Caspase-4 has a role in cell division in epithelial cells through actin depolymerization.

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment.

During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non-neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin-332 (LN-332) as a principle paracrine factor in conditioned medium from mammary epithelium-derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA-MB-231 cells. Then, we carried out a morphology-based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA-MB-231 cells that were induced by conditioned medium from MCF10A cells and LN-332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium-treated MDA-MB-231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.

IRR is involved in glucose-induced endocytosis after insulin secretion.

Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic ß-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3 generation and controls endocytosis after insulin secretion.

GDP-Bound Rab27a Dissociates from the Endocytic Machinery in a Phosphorylation-Dependent Manner after Insulin Secretion.

Glucose-stimulated insulin secretion is controlled by both exocytosis and endocytosis in pancreatic ß-cells. Although endocytosis is a fundamental step to maintain cellular responses to the secretagogue, the molecular mechanism of endocytosis remains poorly defined. We have previously shown that in response to high concentrations of glucose, guanosine 5'-diphosphate (GDP)-bound Rab27a is recruited to the plasma membrane where IQ motif-containing guanosine 5'-triphosphatase (GTPase)-activating protein 1 (IQGAP1) is expressed, and that complex formation promotes endocytosis of secretory membranes after insulin secretion. In the present study, the regulatory mechanisms of dissociation of the complex were investigated. Phosphorylation of IQGAP1 on serine (Ser)-1443, a site recognized by protein kinase Cε (PKCε), inhibited the interaction of GDP-bound Rab27a with IQGAP1 in a Cdc42-independent manner. Glucose stimulation caused a translocation of PKCε from the cytosol to the plasma membrane. In addition, glucose-induced endocytosis was inhibited by the knockdown of IQGAP1 with small interfering RNA (siRNA). However, the expression of the non-phosphorylatable or phosphomimetic form of IQGAP1 could not rescue the inhibition, suggesting that a phosphorylation-dephosphorylation cycle of IQGAP1 is required for endocytosis. These results suggest that IQGAP1 phosphorylated by PKCε promotes the dissociation of the IQGAP1-GDP-bound Rab27a complex in pancreatic ß-cells, thereby regulating endocytosis of secretory membranes following insulin secretion.

PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic ß-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic ß-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

Citron kinase mediates transition from constriction to abscission through its coiled-coil domain.

Cytokinesis is initiated by constriction of the cleavage furrow, and completed with separation of the two daughter cells by abscission. Control of transition from constriction to abscission is therefore crucial for cytokinesis. However, the underlying mechanism is largely unknown. Here, we analyze the role of Citron kinase (Citron-K) that localizes at the cleavage furrow and the midbody, and dissect its action mechanisms during this transition. Citron-K forms a stable ring-like structure at the midbody and its depletion affects the maintenance of the intercellular bridge, resulting in fusion of two daughter cells after the cleavage furrow ingression. RNA interference (RNAi) targeting Citron-K reduced accumulation of RhoA, Anillin, and septins at the intercellular bridge in mid telophase, and impaired concentration and maintenance of KIF14 and PRC1 at the midbody in late telophase. RNAi rescue experiments revealed that these functions of Citron-K are mediated by its coiled-coil (CC) domain, and not by its kinase domain. The C-terminal part of CC contains a Rho-binding domain and a cluster-forming region and is important for concentrating Citron-K from the cleavage furrow to the midbody. The N-terminal part of CC directly binds to KIF14, and this interaction is required for timely transfer of Citron-K to the midbody after furrow ingression. We propose that the CC-domain-mediated translocation and actions of Citron-K ensure proper stabilization of the midbody structure during the transition from constriction to abscission.

Protective effect of hydrogen sulfide on pancreatic beta-cells.

Hydrogen sulfide (H2S) is recognized as a third gaseous signaling molecule behind nitric oxide (NO) and carbon monoxide (CO). In pancreatic beta-cells, H2S inhibits glucose-induced insulin release. There are multiple underlying mechanisms for this inhibitory process. Apart from these inhibitory effects, H2S also protects pancreatic islets from apoptotic cell death induced by high glucose. Moreover, expression of the H2S-producing enzyme, cystathionine γ-lyase (CSE), is induced by glucose stimulation. These observations suggest that H2S is produced in an inducible manner, as are the other two gaseous signaling molecules, NO and CO. We recently reported that a lack of CSE induces apoptotic beta-cell death and promotes the development of high-fat diet (HFD)-induced diabetes. These findings tempt us to suggest that H2S produced by CSE is part of a homeostatic mechanism used by pancreatic beta-cells to inhibit insulin release and reduce cellular stress evoked by glucose, possibly via the anti-oxidant properties of H2S.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Liprin-α controls stress fiber formation by binding to mDia and regulating its membrane localization.

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-α as an mDia-binding protein. The binding is mediated through the central region of Liprin-α and through the N-terminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-α competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-α fragment containing the mDia-binding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-α by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.

EphA4-dependent axon retraction and midline localization of Ephrin-B3 are disrupted in the spinal cord of mice lacking mDia1 and mDia3 in combination.

mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking mDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left-right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3-EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3-EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.

Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

Endogenous hydrogen sulfide protects pancreatic beta-cells from a high-fat diet-induced glucotoxicity and prevents the development of type 2 diabetes.

Chronic exposure to high glucose induces the expression of cystathionine gamma-lyase (CSE), a hydrogen sulfide-producing enzyme, in pancreatic beta-cells, thereby suppressing apoptosis. The aim of this study was to examine the effects of hydrogen sulfide on the onset and development of type 2 diabetes. Middle-aged (6-month-old) wild-type (WT) and CSE knockout (CSE-KO) mice were fed a high-fat diet (HFD) for 8weeks. We determined the effects of CSE knockout on beta-cell function and mass in islets from these mice. We also analyzed changes in gene expression in the islets. After 8weeks of HFD, blood glucose levels were markedly increased in middle-aged CSE-KO mice, insulin responses were significantly reduced, and DNA fragmentation of the islet cells was increased. Moreover, expression of thioredoxin binding protein-2 (TBP-2, also known as Txnip) was increased. Administration of NaHS, a hydrogen sulfide donor, reduced TBP-2 gene levels in isolated islets from CSE-KO mice. Gene levels were elevated when islets were treated with the CSE inhibitor dl-propargylglycine (PPG). These results provide evidence that CSE-produced hydrogen sulfide protects beta-cells from glucotoxicity via regulation of TBP-2 expression levels and thus prevents the onset/development of type 2 diabetes.

Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK-I and ROCK-II.

Rho-associated coiled-coil-forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK-I and ROCK-II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross-mating of ROCK-I(+/-) ROCK-II(+/-) double heterozygous mice showed that all of the ROCK-I(-/-) ROCK-II(-/-) homozygous mice die in utero before 9.5 days post-coitum (dpc) and ROCK-I(-/-) ROCK-II(+/-) homo-heterozygous or ROCK-I(+/-) ROCK-II(-/-) hetero-homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK-I(+/-) ROCK-II(-/-) or ROCK-I(-/-) ROCK-II(+/-) mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild-type embryos treated ex vivo with a ROCK inhibitor, Y-27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.

Rho-mDia1 pathway is required for adhesion, migration, and T-cell stimulation in dendritic cells.

Dendritic cells (DCs) are essential for the initiation of acquired immune responses through antigen acquisition, migration, maturation, and T-cell stimulation. One of the critical mechanisms in this response is the process actin nucleation and polymerization, which is mediated by several groups of proteins, including mammalian Diaphanous-related formins (mDia). However, the role of mDia in DCs remains unknown. Herein, we examined the role of mDia1 (one of the isoforms of mDia) in DCs. Although the proliferation and maturation of bone marrow-derived DCs were comparable between control C57BL/6 and mDia1-deficient (mDia1(-/-)) mice, adhesion and spreading to cellular matrix were impaired in mDia1(-/-) bone marrow-derived DCs. In addition, fluorescein isothiocyanate-induced cutaneous DC migration to draining lymph nodes in vivo and invasive migration and directional migration to CCL21 in vitro were suppressed in mDia1(-/-) DCs. Moreover, sustained T-cell interaction and T-cell stimulation in lymph nodes were impaired by mDia1 deficiency. Consistent with this, the DC-dependent delayed hypersensitivity response was attenuated by mDia1-deficient DCs. These results suggest that actin polymerization, which is mediated by mDia1, is essential for several aspects of DC-initiated acquired immune responses.

A novel mRNA decay inhibitor abolishes pathophysiological cellular transition.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

Involvement of activator protein-1 family members in ß-catenin and p300 association on the genome of PANC-1 cells.

Wnt/ß-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for ß-catenin/CBP but not for ß-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying ß-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased ß-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates ß-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/ß-catenin signaling divergence.

Mechanism of action of non-camptothecin inhibitor Genz-644282 in topoisomerase I inhibition.

Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT), causes replication arrest and cell death. Although CPT has excellent cytotoxicity, it has the disadvantage of instability under physiological conditions. Therefore, new types of TOP1 inhibitor have attracted particular attention. Here, we characterised the effect of a non-camptothecin inhibitor, Genz-644282 (Genz). First, we found that treatment with Genz showed cytotoxicity by introducing double-strand breaks (DSBs), which was suppressed by co-treatment with aphidicolin. Genz-induced DSB formation required the functions of TOP1. Next, we explored the advantages of Genz over CPT and found it was effective against CPT-resistant TOP1 carrying either N722S or N722A mutation. The effect of Genz was also confirmed at the cellular level using a CPT-resistant cell line carrying N722S mutation in the TOP1 gene. Moreover, we found arginine residue 364 plays a crucial role for the binding of Genz. Because tyrosine residue 723 is the active centre for DNA cleavage and re-ligation by TOP1, asparagine residue 722 plays crucial roles in the accessibility of the drug. Here, we discuss the mechanism of action of Genz on TOP1 inhibition.

The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants.

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

Disruption of actin dynamics regulated by Rho effector mDia1 attenuates pressure overload-induced cardiac hypertrophic responses and exacerbates dysfunction.

AIMS: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload and that Rho signalling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy. METHODS AND RESULTS: Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10-12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy-related genes, including immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the filamentous/globular (F/G)-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors and serum response factor were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs. CONCLUSION: mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.

Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion.

The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.