title: R-spondin1 regulates cell proliferation of corneal endothelial cells via the Wnt3a/β-catenin pathway.
abstract: OBJECTIVE
  To evaluate the effect of Roof plate-specific spondin 1 (R-spondin1) on the proliferation of corneal endothelial cells (CECs) and to determine whether the Wnt/β-catenin pathway is involved in the activities of R-spondin1.
  
  
  METHODS
  The proliferation of rabbit CECs (RCECs) and human CECs (HCECs) was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA. The effect of R-spondin1 on CEC density was evaluated in ex vivo organ-cultured rabbit and human corneal tissues. The cell density of HCECs cultured with R-spondin1 was also evaluated in vitro. The subcellular localization of function-associated markers of CECs (zona occludens 1 [ZO-1] and Na+/K+-ATPase) was determined by immunohistocytochemistry. The expression of cell cycle proteins and localization of β-catenin were determined by immunoblotting.
  
  
  RESULTS
  The in vitro proliferation of RCECs and HCECs increased by 1.2- to 1.3-fold in response to R-spondin1. The CEC densities of rabbit and human corneal tissues were increased significantly by R-spondin1 treatment. Na+/K+-ATPase and ZO-1 were well preserved on the plasma membranes. When HCECs were maintained in the presence of R-spondin1 for up to 90 days, the maximum cell density was observed at approximately 50 days, and the cell density was maintained for up to 90 days. R-spondin1 facilitated the nuclear import of β-catenin in RCECs within 30 minutes, which subsequently upregulated cyclin D and downregulated p27, leading to G1/S progression by hyperphosphorylation of the retinoblastoma protein.
  
  
  CONCLUSIONS
  The unique effects of R-spondin1 on the proliferation of CECs, regardless of species, indicate that R-spondin1 may play a key role in maintaining corneal endothelium homeostasis through the Wnt/β-catenin pathway.
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okumura
authors.affiliation: Research Center for Tissue Engineering and Inflammation, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Nakahara
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D006706:Homeostasis; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D011817:Rabbits; D019699:Thrombospondins / Q000494:pharmacology*; D060509:Wnt3A Protein / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism*; D000098907:R-Spondins
chemical_list: D019699:Thrombospondins; D060509:Wnt3A Protein; D051176:beta Catenin; C503243:RSPO1 protein, human; D000098907:R-Spondins; C555510:WNT3A protein, human
keywords: R-spondin; corneal endothelial cells; homeostasis