Knockdown of both p120 catenin and Kaiso promotes expansion of human corneal endothelial monolayers via RhoA-ROCK-noncanonical BMP-NFκB pathway.
OBJECTIVE
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.
Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, Florida.
Ying-Ting
YT
Zhu
Bo
B
Han
Fu
F
Li
Szu-Yu
SY
Chen
Sean
S
Tighe
Suzhen
S
Zhang
Scheffer C G
SC
Tseng
D000293:Adolescent; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D051177:Catenins / Q000235:genetics* / Q000378:metabolism; D002452:Cell Count; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport; D012313:RNA / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D000095002:Delta Catenin
D019485:Bone Morphogenetic Proteins; D051177:Catenins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D014157:Transcription Factors; C492254:ZBTB33 protein, human; D012313:RNA; D020742:rhoA GTP-Binding Protein; D000095002:Delta Catenin; C000722347:CTNND1 protein, human
Kaiso; cornea; endothelium; p120 catenin; proliferation; siRNA; tissue engineering