ABSTRACT
AIM: To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS: Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. RESULTS: hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. CONCLUSION: hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.
Subject(s)
Cell Differentiation , Cornea/metabolism , Endothelial Cells/metabolism , Human Embryonic Stem Cells/metabolism , Collagen Type VIII/biosynthesis , Cornea/cytology , Endothelial Cells/cytology , Human Embryonic Stem Cells/cytology , Humans , Sodium-Potassium-Exchanging ATPase/biosynthesis , Zonula Occludens-1 Protein/biosynthesisABSTRACT
Formation of a heterotrimeric IPP complex composed of integrin-linked kinase (ILK), the LIM domain protein PINCH, and parvin is important for signaling through integrin adhesion receptors. Mammals possess two PINCH genes that are expressed simultaneously in many tissues. PINCH1 and PINCH2 have overlapping functions and can compensate for one another in many settings; however, isoform-specific functions have been reported and it is proposed that association with a PINCH1- or PINCH2-containing IPP complex may provide a bifurcation point in integrin signaling promoting different cellular responses. Here we report that the LIM1 domains of PINCH1 and PINCH2 directly compete for the same binding site on the ankyrin repeat domain (ARD) of ILK. We determined the 1.9A crystal structure of the PINCH2 LIM1 domain complexed with the ARD of ILK, and show that disruption of this interface by point mutagenesis reduces binding in vitro and alters localization of PINCH2 in cells. These studies provide further evidence for the role of the PINCH LIM1 domain in association with ILK and highlight direct competition as one mechanism for regulating which PINCH isoform predominates in IPP complexes. Differential regulation of PINCH1 and PINCH2 expression may therefore provide a means for altering cellular integrin signaling pathways.
Subject(s)
Binding, Competitive , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Models, Molecular , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/genetics , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Ankyrin Repeat/genetics , Crystallization , Gene Expression Regulation/genetics , LIM Domain Proteins , Membrane Proteins , Molecular Sequence Data , Mutagenesis , Protein Serine-Threonine Kinases/geneticsABSTRACT
The heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. We report a 1.6-A crystal structure of the ILK ankyrin repeat domain bound to the PINCH1 LIM1 domain, revealing the molecular basis of ILK-PINCH interactions and providing a structural description of this region of ILK. This structure identifies 5 ankyrin repeats in ILK, explains previous deletion mutagenesis data, permits identification of ILK and PINCH1 point mutations that disrupt the interaction, shows how zincs are coordinated by PINCH1 LIM1, and suggests that conformational flexibility and twisting between the 2 zinc fingers within the LIM1 domain may be important for ILK binding. These data provide an atomic-resolution description of a key interaction in the ILK-PINCH-parvin scaffolding complex.
