Rho-associated Coiled-coil Kinase Inhibition Research Articles

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates’ biomineralizing cells, yet, little is known on ROCK’s role in invertebrates’ biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia.

The current global platelet supply is often insufficient to meet all the transfusion needs of patients, in particular for those with alloimmune thrombocytopenia. To address this issue, we have developed a strategy employing a combination of approaches to achieve more efficient production of functional megakaryocytes (MKs) and platelets collected from cord blood (CB)-derived CD34+ hematopoietic cells. This strategy is based on ex-vivo expansion and differentiation of MKs in the presence of bone marrow niche-mimicking mesenchymal stem cells (MSCs), together with two other key components: (1) To enhance MK polyploidization, we used the potent pharmacological Rho-associated coiled-coil kinase (ROCK) inhibitor, KD045, resulting in liberation of increased numbers of functional platelets both in-vitro and in-vivo; (2) To evade HLA class I T-cell-driven killing of these expanded MKs, we employed CRISPR-Cas9-mediated β-2 microglobulin (β2M) gene knockout (KO). We found that coculturing with MSCs and MK-lineage-specific cytokines significantly increased MK expansion. This was further increased by ROCK inhibition, which induced MK polyploidization and platelet production. Additionally, ex-vivo treatment of MKs with KD045 resulted in significantly higher levels of engraftment and donor chimerism in a mouse model of thrombocytopenia. Finally, β2M KO allowed MKs to evade killing by allogeneic T-cells. Overall, our approaches offer a novel, readily translatable roadmap for producing adult donor-independent platelet products for a variety of clinical indications.

The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes

Oocyte vitrification, while beneficial for research and species conservation applications, has limited success due to cryoinjury to the meiotic spindle. This study aimed to improve meiotic spindle recovery in vitrified bovine oocytes by investigating the effects of treatment with either a microtubule stabilizing agent, or a microtubule recovery agent. In the first two experiments, either paclitaxel or epothilone B were used to treat bovine oocytes before vitrification. Both compounds have microtubule stabilizing properties and are known antimitotic compounds used to disrupt microtubule dynamics in rapidly proliferating cancer cells. Paclitaxel treatment at 2.0 μM significantly increased the proportion of oocytes with normal microtubule distribution and chromosome arrangement after warming. Treatment with 1.0 μM had no effect and 0.5 μM had a negative effect on meiotic spindle recovery. Epothilone B treatment at all concentrations significantly increased the proportion of oocytes with meiotic spindle disruption and abnormally dispersed chromosomes. In the second set of experiments, Rho-associated coiled-coil kinase inhibition and glutathione accumulation were investigated as recovery treatments after vitrification. Oocytes were incubated with either Y-27632 or combinations of cysteine and cysteamine for 4 h after warming. Treatment with 5 μM and 10 μM of Y-27632 to inhibit rho-associated coiled-coil kinase activity significantly increased the proportion of vitrified oocytes with normal microtubule distribution and chromosome arrangement. When oocytes were incubated with 20 μM of Y-27632 there was no effect on spindle recovery. Incubation with 100 μM of cysteamine also had no effect on spindle recovery while 0.6 mM of cysteine and both 0.6 mM of cysteine and 100 μM of cysteamine significantly increased oocytes with normal microtubule distribution and chromosome arrangement.

Rho-Associated Coiled-Coil Kinase 1 Translocates to the Nucleus and Inhibits Human Cytomegalovirus Propagation.

Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus.IMPORTANCE ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCK's antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus.

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules.

Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy.

Multimodal Effects of Small Molecule ROCK and LIMK Inhibitors on Mitosis, and Their Implication as Anti-Leukemia Agents

Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs.

Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor

Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications.

Rescue of Vitrified-Warmed Bovine Oocytes with Rho-Associated Coiled-Coil Kinase Inhibitor1

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

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.

Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability.

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.