SB525334

Transforming growth factor b1-induced collagen production in myofibroblasts is mediated by reactive oxygen species derived from NADPH oxidase 4

Yuma Hotta, Kazuhiko Uchiyama*, Tomohisa Takagi, Saori Kashiwagi, Takahiro Nakano, Rieko Mukai, Yuki Toyokawa, Tomoyo Yasuda, Tomohiro Ueda, Yosuke Suyama, Takaaki Murakami, Makoto Tanaka, Atsushi Majima, Toshifumi Doi, Yasuko Hirai, Katsura Mizushima, Mayuko Morita, Yasuki Higashimura, Ken Inoue, Akifumi Fukui

A B S T R A C T

Intestinal fibrosis with stricture formation is a severe complication of Crohn’s disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-b1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-b1. Subsequently, we investigated the role of NOX4 in TGF-b1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneio- donium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-b1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-b1 receptor and NOX significantly reduced TGF-b1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients.

Keywords:
Crohn’s disease NADPH oxidase 4
Reactive oxygen species Fibrosis
Inflammatory bowel disease Collagen

1. Introduction

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis, is a chronic relapsing-remitting in- flammatory disorder of the gastrointestinal tract. Its pathogenesis remains unclear and primary treatment has not been established. CD is characterized as chronic, relapsing, and refractory inflam- mation affecting all intestines, and its etiology is still unknown [1]. Intestinal fibrosis with stricture formation, caused by chronic in- testinal inflammation, is a severe complication of IBD and espe- cially affects CD patients [2]. Early stage CD may demonstrate inflammatory characteristics, but 60e70% of patients exhibit stricture or perforation 10 years after diagnosis [3] and need sur- gical treatment [4]. During the current decade, rapid progress has been made with respect to anti-inflammatory therapies, including biologics; however, the development of treatments for intestinal fibrosis remains insufficient [5] [6]. Therefore, it is essential to elucidate the mechanism underlying intestinal fibrosis for this process to be appropriately controlled in CD patients.
As in other organs, the mechanism that induces fibrosis in the gut is believed to depend on the tissue repair reaction. Under normal circumstances, this reaction restores the integrity of tissues following inflammatory damage, and involves a controlled response mediated by mesenchymal cells and the extracellular matrix (ECM) [7] [8]. On the other hand, an excessive fibrosis response may occur, defined as the deposition of collagen-rich ECM produced by mesenchymal cells, including fibroblasts, myofibro- blasts, and smooth muscle cells [9]. NADPH oxidase (NOX) 4, a member of the NOX family of enzymes, which catalyze the reduc- tion of O2 to reactive oxygen species (ROS) [10], has recently been reported to play an important role in fibrogenesis in the lung and other organs via ROS production [11e14]. However, it is not known if a similar mechanism exists in intestinal fibrogenesis. In the pre- sent study, we investigated the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-b1.

2. Materials and methods

2.1. Cell culture

LmcMF cells, a mouse intestinal subepithelial myofibroblast line provided by Yamaguchi University (Japan), were cultured in Dul- becco’s modified Eagle’s medium (DMEM) – high glucose (4.5 g/l glucose) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) at 37 ◦C in 5% CO2 and 95% air. Before stimulation with TGF-b1, 1.0 105 cells were plated in a 3.5-cm dish and starved for 12 h in DMEM lacking FBS.

2.2. Collagen assay

As a model of fibrosis, we stimulated LmcMF cells with 1e50 ng/ ml recombinant mouse TGF-b1 (R&D Systems, Minneapolis, MN, USA) and harvested them 24 h later. The cells were then fixed in 95% ethanol and incubated with a Sirius Red/Fast Green Collagen Staining Kit dye solution (Chondrex, Redmond, WA, USA) for 30 min. After removing the dye solution, dye extraction solution was added and subsequently collected, before being subjected to measurements of optical density at 540 nm and 605 nm with a spectrophotometer.

2.3. Immunofluorescence staining

For immunofluorescence staining of collagen, LmcMF cells were cultured on a 35-mm m-Dish (ibidi GmbH, Munich, Germany). The cells were subsequently fixed in 4% paraformaldehyde in PBS and incubated with PBS containing 0.1% Triton X-100. After incubation with Protein Block, Serum-Free (Dako Corp., Carpinteria, CA, USA), the cells were exposed to a mouse monoclonal anti-collagen type I antibody (Chemicon, Temecula, CA, USA), followed by an Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (Invi- trogen, Carlsbad, CA, USA). Finally, they were stained with Hoechst 33342 to label nuclear chromatin, and inspected using a confocal laser scanning microscope.

2.4. Inhibition of TGF-b1 signaling, NOX family members, and NOX4 in LmcMF cells

To investigate the role of NOX4 in collagen production by TGF- b1-stimulated LmcMF cells, we used SB525334 (an SMAD2/3 in- hibitor; Sigma, St. Louis, MO, USA), diphenyleneiodonium (DPI; an NOX inhibitor; TOCRIS, Bristol, UK), and small interfering RNA (siRNA) targeting Nox4 (Invitrogen).

2.5. Inhibition of NOX4

To silence Nox4 in LmcMF cells, the cells were treated with a commercially available Nox4-silencing oligonucleotide (siRNA #AM16708; Invitrogen) or Stealth oligonucleotide (1299001). In all cases, silencing oligonucleotides were complexed with Lipofect- amine RNAiMAX (Invitrogen) in serum-free Opti-MEM (Invi- trogen). LmcMF cells were treated with the complexed oligonucleotides 1 day after plating (at 1 × 104 cells/cm2), at a final concentration of 20 nM silencing oligonucleotides and 0.1 ml Lip- ofectamine RNAiMAX per cm2. The oligonucleotide and Lipofect- amine RNAiMAX were individually diluted in Opti-MEM, before being mixed and allowed to complex for 20 min at room temper- ature. The cell culture medium was then removed and replaced with Opti-MEM and the complexed oligonucleotides. After 60 min, complete medium was added and the cells were returned to the incubator, in which they were kept overnight. Silencing of Nox4 was monitored by examining NOX4 expression using quantitative real- time PCR (qPCR) and western blotting.

2.6. ROS detection assay

LmcMF cells were treated with TGF-b1 (10 ng/ml) alone or with DPI or Nox4 siRNA for 6 h at 37 ◦C. ROS were detected by measuring the fluorescence associated with 5-(and —6)-carboxy-20,70- dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Invitrogen) oxidation due to H2O2 production. Briefly, cells were incubated with 10 mM CM-H2DCFDA for 15 min at 37 ◦C, washed with PBS three times, and fixed with 4% paraformaldehyde. Samples were then mounted for imaging using fluorescence microscopy. Images were obtained following 1 min of exposure to the microscope’s light source at excitation and emission wavelengths of 492 nm and 520 nm, respectively.

2.7. RNA extraction and qPCR

Expression of transcripts encoding NOX family members (NOX1, 2, 3, and 4, and DUOX1 and 2) in LmcMF cells was determined using qPCR. Total RNA was isolated from the homogenized cells with the acid guanidinium phenol-chloroform method using Isogen (Nippon Gene, Tokyo, Japan), before being reverse transcribed. The resultant ditions comprised denaturation at 95 ◦C for 15 s, primer annealing at 60 ◦C for 1 min, and subsequent melting curve analysis, in which the temperature was increased from 60 to 95 ◦C.

2.8. Statistical analysis

The results of this study are expressed as means ± standard er- rors of the means. Overall differences between the groups were determined by one-way analysis of variance (ANOVA). When the results of one-way ANOVA were significant, the differences be- tween individual groups were analyzed using Bonferroni’s multiple comparisons test. P-values <0.05 were considered to indicate sta- tistically significant differences. All analyses were performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA) for Macintosh-based computers. 3. Results 3.1. Induction of collagen production in LmcMF cells by TGF-b1 In LmcMF cells, TGF-b1 stimulation enhanced Sirius red stain- ing, indicative of intracellular collagen levels (Fig. 1A). Moreover, collagen production was increased by TGF-b1 in a dose-dependent manner (Fig. 1B). This TGF-b1-induced increase in collagen in LmcMF cells was also observed using immunofluorescence (Fig.1C). Enhancement of collagen production by TGF-b1 stimulation was negated by treatment with SB525334 (Fig. 1D). 3.2. Induction of Nox4 expression by TGF-b1 We next investigated the expression of Nox gene family mem- bers in LmcMF cells following TGF-b1 stimulation. Whereas expression of Nox4 was significantly induced by TGF-b1 (Fig. 2A), that of other members of the Nox gene family was not affected at the mRNA level. Furthermore, Nox4 expression increased in a TGF- b1 concentration-dependent manner (Fig. 2B). 3.3. Induction of ROS production by TGF-b1 To investigate whether fibrotic reactions involve oxidative stress, we examined ROS production in LmcMF cells after TGF-b1 treatment. ROS production was enhanced 6 h after TGF-b1 stimu- lation, and was suppressed by the broad-spectrum NOX inhibitor DPI (Fig. 3A and B). A significant decrease in NOX4 mRNA and protein expression in TGF-b1-stimulated LmcMF cells was noted following Nox4 siRNA transfection (Fig. 3C), accompanied by a significant reduction in TGF-b1-induced ROS production (Fig. 3D). 3.4. Induction of collagen production by TGF-b1 through NOX4 TGF-b1-induced collagen production in LmcMF cells was significantly decreased by DPI treatment (Fig. 4A and B), as well as by administration of Nox4 siRNA (Fig. 4C and D). 4. Discussion In the present study, we investigated the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with TGF-b1. TGF-b1 induced NOX4 expression, and ROS production increased in proportion to NOX4 levels. Moreover, TGF-b1-derived collagen production in myofibroblasts was dependent on NOX4 activation, as it was inhibited by administration of Nox4 siRNA or DPI. To our knowledge, this study is the first to demonstrate the role of NOX4 in collagen production by intestinal myofibroblasts. Fibrosis is a system enabling the restoration of compromised tissue and ineffective epithelial repair in multiple organ systems, and its incidence increases with age [15]. In mammalian tissues, fibrosis is a well-established mechanism in which mesenchymal cells adopt a tissue barrier function. However, persistent mesen- chymal activation results in uncontrolled and progressive fibrosis. Multiple soluble factors are produced during the inflammatory response and are related to fibrogenesis in several organs. Of these factors, TGF-b1 is one of the most important mediators of fibro- genesis, activating mesenchymal cells to induce collagen produc- tion [16] [17]. NOX4, a member of the NOX family of enzymes, which catalyze the reduction of O2 to ROS [10], has also been re- ported to have an important function in lung fibrogenesis via the activation of mesenchymal cells [11,18]. However, the relationship between TGF-b1 and NOX4 in enhancing fibrogenesis has not been described. In the present study, we demonstrated the role of TGF- b1 in activating NOX4 in colonic myofibroblasts, resulting in in- testinal fibrogenesis. TGF-b is a pleiotropic cytokine that regulates various biological processes in multiple tissues in an autocrine and paracrine manner [19]. It also modulates the tumor microenvironment, principally by contributing to the conversion of fibroblasts to myofibroblasts [20]. Specifically, the compressive forces that develop inside a tumor due to its growth in the confined space of the host tissue can facilitate the conversion of fibroblasts to proto-myofibroblasts. Subse- quently, TGF-b increases levels of collagens I and III and fibronectin, which promote cellular adhesion to extracellular fibers, and thus, enhances the communication of mechanical signals between the tumor ECM and fibroblasts [21]. TGF-b also has an important role in CD, especially with respect to collagen expression and intestinal fibrosis. Increased TGF-b1 and TGF-b1-dependent collagen I pro- duction in intestinal mesenchymal cells result in fibrosis in patients with Montreal B2 fibrostenotic CD [22]. The pathway by which collagen production is induced by TGF- b1 in conjunctival fibroblasts has recently been elucidated and has been shown to involve NOX4-derived H2O2 [23]. In this work, the authors demonstrated that suppression of Nox4 gene expression by Adv-Nox4i completely attenuated TGF-b1-induced H2O2 release and collagen production. In the present study, we also used Nox4 siRNA to reduce expression of this gene, which downregulated TGF- b1-mediated collagen production. As we obtained these results using intestinal myofibroblasts, we believe that this pathway is related to intestinal fibrosis in CD pa- tients. This is the first study to implicate a specific NOX isoform in intestinal fibrogenesis, and these results identify NOX4 as a po- tential therapeutic target in recalcitrant fibrotic disorders of CD patients. References [1] R.B. Sartor, Mechanisms of disease: pathogenesis of Crohn's disease and ul- cerative colitis, Nat. Clin. Pract. Gastroenterol. Hepatol. 3 (2006) 390e407. [2] E. Louis, A. Collard, A.F. Oger, E. Degroote, F.A. Aboul Nasr El Yafi, J. Belaiche, Behaviour of Crohn's disease according to the Vienna classification: changing pattern over the course of the disease, Gut 49 (2001) 777e782. [3] J. Cosnes, S. Cattan, A. Blain, L. Beaugerie, F. Carbonnel, R. Parc, J.P. Gendre, Long-term evolution of disease behavior of Crohn's disease, Inflamm. Bowel Dis. 8 (2002) 244e250. [4] P. Munkholm, E. Langholz, M. Davidsen, V. Binder, Intestinal cancer risk and mortality in patients with Crohn's disease, Gastroenterology 105 (1993) 1716e1723. [5] F. Rieder, C. Fiocchi, G. Rogler, Mechanisms, management, and treatment of fibrosis in patients with inflammatory bowel diseases, Gastroenterology 152 (2017) 340e350, e6. [6] J. Cosnes, A. Bourrier, I. Nion-Larmurier, H. Sokol, L. Beaugerie, P. Seksik, Factors affecting outcomes in Crohn's disease over 15 years, Gut 61 (2012) 1140e1145. [7] G. Latella, J. Di Gregorio, V. Flati, F. Rieder, I.C. Lawrance, Mechanisms of initiation and progression of intestinal fibrosis in IBD, Scand. J. Gastroenterol. 50 (2015) 53e65. [8] F. Rieder, T. Karrasch, S. Ben-Horin, A. Schirbel, R. Ehehalt, J. Wehkamp, C. de Haar, D. Velin, G. Latella, F. Scaldaferri, G. Rogler, P. Higgins, M. Sans, Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing, J. Crohns Colitis 6 (2012) 373e385. [9] F. Rieder, E.M. Zimmermann, F.H. Remzi, W.J. Sandborn, Crohn's disease complicated by strictures: a systematic review, Gut 62 (2013) 1072e1084. [10] Y. Nisimoto, B.A. Diebold, D. Cosentino-Gomes, J.D. Lambeth, Nox4: a hydrogen peroxide-generating oxygen sensor, Biochemistry 53 (2014) 5111e5120. [11] L. Hecker, R. Vittal, T. Jones, R. Jagirdar, T.R. Luckhardt, J.C. Horowitz, S. Pennathur, F.J. Martinez, V.J. Thannickal, NADPH oxidase-4 mediates myo- fibroblast activation SB525334 and fibrogenic responses to lung injury, Nat. Med. 15 (2009) 1077e1081.
[12] C.D. Bondi, N. Manickam, D.Y. Lee, K. Block, Y. Gorin, H.E. Abboud, J.L. Barnes, NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofi- broblasts, J. Am. Soc. Nephrol. 21 (2010) 93e102.
[13] Q. Wang, X. Sui, R. Chen, P. Ma, T. Ding, D. Sui, P. Yang, Anti-fibrotic actions of Ghrelin by inhibition of the NADPH oxidase-ROS signaling pathway, Clin. Exp. Pharmacol. Physiol. (2018), https://doi.org/10.1111/1440-1681.12948.
[14] M. Pan, Z. Zheng, Y. Chen, N. Sun, B. Zheng, Q. Yang, Y. Zhang, X. Li, Y. Meng, Angiotensin-(1-7) attenuated cigarette smoking-related pulmonary fibrosis via improving the impaired autophagy caused by NOX4-dependent ROS, Am. J. Respir. Cell Mol. Biol. (2018), https://doi.org/10.1165/rcmb.2017-0284OC.
[15] G. Raghu, D. Weycker, J. Edelsberg, W.Z. Bradford, G. Oster, Incidence and prevalence of idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 174 (2006) 810e816.
[16] M.C. Manresa, M.M. Tambuwala, P. Radhakrishnan, J.M. Harnoss, E. Brown, M.A. Cavadas, C.E. Keogh, A. Cheong, K.E. Barrett, E.P. Cummins, M. Schneider, C.T. Taylor, Hydroxylase inhibition regulates inflammation-induced intestinal fibrosis through the suppression of ERK-mediated TGF-beta1 signaling, Am. J. Physiol. Gastrointest. Liver Physiol. 311 (2016) G1076eG1090.
[17] Y. Wei, T.J. Kim, D.H. Peng, D. Duan, D.L. Gibbons, M. Yamauchi, J.R. Jackson, C.J. Le Saux, C. Calhoun, J. Peters, R. Derynck, B.J. Backes, H.A. Chapman, Fibroblast-specific inhibition of TGF-beta1 signaling attenuates lung and tumor fibrosis, J. Clin. Invest. 127 (2017) 3675e3688.
[18] N. Amara, D. Goven, F. Prost, R. Muloway, B. Crestani, J. Boczkowski, NOX4/ NADPH oxidase expression is increased in pulmonary fibroblasts from pa- tients with idiopathic pulmonary fibrosis and mediates TGFbeta1-induced fibroblast differentiation into myofibroblasts, Thorax 65 (2010) 733e738.
[19] P. Papageorgis, T. Stylianopoulos, Role of TGFbeta in regulation of the tumor microenvironment and drug delivery (review), Int. J. Oncol. 46 (2015) 933e943.
[20] P.J. Wipff, D.B. Rifkin, J.J. Meister, B. Hinz, Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix, J. Cell Biol. 179 (2007) 1311e1323.
[21] G.S. Karagiannis, T. Poutahidis, S.E. Erdman, R. Kirsch, R.H. Riddell, E.P. Diamandis, Cancer-associated fibroblasts drive the progression of metastasis through both paracrine and mechanical pressure on cancer tissue, Mol. Canc. Res. 10 (2012) 1403e1418.
[22] C. Li, A. Iness, J. Yoon, J.R. Grider, K.S. Murthy, J.M. Kellum, J.F. Kuemmerle, Noncanonical STAT3 activation regulates excess TGF-beta1 and collagen I expression in muscle of stricturing Crohn’s disease, J. Immunol. 194 (2015) 3422e3431.
[23] K.D. Brown, M.H. Shah, G.S. Liu, E.C. Chan, J.G. Crowston, H.M. Peshavariya, Transforming growth factor beta1-induced NADPH oxidase-4 expression and fibrotic response in conjunctival fibroblasts, Invest. Ophthalmol. Vis. Sci. 58 (2017) 3011e3017.