Knockdown of both p120 catenin and Kaiso promotes expansion of human corneal endothelial monolayers via RhoA-ROCK-noncanonical BMP-NFκB pathway - PubMed (original) (raw)

Knockdown of both p120 catenin and Kaiso promotes expansion of human corneal endothelial monolayers via RhoA-ROCK-noncanonical BMP-NFκB pathway

Ying-Ting Zhu et al. Invest Ophthalmol Vis Sci. 2014.

Abstract

Purpose: To determine the signaling pathway involved in expanding contact-inhibited human corneal endothelial cells (HCECs) using p120 and Kaiso small interfering RNAs (siRNAs).

Methods: Expansion of HCEC monolayers on collagen IV in SHEM using p120 siRNA was optimized regarding various dosage, frequency, and starting date before being added Kaiso siRNA or various inhibitors of Rho, ROCK, NFκB, and TAK1. Phase contrast micrographs were used for monitoring cell shape, monolayer size, and cell density. Immunostaining was used to determine cytolocalization of BrdU, p120, pNFkB, F-actin, α-catenin, β-catenin, LEF1, Na+/K+-ATPase, N-cadherin, ZO-1, and S100A4. Western blotting was used to determine the protein level of RhoA and RhoA-guanosine-5'-triphosphate (GTP).

Results: The HCEC monolayer size in diameter was expanded from 2.1 ± 0.4 mm to 4.3 ± 0.3 mm (P < 0.05) by increasing p120 siRNA from 40 nM to 100 nM starting at day 7, to 5.0 ± 0.4 mm (P < 0.05) by adding 100 nM Kaiso siRNA, to 6.8 ± 0.3 mm by using one-fourth corneoscleral rim (P < 0.05), and to 8.1 ± 0.5 mm by using one-half corneoscleral rim (P < 0.05). Such proliferative effect required activation of RhoA-ROCK-noncanonical bone morphogenic protein (BMP) signaling and nuclear translocation of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (pNFκB). After withdrawal of siRNAs for 1 week, the resultant HCEC monolayer maintained a hexagonal shape, the average cell density of 2254 ± 87 mm(2) (n = 3), and normal expression patterns of F-actin, α-catenin, β-catenin, N-cadherin, ZO-1, and Na+/K+-ATPase without S100A4 and alpha-smooth muscle actin (α-SMA).

Conclusions: The optimized knockdown with p120 and Kaiso siRNAs further expands the size of HCEC monolayers without endothelial mesenchymal transition (EMT) via selective activation of p120/Kaiso signaling that requires the RhoA-ROCK-noncanonical BMP-NFkB signaling.

Keywords: Kaiso; cornea; endothelium; p120 catenin; proliferation; siRNA; tissue engineering.

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Figures

Figure 1

Optimization of p120 siRNA knockdown regimen based on BrdU labeling. BrdU labeling was performed in HCEC cultures treated with scRNA (○) or p120 siRNA (•) at concentrations from 10 to 160 nM starting from day 14 of culturing ([A], *P < 0.05 for each concentration between scRNA and p120 siRNA, and #_P_ < 0.05 between 40–100 nM p120 siRNA, _n_ = 3), or with 100 nM of p120 siRNA starting from day 14 of culturing once, twice, or three times a week ([B**], P > 0.05 between groups, n = 3), or starting from day 7, 10, 14, and 17 of culturing ([C**], P > 0.05 between each group, n = 3). All cultures were treated for 48 hours.

Figure 2

Human corneal epithelial cell monolayer sizes expanded by optimized p120 siRNA knockdown regimen. Human corneal epithelial cell cultures were treated weekly with 40 nM of scRNA (○) or p120 siRNA ( formula image ) starting from day 14 or with 100 nM of scRNA (•) or p120 siRNA (▪) starting from day 7 for a total of 5 weeks of culturing. (*P < 0.05, n = 3, at each time point between scRNA and p120 siRNA; #P < 0.05 between 40 nM p120 siRNA and 100 nM p120 siRNA, n = 3).

Figure 3

Further expansion of HCEC monolayer size by Kaiso siRNA, nocodazole, or increasing the initial size. The HCEC monolayer size in diameter was measured weekly when treated with the optimized p120 siRNA knockdown regimen with (○) or without (•) 100 nM Kaiso siRNA ([A], *P < 0.05, n = 3). The size achieved by knockdown with both p120 siRNA and Kaiso siRNA (•) was inhibited by 5 μg/mL nocodazole (○; [B], *P < 0.05 n = 3) but promoted without nocodazole by increasing initial HCEC aggregates from one-eighth (•) to one-fourth (○) or one-half ( formula image ) of the corneoscleral rim ([C], *P < 0.05 when compared with one-eighth corneoscleral group, #P < 0.05 when compared to one-fourth corneoscleral group, n = 3).

Figure 4

RhoA-ROCK-pNFkB signaling involved in proliferation activated by p120-Kaiso siRNAs. Human corneal epithelial cell monolayers cultured at 1 week were treated by 2 days knockdown by scRNA, p120 siRNA, or p120-Kaiso siRNAs, The RhoA-GTP level was measured in respective cell extracts by Western blot ([A], P < 0.05 and P < 0.01 for p120 siRNA and p120-kaisos, respectively, n = 3 when compared with scRNA and normalized by α-tubulin). In HCEC monolayers cultured for 2 weeks, nuclear translocation of pNFκB (p65) was induced only p120 siRNA and p120-Kaiso siRNAs, of which the latter was abolished by simultaneous addition for 2 days of 5 μg/mL CT-04, 20 μM Y27632, 1 μM CAY 10512, 5 μg/mL noggin, or 1 μM 5Z-7-Oxozeaenol (B). Double immunostaining of p120 and BrdU also revealed nuclear colocalization of p120 and BrdU induced only both p120 siRNA and p120-Kaiso siRNAs, of which the latter was abolished by simultaneous addition of 5 μg/mL CT-04, 20 μM Y27632, 1 μM CAY 10512, 5 μg/mL Noggin, or 1 μM 5Z-7-Oxozeaenol (C). Scale bar: 100 μm.

Figure 5

Activation of the Noncanonical BMP signaling by p120-Kaiso siRNAs. Human corneal epithelial cell monolayers treated by different knockdown regimens for 2 days before qRT-PCR analysis of transcript expression of BMP ligands and bone morphogenic protein receptors ([A], *P < 0.05 and **_P_ < 0.01 compared with the scRNA control), ID1-4 ([B**], all P > 0.05 compared with the scRNA control), and immunostaining of pSMAD1/5/8 [C], scale bar: 100 μm). Over-expression of BMP2, BMP4, and BMPR1B transcript by p120-Kaiso siRNAs was challenged by various small molecular weight inhibitors and Noggin ([D**], *P < 0.05 and **P < 0.01 compared with the scRNA control).

Figure 6

Maintenance of normal hexagonal shape, density, and phenotype of HCEC monolayers 1 week after withdrawal of p120-Kaiso siRNA. Human corneal epithelial cell cultures were treated with the optimized knockdown regimen of p120-Kaiso siRNAs weekly starting from day 7 for a total of 5 weeks of culturing. One week after withdrawal of p120 siRNA, the resultant HCEC monolayers retained the normal hexagonal shape (A), the in vivo cell density ( formula image , scRNA, ○, p120-Kaiso siRNA; [B], P > 0.05 when compared with the size of in vivo HCEC, *P < 0.05 when compared with the size of HCEC treated with scRNA; #P < 0.05 when compared with the size of in vivo HCEC) and the in vivo immunosfluorescence positive staining patterns of p120, N-cadherin, α-catenin, β-catenin, ZO-1, F-actin, and Na-K-ATPase, and negative staining patterns of LEF1 and S100A4 ([C], scale bar: 100 μm for [A, C]). Scale bar: 100 μm.

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Knockdown of both p120 catenin and Kaiso promotes expansion of human corneal endothelial monolayers via RhoA-ROCK-noncanonical BMP-NFκB pathway - PubMed (original) (raw)