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Basic research

Neutrophil elastase stimulates MUC5AC expression in human biliary epithelial cells: a possible pathway of PKC/Nox/ROS

Yu Tian
,
Min Li
,
Shuodong Wu
,
Duoliang Wang
,
Ben Sun
,
Junqing Xie
,
Hong Wang

Arch Med Sci 2017; 13, 3: 677–685
Online publish date: 2017/04/20
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Introduction

Hepatolithiasis is a prevalent disease in Asian regions, particularly in China [1]. Despite the therapeutic effects of surgical and nonsurgical procedures [2], stone recurrence is observed in most hepatolithiasis patients, resulting in serious complications, such as cholangitis, biliary stenosis, biliary fibrosis, and even cholangiocarcinoma, which always necessitates reoperation and significantly limits long-term survival [3]. Therefore, investigating the mechanism underlying stone formation and identifying potential targets to prevent stone recurrence remain a priority.
Although hepatolithiasis is a multifactorial disease, bacterial infection and bile flow retardation form a vicious cycle which promotes stone formation and recurrence, and mucin overexpression plays important roles in this process [4]. Bile is normally sterile, while bacteria, the most common of which are Escherichia coli and Klebsiella, are frequently found in the bile of patients with hepatolithiasis [5, 6]. Mucin distributed in epithelial mucosae has tissue and cell-specific expression, and functions to protect the epithelial surface and lubricate. To date, more than 20 types of mucin have been reported, six of which have been found in the biliary tract. During hepatolithiasis development, bacterial infection increases mucin 5AC (MUC5AC) expression, which is marginally present in normal bile, resulting in increased mucus viscosity and bile flow retardation, which promotes stone formation and recurrence [7]. Therefore, investigating the regulatory mechanism of MUC5AC overexpression and finding potential targets to inhibit this process might be a strategy for preventing stone recurrence and will improve the long-term survival in cases of hepatolithiasis.
Polymorphonuclear neutrophil (PMN) infiltration is a common phenomenon in the process of bacterial infection. During the process, PMNs release several mediators, and one, neutrophil elastase (NE; EC 3.4.21.37), is a serine protease that is thought an “end-effect” factor of inflammatory pathologies [8]. Neutrophil elastase is considered a significant factor in hepatic ischemia-reperfusion injury (IRI), nonalcoholic fatty liver disease (NAFLD) and many other diseases [9, 10]. Several studies have shown that NE can induce the overexpression of MUC5AC in human airway epithelial cells [11–16]. These studies showed that the PKC-Nox pathway, platelet activating factor (PAF), interleukin (IL), tumor necrosis factor  (TNF-), and prostaglandin can increase expression of MUC5AC [10, 17, 18]. Studies on airway epithelial cells also showed that NE could activate protein kinase C (PKC) as well as downstream signaling molecules including dual oxidase 1 (Duox1), reactive oxygen species (ROS) and other factors that increase MUC5AC expression [12–14, 16].
As the biliary tract and the respiratory tract harbor similar embryological origins, our current study was undertaken to elucidate whether there exists a similar pathway of NE-induced MUC5AC expression in human intrahepatic biliary epithelial cells (HIBEpiC).

Material and methods

Basic cell culture and passage

Human intrahepatic biliary epithelial cells (HIBEpiC) were purchased from Sciencell (No. 5100) and were cultured in epithelial cell medium (consisting of 500 ml of basal medium, 10 ml of fetal bovine serum, 5 ml of epithelial cell growth supplement, and 5 ml of penicillin/streptomycin solution, all purchased from Sciencell) in a humidified atmosphere of 5% CO2 at 37°C. The medium was refreshed every 2 days, and cells were passaged at a ratio of 1 : 2–1 : 3 every 3–5 days according to the cell condition.

Cell treatment with neutrophil elastase (NE) and inhibitors

HIBEpiC cells were treated with 50 ng/ml NE (Sigma). For inhibitor studies, cells were pretreated with inhibitors for 30 min before exposing the cells to NE. Concentrations of DMTU (inhibitor of ROS), apocynin (inhibitor of Nox) and bisindolylmaleimide I (inhibitor of PKC) were 25 mM, 1 mM and 5 µM respectively according to our previous work [16].

H2O2 measurement

Cells were treated with NE (50 ng/ml, 100 ng/ml, 1 µg/ml or 10 µg/ml) for 2 h, with or without selective inhibitors pretreated for 30 min, and H2O2 production in the cell supernatants was measured by using the Hydrogen Peroxide Assay kit (Invitrogen) according to the manufacturer’s instructions.

Real-time polymerase chain reaction (PCR)

Total RNA was isolated from cultured cells using Trizol reagent (Takara) following the manufacturer’s instructions, and RNA was quantified by spectrophotometry. cDNA was prepared using the PrimeScript RT reagent kit with gDNA Eraser (Takara). Real-time PCR was performed using SYBR Green Premix Ex Taq (Takara). Sequences for the primers used were as follows: MUC5AC: (forward) 5-TGGACACCAAATACGCCAACAAG-3, (reverse) 5-CTGCTCACAGATGCCAAAGCC-3, Duox1: (forward) 5-ATCGCCACCTACCAGAACATC-3, (reverse) 5- GGAGACACTTGAGTTCCGATTG-3, -actin: (forward) 5-CTTAGTTGCGTTACACCCTTTCTTG-3, (reverse) 5- CTGTCACCTTCACCGTTCCAGTTT-3.
For real-time PCR, the PCR mixture was denatured at 95°C for 10 s, annealed at 60°C for 20 s and then extended at 72°C for 30 s. This cycle was repeated for a total of 40 cycles. The fold change in expression of MUC5AC mRNA relative to -actin was calculated based on the threshold cycle (Ct) values.

Immunohistochemical staining of MUC5AC protein

Cells were fixed in 4% paraformaldehyde, incubated with 0.5% TritonX-100 for 10 min, peroxi­dase-blocked in 3% H2O2 for 15 min to quench endogenous peroxidases, blocked by normal goat serum for 15 min, and then incubated with MUC5AC antibody (1 : 200 Santa Cruz) overnight at 4°C. The next morning, after removing excess antibody by washing with PBS, cells were incubated with biotinylated goat anti-mouse immunoglobulin G (1 : 200 dilution) for 30 min at room temperature, and then the cells were incubated for another 30 min in horseradish peroxidase (HRP, Beyotime). Cells were developed for 3 min with diaminobenzidine as the chromogen substrate, and the cells were counterstained with hematoxylin (Solarbio). Finally, the cells were observed by microscopy.

Western blot

Protein concentrations were measured by using the bicinchoninic acid protein assay. Protein samples were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). Non-specific binding to the membrane was blocked for 1 h at room temperature with 5% fat-free milk in TBST, and then the membranes were incubated with 1 : 400 MUC5AC primary antibody (Santa Cruz, USA) at 4°C overnight. Then, the membrane was washed 4 times with TBST and then incubated with a 1 : 5000 dilution of the appropriate secondary antibody at room temperature for 45 min. After the membrane was washed twice with TBST, membrane-bound antibody was visualized by using an enhanced chemiluminescent kit (Millipore) according to the manufacturer’s instructions.

Transforming growth factor α (TGF-α) ELISA

Cells were treated with 50 ng/ml NE for 4 h with or without pretreatment of inhibitors of ROS, Nox or PKC. The TGF- level in the cell supernatants was measured using the ELISA kit (R&D) according to the manufacturer’s instructions.

Statistical analysis

Values are given as the mean ± SD. Differences between multiple groups were compared using one-way analysis of variance (ANOVA). When statistical significance was identified based on ANOVA, the Student-Newman-Keuls test was used for multiple comparisons. P-values < 0.05 were regarded as statistically significant.

Results

NE-induced H2O2 production

Cells were treated with different concentrations of NE (0, 50 ng/ml, 100 ng/ml, 1 µg/ml, 10 µg/ml) for 2 h to determine the optimal dose of NE. H2O2 production was 0.13 ±0.04, 1.46 ±0.04, 1.52 ±0.08, 1.68 ±0.04 and 1.72 ±0.08 µmol/l respectively. P < 0.001 for each group compared with the appropriate control. Moreover, 50 ng/ml NE showed statistically significant induction of H2O2 (Figure 1).

ROS are necessary for NE-induced MUC5AC expression

To determine whether ROS were involved in NE-induced MUC5AC expression, we assessed the effect of altering ROS levels in HIBEpiC cells. DMTU (25 nM), an ROS scavenger, attenuated NE-induced MUC5AC expression at the mRNA level based on real-time PCR as shown in Figure 2 A (1.00 ±0.03, 3.27 ±0.17 and 1.90 ±0.05, p < 0.01, expressed in 2–Ct, respectively). It was found that MUC5AC protein increased at 6 h and peaked at 24 h in airway epithelial cells [19]. Therefore, we determined MUC5AC protein expression by western blot analysis (Figures 2 B, C) and immunohistochemistry (Figures 2 D, E) after NE stimulation for 24 h. Figure 2 C shows grey analysis for western blot, and values were 1.00, 2.25 ±0.08, 1.62 ±0.03 respectively, p < 0.001 for each group compared with the appropriate control. Figure 2 E shows mean density for MUC5AC, and values were 0.29556 ±0.000573, 0.30828 ±0.0024015 and 0.29898 ±0.000968, p < 0.01 for each group compared with the control. Taken together, these data indicate that ROS are involved in NE-induced MUC5AC expression in HIBEpiC cells.

PKC and NADPH oxidase play important roles in NE-induced upregulation of MUC5AC

As H2O2 production is regulated by NADPH oxidase (Nox), and Nox can be activated by PKC to generate ROS [20], we hypothesized that Nox and PKC may be involved in NE-induced MUC5AC expression. Apocynin, a Nox inhibitor, and bisindolylmaleimide I, a PKC inhibitor, were used respectively to determine the involvement of Nox and PKC in NE-mediated MUC5AC expression. Cells were treated with different doses of NE (0, 50 ng/ml, 100 ng/ml, 1 µg/ml, 10 µg/ml) for 2 h, while two of the four groups of cells were pretreated with inhibitors respectively for 30 min. We found that both apocynin and bisindolylmaleimide I inhibit NE-induced ROS generation, as shown in Figure 3 A. H2O2 production in the normal group was 0.13 ±0.04 µmol/l, and in the “NE” group was 1.46 ±0.04, 1.52 ±0.08, 1.68 ±0.04 and 1.72 ±0.08 µmol/l respectively as the concentration of NE increased. H2O2 production in the “NE + apocynin” group was 1.06 ±0.08, 1.13 ±0.04, 1.26 ±0.10 and 1.35 ±0.10 µmol/l and in the “NE + bisindolylmaleimide I” group was 1.19 ±0.04, 1.21 ±0.08, 1.37 ±0.07 and 1.43 ±0.07 µmol/l respectively as the concentration of NE increased (p < 0.05 for each group compared with the control group). Furthermore, both agents blocked NE-induced MUC5AC expression at the mRNA level (Figure 3 B); it was 1.00 ±0.03, 3.27 ±0.17, 2.00 ±0.04 and 2.05 ±0.10, p < 0.01, expressed in 2–Ct, respectively. Furthermore, upregulation of MUC5AC protein by NE was inhibited by apocynin and bisindolylmaleimide I treatment (Figures 3 C–F). Figure 3 D shows grey analysis for Western blot, and values were 1.00, 2.25 ±0.08, 1.63 ±0.01 and 1.47 ±0.06, p < 0.001 for each group compared with the control. Figure 3 F shows mean density for MUC5AC (values were 0.29556 ±0.000573, 0.30828 ±0.0024015, 0.30692 ±0.0024974 and 0.30508 ±0.0034838, p < 0.001 for each group compared with the control). Thus, we believe that PKC and Nox generate ROS in response to NE stimulation, which results in the upregulation of MUC5AC expression.

PKC/Nox/ROS were involved in NE-induced TGF-α release

To determine whether PKC/Nox/ROS were involved in NE-induced TGF- release in HIBEpiC, we assessed the effect on TGF- release of altering PKC, Nox and ROS with bisindolylmaleimide, apocynin and DMTU respectively, and determined TGF- level by ELISA (Figure 4). These data (1.45 ±0.35, 4.58 ±0.35, 2.37 ±1.40, 2.72 ±1.40 and 3.07 ±0.88 pg/ml, p < 0.05 for each group compared with the control) indicate that NE induced TGF- release in HIBEpiC, and PKC/Nox/ROS were involved in this process.

Discussion

Inflammation is a pathophysiological reaction of the host to protect itself from pathogens. This complex and dynamic process is characterized by an innate immune response, which involves coordinated expression of inflammatory cytokines and implication of various cell types particularly immune cells aimed at clearing the pathogenic agent. In the setting of biliary bacterial infections (e.g. E. coli or Klebsiella), the host innate immune response is characterized by the initial recognition of invading microbes by the host via Toll-like receptors (TLRs) or other pattern recognition molecules [21]. Subsequently, this results in the production of an array of inflammatory mediators including early responsive cytokines. Another hallmark of innate host biliary defense, especially when the first lines of defense – the epithelial barrier and resident macrophages – are breached, is the massive recruitment of polymorphonuclear neutrophils (PMN) to the infected site. The PMNs are efficient phagocytes whose main function upon activation is thought to be the clearance of infecting bacteria. To do so, these cells are equipped with a myriad of antimicrobial molecules grouped into oxidative and nonoxidative systems [22, 23]. The NE, the PMN-specific serine protease, has been identified as a key antimicrobial enzyme [24]. As a cationic glycoprotein, it is stored in a readily active form in PMN primary granules at concentrations exceeding the millimolar range, making it a major component of PMN. Our previous studies have found that exogenous LPS could stimulate HIBEpiC MUC5AC expression [16, 25]. The NE is a 30 kD neutral serine protease, stored in an active form in the azurophilic granules of neutrophils, and can be released when neutrophils are exposed to LPS. Whether NE could also induce MUC5AC expression remains unknown.
Reactive oxygen species (ROS) are generated from biological aerobic metabolism. They play a physiological role in cells, as well as being risk factors for several diseases [26–28]. Fischer et al. [12] found that ROS mediated NE-induced MUC5AC gene expression in A549 cells. In this research, we found that NE induced H2O2 production in a dose-dependent manner, subsequently upregulating MUC5AC gene and protein expression. 30-min preincubation of cells with DMTU, a ROS scavenger, could inhibit this upregulation. Thus, we thought that ROS may participate in NE-induced MUC5AC expression in HIBEpiC. In studies in NCI-H292 airway epithelial cells, it has been reported that NE-induced ROS production activated tumor necrosis factor--converting enzyme (TACE), resulting in conversion of pro-TGF- into soluble TGF-, and epidermal growth factor receptor (EGFR) phosphorylation [14, 20]. Moreover, the EGFR pathway has been thought to be a convergent pathway activated by various stimuli which induce mucin gene expression and synthesis [29]. In our experiment, we also found that NE could induce TGF- release and DMTU inhibits TGF- production (Figure 4), suggesting that TGF-, produced by activated ROS, may take part in NE-induced MUC5AC expression.
It has been reported that ROS were generated by Nox of phagocytes (Phox), and the catalytic core of Nox was the six-transmembrane glycoprotein p91phox. Duox1 is a homologue of p91phox, and was first reported in airway epithelial cells [13]. In our experiment, we found that using apocynin to block Nox, the increased H2O2, TGF- as well as MUC5AC gene and protein expression all decreased (Figures 3, 4). Thus, we believed that Nox might active ROS that induced MUC5AC expression.
PKC activation has been implicated in mucin secretion in human epithelial cells [30]. PKC exists as isoforms  and , and it has been found that PKC might take part in the NE-induced MUC5AC expression in airway epithelial cells. The activated PKC caused p47phox and p67phox translocated from cytosol to plasma membrane to form the complete enzyme Nox with gp91phox [13, 31]. Then Nox is activated, and consequently other downstream signaling molecules. In this experiment, we found that using a PKC inhibitor, bisindolylmaleimide I, could attenuate the increased H2O2, TGF- and MUC5AC gene and protein expression stimulated by NE (Figures 3, 4). So it seemed that PKC also participates in NE-induced MUC5AC expression. NE is a native ligand for protease-activated receptors (PARs) and Ca2+ plays an important role in regulating the final steps of exocytosis in goblet and other secretory cells. Zhou et al. [32] found NE acting on PAR2, increasing the cytosolic Ca2+ concentration, and subsequently activating PKC. Whether NE has a similar pathway in activating PKC and whether other factors such as annexin II (ANXII) and ezrin [33, 34], which were reported to regulate NE-induced MUC5AC expression in airway epithelial cells, have a similar effect in HIBEpiC requires more intensive research.
Based on our results, we also found that all of the three inhibitors could attenuate the upregulation, while none of them could attenuate the upregulation completely, and it seems that DMTU was more “effective”. We suppose there might exist other pathways or cytokines that participate in this process, such as PG2, TNF-, IL or something else, as in other epithelial cells.
There were some weaknesses in our experiment. First, when we detected H2O2 production, we did not collect cells that were treated without NE at different time points. Moreover, we did not use the TUNEL assay or LDH assay or another method to confirm that there had not been any cell death in our experimental set-up.
In conclusion, we have shown that NE could induce H2O2 production in a dose-dependent manner. Moreover, using an ROS inhibitor could reduce NE-induced MUC5AC expression. We then found that using a Nox inhibitor could reduce NE-induced MUC5AC expression. As Nox is activated by PKC, we detected that the PKC inhibitor also has the ability to reduce MUC5AC expression. All considered, we concluded that NE-induced ROS participated in the upregulation of MUC5AC production, and, moreover, PKC and Nox have a role in NE-challenged human biliary epithelial cell MUC5AC expression.

Acknowledgments

Yu Tiana and Min Li contributed equally.
This work was supported in part by grants from the National Natural Science Foundation of China (No. 81070365).

Conflict of interest

The authors declare no conflict of interest.

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