Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells - PubMed (original) (raw)

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells

Ying-Ting Zhu et al. J Cell Biol. 2014.

Abstract

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-β signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell-cell junctions and instead activates Ras homologue gene family A (RhoA)-Rho-associated protein kinase (ROCK)-canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest-like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.

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Figures

Figure 1.

Activation of canonical Wnt signaling by trypsin/EDTA but not by p120-Kaiso knockdown. Contact-inhibited HCEC monolayers cultured at 14 d in SHEM were treated with trypsin/EDTA for 5 min, or knockdown with p120 siRNA or p120-Kaiso siRNAs weekly for 4 wk. The resultant cells were investigated by phase-contrast microscopy, immunofluorescence staining of β-catenin and LEF1 (both green; A), qRT-PCR of β-catenin and LEF1 transcripts (48 h later; B), Western blotting of nuclear extracts (48 h later, using histone as the loading control; C), and TCF/LEF promoter activity (24 h later; D). *, P < 0.05; **, P < 0.01. n = 4. Error bars indicate ±SEM. Bar, 25 µm.

Figure 2.

p120-Kaiso knockdown in MESCM activates RhoA-ROCK-canonical BMP signaling to unlock the mitotic block. (A) HCEC monolayers obtained by weekly knockdown with p120-Kaiso siRNAs after 1 wk of culturing for 5 wk. BrdU-labeled nuclei (red) with double labeling with p120 (green) were abolished by CT-04, ROCK1/ROCK2 siRNAs, or Noggin. (B) RhoA-GTP was increased by p120 siRNA and further increased by p120-Kaiso siRNAs (*, P < 0.05; **, P < 0.01). n = 4, compared with scRNA and normalized by α-tubulin. (C and D) Expression of BMP ligands, receptors (C), and BMP downstream genes, ID1-4 (D), was measured by qRT-PCR and Western blotting (*, P < 0.05; **, P < 0.01; n = 4, compared with scRNA). Error bars indicate ±SEM. (E and F) Immunofluorescence staining of pSMAD1/5/8 (green) and pNF-κB (green in MESCM, red in SHEM) was performed to examine the canonical BMP signaling (E) and noncanonical BMP signaling (F), respectively. In E, CT-04, ROCK1/2 siRNA, or Noggin were added to p120-Kaiso siRNA. Bars, 25 µm.

Figure 3.

Successful expansion of HCEC monolayers by knockdown with p120-Kaiso siRNAs in MESCM. (A and B) The size of HCEC monolayers in diameter was measured when cultured in SHEM and MESCM without (A; *, P < 0.05, n = 4, when compared with SHEM) or with knockdown by 100 nM of scRNA or p120-Kaiso siRNAs (B; *, P < 0.05; **, P < 0.01 when compared with their corresponding scRNA controls; #, P < 0.05; ##, P < 0.01 when compared with SHEM, n = 4). Error bars indicate ±SEM. (C) BrdU-labeled nuclei (red) were only detected in those treated with p120-Kaiso siRNAs in MESCM (P < 0.01, compared with their corresponding scRNA control in MESCM in zone 3 and zone 4, which were 50% and 40%, respectively, when equally subdivided into four zones from the center after 6 wk of culturing, i.e., 5 wk after weekly knockdown). Bar, 25 µm.

Figure 4.

Expression of markers of neural crest and ESCs and miR302 by knockdown with p120 or p120-Kaiso siRNAs in MESCM. HCEC monolayers were cultured in MESCM under weekly knockdown by p120 siRNA or p120-Kaiso siRNAs starting from 1 wk of culturing. (A–C) Transcript expression of ESC markers (A), neural crest cell markers (B), and miR302 (C) was measured by qRT-PCR after 5 wk of culturing (*, P < 0.05, **, P < 0.01, n = 4, when compared with the corresponding scRNA control). Such overexpression was abolished by Noggin added in the final week of culturing (A and B). Error bars indicate ±SEM. (D) Immunostaining of nuclear expression of Nanog, Oct4, and Sox 2 as well as membranous and cytoplasmic expression of Nestin, SSEA4, SOX9, and FOXD3 was also compared. Bar, 25 µm.

Figure 5.

Reprogramming and expression of miR302b are attenuated by the Rho inhibitor CT-04, ROCK1/2 siRNAs, or Noggin. HCEC monolayers were cultured in MESCM under weekly knockdown by p120 siRNA or p120-Kaiso siRNAs starting from 1 wk, and various inhibitors were added for the last week of culturing. Transcript expression of BMPs, BMPRs, IDs (A), miR302b and miR302c (B), ESC markers (C), and neural crest cell markers (D) was measured by qRT-PCR after 5 wk of culturing (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4, when compared with the p120-Kaiso siRNAs by setting the expression level of scRNA as 1). Error bars indicate ±SEM.

Figure 6.

Maintenance of normal HCEC shape, density, and phenotype after withdrawal of p120-Kaiso siRNAs. (A and B) The morphology (A) and the density (B; *, P < 0.05 compared with the scRNA control; #, P < 0.05 compared with the in vivo density; n = 4) of HCEC monolayers were monitored throughout the entire period. Error bars indicate ±SEM. (C) Immunofluorescence staining to acetyl-α-tubulin (red) and other markers (all green) was performed in HCEC monolayers 1 wk after withdrawal. Bars, 25 µm.

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Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells - PubMed (original) (raw)