RESUMO
PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. MATERIALS AND METHODS: Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. RESULTS: Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. CONCLUSION: The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.
RESUMO
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. FGF2-induced 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.
Assuntos
Endotélio Corneano , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Células Cultivadas , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Interferente Pequeno , Fatores de Transcrição Sp/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Dedos de ZincoRESUMO
Purpose: To determine whether the mouse corneal endothelium enters endothelial to mesenchymal transition (EndoMT) following surgical injury in vivo. Methods: The corneal endothelium in anesthetized mice was surgically injured in vivo under direct visualization. The secretion of interleukin-1 beta (IL-1ß) and fibroblast growth factor 2 (FGF2) into the aqueous humor was analyzed with western blotting. The expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, Ccne1, and Cdh1 was analyzed with semiquantitative RT-PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. Col8a2 was used as a corneal endothelial marker, and Keratocan (Ktcn) was used as a stromal marker. ß-actin was used as a loading control. Results: Sequential expression of IL-1ß and FGF2 was detected in the aqueous humor after surgical injury. FGF2 treatment induced expression of endothelial to mesenchymal transition-related genes including Snai1, and Zeb1 in the mouse ex vivo corneal endothelium. This led to increased expression of Col1a1, Col1a2, Fn1, and Vim and suppression of Cdh1 in a time-dependent manner. Expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced FGF2 expression in the in vivo mouse corneal endothelium. The injury-dependent expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 and the suppression of Cdh1 were inhibited by siRNA knockdown of FGF in the mouse corneal endothelium in vivo. Moreover, siRNA knockdown of FGF2 inhibited the formation of the injury-dependent retrocorneal membrane in the in vivo mouse corneal endothelium. Conclusions: These findings suggest that after surgical injury, FGF2 induces the expression of EndoMT-related genes Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.
Assuntos
Córnea/metabolismo , Lesões da Córnea/genética , Endotélio/metabolismo , Transição Epitelial-Mesenquimal/genética , Fator 2 de Crescimento de Fibroblastos/genética , Interleucina-1beta/genética , Animais , Humor Aquoso/química , Humor Aquoso/metabolismo , Proteínas Cdh1/genética , Proteínas Cdh1/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Córnea/patologia , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/lesões , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Técnicas de Cultura de Tecidos , Vimentina/genética , Vimentina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
We evaluated cortisol response to buspirone in extended abstinent alcoholic patients to determine 5-HT1A receptor sensitivity in alcoholism. Alcoholic patients were inpatients with an extended abstinent period of at least 3 months. Alcoholics had a significantly lower cortisol level than did the normal controls from 60 min through to 150 min after administration of 30 mg buspirone. Our results show that cortisol response to buspirone was significantly decreased in alcoholic patients compared to normal controls, reflecting decreased 5-HT1A receptor sensitivity.
