Activation of ERK1/2-mTORC1-NOX4 mediates TGF-β1-induced epithelial-mesenchymal transition and fibrosis in retinal pigment epithelial cells

https://doi.org/10.1016/j.bbrc.2020.06.034Get rights and content

Highlights

  • TGF-β induces EMT and fibrosis in retinal pigment epithelial cells via ERK1/2-mTORC1-NOX4 axis.

  • TGF-β1 increases both cytosolic and mitochondrial ROS production through NOX4 upregulation.

  • Scavenging oxidative stress or NOX inhibition successfully prevents TGF-β-induced EMT and fibrosis.

  • FDA-approved drug trametinib effectively prevents EMT and fibrosis by inhibition of ERK-mTOR signaling.

Abstract

Transforming growth factor-β (TGF-β) plays a crucial role in the development of epithelial to mesenchymal transition (EMT) and fibrosis, particularly in an ocular disorder such as proliferative vitreoretinopathy (PVR). However, the key molecular mechanism underlying its pathogenesis remains unknown. In the present study, using cultured ARPE-19 cells, we determined that TGF-β initiates a signaling pathway through extracellular signal-regulated kinase (ERK)-mammalian target of rapamycin complex 1 (mTORC1) that stimulates trans-differentiation and fibrosis of retinal pigment epithelium. Blocking this pathway by a TGF-βRI, ERK or mTORC1 inhibitor protected cells from EMT and fibrotic protein expression. TGF-β1 treatment increased reactive oxygen species (ROS) via NOX4 upregulation, which acts downstream of ERK and mTORC1, as the ROS scavenger N-acetylcysteine and a pan-NADPH oxidase (NOX) inhibitor DPI dissipated excess ROS generation. TGF-β1-induced oxidative stress resulted in EMT and fibrotic changes, as NAC and DPI prevented α-SMA, Col4α3 expression and cell migration. All these inhibitors blocked the downstream pathway activation in addition to clearly preventing the activation of its upstream molecules, indicating the presence of a feedback loop system that may boost the upstream events. Furthermore, the FDA-approved drug trametinib (10 nM) blunted TGF-β1-induced mTORC1 activation and downstream pathogenic alterations through ERK1/2 inhibition, which opens a therapeutic avenue for the treatment of PVR in the future.

Introduction

Retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells, form the outer blood-retinal barrier between the choroids and the neurosensory retina [1]. The physiologic function of the RPE is essential for the survival of photoreceptors and capillaries in choroids [2]. Previous studies have suggested that RPE cells play an important role in the pathogenesis of proliferative vitreoretinopathy (PVR) [3,4]. PVR is the most common cause of surgical failure upon retinal detachment leading to blindness. During PVR, RPE cells undergo transformation into fibroblast-like cells through epithelial to mesenchymal transition (EMT), losing cell-to-cell contact and acquiring migratory mesenchymal properties [[4], [5], [6], [7]]. Excessive fibrosis in both surfaces of the retina and within the vitreous cavity occurs during the course of progression of PVR. RPE cells secrete various growth factors including transforming growth factor β (TGF-β), which is considered as the primary regulator of EMT [[8], [9], [10]]. Therefore, TGF-β released from RPE cells induces the EMT and fibrosis of these cells through autocrine signaling, hence contributing to the development of PVR.

TGF-β is a member of a superfamily of multifunctional cytokines that regulate pivotal biological processes, such as development and wound repair [11,12]. There are two pathways activated by TGF-β receptors: canonical and non-canonical. Smad-independent non-canonical pathways are known to mediate the p38 MAPK and PI3K/AKT pathways. The activation of extracellular signal-regulated kinase (ERK) is involved in TGF-β-induced EMT; however, activation has not been clearly elucidated upstream and downstream of ERK1/2 [13]. In kidney podocytes, we have identified the existence of TGF-β1-ERK1/2-mammalian target of rapamycin complex 1 (mTORC1) axis, which is involved in the pathogenesis of glomerulosclerosis [14]. In this process, TGF-β produces reactive oxygen species (ROS) mediated by NADPH oxidase 4 (NOX4) upregulation [[15], [16], [17]]. The suppression of oxidative stress attenuates TGF-β-induced fibrosis and apoptosis in podocytes [18].

In the present study, we examined the pathogenic role of TGF-β1-ERK1/2-mTORC1-NOX4 axis in the EMT and fibrosis of RPE cells. We have demonstrated the protective effect generated by the pharmacologic inhibition of this axis, suggesting a novel therapeutic target for PVR.

Section snippets

Cell culture

ARPE-19 cells were obtained from American Type Cell Culture (ATCC; CRL2302) and maintained in DMEM/F-12 medium (a mixture of Dulbecco’s modified Eagle’s minimum essential medium and Hank’s Balanced salt solution, Hyclone, Themo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Themo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (HyClone).

Experimental reagents

SB431542 (catalog no. S4317), rapamycin (catalog no. R8781), N-acetylcysteine (NAC) (catalog no.

TGF-β1 induces EMT and fibrosis through ERK1/2 and mTORC1 signaling in ARPE-19 cells

ARPE-19 cells were treated with 1 ng/ml of TGF-β1 revealed activation of ERK1/2 and mTOR (Ser2448) at 6 h and sustained activations were clearly observed till 72 h time point and α-smooth muscle actin (α-SMA), collagen 4α3 (Col4α3) and plasminogen activator inhibitor-1 (PAI-1) were clearly increased at 48 h. In addition, TGF-β1 augmented NOX4 at this time point indicating a possible role of oxidative stress causing EMT in ARPE-19 cells (Fig. 1A). To confirm the involvement of TGF-β signaling to

Discussion

In the present study, we demonstrated that the TGF-β1–ERK1/2–mTORC1–NOX4 axis plays a critical role in the EMT and the fibrosis of ARPE cells, which are known to be correlated with retinal disorders, such as PVR and age-related macular degeneration. We observed that TGF-β1 markedly increased both cytosolic and mitochondrial ROS production through NOX4, which is linked to cellular trans-differentiation. The inhibition of TGF-βR1, ERK1/2, mTORC1, NOX4, and scavenging oxidative stress prevented

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Medical Research Center Program (2017R1A5A2015369) from the Ministry of Science and ICT, and the Korea Health Technology R&D Project through the KHIDI (HI18C2196) from the Ministry of Health & Welfare, and Myung Sun Kim Memorial Foundation (2016).

References (26)

  • H. Sui et al.

    Epithelial-mesenchymal transition and drug resistance: role, molecular mechanisms, and therapeutic strategies

    Oncol Res Treat

    (2014)
  • M. Li et al.

    Epithelial-mesenchymal transition: an emerging target in tissue fibrosis

    Exp. Biol. Med.

    (2016)
  • C.N. Nagineni et al.

    Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

    J. Cell. Physiol.

    (2003)
  • Cited by (21)

    • Complanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy

      2022, Phytomedicine
      Citation Excerpt :

      NOX4 is the major source of superoxide anions and plays a non-negligible role in the development of DN (Thallas-Bonke et al., 2015; Yang et al., 2018). As reported, NOX4 protein activity leads to concomitant ROS generation and plays a crucial role in inducing EMT (Kim et al., 2020). Abnormally elevated NOX4 has been reported in various animal models of diabetic nephropathy, including streptozotocin (STZ)-induced models of diabetic rats and mice and db/db mice (Block et al., 2009; Etoh et al., 2003; Gorin et al., 2005).

    • Demethoxycurcumin inhibits the cell migration and MMP-2 expression in human retinal pigment epithelial cells by targeting the STAT-3 pathway

      2021, Experimental Eye Research
      Citation Excerpt :

      Patients with chronic rhegmatogenous retinal detachment (RRD) may also develop problems, such as raised intraocular pressure and ocular inflammatory conditions (Ni et al., 2020). Several inflammatory factors, such as growth factors and cytokines, have been implicated in the activation RPE cell migration, proliferation and EMT (Balogh et al., 2020; Kim et al., 2020). Moreover, matrix metalloproteinases (MMPs) that participate in RPE cell migration and EMT have been characterised (Matoba et al., 2017).

    • Backstage players of fibrosis: NOX4, mTOR, HDAC, and S1P; companions of TGF-β

      2021, Cellular Signalling
      Citation Excerpt :

      Interestingly it has been reported that this pathway occurs in fibroblasts rather than epithelial cells [147]. In addition to direct induction, some evidence indicates that NOX4 mediates mTORC1 activation in cardiac and retinal epithelial cells [124,148,149]. On the other hand, in cardiac and kidney cells, the expression and activation of mTORC1 are mediated by the B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a transcriptional repressor associated with fibrosis development [150,151].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text