Journal of Trace Elements in Medicine and Biology 44 (2017) 247–255

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Pathobiochemistry

The copper dependent-lysyl oxidases contribute to the pathogenesis of pulmonary emphysema in chronic obstructive pulmonary disease patients Neziha Besiktepea, Ozgecan Kayalara, Ezel Ersenb, Fusun Oztaya, a b

MARK



Department of Biology, Faculty of Science, Istanbul University, 34134 Vezneciler, Istanbul, Turkey Department of Thoracic Surgery, Cerrahpasa Medical Faculty, Istanbul University, 34098 Kocamustafapasa, Istanbul, Turkey

A R T I C L E I N F O

A B S T R A C T

Keywords: Pulmonary emphysema LOX LOXL1 LOXL2 HIF-1 COMMD1 PTEN

Abnormalities in the elastic fiber biology are seen in pulmonary emphysema (PE). The copper-dependent lysyl oxidases regulate the production and accumulation of elastic fibers in the connective tissue. This study focused on the relationship between lysyl oxidase (LOX), LOX-like protein 1 (LOXL1), and LOXL2 and PE pathogenesis. Lung samples with or without PE from patients with chronic obstructive lung disease (n = 35) were used. Protein levels of elastin, LOX, LOXL1, LOXL2, hypoxia inducible factor 1-alpha (HIF-1α), copper metabolism domain containing-1 (COMMD1), and phosphatase and tensin homolog (PTEN) were assayed using microscopic and biochemical methods The emphysematous areas were characterized by enlargement of the alveoli, destruction of the alveolar structure, accumulation of macrophages in the alveolar lumens, and showed increased HIF-1α immunoreactivity. Additionally, the emphysematous areas had significantly lower elastin, LOX, LOXL1, LOXL2, HIF-1α, COMMD1, and PTEN protein levels than the non-emphysematous areas. We suppose that the reductions in the HIF-1α levels led to decreases in the protein levels of active LOX, LOXL1, and LOXL2. These decreases might cause abnormalities in the elastic fiber biology. HIF-1α activation induced by decreased COMMD1 and protease activation induced by decreased PTEN might contribute to the development of PE. Finally, methods aimed at increasing the protein levels of LOXs, COMMD1 and PTEN might be effective for treating PE.

1. Introduction Chronic obstructive pulmonary disease (COPD) is a complex disease that includes pulmonary emphysema (PE), chronic bronchitis, certain types of bronchiectasis, and occasionally asthma. PE is observed in approximately 20% of COPD patients. It is characterized by the enlargement of the alveoli distal to the terminal bronchioles, destruction of the alveolar wall, abnormalities in the accumulation and degradation of elastic fiber, loss of lung elasticity, and a progressive decline in the forced expiratory volume in the first second of inspiration (FEV1) [1]. Experimental and clinical studies have indicated that increased oxidative stress, the proteinase/anti-proteinase imbalance, and apoptosis of the epithelial and endothelial cells of the pulmonary airways contribute to the development of PE [2]. Additionally, emphysematous lesions have been observed to occur after the intratracheal administration of matrix degradation enzymes such as elastase or papain to murine. The relationship between the destruction of the elastic fibers and the enlargement of the alveoli can thus be explained [3]. On the other hand, gene−targeting studies have reported that PE may develop in cases ⁎

wherein the elastic fibers cannot be correctly synthesized. For example, mice null for fibulin-5, which is required for elastic fiber remodeling, possess reduced secondary septation with enlarged alveolar sacs at 2 weeks that evolve into bullous emphysema in adult life [4], while mice null for elastin display markedly enlarged air sacs with no secondary septation and impaired distal airway development [5]. Elastic fibers consist of a core of elastin protein surrounded by microfibrils. For the polymerization of the elastin protein, adjacent tropoelastin monomers are spontaneously clustered; thereafter, these monomers are crosslinked by one or more members of the enzymes of the lysyl oxidase family [6]. These enzymes are copper-dependent amine oxidases. Healthy mammalian lungs express three members of these amine oxidases: lysyl oxidase (LOX), LOX-like protein 1 (LOXL1) and LOX-like protein 2 (LOXL2) [7]. The critical role of LOX in PE development has been reported in several experimental studies. These studies have reported the development of emphysematous areas in lungs of rats and hamsters fed with a copper-deficient diet [8,9]. Patients with Menkes disease, an X-linked recessive disorder of copper transport associated with LOX deficiency, developed severe diffuse

Corresponding author. E-mail addresses: [email protected] (N. Besiktepe), [email protected] (O. Kayalar), [email protected], [email protected] (E. Ersen), [email protected] (F. Oztay). http://dx.doi.org/10.1016/j.jtemb.2017.08.011 Received 20 June 2017; Received in revised form 15 August 2017; Accepted 17 August 2017 0946-672X/ © 2017 Elsevier GmbH. All rights reserved.

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emphysema leading to respiratory failure and early death [10]. Other studies have reported emphysematous lesions accompanied by the inhibition of elastin gene expression caused by the down-regulation of LOX at the mRNA, protein, and catalytic levels in cell models and in the lung tissue in animal models [11,12]. Liu et al. reported enlarged air sacs resulting from decreased elastin content in LOXL1-null mice [13]. To the best of our knowledge, no previous studies have assessed the role of the enzymes of the lysyl oxidase family in the development of PE in humans. Therefore, the present study focused on the relationship between the lysyl oxidases (LOX, LOXL1, LOXL2) and PE pathogenesis in COPD patients. Alterations at the protein levels of LOX, LOXL1, LOXL2, copper metabolism domain containing-1 (COMMD1, a regulator protein of copper homeostasis), hypoxia inducible factor 1-alpha (HIF-1α, a transcription factor of lysyl oxidase genes), and phosphatase and tensin homolog (PTEN, a regulator protein for HIF-1α) were for the first time investigated to determine their role in the pathogenesis of emphysema in the lungs of COPD patients.

3% hydrogen peroxide in 1:1 methanol/phosphate-buffered saline mixture. The sections were incubated with rabbit anti-HIF-1α antibody at a 1:100 dilution overnight at +4 °C and rabbit anti-cluster of differentiation 68 (CD68) antibody (a macrophage marker) at a 1:2500 dilution for 32 min at +37 °C. Later on, the sections were incubated in Histostain Plus-peroxidase kit according to the manufacturer’s instructions. The 3-amino-9-ethylcarbazole substrate kit was used as final staining. Slides were counterstained with Mayer’s hematoxylin. Negative controls were those in which phosphate-buffered saline solution was used instead of the primary antibodies. Five microscopic fields were randomly selected from alveolar areas without bronchioles from sections of each sample. Digital images of these fields were captured at a magnification of 400 and overlaid with transparent grids (1 mm2). The number of HIF-1α-immunoreactive cells having immunoreactivity localized in cytoplasm or nuclei was calculated as percentages of the immunoreactive cells in the total number of cells.

2. Materials and methods

2.3. Western blotting

2.1. Study design

Lung samples were snap frozen in liquid nitrogen and stored at −86 °C. Lung samples weighing 200 mg were homogenized using a MagNA Lyser instrument (Roche, Mannheim, Germany), in cold radioimmunoprecipitation assay buffer (Santa Cruz, CA, USA). The lysates were then centrifuged at 13,000g for 10 min at 4 °C, and the supernatants were collected and stored at −20 °C. The total protein concentrations were determined using Bradford method [15]. The samples (80 μg) were mixed with loading buffer, denatured by heating at 95 °C for 5 min, resolved via a SDS-polyacrylamide gel, and transferred to nitrocellulose membranes. The membranes were treated with in 4% neutral formalin for 5 min. And then, they were blocked with 5% non-fat dried milk for 1 h and incubated with rabbit primary antibodies against elastin, LOX, LOXL1, LOXL2, HIF-1α, COMMD1 and PTEN diluted 1:500 for overnight at 4 °C. Finally, the blots were developed with luminol reagent (Santa Cruz, CA, USA). The intensities of the protein bands were quantified using molecular imaging software (Kodak GL 1500, CT, USA) and normalized with β-actin protein bands. Anti-LOX and anti-HIF-1α antibodies were purchased from Novus Biologicals, Littleton, CO, USA. Anti-COMMD1 antibody was supplied from Bioss, USA. The remaining antibodies were purchased from Santa Cruz, CA, USA.

The study protocol was ethically reviewed and approved by the Clinical Research Ethics Committee in Cerrahpasa Medical Faculty of Istanbul University, Turkey (Diary No a−15/01.10.2013). The study was conducted in accordance with the Declaration of Helsinki. Populations of the present study were COPD patients with diagnosed of lung cancer (Table 1). Gold 2016 guideline was used for COPD diagnosis. Lung samples around the tumor areas were removed by thoracoscopic surgery from male patients (n = 35). Each sample was divided into two parts. One of them was used for microscopic analysis, while the other one was used for Western blot analysis. 2.2. Histology and immunohistochemistry Fresh lung samples were fixed in 10% neutral formalin buffered phosphate for 24 h at 4 °C. The tissues were dehydrated in graded ethanol series and embedded in paraffin wax. Sections 5 μm thick were taken serially and stained with hematoxylin-eosin and Verhoeff's elastic stain. Lung samples with and without emphysema were identified under a light microscope. Emphysematous alterations were evaluated using the following criteria: presence of big and dilated alveoli, and thinning of their wall, breakage and disruption of alveolar walls and/or epithelium in the lung. Lung samples without emphysema were accepted as control tissue. The lung sections were dipped in a 10 mM citrate buffer (pH 6.0) and heated in a microwave oven for 15 min at 650-W to reveal masked antigens [14]. Endogenous peroxidase was eliminated by incubation in

2.4. Statistical analyses The statistical analysis was performed using GraphPad Prism software, version 6.00 (San Diego, CA). The results were analyzed by Student T- test to compare statistical differences among groups. P-values of < 0.05 were considered significant. 3. Results

Table 1 Main clinical characteristics of the study population, by phenotype/subgroup. Number of samples Gender Mean age (years) Diagnosis

Mean FEV1% Current smoking Smoking Rate (Pack/Year)

3.1. Lungs with emphysema were characterized by destruction of the alveolar areas and decreased elastin protein levels

Control (n = 6) Emphysema (n = 10) Male (n = 16) Female (n = 0) Control (57) Emphysema (62) Adenocarcinoma (13) Organising Pneumonia (1) Irregular Emphysema (1) Squamous Cell Carcinoma (1) Control (64) Emphysema (77) Yes (8) No (8) Control (26) Emphysema (35)

Since lung samples were generally collected from the distal regions of the airway, bronchi and bronchioles were very rarely observed in the lung sections. Hemorrhage and anthracosis were observed in the interstitial areas throughout all the lung tissues. Although the lung samples without emphysema have a relatively conserved alveolar structure, leukocyte infiltration, partial damage of the alveolar epithelium, as well as erythrocytes, macrophages, and cellular residues were detected in the alveolar lumens (Fig. 1a). Enlarged alveoli and destruction of the alveolar structure including thinning and breakage of the alveolar epithelium, were commonly observed in the lung samples with emphysema (Fig. 1b; Table 2). Particularly, alveolar wall in the emphysematous areas contained more less elastic fibers than the non248

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Fig. 1. Histological section of human lung stained with hematoxylin-eosin. a) Lung sample without emphysema; (*) alveoli, ( ) erythrocyte in the alveolar lumen, ( ) anthracosis areas; b) Lung sample with emphysema; (*) wide and large alveoli, ( ) several erythrocytes in the alveolar lumen, (●) leucocyte infiltration in the tissue. Scale bars = 200 μm and 500 μm.

emphysematous areas. These elastic fibers were discontinuous rather than harmonious in the alveolar wall (Fig. 2a and b). Western blot analysis showed a decrease in the elastin protein levels of emphysematous areas versus that in non-emphysematous areas (Fig. 3a and b; p < 0.001). Additionally, several erythrocytes, macrophages (CD68+ cells) and cellular residues were present in the alveolar lumens of emphysematous lungs compared to those in the control lung tissues (Fig. 4).

Table 2 Grades of destruction index in the lungs. Samples

Presence of enlargement alveoli

Distribution of emphysematous areas

Presence of cell released to alveolar lumen

C1 C2 C3 C4 C5 C6 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10

+ + ++ + ++ + +++ ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++

+ + ++ + ++ + +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

++ +++ ++ + ++ + +++ +++ ++++ ++ ++ ++++ ++++ +++ +++ +++

3.2. Levels of active LOX, LOXL1 and LOXL2 proteins were reduced in the lung with emphysema The lungs of COPD patients diagnosed with lung cancer produced a higher level of LOXL2 proteins than LOX and LOXL1 proteins. We found a decrease in the levels of active LOX (p < 0.05), LOXL1 (p < 0.001) and LOXL2 (p < 0.05) in the emphysematous areas (Fig. 5). 3.3. Level of HIF-1α protein was low in the lung with emphysema; however, HIF-1α activation was induced

C: Control, E: Emphysema, +: rare, ++: low, +++: much, +++ +: abundant.

HIF-1α immunoreactivity in the human lung was clearly observed in the nucleus and cytoplasm of macrophages, leukocytes, connective tissue cells, and alveolar epithelial cells of the human lung (Fig. 6). HIF1α immunoreactivity was increased and mostly detected in the nuclei of

Fig. 2. Elastic fibers of the human lung are blue-black as stained with Verhoeff’s Elastic Stain. Alveolar wall in the emphysematous areas contained more less elastic fibers than the nonemphysematous areas. These elastic fibers were discontinuous rather than harmonious. a) Lung sample without emphysema; b) Lung sample with emphysema. Scale bars = 100 μm.

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Fig. 3. Alterations of elastin protein levels in the lungs with and without emphysema of COPD patients. a) intensity analysis of the protein bands and b) elastin protein levels in the lung samples compared to the control group (***p < 0.001).

Fig. 4. CD68+ cells (macrophages) are present in the alveolar lumens of COPD patients without (a) and with emphysema (b). Scale bars = 50 μm and 100 μm. Mayer’s hematoxylin stain.

that the unorganized large masses of elastic fibers in the extracellular matrix (ECM) would be rapidly digested by the proteases. HIF-1α activation induced by decreased COMMD1 level and protease activation induced by decreased PTEN level can contribute to the generation of the disease pathogenesis. We concluded that methods aimed at increasing LOX, LOXL1, LOXL2, COMMD1 and PTEN protein levels can reduce the undesirable emphysematous alterations in COPD patients. PE is seen as a reaction of COPD in cigarette smoking patients who are 45–60 years old. In some studies, 82% of COPD cases and 80% of lung cancer cases are attributed to smoking [16,17]. A study involving approximately 1000 subjects reported a 3–4 fold increase in the risk of lung cancer in younger subjects with PE who were chronic cigarette smokers [16]. However, recent studies have also revealed that the risk of lung cancer in COPD patients is 5-fold higher than that in smokers without COPD [17]. The present study established the presence of typical emphysematous areas in the lung of COPD patients with lung cancer. Elastolytic activity contributes to the generation of these structural alterations observed in PE by inducing the destruction of the elastic fibers in the alveolar wall through the activation of proteases. When several proteolytic enzymes such as papain, pancreatic elastase and human neutrophil elastase are administered to animals via the intratracheal route or inhalation, they lead to the digestion of the elastic fibers in the alveolar wall within 24 h, destruction of the alveolar walls, and alveolar enlargement over time [3,5]. It has been established that elastases, serine proteases, and several matrix metalloproteases (MMPs) released from the macrophages, neutrophils, and lymphocytes

cells in the emphysematous areas rather than in the non-emphysematous areas. Additionally, the number of cells showing nuclear HIF-1α immunoreactivity was higher in the emphysematous areas. In contrast, the HIF-1α levels were significantly decreased in the lung tissues with emphysema (Fig. 7; p < 0.05). 3.4. Levels of COMMD1 and PTEN proteins were reduced in the lung with emphysema Western blot analysis showed a decrease in the levels of COMMD1 (p < 0.01) and PTEN (p < 0.05) proteins in emphysematous tissues as compared to those in the non−emphysematous tissues (Fig. 8). 4. Discussion The present study provided the first data on the relationship between LOX, LOXL1, LOXL2 and PE pathogenesis in COPD patients with diagnosis of lung cancer. The prominent findings of the present study are as follows: the decreases in the levels of LOX, LOXL1, LOXL2, HIF1α, COMMD1 and PTEN proteins might be associated with the pathogenesis of PE. The decrease in the HIF-1α and COMMD1 levels, despite of the increased nuclear localization of HIF-1α, results in the reduction of expression and activation of LOX, LOXL1 and LOXL2 proteins in emphysematous lungs. Thereby, the development of PE is stimulated by the mediation of some anomalies in the biogenesis and organization of the elastic fibers in the lungs of COPD patients. Moreover, it is possible 250

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Fig. 5. Alterations in the protein levels of the lysyl oxidase enzyme family in the lungs with and without emphysema of COPD patients. Data showing the intensity analysis of active LOX (a.1), LOXL1 (b.1), and LOXL2 (c.1) bands; Western blot analysis showing changes in the levels of active LOX (a.2), LOXL1 (b.2) and LOXL2 (c.2) compared to the control group (*p < 0.05 and ***p < 0.001).

and experimental studies mentioned above, we can conclude that the stimulation of inflammation and activation of the proteases can cause PE in COPD patients. Moreover, in our study, Western blot analysis clearly showed a decrease in the elastin protein levels (p < 0.001) in the emphysematous lungs. We conclude that the depletion of elastin protein may be dependent on the digestion of the elastic fibers by the proteases in emphysematous lungs. Elastic fiber formation and their abnormal organization in the ECM, as well as the digestion of elastic fibers by elastases, play an important

are affected by the degradation of ECM components, including elastic fibers in the lungs of COPD patients who smoked cigarettes [18]. MMP12 is a kind of elastase. Rats with depleted neutrophils and alveolar macrophages as well as MMP-12-knockdown mice are protected from the development of cigarette smoke-induced emphysema [19]. In the present study, we observed several macrophages and few neutrophils in the lumens of the enlarged alveoli in the emphysematous areas. Furthermore, the majority of the subjects in the current study were chronic, long term cigarette smokers. Considering the results of the past clinical 251

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Fig. 6. Nuclear and cytoplasmic localization of anti-HIF-1α immunoreactivity marked by an arrow in the alveolar areas of the non-emphysematous areas (a) and the emphysematous areas (b). Scale bars = 50 μm. Mayer’s hematoxylin counterstain.

Fig. 7. Alterations of HIF-1α protein levels in the lungs with and without emphysema of COPD patients. Data showing the intensity analysis of the HIF-1α bands (a), and changes in the HIF-1α level (b) compared to the control group (*p < 0.05).

down-regulation of LOX at the mRNA, protein, and catalytic levels in fetal lung fibroblasts and in rat lungs [12,16,21,22]. Premature deaths were observed because of intensive enlargement of the airways in elastin gene-deficient mice [5]. Liu et al. reported enlarged pulmonary air spaces resulting from decreased elastin content in the LOXL1-null mice [13]. Patients with Menkes disease, an X-linked recessive disorder of copper transport associated with LOX deficiency, developed severe diffuse emphysema leading to respiratory failure and early death [10]. Immunofluorescence labeling showed the colocalization of both LOX and LOXL1 to elastin at the earliest stages of elastic fiber assembly [23]. Thomassin et al. demonstrated that the pro-regions of LOX and LOXL1 are required for deposition onto elastic fibers by the mediating interactions with tropoelastin [23]. The present study shows that in the

role in the development of PE. Few experimental studies carried out on animals provide some information regarding this. However, to the best of our knowledge, the alterations in the formation and organization of elastic fibers in the humans that could explain the development of PE have not been investigated. Therefore, the present study investigated the relationship between the lysyl oxidases (LOX, LOXL1, LOXL2) and PE pathogenesis in COPD patients. In the elastic fiber assembly, LOXL1 regulates the accumulation of elastin protein, while LOXL2 is involved in both the accumulation of the elastin protein and the cross-linking among their monomers. LOX catalyzes the cross-linking between the elastic fibers and the collagen fibers, thus organizing the elastic fibers in the ECM [20]. Experimental studies have reported emphysematous lesions accompanied by the inhibition of elastin expression caused by the

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Fig. 8. Alterations of COMMD1 and PTEN protein levels in the lungs with and without emphysema of COPD patients. Data showing the intensity analysis of COMMD1 (a.1) and PTEN (b.1) protein bands; and the alterations at the protein levels of COMMD1 (a.2) and PTEN (b.2) compared to the control group (*p < 0.05 and **p < 0.01).

copper export in the emphysematous lungs and correlates with the reduction of active LOX, LOXL1 and LOXL2 protein levels. Consequently, copper, which cannot be released from the cell due to the depletion of COMMD1 proteins, can cause a decrease in the activation of the copperdependent functions of LOX, LOXL1, and LOXL2 in the ECM. These reductions in the activation of the enzymes can contribute to the development of PE by the deterioration in the biogenesis and organization of the elastic fibers in the ECM. HIF-1 comprising α and β subunits is an important transcription factor. The hypoxic conditions stops the destruction of HIF-1α. Thus, HIF-1α is transported from cytoplasm to the nucleus, dimerizes with HIF-1β, and then this complex stimulates transcription of various target genes. HIF-1α controls tumor vasculogenesis and invasion under hypoxic conditions in lungs with adenocarcinoma [27]. Most of the lung samples used in the present study were taken from COPD patients with lung adenocarcinoma. However, these samples were from non-cancerous tissues surrounding a tumor. The levels of HIF-1α in lung samples with emphysema were lower than those in lung samples without emphysema. Based on this finding, we believe that HIF-1α expression profiles can differ in various regions of the lung in COPD patients with lung cancer. Yasuo et al. reported decreased HIF-1α expressions in the emphysematous areas in the lungs of COPD patients [28]. Another study reported a reduction in the HIF-1α immunoreactivity in COPD rat models exposed to cigarette smoking [29]. In agreement with these results, our study found a reduction in the HIF-1α protein levels in the emphysematous lungs and an increase HIF-1α immunoreactivity localized in the nuclei of macrophages, leukocytes, connective tissue cells and alveolar epithelial cells. Thus, we can conclude that emphysematous lungs are characterized by a decrease in the HIF-1α protein levels and an increase in HIF-1α activation. Jiang et al. demonstrated that the stimulated HIF-1α activation contributed to COPD pathogenesis in an experimental COPD model rats [30]. Experimental studies on human lung fibroblast cell lines revealed that the insufficient HIF-1α

lungs of COPD patients, LOXL2 proteins are present in the high concentration, followed by the LOXL1 and LOX proteins. The decreases in the active LOX (p < 0.05), LOXL1 (p < 0.001) and LOXL2 (p < 0.05) proteins was associated with the reduction in the elastin protein in the emphysematous areas. The LOXL1 protein levels reduced considerably. LOXL1 is responsible for elastin accumulation; therefore, the decrease in the LOXL1 protein levels in the emphysematous areas results in the failure of elastin accumulation, an important step in the elastic fiber assembly. The decreased active LOX and LOXL2 protein levels can also remark some disruptions in the cross-linking between the elastin monomers and the microfibers. Thereby, the reduction of elastin, LOX, LOXL1 and LOXL2 protein levels and disruptions in the cross-linking among the elastic fibers can contribute to the development of PE by mediating some of the alterations in the biogenesis and the organization of elastic fiber in COPD patients. Moreover, it is possible that the unorganized large masses of elastic fibers in the ECM would be rapidly digested by the proteases. PE was induced in murine fed a copper-deficient diet [9,24]. Copper is required for the activation of LOX, LOXL1 and LOXL2 precursor proteins involved in the organization and biogenesis of elastic fibers [20]. PE was observed in the hereditary copper deficiency in Menkes and Wilson patients, due to disruption in the activations of the LOX enzymes and elastin biogenesis, as well as in mice with stained skin disease (Blotchy mice). These studies showed that the reductions in the level and activation of LOX enzymes due to copper deficiency induced the development of PE. COMMD1 is an effective protein for copper metabolism and adaptation to hypoxia. It regulates cellular copper concentration and export [25]. Liver-specific COMMD1 knockout mice were susceptible to hepatic copper accumulation and liver toxicity [26]. We found that lung samples with emphysema had lower COMMD1 levels than the lung samples without emphysema (p < 0.01). On the basis of our results, we can suggest that PE can occur in copper deficiency. The decrease in the COMMD1 protein levels can lead to cellular

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References

expression resulted in the depletion of LOX transcription, matrix protein synthesis and matrix protein cross-linking in human lung fibroblasts [24]. We hypothesized that the decreased HIF-1α levels in the emphysematous lungs of COPD patients could not have sufficiently stimulated the expression of LOX, LOXL1 and LOXL2 genes, despite the increased nuclear localization of HIF-1α. Consequently, the sufficient levels of active LOX, LOXL1 and LOXL2 proteins might not have been converted from their precursor proteins, resulting in the development of PE owing to some anomalies in the biogenesis and organization of the elastic fibers. Berk et al. reported that hypoxic signaling reduced de novo elastin production to 70% in rat lung fibroblasts [31]. On the other hand, we believe that the stimulation of HIF-1α activation can contribute to PE pathogenesis by inducing elastolytic activity in COPD patients. HIF-1α secreted from the alveolar epithelial cells, macrophages and neutrophils, stimulates the release of elastases such as MMP-9 and MMP-12 from these cells under hypoxic conditions [32]. The COMMD1 protein regulates the intracellular protein stabilization of HIF-1α and dimerization of HIF-1α/HIF-1β. For example, when COMMD1 protein expression decreases, intracellular protein stabilization of HIF-1α increases, inducing HIF-1α activation [33]. Another study showed that the COMMD1 protein inhibited HIF-1 activation and HIF-1-DNA binding via the disruption of HIF-1α/HIF-1β dimerization [34]. In the present study, the decreased COMMD1 protein may have contributed to the activation of HIF-1α. As a result of this, the stimulation of HIF-1α activation by the reduction of the COMMD1 protein could have contributed to the formation of PE in COPD patients, by disrupting the action of the copper/lysyl oxidases and by modulating the elastolytic activity. PTEN suppresses HIF-1α expression by inhibiting the PI3K-Akt pathway [35]. It was reported that the depletion of the PTEN protein resulted in the Akt-induced hypoxic signaling [36]. Therefore, the induction of PTEN protein synthesis is believed to be useful for reducing the hypoxic signaling in cancer treatment. However, we detected a decrease in the levels of both PTEN and HIF-1α in the present study; this was not in agreement with the previous results of the inverse relationship between PTEN and HIF-1 signaling. In a study designed to understand the etiology of COPD, large-scale target gene screening was performed on 107 patients. The findings suggest that PTEN may be a prognostic risk factor for COPD patients and lead to lung cancer in these patients [37]. Yamada et al. reported reduced PTEN mRNA levels in bronchoscopic biopsy specimens of COPD patients who smoked cigarettes [38]. On the other hand, in patients with stomach cancer, a close relationship was found between the decreased PTEN expression and increased MMP-7 expression [39]. MMP-7 is a protein responsible for the degradation of the ECM proteins such as elastin, collagen, and fibronectin. Increased PTEN expression in U87MG glioblastoma cells has been shown to arrest the expression of MMP-9, an elastase [40]. This shows that the PTEN protein is effective in reducing the expressions of various elastases such as MMP-7 and MMP-9. Based on these findings, we can conclude that the reduced PTEN protein levels in the lung of COPD patients caused the destruction of the elastic fibers and formation of emphysematous lesions by inducing the expressions of elastases such as MMP-7 and MMP-9. A decreased PTEN protein levels in COPD patients may contribute to the development of PE in these patients.

[1] R.M. Tuder, S. McGrath, E. Neptune, The pathobiological mechanisms of emphysema models: what do they have in common? Pulm. Pharmacol. Ther. 16 (2003) 67–78. [2] A. Sharafkhaneh, N.A. Hanania, V. Kim, Pathogenesis of emphysema: from the bench to the bedside, Proc. Am. Thorac. Soc. 5 (2008) 475–477. [3] M.A. Antunes, P.R. Rocco, Elastase-induced pulmonary emphysema: insights from experimental models, An. Acad. Bras. Cienc. 83 (2011) 1385–1396. [4] H. Yanagisawa, E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, E.N. Olson, Fibulin-5 is an elastin-binding protein essential for elastic fiber development in vivo, Nature 415 (2002) 168–171. [5] D.P. Wendel, D.G. Taylor, K.H. Albertine, M.T. Keating, D.Y. Li, Impaired distal airway development in mice lacking elastin, Am. J. Respir. Cell Mol. Biol. 23 (2000) 320–326. [6] C.M. Kielty, Elastic fibers in health and disease, Expert. Rev. Mol. Med. 8 (2006) 1–23. [7] H. Ovet, F. Oztay, The copper chelator tetrathiomolybdate regressed bleomycininduced pulmonary fibrosis in mice, by reducing lysyl oxidase expressions, Biol. Trace Elem. Res. 162 (2014) 189–199. [8] B.L. O’Dell, K.H. Killburn, W.N. Mckenzie, R.J. Thurston, The lung of teh copperdeficient rat, Am. J. Pathol. 91 (1978) 413–424. [9] N.T. Soskel, S. Watanabe, L.B. Sandberg, Mechanisms of lung injury in the copperdeficient hamster model of emphysema, Chest 85 (1984) 70–72. [10] D.K. Grange, S.G. Kaler, G.M. Albers, J.A. Petterchak, C.M. Thorpe, D.E. DeMello, Severe bilateral panlobular emphysema and pulmonary arterial hypoplasta: unusual manifestations of menkes disease, Am. J. Med. Genet. A 139A (2005) 151–155. [11] K. Kida, T.M. Thurlbeck, The effect of beta-aminopropionitrile on the growing rat lung, Am. J. Pathol. 101 (1980) 693–710. [12] Y. Zhao, S. Gao, I.N. Chou, P. Toselli, P. Stone, W. Li, Inhibition of the expression of lysyl oxidase and its substrates in cadmium-resistant rat fetal lung fibroblasts, Toxicol. Sci. 90 (2006) 478–489. [13] X. Liu, Y. Zhao, J. Gao, J.B. Pawlyk, B. Starcher, J.A. Spencer, H. Yanagisawa, J. Zuo, T. Li, Elastic fiber homeostasis requires lysyl oxidase-like 1 protein, Nat. Genet. 36 (2004) 178–182. [14] G. Liguori, L.M. Pavone, L. Assisi, E. Langella, S. Tafuri, N. Mirabella, A. Costagliola, A. Vittoria, Expression of orexin B and its receptor 2 in rat testis, Gen. Comp. Endocrinol. 242 (2017) 66–73. [15] M.M. Bradford, A rapid and sensitive method for quanitation of microgram quantties of protein utilizing the princible of protein-dye bindig, Anal. Biochem. 72 (1976) 248–254. [16] W. Li, J. Zhou, L. Chen, Z. Lou, Y. Zhao, Lysyl oxidase: a critical intra- and extracellular target in the lung for cigarette smoke pathogenesis, Int. J. Environ. Res. Public Health 8 (2011) 161–184. [17] S. Raviv, K.A. Hawkins, M.M. DeCamp, R. Kalhan, Lung cancer in chronic obstructive pulmonary disease enhancing surgical options and outcomes, Am. J. Respir. Crit. Care Med. 183 (2011) 1138–1146. [18] C.A. Owen, Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease, Int. J. Chron. Obstruct. Pulmon. Dis. 3 (2008) 253–268. [19] A.F. Oflue, K.H. Ko, Effects of depletion of neutrophils or macrophages on development of cigarette smoke-induced emphysema, Am. J. Physiol. 277 (1999) 97–105. [20] H.M. Kagan, F. Ryvkin, Lysyl oxidase and lysyl oxidase-like enzymes, in: R.P. Mecham (Ed.), In the Extracellular Matrix: An Overview, Biology of Extracellular Matrix, Springer-Verlag, Berlin Heidelberg, 2011, pp. 303–335, , http://dx.doi.org/10.1007/978-3-642-1655-9_9 Chapter 9. [21] S. Gao, K. Chen, Y. Zhao, B. Richc, L. Chen, S.J. Li, P. Taselli, P. Stone, W. Li, Transcriptional and posttranscriptional inhibition of lysyl oxidase expression by cigarette smoke condensate in cultured rat fetal lung fibroblast, Toxicol. Sci. 87 (2005) 197–203. [22] L.J. Chen, Y. Zhao, S. Gao, I.N. Chou, P. Taselli, P. Stone, W. Li, Downregulation of lysyl oxidase and upregulation of cellular thiols in rat fetal lung fibroblasts treated with cigarette smoke condensate, Toxicol. Sci. 83 (2005) 372–379. [23] L. Thomassin, C.C. Werneck, T.J. Broekelmann, C. Gleyzal, I.K. Hornstra, R.P. Mecham, P. Sommer, The pro-regions of lysyl oxidase and lysyl oxidase-like 1 are required for deposition onto elastic fibers, J. Biol. Chem. 52 (2005) 42848–42855. [24] S. Mizuno, M. Yasuo, H.J. Bogaard, D. Kraskauskas, A. Alhussaini, J. Gomez-Arroya, D. Farkas, L. Ferbas, I.N.F. Voelke, Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation, PLoS One 7 (2012) e30678. [25] F.J. McDonald, COMMD1 and ion transport proteins: what is the COMMection? Focus on COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination, Am. J. Physiol. Cell Physiol. 305 (2013) 129–130. [26] W.I. Vonk, P. Bartuzi, P. Bie, N. Kloosterhuis, C.G. Wichers, R. Berger, S. Haywood, L.W. Klomp, C. Wijmenga, B. Van de Sluis, Liver-spesific COMMD1 knockout mice are susceptible to hepatic copper accumulation, Plos. One (2011) e29183. [27] K.G. Shyu, F.L. Hsu, M.F. Wang, B.M. Wang, S. Lin, Hypoxia-inducible factor 1 alpha regulates lung adenocarcinoma cell invasion, Exp. Cell Res. 313 (2007) 1181–1191. [28] M. Yasuo, S. Mizuno, D. Kraskauskas, H.J. Bogaard, R. Natarajan, C.D. Cool, M. Zamora, N.F. Voelkel, Hypoxia inducible factor-1α in human emphysema lung tissue, Eur. Respir. J. 37 (2011) 775–783. [29] D. Xiao Qian, G. Jian, The significance and expression on HIF-1α and VEGF in rats model with COPD and the chronic bronchitis, J. Prog. Mod. Biomed. 10 (2010)

Conflicts of interest None. Acknowledgements This work was supported by Scientific Research Projects Coordination Unit of Istanbul University with the project numbers 47811, 47792 and 26451. We sincerely thank to Dr. Anna Costagliola for making the English language edition of the text. 254

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391–396. [36] J.C. Soria, H.Y. Lee, J.I. Lee, L. Wang, J.P. Issa, B.L. Kemp, D.D. Liu, J.M. Kurie, L. Mao, F.R. Khuri, Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation, Clin. Cancer Res. 8 (2002) 1178–1184. [37] H.D. Hosgood III, I. Menashe, X. He, S. Chanock, Q. Lan, PTEN identified as important risk factor of chronic obstructive pulmonary disease, Respir. Med. 103 (2009) 1866–1870. [38] K. Yamada, K. Asai, Y. Ohara, Y. Sugiyama, A.U. Shirai, K. Sato, N. Yamamoto, G. Tamagaki, T.S. Watanabe, K. Kohnishi, Y. Tochino, M. Uji, H. Kanazawa, K. Hirata, The effect of smoking on phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) mRNA expression in human airway epithelial cells, Eur. Respir. J. 44 (2014) 3900. [39] T. Zheng, Z. Zhu, Z. Wang, R.J. Homer, B. Ma, R.J. Riese Jr., H.A. Chapman Jr., S.D. Shapiro, J.A. Elias, Inducible targeting of IL-13 to the adult lung cause matrix metalloproteinase and cathepsin dependent emphysema, J. Clin. Invest. 106 (2000) 1081–1093. [40] M.J. Park, M.S. Kim, I.C. Park, H.S. Kang, H. Yoo, S.H. Park, C.H. Rhee, S.I. Hong, S.H. Lee, PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation, Cancer. Res. 62 (2002) 6318–6322.

478–480. [30] H. Jiang, Y. Zhu, H. Xu, Y. Sun, Q. Li, Activation of hypoxia-inducible factor-1 alpha via nuclear factor-kappa B in rats with chronic obstructive pulmonary disease, Acta Biochim. Biophys. Sin. (Shanghai) 42 (2010) 483–488. [31] J.L. Berk, C.A. Hatch, S.M. Morris, P.J. Stone, R.H. Goldstein, Hypoxia suppresses elastin repair by rat lung fibroblast, Am. J. Physiol. Lung Cell Mol. Physiol. 289 (2005) 931–936. [32] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721–732. [33] B. Van de Sluis, P. Muller, K. Duran, A. Chen, A.J. Groot, L.W. Klomp, P.P. Liu, C. Wijmenga, Increased activity of hypoxia-inducible factor 1 is associated with early embryonic lethality in Commd1 null mice, Mol. Cell Biol. 27 (2007) 4142–4156. [34] B. Van de Sluis, X. Mao, Y. Zhai, P.J. Diest, M.H. Hofker, C. Wijmenga, L.W. Klomp, K.R. Cho, E.R. Fearon, M. Vooijs, E. Burstein, COMMD1 disrupts HIF-1alpha/beta dimerization and inhibits human tumor cell invasion, J. Clin. Invest. 120 (2010) 2119–2130. [35] W. Zundel, C. Schindler, D. Haas-Kogan, A. Koong, F. Kaper, E. Chen, A.R. Gottchalk, H.E. Ryan, R.S. Johnson, A.B. Jefferson, D. Stokoe, A.J. Giaccia, Loss of PTEN facilitates HIF-1-mediated gene expression, Genes Dev. 14 (2000)

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The copper dependent-lysyl oxidases contribute to the pathogenesis of pulmonary emphysema in chronic obstructive pulmonary disease patients.

Abnormalities in the elastic fiber biology are seen in pulmonary emphysema (PE). The copper-dependent lysyl oxidases regulate the production and accum...
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