Search

Ras homolog family member A (RhoA) and Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) are key regulators of focal adhesion, actomyosin contraction and cell motility. RhoA/ROCK signaling has emerged as an attractive target for the development of new cancer therapeutics. Whether RhoA/ROCK is involved in regulating the formation of tumor cell vasculogenic mimicry (VM) is largely unknown. To confirm this hypothesis, we performed in vitro experiments using hepatocellular carcinoma (HCC) cell lines. Firstly, we demonstrated that HCC cells with higher active RhoA/ROCK expression were prone to form VM channels, as compared with RhoA/ROCK low-expressing cells. Furthermore, Y27632 (a specific inhibitor of ROCK) rather than exoenzyme C3 (a specific inhibitor of RhoA) effectively inhibited the formation of tubular network structures in a dose-dependent manner. To elucidate the possible mechanism of ROCK on VM formation, real-time qPCR, western blot and immunofluorescence were used to detect changes of the key VM-related factors, including VE-cadherin, erythropoietin-producing hepatocellular carcinoma-A2 (EphA2), phosphoinositide 3-kinase (PI3K), matrix metalloproteinase (MMP)14, MMP2, MMP9 and laminin 5γ2-chain (LAMC2), and epithelial-mesenchymal-transition (EMT) markers: E-cadherin and Vimentin. The results showed that all the expression profiles were attenuated by blockage of ROCK. In addition, in vitro cell migration and invasion assays showed that Y27632 inhibited the migration and invasion capacity of HCC cell lines in a dose-dependent manner markedly. These data indicate that ROCK is an important mediator in the formation of tumor cell VM, and suggest that ROCK inhibition may prove useful in the treatment of VM in HCC.

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.

Dormant carcinoma cancer cells showing epithelial characteristics can be activated to dissipate into the surrounding tissue or organs through epithelial-mesenchymal transition (EMT). However, the molecular details underlying the activation of dormant cancer cells have been less explored. In this study, we examined the molecular pathway to activate dormant breast cancer cells. Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates. The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition. Migration / invasion also increased upon ROCK inhibition. However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug. Based on these results, we suggest the potential risk for dormant cancer cells to dissipate through non-typical EMT when ROCK activity is down-regulated.

In native fibrocartilage, mechanotransduction allows the cells to perceive the physical microenvironment not only through topographical cues from the extracellular matrix, but also through mechanical cues, such as interstitial flow. To create a microenvironment that simultaneously integrates nanotopography and flow stimulus, we developed a biomimetic microfluidic device embedded with aligned nanofibers to contain microchambers of different angles, which enabled the flow direction to form different angles with the fibers. Using this device, we investigated the effects of microfluidic and nanotopographical environment on the morphology and fibrochondrogenesis of mesenchymal stem cells (MSCs) and the involvement of RhoA/ROCK pathway and Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ). The results showed that the flow direction perpendicular to aligned nanofibers was conducive to fibrochondrogenesis of MSCs. In addition, ROCK inhibitor and knockdown of YAP/TAZ disrupted fibrochondrogenic differentiation of MSCs. In conclusion, our data suggest the crucial role of mechanotransduction in regulating fibrochondrogenic differentiation of MSCs, which may be mediated by RhoA/ROCK pathway and YAP/TAZ.

Background: ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration., Methodology/principal Findings: In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration., Conclusions: ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.

Background: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity, molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored., Methodology/principal Findings: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock, a major effector kinase downstream of Rho, played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore, a novel culture system combining a single synthetic matrix, defined medium, and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells as determined by teratoma formation assay., Conclusions/significance: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells, and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells.

Background: The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-beta (TGF-beta) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-beta/Smad pathway in atherosclerosis and vascular cells., Methodology: In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-beta. In addition, statins upregulated TGF-beta receptor type II (TRII), and increased TGF-beta synthesis and TGF-beta/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-beta induced apoptosis and increased TGF-beta-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-beta/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected., Conclusions: Statins enhance TGF-beta/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-beta/Smad pathway is essential for statins-dependent actions in VSMCs.