title: Establishment of functioning human corneal endothelial cell line with high growth potential.
abstract: Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+)- and K(+)-dependent ATPase (Na(+)/K(+)-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+)/K(+)-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.
authors.affiliation: Department of Ophthalomology, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yokoi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Seko
authors.forename: Tae
authors.initials: T
authors.lastname: Yokoi
authors.forename: Hatsune
authors.initials: H
authors.lastname: Makino
authors.forename: Shin
authors.initials: S
authors.lastname: Hatou
authors.forename: Masakazu
authors.initials: M
authors.lastname: Yamada
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
authors.forename: Akihiro
authors.initials: A
authors.lastname: Umezawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishina
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Azuma
mesh_terms: D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000166:cytology*; D052163:Human papillomavirus 6 / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D062826:Zonula Occludens-1 Protein
chemical_list: D015820:Cadherins; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D019938:Cyclin D1; D000254:Sodium-Potassium-Exchanging ATPase