Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

Abstract

Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness due to endothelial dysfunction. We previously reported that IL-1β stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-boxbinding homeobox 1 (ZEB1), and ZEB2. This, in turn, induced expression of fibronectin, vimentin and, type I collagen and suppressed E-cadherin in CECs in vitro and ex vivo. siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.The cornea is the anterior, transparent tissue of the human eye and consists of epithelium, stroma and endothelium. The corneal endothelium is composed of a monolayer of cells and plays a critical role in maintaining corneal transparency that is critical for sharp vision through its pump function (1,2). Adult human corneal endothelial cells (CEC) are usually mitotically inactive and arrested at the G 1 phase of the cell cycle (3,4). Because of the cell cycle arrest, there is a progressive decline in CEC density that can be further accelerated by injury, such as intraocular surgery or infection. When the density decreases below a critical threshold, corneal edema ensues, leading to loss of transparency and vision. Vision loss secondary to endothelial dysfunction is a common indication for corneal transplantation in developed nations.There have been many attempts to overcome the cell cycle arrest using various approaches including the use of growth factors such as FGF, EGF, and TGF-β, the use of endothelial cell growth supplements (5-9), and disruption of contact inhibition using EDTA (10). Moreover, severely injured CEC can undergo mesenchymal transition through which they lose their polarity and assume a fibroblastic phenotype. These CEC a...

ZEB1 Mediates Fibrosis in Corneal Endothelial Mesenchymal Transition Through SP1 and SP3

“…We previously reported that ZEB1 mediates FGF2-dependent fibrosis in the human corneal endothelium ex vivo, 19 and it has also previously reported that SP1 activates expression of type I collagen. 31,32 To investigate the role of SP1 in ZEB1-mediated fibrosis in human corneal endothelium ex vivo, siRNA-mediated ZEB1 knockdown was performed.…”

“…FGF2 induces EnMT as evidenced by changes in cell morphology and expression of mesenchymal transition markers SNAI1 and ZEB1 in human and mouse corneal endothelium. 17,19 SNAI1 serves as a bifurcation point in the regulatory network, below which pathways for regulation of cell proliferation and fibrosis diverge. ZEB1 functions downstream of SNAI1 and promotes fibrosis by activating expression of type I collagen, a major component of retrocorneal membranes.…”

“…Interleukin-1β induces FGF2 expression, which in turn, activates SNAI1 and stimulates both proliferation and fibrosis in the corneal endothelium. 11,19 Downstream of SNAI1, CDK2 regulates proliferation whereas the transcription factor ZEB1 was shown to regulate fibrosis through increased expression of type I collagen. 19 The promoters of the fibrosis-related genes such as fibronectin (FN1), vimentin (VIM), and alpha 1 (COL1A1), and alpha 2 (COL1A2) chains of type I collagen do not contain putative ZEB1 binding sites.…”

Human CardioChimeras: Creation of a Novel ‘Next Generation’ Cardiac Cell

“…SDF expression was downregulated in D6 and G4 and 6 undetectable in B3 ( Figure 5C). FGF2, an important growth factor for wound healing, angiogenesis 7 and cellular proliferation 29 was highly upregulated in B3. hCC F1 and D2 increased FGF2 8 expression relative to cCICs and were more reminiscent of MSCs, while hCC D6 and G4 expressed 9 lower levels of FGF2 compared to both parent cells ( Figure 5D).…”

Fibroblast Growth Factor 2 Augments Transforming Growth Factor Beta 1 in Inducing Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells

“…Among various FGFs, FGF2 and FGF4 are key regulators of EMT during development and cancer progression in the lung [38,39]. It has been reported that FGF2 reduces Ecadherin in human ovarian cancer cells [40], and induces the expression of mesenchymal markers (VIM, FSP1, α-SMA and SNAI1) in corneal endothelial cells [41] and proximal tubular epithelial cells [39,42]. A number of studies have shown the synergistic effect of combined treatment of TGFβ1 and FGF2 in inducing EMT in mouse normal mammary epithelial (NMuMG) cells [43], rat Hertwig's epithelial root sheath (HERS) cells [44], mouse lung epithelial type II cell line MLE-12 [45], human non-small cell lung cancer cell lines NCI-H1975 and NCI-H165 [46], and human lung adenocarcinoma cell lines PC-9, HCC-827 and A549 [47,48].…”