (92). Chronic infection with human papilloma virus develops cervical carcinoma and is a leading cause of death among women worldwide and in the United States (94). Patients who are diagnosed with cervical squamous cell carcinoma have higher DUOX1 expression than those with endocervical adenocarcinoma (92). Furthermore, high DUOX1 gene expression is a poor prognostic sign in gastric carcinoma (93). Compared to common silencing of DUOX1 that happens in various malignancies according to both TCGA data and immunohistochemical data in the Human Protein Atlas, these data often show positive DUOXA1 protein expression in various cancer tissues (e.g. neuroendocrine, head and neck, testicular cancers) as well as mixed immunostaining in multiple other cancers (84). Overall, these studies indicate that DUOX1 and DUOXA1 might function independently, and changes of DUOXA1 expression in malignancies might impact tumor pathology by DUOX1-independent mechanisms, associated with p53 or Numb/Notch pathways, in addition to regulating the actions of DUOX1. Further studies are needed using DUOX1 and DUOXA1 knockout animal models (global or conditional) to explore the potential mechanisms by which DUOX1 and DUOXA1 suppress or promote tumor growth. A better understanding of these mechanisms may provide valuable new diagnostic insights and novel therapeutic opportunities in cancer research. Likewise, it would be of interest to assess whether DUOX1 and DUOX1A gene mutations are associated with or protect from any form of cancer.

DUOX1 in other diseases

DUOX1 plays an important role in H₂O₂ production, which might act as predisposing factor for several diseases including irradiation mediated heart damage (96–98). Irradiation-induced fibrogenesis is a predisposing factor for several cardiac diseases such as coronary and carotid artery fibrosis, hyperatrophy, ischemia and pericarditis (48). A study indicated that DUOX1 plays an important role in stimulating the Smad-dependent TGF-β1 signalling pathway, which promotes irradiation-mediated pulmonary fibrogenesis after inflammation (47). Radiation-induced heart disease (RIHD) is a common side effect of radiation therapy for the management of multiple malignancies, and RIHD increases mortality rate among patients under radiotherapy (99). Histological studies have shown that the recruitment of inflammatory cells such as neutrophils via DUOX1 plays an important function in chronic oxidative damage, inflammation and fibrosis, causing alterations in the cardiac tissue structures and elevate the risk of cardiac attack (49, 50). In addition, DUOX1-mediated neutrophil recruitment could contribute to fibrosis due to elastase production (37). Duox1-deficient mice have lower neutrophil elastase levels (47). A function for H₂O₂-mediated pulmonary fibrosis was previously proven by the protective effect of catalase, an enzyme that degrades H₂O₂ (52). TGF-β1-mediated myofibroblast differentiation was previously proven to be linked to extracellular H₂O₂ production which could damage the heart (47). In a study using rats, metformin attenuated the upregulation of Duox1 after irradiation exposure and lowered the recruitment of inflammatory cells (100). Metformin is an oral diabetes type 2 medicine that helps control blood glucose levels (101). Lowering Duox1 gene expression via metformin could potentially play an important role in the protection against irradiation’s toxic effects on the heart (100). In another study also using rats, melatonin was used to attenuate Duox1 expression and lower irradiation-mediated lung injury (102). Subsequently, these studies reveal the potential importance of DUOX1 expression in promoting fibrotic changes. Selective DUOX1 inhibitors could improve the outcome of these diseases. Since DUOX1 is mostly expressed in the respiratory system, DUOX1 might also have a role in the pathogenesis of cystic fibrosis (103). Cystic fibrosis (CF), is an autosomal recessive disorder that mostly occurs in Caucasians. It is due to mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator protein. Pathology of CF is mainly caused by abnormal Na⁺ and Cl⁻ ion transport in various tissues such as the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system. Patients with CF have high morbidity and mortality due to progressive pulmonary disease. DUOX1-mediated recruitment of immune cells such as neutrophils and macrophages to the respiratory epithelial surface might contribute to the oxidative and proteolytic stress implicated in the pathogenesis of CF (103). The function of DUOX1 enzyme in the molecular mechanisms of these diseases need to be explored in more detail.

Concluding Remarks

High level of DUOX1 expression has been linked to many disease pathologies and has been successfully marked as a good prognostic factor in patients with hepatocellular carcinoma (88) (Fig. 1). Recent research showed a correlative association between low DUOX1 expression and epithelial-to-mesenchymal transition (EMT), cancer stem cell characteristics and resistance of epidermal growth factor receptor-targeted therapy (41). Most of the data proposing a link between DUOX1 and disease conditions remain, however, correlative, rely on mRNA analyses and do not provide a causative relationship. While results obtained on human clinical samples are essential to establish the potential clinical relevance of DUOX1, mechanistic studies using animal models or in vitro experimental systems are needed to better understand whether and how DUOX1 promotes or attenuates the pathogenesis of a variety of diseases. While these tools such as Duox1-deficient mice are available, most of the correlative data between DUOX1 and diseases has not been confirmed in mechanistic studies. Generating mouse models with tissue-specific deficiencies in Duox1 is highly desired to better understand its role in different diseases. Creating Duox1-deficient animals in mammalian species other than the mouse would solve problems and limitations related to using mice as an experimental model. Duox1 overexpression in mice could also provide a new, independent approach to help better understand the potential role of DUOX1 overexpression in certain pathophysiologies.

Figure 1:

Conditions associated with either high or low expression of the NADPH oxidase DUOX1.

Another important chapter in this field is the absence of information regarding existing DUOX1 single nucleotide polymorphisms (SNPs) and their correlation with different diseases. While several studies have linked DUOX2 SNPs to hypothyroidism (104, 105), and numerous studies indicate that the development of hypothyroidism is a predisposing factor for the onset of thyroid cancer (106–108), little is known about DUOX1 SNPs. Only one study reported two DUOX1 SNPs on exons 27 and 28 without any link to disease pathology (109).

Appropriate progress in the field is also delayed by the lack of DUOX1-specific antibodies. Specific detection of DUOX1 on the protein level, and its distinction from DUOX2, is very important and highly desired but remain largely problematic. Most antibodies used in the field detect both isoforms. While several companies sell antibodies claimed to be specific to DUOX1, they fail to characterize their specificities and to provide experimental evidence for it. Testing these antibodies on Duox1-deficient murine tissues/cells or on human cell lines overexpressing DUOX1 and DUOXA1 should be a routine and mandatory exercise prior to product launch to the market. DUOX1 functions in a complex with DUOXA1. While recent studies emerged studying this protein-protein interaction in details, more work is needed to better understand the subcellular localization and molecular interaction of DUOX1 and DUOXA1, especially in the complex situation of an in vivo organism.

In summary, DUOX1 gene expression has been linked to a range of clinical conditions and the contribution of DUOX1 to airway diseases has been started to be explored in murine models, several challenges remain though in the field that need to be addressed in the future.

Abbreviations

DUOX1

Dual oxidase 1

NOX

NADPH oxidase

ROS

reactive oxygen species