JNK1/β-catenin axis regulates H2O2-induced epithelial-to-mesenchymal transition in human lens epithelial cells

https://doi.org/10.1016/j.bbrc.2019.02.049Get rights and content

Abstract

Epithelial-mesenchymal transition (EMT) is the main cause of fibrotic cataracts. Oxidative stress was recently shown to trigger epithelial-mesenchymal transition in human lens epithelial cells (hLECs). However, the underlying mechanism is not fully understood. Here we reported that exposure to low doses (100 μM) of H2O2 led to EMT in hLECs, as indicated by simultaneous down-regulated of E-cadherin and ZO-1, and up-regulated of alpha smooth muscle actin (α-SMA). H2O2-induced EMT was accompanied by accumulation of phosphorylated JNK1. In contrast, knockdown of JNK1 via siRNA reversed H2O2-induced EMT. Of interest, in human lens capsules of anterior subcapsule cataracts, the expressions of JNK1, as well as β-catenin and its downstream effectors cyclin D and c-Myc, were augmented compared to that in normal lens capsules. Mechanistically, activated JNK1 dislodged β-catenin from the cell membrane, which subsequently translocated to the nuclei and triggered transcription of its effectors. Nuclei β-catenin, cyclin D and c-Myc were accumulated in H2O2-induced EMT and JNK1 depletion abrogated these trend in hLECs. In conclusion, our data suggest that JNK1 is essential for H2O2-induced EMT in hLECs via mediating the translocation of β-catenin.

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.

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