Biochemical and Biophysical Research Communications
Activation of ERK1/2-mTORC1-NOX4 mediates TGF-β1-induced epithelial-mesenchymal transition and fibrosis in retinal pigment epithelial cells
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-1 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)
- et al.
Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration
Neurobiol. Aging
(2014) - et al.
Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy
Exp. Eye Res.
(2016) - et al.
Pathophysiology of proliferative vitreoretinopathy in retinal detachment
Surv. Ophthalmol.
(2013) - et al.
Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro
Neoplasia
(2004) - et al.
Inhibition of the ERK1/2-mTORC1 axis ameliorates proteinuria and the fibrogenic action of transforming growth factor-beta in Adriamycin-induced glomerulosclerosis
Kidney Int.
(2019) - et al.
Reciprocal regulation of TGF-beta and reactive oxygen species: a perverse cycle for fibrosis
Redox Biol
(2015) - et al.
Transforming growth factor beta1-induced apoptosis in podocytes via the extracellular signal-regulated kinase-mammalian target of rapamycin complex 1-NADPH oxidase 4 Axis
J. Biol. Chem.
(2015) - et al.
MEK inhibitors induce apoptosis via FoxO3a-dependent PUMA induction in colorectal cancer cells
Oncogenesis
(2018) - et al.
Retinal pigment epithelium transplantation: concepts, challenges, and future prospects
Eye
(2015) - et al.
Pathogenesis of proliferative vitreoretinopathy. Modulation of retinal pigment epithelial cell functions by vitreous and macrophages
Dev. Ophthalmol.
(1989)
Epithelial-mesenchymal transition and drug resistance: role, molecular mechanisms, and therapeutic strategies
Oncol Res Treat
Epithelial-mesenchymal transition: an emerging target in tissue fibrosis
Exp. Biol. Med.
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.
Cited by (21)
Coenzyme Q<inf>0</inf>, a quinone derivative from Antrodia camphorata, inhibits epithelial-mesenchymal transition by activating the Nrf2 signaling pathway in TGF-β-stimulated adult retinal pigment epithelial cells to improve age-related macular degeneration and proliferative vitreoretinopathy
2024, Journal of Functional FoodsComplanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy
2022, PhytomedicineCitation 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 ResearchCitation 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 SignallingCitation 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].
- 1
These authors contributed equally to this work.