Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.     

The stimulatory effect of ROCK inhibitor on bovine corneal endothelial cells

Reagents which can promote the proliferation, adhesion and migration of cultured corneal endothelial cells (CECs) will be helpful for the treatment of reduced visual acuity due to CECs deficiency. The objectives of this study were to investigate the potential use of an inhibitor of Rho-associated protein kinase (ROCK), Y-27632, to cultured bovine corneal endothelial cells (B-CECs) and evaluated its effects on the proliferation, adhesion and migration of B-CECs. The proliferation of cultured B-CECs was moderately enhanced by 10 μM Y-27632. Y-27632 induced fibroblast-like morphological changes in the cultured B-CECs and normal cell morphology could recover after Y-27632 removal. In addition, Y-27632 was found to significantly enhance the adhesion and migration of B-CECs. Furthermore, the hanging drop aggregation assay showed that Y-27632 promoted B-CECs to form cellular networks and sheets, which proliferated along the liquid–air interface and migrated to the surface of the lid of dish. Our study demonstrated that Y-27632 is a potentially powerful reagent which can enhance the proliferation of cultured B-CECs. Y-27632 will be useful in CEC injection therapy and topical application for CEC deficiency.     

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 μM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.     

A role of activated Sonic hedgehog signaling for the cellular proliferation of oral squamous cell carcinoma cell line

Sonic hedgehog (Shh) is a secreted morphogen crucial for appropriate cellular proliferation during mammalian development. The activated Shh signaling is known to predispose to human tumors such as medulloblastoma and basal cell carcinoma, while a role of Shh signaling in the other common tumors is still controversial. Here we showed the overexpression of Shh in five cell lines among 14 human oral squamous cell carcinoma (OSCC) cell lines. One of the Shh-expressing OSCC cell lines HSQ-89 showed the inhibition of G1/S transition and apoptotic cell death by treatment with Cyclopamine, a steroidal alkaloid that blocks the intracellular Shh signaling. Furthermore, we found that treatment with Y-27632, a specific inhibitor of Rho-associated kinase, mimicked the effect of Cyclopamine on the cell cycle progression of HSQ-89. Our study revealed the involvement of activated Shh signaling in the cellular proliferation of OSCC cells, indicating Shh signaling might be a good therapeutic target for OSCC.     

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.     

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.     

Honokiol causes the p21WAF1-mediated G(1)-phase arrest of the cell cycle through inducing p38 mitogen activated protein kinase in vascular smooth muscle cells

Honokiol, an active component in extracts of Magnolia officinalis, has been proposed to play a role in anti-inflammatory, antioxidant activity, anti-angiogenic and anti-tumor activity. Although honokiol has a variety of pharmacological effects on certain cell types, its effects on vascular smooth muscle cells (VSMC) are unclear. This issue was investigated in the present study, honokiol was found to inhibit cell viability and DNA synthesis in cultured VSMC. These inhibitory effects were associated with G1 cell cycle arrest. Treatment with honokiol blocks the cell cycle in the G1 phase, down-regulates the expression of cyclins and CDKs and up-regulates the expression of p21WAF1, a CDK inhibitor. While honokiol did not up-regulate p27, it caused an increase in the promoter activity of the p21WAF1 gene. Immunoblot and deletion analysis of the p21WAF1 promoter showed that honokiol induced the expression of p21WAF1 and that this expression was independent of the p53 pathway. Furthermore, the honokiol-mediated signaling pathway involved in VSMC growth inhibition was examined. Among the relevant pathways, honokiol induced a marked activation of p38 MAP kinase and JNK. The expression of dominant negative p38 MAP kinase and SB203580, a p38 MAP kinase specific inhibitor, blocked the expression of honokiol-dependent p38 MAP kinase and p21WAF1. Consistently, blockade of p38 MAPK kinase function reversed honokiol-induced VSMC proliferation and cell cycle proteins. These data demonstrate that the p38 MAP kinase pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E/CDK2 complexes and honokiol-dependent VSMC growth inhibition. In conclusion, these findings concerning the molecular mechanisms of honokiol in VSMC provides a theoretical basis for clinical approaches to the use therapeutic agents in treating atherosclerosis.