JNK1/β-catenin axis regulates H2O2-induced epithelial-to-mesenchymal transition in human lens epithelial cells
Introduction
Fibrotic cataract is a special type of cataract, in which opaque plaques form only in the subcapsule. These plaques consist of aggregates of myofiber-like cells with excessive extracellular matrix, and are thus considered fibrotic. Fibrotic cataracts are now generally considered as a result of the pathologic epithelial-mesenchymal transition (EMT) in lens epithelial cells [1,2]. The transition is initiated through progressive loss of epithelial junctions formed by E-cadherin and ZO-1, accompanied by acquired expression of mesenchymal factors such as α-smooth muscle actin (α-SMA) and Fibronectin [3,4]. EMT is activated by various stimuli, including growth factors, cytokines, and changes in microenvironment [5,6].
Oxidative stress, through oxidative modifications of redox sensitive proteins, was supposed to mediate EMT in lens [[6], [7], [8], [9]]. Pioneering surveys reported that H2O2, as well as oxygen levels, significantly accumulated in the aqueous humor after cataract surgery, which might involve in the process of EMT in the left-over LECs [[10], [11], [12], [13]]. Recently, Fan's group reported that declined GSH levels triggered EMT in lens epithelial cells [7], confirming the role of oxidative stress in EMT. While the underlying mechanism of oxidative stress-induced EMT in lens is not fully understood.
C-Jun N-terminal kinase (JNK) owns three isoforms, JNK1-3. Oxidative stress can directly or indirectly phosphorylate/activate JNK through oxidize proteins such as thioredoxin and glutaredoxin [14,15]. In turn, JNK phosphorylates downstream proteins to elict various cellular responses. The role of JNK in EMT has been illustrated [16]. Moreover, JNK was found to regulate the expression of E-cadherin in human LECs (hLECs), implying JNK acted a role in epithelial junctions diminishment, which initiated EMT process [17]. However, whether JNK links oxidative stress to epithelial-mesenchymal transition has not been determined.
We now tested whether JNK cascades are required for oxidative stress-induced EMT in lens epithelial cells, with a view to investigate mechanisms that drive the formation of anterior/posterior polar cataracts and to identify new therapeutic strategies.
Section snippets
Antibodies
Mouse antibodies to α-SMA (1:500) and phosphorylated JNK (1:200) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), along with rabbit antibodies to c-Myc (1:200). Rabbit antibodies to SAPK/JNK (1:1000) cyclin D (1:1000), Smad2/3 (1:1000), GAPDH (1:1000), E-cadherin (1:1000), ZO-1 (1:1000), and non-phosphorylated β-catenin (1:1000) were obtained from Cell Signaling (Danvers, MA, USA). Donkey anti-rabbit, anti-mouse, or anti-goat IgG conjugated to Alexa Fluor 488 or Alexa Fluor 568
Low dose of H2O2 triggered EMT and activated JNK1 in hLECs
H2O2 is the only superoxide that can diffuse through biological membranes and is relatively resistant to degradation [18]. Hence we first tested whether direct exposure to H2O2 triggers EMT in hLECs. We previously reported that 200 μM H2O2 induced apoptosis in hLECs [11,19,20]. Accordingly, lower doses were tested [21]. We found that after exposed to 100 μM H2O2 for 48 h, hLECs lost the cubic shape, extended, and became disordered (Fig. 1A). Cells treated at a dose of 50 μM H2O2 for 48 h
Discussion
Oxidative stress has been recently found to be EMT-inducer in lens. In present study, we reported that directly exposure to low dose of H2O2 (100 μM) triggered EMT in hLECs and demonstrated the underlying JNK1/β-catenin axis in H2O2-induced EMT in hLECs for the first time. These results suggested a potential target in preventing fibrotic cataracts.
In the present study, a low dose of H2O2 significantly downregulates proteins that form epithelial cell junctions, at the same time boosts
Conflicts of interest
There is no conflict of interest in this manuscript.
References (36)
- et al.
Fibrosis in the lens. Sprouty regulation of TGFbeta-signaling prevents lens EMT leading to cataract
Exp. Eye Res.
(2016) - et al.
Elevated hyaluronan production induces mesenchymal and transformed properties in epithelial cells
J. Biol. Chem.
(2003) - et al.
Reduced glutathione level promotes epithelial-mesenchymal transition in lens epithelial cells via a Wnt/beta-catenin-mediated pathway: relevance for cataract therapy
Am. J. Pathol.
(2017) - et al.
Nox-2 is a modulator of fibrogenesis in kidney allografts
Am. J. Transplant.
(2009) - et al.
Epithelial-to-mesenchymal transition and oxidative stress in chronic allograft nephropathy
Am. J. Transplant.
(2005) - et al.
Hydrogen peroxide and human cataract
Exp. Eye Res.
(1981) - et al.
Regulation of apoptosis signal-regulating kinase 1 in redox signaling
Methods Enzymol.
(2010) - et al.
Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases
Cell
(2005) - et al.
Alpha lipoic acid protects lens from H(2)O(2)-induced cataract by inhibiting apoptosis of lens epithelial cells and inducing activation of anti-oxidative enzymes
Asian Pac J Trop Med
(2013) Signal transduction by the JNK group of MAP kinases
Cell
(2000)
Myofibroblast transdifferentiation: the dark force in ocular wound healing and fibrosis
Prog. Retin. Eye Res.
Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling
Biochim. Biophys. Acta
Should I stay or should I go: beta-catenin decides under stress
Biochim. Biophys. Acta
Redox regulation of beta-actin during integrin-mediated cell adhesion
J. Biol. Chem.
Non-Smad pathways in TGF-beta signaling
Cell Res.
Complex networks orchestrate epithelial-mesenchymal transitions
Nat. Rev. Mol. Cell Biol.
Cell adhesion: integrating cytoskeletal dynamics and cellular tension
Nat. Rev. Mol. Cell Biol.
Epithelial-mesenchymal transition: from molecular mechanisms, redox regulation to implications in human health and disease
Antioxidants Redox Signal.
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