R-spondin1 regulates cell proliferation of corneal endothelial cells via the Wnt3a/β-catenin pathway

Invest Ophthalmol Vis Sci. 2014 Oct 2;55(10):6861-9. doi: 10.1167/iovs.14-14091.

Abstract

Purpose: 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.

Keywords: R-spondin; corneal endothelial cells; homeostasis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Proliferation / drug effects*
  • Cells, Cultured
  • Endothelium, Corneal / drug effects
  • Endothelium, Corneal / metabolism
  • Endothelium, Corneal / pathology*
  • Enzyme-Linked Immunosorbent Assay
  • Homeostasis
  • Humans
  • Immunoblotting
  • Immunohistochemistry
  • Rabbits
  • Thrombospondins / pharmacology*
  • Wnt3A Protein / metabolism*
  • beta Catenin / metabolism*

Substances

  • RSPO1 protein, human
  • Thrombospondins
  • WNT3A protein, human
  • Wnt3A Protein
  • beta Catenin