Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood–brain barrier

We have investigated the role of the Rho/ROCK signaling pathway in the interaction of metastatic melanoma cells with the brain endothelium. ROCK inhibition induced a shift of melanoma cells to the mesenchymal phenotype, increased the number of melanoma cells attached to the brain endothelium, and strengthened the adhesion force between melanoma and endothelial cells. Inhibition of ROCK raised the number of melanoma cells migrating through the brain endothelial monolayer and promoted the formation of parenchymal brain metastases in vivo. We have shown that inhibition of the Rho/ROCK pathway in melanoma, but not in brain endothelial cells, is responsible for this phenomenon. Our results indicate that the mesenchymal type of tumor cell movement is primordial in the transmigration of melanoma cells through the blood–brain barrier.     

Caveolin-1 knockdown increases the therapeutic sensitivity of lung cancer to cisplatin-induced apoptosis by repressing Parkin-related mitophagy and activating the ROCK1 pathway

Chemotherapy is the first-line treatment option for patients with lung cancer. However, therapeutic resistance occurs through an incompletely understood mechanism. Our research wants to investigate the influence of Caveolin-1 (Cav-1) on the therapeutic sensitivity of lung cancer in vitro. Results in this study demonstrated that Cav-1 levels were markedly inhibited in A549 lung cancer cells after exposure to cisplatin. Knockdown of caveolin further enhanced cisplatin-triggered cancer death in A549 cells. The functional investigation demonstrated that Cav-1 inhibition amplified the mitochondrial stress signaling induced by cisplatin, as evidenced by the mitochondrial reactive oxygen species burst, cellular metabolic disruption, mitochondrial membrane potential reduction, and mitochondrial caspase-9-related apoptosis activation. At the molecular level, cav-1 augmented cisplatin-mediated mitochondrial damage by inhibiting Parkin-related mitochondrial autophagy. Mitophagy activation effectively attenuated the promotive impact of Cav-1 knockdown on mitochondrial damage and cell death. Furthermore, our data indicated that Cav-1 affected Parkin-related mitophagy by activating the Rho-associated coiled-coil kinase 1 (ROCK1) pathway; inhibition of the ROCK1 axis prevented cav-1 knockdown-mediated cell death and mitochondrial damage. Taken together, our results provide ample data illuminate the necessary action exerted by Cav-1 on affecting cisplatin-related therapeutic resistance. Silencing of Cav-1 inhibited Parkin-related mitophagy, thus amplifying cisplatin-mediated mitochondrial apoptotic signaling. This finding identifies the Cav-1/ROCK1/Parkin/mitophagy axis as a potential target to overcome cisplatin-related resistance in lung cancer cells.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

Involvement of a Rho-ROCK-JNK pathway in arsenic trioxide-induced apoptosis in chronic myelogenous leukemia cells

The apoptotic signals activated by As(2)O(3) in the chronic myelogenous leukemia (CML) cell lines K562 and KCL22 were investigated. As(2)O(3) was found to induce apoptosis in these cells via the intrinsic pathway. As(2)O(3) also induced a sustained c-Jun NH2-terminal kinase (JNK) activation which preceded and was necessary for caspase-9 activation. We established that Rho and its effector, the kinase ROCK, are activated by As(2)O(3). Inhibition of either Rho or ROCK prevented JNK activation and protected against apoptosis. Thus, in CML cells, apoptosis induced by As(2)O(3) is mediated, at least in part, via a Rho-ROCK-JNK axis. These findings define a novel signaling pathway for As(2)O(3)-induced apoptosis.     

Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.     

JNK interaction with Sab mediates ER stress induced inhibition of mitochondrial respiration and cell death

Our aim was to better understand the mechanism and importance of sustained c-Jun N-terminal kinase (JNK) activation in endoplasmic reticulum (ER) stress and effects of ER stress on mitochondria by determining the role of mitochondrial JNK binding protein, Sab. Tunicamycin or brefeldin A induced a rapid and marked decline in basal mitochondrial respiration and reserve-capacity followed by delayed mitochondrial-mediated apoptosis. Knockdown of mitochondrial Sab prevented ER stress-induced sustained JNK activation, impaired respiration, and apoptosis, but did not alter the magnitude or time course of activation of ER stress pathways. P-JNK plus adenosine 5′-triphosphate (ATP) added to isolated liver mitochondria promoted superoxide production, which was amplified by addition of calcium and inhibited by a blocking peptide corresponding to the JNK binding site on Sab (KIM1). This peptide also blocked tunicamycin-induced inhibition of cellular respiration. In conclusion, ER stress triggers an interaction of JNK with mitochondrial Sab, which leads to impaired respiration and increased mitochondrial reactive oxygen species, sustaining JNK activation culminating in apoptosis.     

Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

The differentiation of valvular interstitial cells (VICs) to a myofibroblastic or osteoblast-like phenotype is commonly found in calcific valvular stenosis, although the molecular-level mechanisms of this process remain poorly understood. Due to the role of the Rho pathway in various vascular diseases and in the expression of a myofibroblast phenotype, the present study was inspired by the hypothesis that Rho activation is involved in regulating cellular processes related to valve calcification. It was found that increased RhoA and Rho kinase (ROCK) activity was associated with increased nodule formation in VIC cultures in vitro, and intentional induction of RhoA activity led to a further increase in nodules and expression of α-smooth muscle actin. VICs treated with ROCK inhibitors were also examined for nodule formation, proliferation, apoptosis, and expression of myofibroblastic or osteoblastic markers. ROCK inhibition dramatically reduced myofibroblast-regulated nodule formation in VIC cultures, as evidenced by a decrease in nodule number, total nodule area, α-smooth muscle actin-positive stress fibers, apoptosis, and gene expression of myofibroblast-related phenotypic markers. Meanwhile, ROCK inhibition was less effective at reducing nodule formation associated with osteogenic activity. In fact, ROCK inhibition increased the expression of alkaline phosphatase and effected only a modest decrease in nodule number when applied to VIC cultures with higher osteogenic activity. Thus, the Rho pathway possesses a complex role in regulating the VIC phenotype and nodule formation, and it is hoped that further elucidation of these molecular-level events will lead to an improved understanding of valvular disease and identification of potential treatments.     

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.     

Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.     

Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells

Background

The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.

Methodology/Principal Findings

P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a ‘ground’ state after the inhibition had been removed.

Conclusions/Significance

Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular ‘fate’ decision making processes.     

Localized Rho GTPase activation regulates RNA dynamics and compartmentalization in tumor cell protrusions

mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK), and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.     

Rho/ROCK-dependent pseudopodial protrusion and cellular blebbing are regulated by p38 MAPK in tumour cells exhibiting autocrine c-Met activation

BACKGROUND INFORMATION: The c-Met-dependent, beta-actin-rich, blebbed pseudopodia of MSV-MDCK-INV (invasive Moloney-sarcoma-virus-transformed Madin-Darby canine kidney) cells are induced by Rho/ROCK (Rho kinase) activation, and are morphologically distinct from flat extended lamellipodia. RESULTS: Microtubules were shown to extend to these actin-rich pseudopodial domains, and microtubule depolymerization by nocodazole treatment resulted in progressive cellular blebbing, initiating in the pseudopodial domains and resulting in transient cellular rounding and blebbing after 30 min. The blebbing response was dependent on autocrine HGF (hepatocyte growth factor) activation of c-Met and prevented by inhibition of RhoA, ROCK and p38 MAPK (p38 mitogen-activated protein kinase), but not ERK (extracellular-signal-regulated kinase) or PI3K (phosphoinositide 3-kinase). Phospho-p38 MAPK was present in pseudopodia, localizing activation of this signalling pathway to this protrusive membrane structure. In serum-starved cells, LPA (lysophosphatidic acid) activation of RhoA induced p38 MAPK-dependent pseudopodial protrusions, and inhibition of p38 MAPK prevented pseudopodial protrusion and displacement of MSV-MDCK-INV cells. MSV-MDCK-INV cells exhibited intermittent blebbing and rounding, which may represent an integral part of their motile behaviour. CONCLUSIONS: The localized activation of an autocrine HGF/c-Met loop regulates Rho/ROCK activation of p38 MAPK signalling to stimulate both membrane blebbing and pseudopod formation.