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Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes ( MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2 ) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2 , DLC1 , or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2 - L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets. Nephrotic syndrome is the second most common chronic kidney disease but there are no targeted treatment strategies available. Here the authors identify mutations of six genes codifying for proteins involved in cytoskeleton remodelling and modulation of small GTPases in 17 families with nephrotic syndrome.

Abstract BACKGROUND More than a million Americans harbor a cerebral cavernous angioma (CA), and those who suffer a prior symptomatic hemorrhage have an exceptionally high rebleeding risk. Preclinical studies show that atorvastatin blunts CA lesion development and hemorrhage through inhibiting RhoA kinase (ROCK), suggesting it may confer a therapeutic benefit. OBJECTIVE To evaluate whether atorvastatin produces a difference compared to placebo in lesional iron deposition as assessed by quantitative susceptibility mapping (QSM) on magnetic resonance imaging in CAs that have demonstrated a symptomatic hemorrhage in the prior year. Secondary aims shall assess effects on vascular permeability, ROCK activity in peripheral leukocytes, signal effects on clinical outcomes, adverse events, and prespecified subgroups. METHODS The phase I/IIa placebo-controlled, double-blinded, single-site clinical trial aims to enroll 80 subjects randomized 1-1 to atorvastatin (starting dose 80 mg PO daily) or placebo. Dosing shall continue for 24-mo or until reaching a safety endpoint. EXPECTED OUTCOMES The trial is powered to detect an absolute difference of 20% in the mean percent change in lesional QSM per year (2-tailed, power 0.9, alpha 0.05). A decrease in QSM change would be a signal of potential benefit, and an increase would signal a safety concern with the drug. DISCUSSION With firm mechanistic rationale, rigorous preclinical discoveries, and biomarker validations, the trial shall explore a proof of concept effect of a widely used repurposed drug in stabilizing CAs after a symptomatic hemorrhage. This will be the first clinical trial of a drug aimed at altering rebleeding in CA.

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.

Triple-negative breast cancer is an aggressive subtype of breast cancer with poor clinical outcomes and poor prognosis. Hesperetin is an active component extracted from Citrus fruits and Traditional Chinese Medicine has a wide range of pharmacological effects. Here, we assessed the anti-migration and anti-invasive effects and explored inhibitory mechanisms of hesperetin on metastasis of human triple negative breast cancer MDA-MB-231 cells. Cell viability experiments revealed that 200 μM hesperetin has a clear inhibitory effect on MDA-MB-231 cells. TGF-β1 treatment induces apparent tumor progression in MDA-MB-231 cells including aberrant wound-healing and invasion ability, which is effectively suppressed by hesperetin co-treatment. Additionally, hesperetin inhibited the TGF-β1-mediated actin stress fiber formation. Western blot results showed that hesperetin suppressed the TGF-β1-mediated (i) activation of Fyn, (ii) phosphorylation of paxillin at Y31, Y88, and Y118 sites, (iii) the increased expression of RhoA, and (iv) activation of Rho-kinase. We demonstrated the increased interaction of Fyn with paxillin and RhoA protein in the TGF-β1-induced metastasis of MDA-MB-231 cells. Small interfering RNA Fyn inhibited phosphorylation of paxillin (Y31) and activation of Rho-kinase induced by TGF-β1. In conclusion, hesperetin has a significant inhibitory effect on migration and invasion of MDA-MB-231 cells induced by TGF-β1, which might be attributed to inhibiting the Fyn/paxillin/RhoA pathway.

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition. 16p11.2 CNVs are associated with neurodevelopmental disorders. Here, the authors show that 16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.

RhoA is a ubiquitously expressed cytoplasmic protein that belongs to the family of small GTPases. RhoA acts as a molecular switch that is activated in response to binding of chemokines, cytokines, and growth factors, and via mDia and the ROCK signaling cascade regulates the activation of cytoskeletal proteins, and other factors. This review aims to summarize our current knowledge on the role of RhoA as a general key regulator of immune cell differentiation and function. The contribution of RhoA for the primary functions of innate immune cell types, namely neutrophils, macrophages, and conventional dendritic cells (DC) to (i) get activated by pathogen-derived and endogenous danger signals, (ii) migrate to sites of infection and inflammation, and (iii) internalize pathogens has been fairly established. In activated DC, which constitute the most potent antigen-presenting cells of the immune system, RhoA is also important for the presentation of pathogen-derived antigen and the formation of an immunological synapse between DC and antigen-specific T cells as a prerequisite to induce adaptive T cell responses. In T cells and B cells as the effector cells of the adaptive immune system Rho signaling is pivotal for activation and migration. More recently, mutations of Rho and Rho-modulating factors have been identified to predispose for autoimmune diseases and as causative for hematopoietic malignancies.

Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro , RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo , through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.

A multifaceted and widely prevalent neurodegenerative disease, Parkinson's disease (PD) is typified by the loss of dopaminergic neurons in the midbrain. The discovery of novel treatment(s) that can reverse or halt the course of the disease progression along with identifying the most reliable biomarker(s) in PD remains the crucial concern. RhoA in its active state has been demonstrated to interact with three distinct domains located in the central coiled-coil region of ROCK. RhoA appears to activate effectors most frequently by breaking the intramolecular autoinhibitory connections, which releases functional domains from the effector protein. Additionally, RhoA is highly expressed in the nervous system and it acts as a central molecule for its several downstream effector proteins in multiple signalling pathways both in neurons and glial cells. Mitochondrial dysfunction, vesicle transport malfunction and aggregation of α-Synuclein, a presynaptic neuronal protein genetically and neuropathologically associated with PD. While the RhoA-ROCK signalling pathway appears to have a significant role in PD symptoms, suggesting it could be a promising target for therapeutic interventions. Thus, this review article addresses the potential involvement of the RhoA-ROCK signalling system in the pathophysiology of neurodegenerative illnesses, with an emphasis on its biology and function. We also provide an overview of the state of research on RhoA regulation and its downstream biological activities, focusing on the role of RhoA signalling in neurodegenerative illnesses and the potential benefits of RhoA inhibition as a treatment for neurodegeneration. Graphical abstract

Hippo effectors YAP/TAZ act as on–off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues. The transcriptional co-activator YAP is known to operate downstream of mechanical signals arising from the cell niche. Here the authors demonstrate that YAP controls cell mechanics, force development and adhesion strength by promoting the transcription of genes related to focal adhesions.