Role of microRNA-381 in bladder cancer growth and metastasis with the involvement of BMI1 and the Rho/ROCK axis.

BACKGROUND: Emerging evidence has noted the important participation of microRNAs (miRNAs) in several human diseases including cancer. This research was launched to probe the function of miR-381 in bladder cancer (BCa) progression. METHODS: Twenty-eight patients with primary BCa were included in this study. Cancer tissues and the adjacent normal tissues were obtained. Aberrantly expressed miRNAs in BCa tissues were analyzed using miRNA microarrays. miR-381 expression in the bladder and paired tumor tissues, and in BCa and normal cell lines was determined. The target relationship between miR-381 and BMI1 was predicted online and validated through a luciferase assay. Gain-of-functions of miR-381 and BMI1 were performed to identify their functions on BCa cell behaviors as well as tumor growth in vivo. The involvement of the Rho/ROCK signaling was identified. RESULTS: miR-381 was poor regulated in BCa tissues and cells (all p < 0.05). A higher miR-381 level indicated a better prognosis of patients with BCa. Artificial up-regulation of miR-381 inhibited proliferation, invasion, migration, resistance to apoptosis, and tumor formation ability of BCa T24 and RT4 cells (all p < 0.05). miR-381 was found to directly bind to BMI1 and was negatively correlated with BMI1 expression. Overexpression of BMI1 partially blocked the tumor suppressing roles of miR-381 in cell malignancy and tumor growth (all p < 0.05). In addition, miR-381 led to decreased RhoA phosphorylation and ROCK2 activation, which were also reversed by BMI1 (all p < 0.05). Artificial inhibition of the Rho/ROCK signaling blocked the functions of BMI1 in cell growth and metastasis (all p < 0.05). CONCLUSION: The study evidenced that miR-381 may act as a beneficiary biomarker in BCa patients. Up-regulation of miR-381 suppresses BCa development both in vivo and in vitro through BMI1 down-regulation and the Rho/ROCK inactivation.

Mutations in the Drosophila tricellular junction protein M6 synergize with RasV12 to induce apical cell delamination and invasion.

Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, we performed a genetic screen in Drosophila and discovered that mutations in the protein M6 synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, we observed that M6-deficient RasV12 clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, we show that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

NO-Releasing Nanoparticles Ameliorate Detrusor Overactivity in Transgenic Sickle Cell Mice via Restored NO/ROCK Signaling.

Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS-/-), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS-/- mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity. SIGNIFICANCE STATEMENT: Voiding abnormalities commonly affect patients with sickle cell disease (SCD) but are problematic to treat. Clarification of the science for this condition in an animal model of SCD may lead to improved interventions for it. Our findings suggest that novel topical delivery of a vasorelaxant agent nitric oxide into the bladder of these mice corrects overactive bladder by improving deranged bladder physiology regulatory signaling.

Cytoskeletal Associated Filamin A and RhoA Affect Neural Progenitor Specification During Mitosis.

Neural progenitor proliferation and cell fate decision from self-renewal to differentiation are crucial factors in determining brain size and morphology. The cytoskeletal dependent regulation of these processes is not entirely known. The actin-binding filamin A (FlnA) was shown to regulate proliferation of progenitors by directing changes in cell cycles proteins such as Cdk1 during G2/M phase. Here we report that functional loss of FlnA not only affects the rate of proliferation by altering cell cycle length but also causes a defect in early differentiation through changes in cell fate specification. FlnA interacts with Rho GTPase RhoA, and FlnA loss impairs RhoA activation. Disruption of either of these cytoskeletal associated proteins delays neurogenesis and promotes neural progenitors to remain in proliferative states. Aurora kinase B (Aurkb) has been implicated in cytokinesis, and peaks in expression during the G2/M phase. Inhibition of FlnA or RhoA impairs Aurkb degradation and alters its localization during mitosis. Overexpression of Aurkb replicates the same delay in neurogenesis seen with loss of FlnA or RhoA. Our findings suggest that shared cytoskeletal processes can direct neural progenitor proliferation by regulating the expression and localization of proteins that are implicated in the cell cycle progression and cell fate specification.

Heparan sulfate proteoglycan (HSPG) can take part in cell division: inside and outside.

Prior to the cytokinesis, the cell-matrix interactions should be disrupted, and the mitotic cells round up. Prerequisite of mitosis, the centrosomes duplicate, spindle fibers are generated and move away from each other to opposite sides of the cells marking the cell poles. Later, an invagination in the plasma membrane is formed a few minutes after anaphase. This furrow ingression is driven by a contractile actomyosin ring, whose assembly is regulated by RhoA GTPase. At the completion of cytokinesis, the two daughter cells are still connected by a thin intercellular bridge, which is subjected to abscission, as the terminal step of cytokinesis. Here, it is overviewed, how syndecan-4, a transmembrane, heparan sulfate proteoglycan, can contribute to these processes in a phosphorylation-dependent manner.

The Bardet-Biedl syndrome protein complex regulates cell migration and tissue repair through a Cullin-3/RhoA pathway.

Cell motility and migration play critical roles in various physiological processes and disease states. Here, we show that the BBBsome, a macromolecule composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1, is a critical determinant of cell migration and wound healing. Fibroblast cells derived from mice or humans harboring a homozygous missense mutation (BBS1M390R/M390R) that disrupt the BBSome exhibit defects in migration and wound healing. Furthermore, we demonstrate that BBS1M390R/M390R mice have significantly delayed wound closure. In line with this, we provide data suggesting that BBS1M390R/M390R fibroblasts have impaired platelet-derived growth factor-AA (PDGF) receptor-α signaling, a key regulator of directional cell migration acting as a chemoattractant during postnatal migration responses such as wound healing. In addition, we show that BBS1M390R/M390R fibroblasts have upregulated RhoA expression and activity. The relevance of RhoA upregulation is demonstrated by the ability of RhoA-kinase inhibitor Y27632 to partially rescue the migration defect of BBS1M390R/M390R fibroblasts cells. We also show that accumulation of RhoA protein in BBS1M390R/M390R fibroblasts cells is associated with reduction and inactivation of the ubiquitin ligase Cullin-3. Consistent with this, Cullin-3 inhibition with MLN4924 is sufficient to reduce migration of normal fibroblasts. These data implicate the BBSome in cell motility and tissue repair through a mechanism that involves PDGF receptor signaling and Cullin-3-mediated control of RhoA.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCß pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

The RhoA/ROCK pathway mediates high glucose-induced cardiomyocyte apoptosis via oxidative stress, JNK, and p38MAPK pathways.

AIMS: To understand the roles of the RhoA/ROCK and mitogen-activated protein kinase (MAPK) pathways in high glucose (HG)-induced apoptosis and oxidative stress in cardiomyocytes. MATERIALS AND METHODS: Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30 mmol/L D-glucose, in the presence or absence of fasudil (50 or 100 µM), SB203580, SP600125, or PD98059 (10 µM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl-2 mRNA levels were determined by quantitative real-time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl-2, and cleaved caspase-3 was analysed by Western blot. RESULTS: Fasudil, SB203580, and SP600125 effectively inhibited the HG-induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl-2 protein expression ratio, and cleaved caspase-3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl-2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG-induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK. CONCLUSIONS: The RhoA/ROCK pathway mediates HG-induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG-induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways.

Bistable front dynamics in a contractile medium: Travelling wave fronts and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions.

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.

Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation.

Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

[Effects of RhoA on the adherens junction of murine ameloblasts].

OBJECTIVE: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoADominant Negative (EGFP-RhoADN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects. METHODS: The enamel thickness of mandibular first molars of EGFP-RhoADN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoADN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoADN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. RESULTS: The EGFP-RhoADN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) µm vs. (106.24±0.24) µm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoADN transgenic mice molars were down-regulated, and the protein level of ß-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of ß-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of ß-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05. CONCLUSION: In the mandibular first molars of EGFP-RhoADN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoADN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.

A MAPK-Driven Feedback Loop Suppresses Rac Activity to Promote RhoA-Driven Cancer Cell Invasion.

Cell migration in 3D microenvironments is fundamental to development, homeostasis and the pathobiology of diseases such as cancer. Rab-coupling protein (RCP) dependent co-trafficking of α5ß1 and EGFR1 promotes cancer cell invasion into fibronectin (FN) containing extracellular matrix (ECM), by potentiating EGFR1 signalling at the front of invasive cells. This promotes a switch in RhoGTPase signalling to inhibit Rac1 and activate a RhoA-ROCK-Formin homology domain-containing 3 (FHOD3) pathway and generate filopodial actin-spike protrusions which drive invasion. To further understand the signalling network that drives RCP-driven invasive migration, we generated a Boolean logical model based on existing network pathways/models, where each node can be interrogated by computational simulation. The model predicted an unanticipated feedback loop, whereby Raf/MEK/ERK signalling maintains suppression of Rac1 by inhibiting the Rac-activating Sos1-Eps8-Abi1 complex, allowing RhoA activity to predominate in invasive protrusions. MEK inhibition was sufficient to promote lamellipodia formation and oppose filopodial actin-spike formation, and led to activation of Rac and inactivation of RhoA at the leading edge of cells moving in 3D matrix. Furthermore, MEK inhibition abrogated RCP/α5ß1/EGFR1-driven invasive migration. However, upon knockdown of Eps8 (to suppress the Sos1-Abi1-Eps8 complex), MEK inhibition had no effect on RhoGTPase activity and did not oppose invasive migration, suggesting that MEK-ERK signalling suppresses the Rac-activating Sos1-Abi1-Eps8 complex to maintain RhoA activity and promote filopodial actin-spike formation and invasive migration. Our study highlights the predictive potential of mathematical modelling approaches, and demonstrates that a simple intervention (MEK-inhibition) could be of therapeutic benefit in preventing invasive migration and metastasis.

Sensing of micropillars by osteoblasts involves complex intracellular signaling.

Topographical material surface features are sensed by cells and provoke a large range of cellular responses. We recognized earlier, that at micropillar topographies in the range of 5 µm, the osteoblasts attempt to phagocytize the pillars resulted in increased energy requirements and reduced osteoblast marker expression, e.g., collagen type I and osteocalcin. However, the precise cellular signaling transducing the topographic information into the cell and evoking phagocytic processes remained unknown. Here, we could show that the RhoA/ROCK signaling is involved in the transduction of the topography-mediated cellular reactions. After inhibition of ROCK-2 with Y27632 for 24 h, no caveolae-mediated micropillar assembly of the cell membrane domain component caveolin-1 (Cav-1) was found. ROCK inhibition was also able to attenuate the pillar-induced decrease in ß-actin. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibition with LY294002 for 24 h did not influence the Cav-1 clustering on micropillars. Our results illustrate the importance of the integrin down-stream signaling of RhoA/ROCK in the recognition of and adaption to surface microtopographies by osteoblasts and extend our understanding about the complex mechanism of action inside the cells.

Lower urinary tract symptoms/benign prostatic hypertrophy and vascular function: Role of the nitric oxide-phosphodiesterase type 5-cyclic guanosine 3',5'-monophosphate pathway.

It is well known that there is an association of lower urinary tract symptoms/benign prostatic hypertrophy with cardiovascular disease, suggesting that lower urinary tract symptoms/benign prostatic hypertrophy is a risk factor for cardiovascular events. Vascular function, including endothelial function and vascular smooth muscle function, is involved in the pathogenesis, maintenance and development of atherosclerosis, leading to cardiovascular events. Vascular dysfunction per se should also contribute to lower urinary tract symptoms/benign prostatic hypertrophy. Both lower urinary tract symptoms/benign prostatic hypertrophy and vascular dysfunction have cardiovascular risk factors, such as hypertension, dyslipidemia, diabetes mellitus, aging, obesity and smoking. Inactivation of the phosphodiesterase type 5-cyclic guanosine 3',5'-monophosphate-nitric oxide pathway causes lower urinary tract symptoms/benign prostatic hypertrophy through an enhancement of sympathetic nervous activity, endothelial dysfunction, increase in Rho-associated kinase activity and vasoconstriction, and decrease in blood flow of pelvic viscera. Both endogenous nitric oxide and exogenous nitric oxide act as vasodilators on vascular smooth muscle cells through an increase in the content of cyclic guanosine 3',5'-monophosphate, which is inactivated by phosphodiesterase type 5. In a clinical setting, phosphodiesterase type 5 inhibitors are widely used in patients with lower urinary tract symptoms/benign prostatic hypertrophy. Phosphodiesterase type 5 inhibitors might have beneficial effects on vascular function through not only inhibition of cyclic guanosine 3',5'-monophosphate degradation, but also increases in testosterone levels and nitric oxide bioavailability, increase in the number and improvement of the function of endothelial progenitor cells, and decrease in insulin resistance. In the present review, the relationships between lower urinary tract symptoms/benign prostatic hypertrophy, the phosphodiesterase type 5-nitric oxide-cyclic guanosine 3',5'-monophosphate pathway, vascular function and cardiovascular outcomes are examined.

Open questions: what about the 'other' Rho GTPases?

Rho GTPases have many and diverse roles in cell physiology, and some family members are very well studied, including RhoA, Rac1 and Cdc42. But many are relatively neglected, and fundamental questions about their mechanisms and functions remain open.

G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/ß-catenin signaling and are essential for head formation in Xenopus.

Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/ß-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/ß-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/ß-catenin signaling by promoting ß-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/ß-catenin signaling pathway.