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

Abstract

PURPOSE. ZEB1 is induced during endothelial-mesenchymal transition (EnMT) in the cornea. Induction of SP1 and SP3 by ZEB1 along with identification of putative SP1 and SP3 binding sites in promoters of EnMT-associated gene lead us to investigate their roles in retrocorneal membrane formation in the corneal endothelium. METHODS. Expressions of SP1, SP3, and EnMT associated genes were analyzed by immunoblotting and semiquantitative reverse transcription polymerase chain reaction. Accell SMARTpool siRNAs targeting ZEB1, SP1, and SP3 were used for gene knockdown. SP1 and SP3 binding to promoters of EnMT associated genes was investigated by chromatin immunoprecipitation assay. Corneal endothelium in mice was surgically injured in vivo under direct visualization. RESULTS. Transient Fibroblast Growth Factor 2 stimulation increased the expression of both SP1 and SP3 in the human corneal endothelium ex vivo. ZEB1 siRNA knockdown inhibited FGF2-induced SP1 mRNA and protein but not the expression of SP3. FGF2induced expression of EnMT-related genes, such as fibronectin, vimentin, and type I collagen, was reduced by both SP1 and SP3 siRNA knockdown, with inhibition of SP1 having a greater inhibitory effect than SP3. Additionally, although SP1 and SP3 proteins were found to bind together, SP1 and SP3 could bind to the same promoter binding sites of EnMT-related genes in the absence of the other. Moreover, siRNA knockdown of Zeb1 inhibited injury-dependent RCM formation in mouse corneal endothelium in vivo. CONCLUSIONS. Zeb1, through SP1 and SP3, plays a central role in mesenchymal transition induced fibrosis in the corneal endothelium and suggests that Zeb1 could be targeted to inhibit anterior segment fibrosis.

Expression and Function of ZEB1 in the Cornea

“…In adults, ZEB1 plays an important role in stem cell homeostasis, immune response, and angiogenesis [ 21 , 64 , 65 ]. However, abnormal activation may cause cellular transformation like EMT leading to tumorigenesis and fibrosis [ 8 , 56 ]. Therefore, targeting ZEB1 is a potential avenue to generate therapeutics for treatments of various ZEB1-related disorders.…”

“…Knockdown of Fgf2 in mouse corneal endothelial cells by siRNA inhibited the injury-dependent expression of Fgf2, Snai1, Zeb1, and Cdh1, indicating that corneal EnMT is under the control of the Fgf2-activated EMT factor Zeb1 [ 55 ]. Transient FGF2 stimulation could increase the expression of SP1 and SP3 in human corneal endothelial cells whereas knockdown of ZEB1 by siRNA only reduced the expression of the FGF2-induced SP1 mRNA and protein, but not SP3 [ 56 ]. The expression of the FGF2-induced EnMT genes such as FN1, VIM and COLI was reduced by siRNA knockdown of SP1 and SP3.…”

“…In addition, siRNA knockdown of Zeb1 inhibited the formation of mouse corneal endothelial injury-dependent fibrosis in vivo. They concluded that ZEB1, through activation of SP1, plays an important role in corneal endothelial fibrosis induced by mesenchymal transition [ 56 ].…”

Cell therapy in corneal endothelial disease

“…Proteomic analysis may provide insights into the ideal micro-environmental milieu for CEC behaviour and throw up clues for further refining CEC culture or in-vivo regeneration [28,29]. ZEB1 has been implicated in EnMT via induction of SP1 and SP3, and may be a target for therapies to prevent EnMT during CEC proliferation [30]. Recently, it was reported that microRNA-30c-1-transfected CECs showed increased resistance to TGF-b1-induced senescence and had better cell viability and proliferation [31].…”

Fibröse retrokorneale und perilentale Membran nach perforierender Keratoplastik

“…Die Membrandicke variierte zwischen 2 μm und 520 μm [1]. Als Ursprung für die fibröse RCM, die erstmals 1901 durch den österreichischen Augenarzt Professor Ernst Fuchs beschrieben wurde [2,6], wurden eine stromale Zellinvasion durch eine nicht vollständige Spender-Empfänger-Adhärenz [10] und/oder endothelial-mesenchymale Transformation vermutet [1,3,5,6]. In einer aktuellen Studie von Naranjo et al [7] waren die immunhistologischen Marker für die endothelial-mesenchymale Transformation positiv, was die wichtige Rolle dieses Mechanismus für die Pathogenese der fibrösen RCM unterstreicht.…”