RhoA Activation Research Articles

RhoA-LIMK Signaling Axis Reveals Rostral-Caudal Plane and Spatial Dysregulation in the Brain of Alzheimer's Disease Mouse Models.

RhoA signaling is widely reported to be dysregulated in Alzheimer's disease (AD), but its therapeutic targeting demonstrated mixed outcomes. We hypothesize that the activation and inactivation states of RhoA and LIMK are different in the cortex and in subregions of hippocampus along the rostral-caudal dimensions. We intended to elucidate the plane and spatial dependent RhoA signaling in association with AD. We applied antibody pRhoA that recognizes an inactive state of RhoA (S188 phosphorylation) and antibody pLIMK against an active state of LIMK (T508 phosphorylation) to investigate RhoA signaling in wildtype (WT) and triple transgenic AD (3xTg-AD) mouse model. We prepared serial sections from the rostral to caudal coronal planes of the entire mouse brain followed by immunofluorescence staining with pRhoA and pLIMK antibodies. Both pRhoA and pLIMK elicited a shift of expression pattern from rostral to caudal planes. Additionally, pRhoA demonstrated dynamic redistribution between the nucleus and cytoplasm. pLIMK did not show such nucleus and cytoplasm redistribution but the expression level was changed from rostral to caudal planes. At some planes, pRhoA showed an increasing trend in expression in the cortex but a decreasing trend in the dentate gyrus of the 3xTg-AD mouse hippocampus. pLIMK tends to decrease in the cortex but increase in the dentate gyrus of 3xTg-AD mouse hippocampus. RhoA activation is dysregulated in both human and mouse AD brains, and the RhoA-LIMK signaling axis reveals spatial dysregulation along the rostral-caudal plane dimensions.

Vav2 promotes ductus arteriosus anatomic closure via the remodeling of smooth muscle cells by Rac1 activation.

The ductus arteriosus (DA), bridging the aorta and pulmonary artery, immediately starts closing after birth. Remodeling of DA leads to anatomic obstruction to prevent repatency. Several histological changes, especially extracellular matrices (ECMs) deposition and smooth muscle cells (SMCs) migration bring to anatomic closure. The genetic etiology and mechanism of DA closure remain elusive. We have previously reported a novel copy number variant containing Vav2 in patent ductus arteriosus (PDA) patients, but its specific role in DA closure remains unknown. The present study revealed that the expression of Vav2 was reduced in human patent DA, and it was less enrichment in the adjacent aorta. Matrigel experiments demonstrated that Vav2 could promote SMC migration from PDA patient explants. Smooth muscle cells with Vav2 overexpression also presented an increased capacity in migration and downregulated contractile-related proteins. Meanwhile, SMCs with Vav2 overexpression exhibited higher expression of collagen III and lessened protein abundance of lysyl oxidase, and both changes are beneficial to DA remodeling. Overexpression of Vav2 resulted in increased activity of Rac1, Cdc42, and RhoA in SMCs. Further investigation noteworthily found that the above alterations caused by Vav2 overexpression were particularly reversed by Rac1 inhibitor. A heterozygous, rare Vav2 variant was identified in PDA patients. Compared with the wild type, this variant attenuated Vav2 protein expression and weakened the activation of downstream Rac1, further impairing its functions in SMCs. In conclusion, Vav2 functions as an activator for Rac1 in SMCs to promote SMCs migration, dedifferentiation, and ECMs production. Deleterious variant potentially induces Vav2 loss of function, further providing possible molecular mechanisms about Vav2 in PDA pathogenesis. These findings enriched the current genetic etiology of PDA, which may provide a novel target for prenatal diagnosis and treatment. KEY MESSAGES: Although we have proposed the potential association between Vav2 and PDA incidence through whole exome sequencing, the molecular mechanisms underlying Vav2 in PDA have never been reported.This work, for the first time, demonstrated that Vav2 was exclusively expressed in closed DAs. Moreover, we found that Vav2 participated in the process of anatomic closure by mediating SMCs migration, dedifferentiation, and ECMs deposition through Rac1 activation. Our findings first identified a deleterious Vav2 c.701C>T variant that affected its function in SMCs by impairing Rac1 activation, which may lead to PDA defect. Vav2 may become an early diagnosis and an effective intervention target for PDA clinical therapy.

THU412 Role of Glucocorticoid On Bone Metabolism Through AKAP13

Abstract Disclosure: K. Igarashi: None. A. Ishida: None. H. Koide: None. K. Yokote: None. It is not completely clear to what extent glucocorticoids are involved in which stages and sites of bone differentiation, development, and maintenance. we investigated the mechanism of glucocorticoid regulation through the regulation of Rho activation using a regulatory system for the expression of the RhoA activator AKAP13. To examine the effect of glucocorticoids on preosteoblasts, immortalized bone marrow mesenchymal stem cells (imBMSCs) were sown in 6-well plates at a concentration of 2x105/well and treated with various concentrations (0-1000 nM) of dexamethasone (Dex) 24 hr later, and The expression of GILZ, osteoblast and adipocyte-associated markers were evaluated using qRT-PCR. AKAP13 expression vector and AKAP13 siRNA were used to examine the response to Dex. GILZ was induced in a Dex-dependent manner on both day1 and day4. In the differentiation phase, PPARG2 expression was not changed by Dex, but GILZ was induced in a similar Dex-dependent manner. In proliferating imBMSCs, Dex treatment tended to enhance PPARG2 expression in a dose-dependent manner by introducing AKAP13 expression vectors. On the other hand, AKAP13 gene expression was significantly down-regulated in imBMSCs treated with AKAP13 siRNA, and Alp2 mRNA was up-regulated in imBMSCs treated with Dex compared to those not treated with Dex. It has a nuclear receptor binding domain and is known to be involved in osteogenesis. On the other hand, AKAP13 expression was decreased by osteoblast differentiation in an in vitro system, suggesting that AKAP13 may influence the regulation of GC action between the proliferative and differentiation phases of BMSCs. Presentation: Thursday, June 15, 2023

Phosphoprotein phosphatase activity positively regulates oligomeric pyrin to trigger inflammasome assembly in phagocytes.

Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like Yersinia's YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with Yersinia using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.

Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

Breakdown of endothelial barrier integrity determines organ dysfunction and outcome of patients with sepsis. Increased levels of soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We provide evidence for a causative role of sVE-cadherin to induce loss of endothelial barrier function. In patients with sepsis, sVE-cadherin levels were associated with organ dysfunction and the need for volume resuscitation. Similarly, LPS-induced systemic inflammation in rats with microvascular dysfunction was paralleled by augmented sVE-cadherin levels. Newly generated recombinant human sVE-cadherin (extracellular domains EC1-5) induced loss of endothelial barrier function in both human microvascular endothelial cells invitro and in rat mesenteric microvessels invivo and reduced microcirculatory flow. sVE-cadherinEC1-5 disturbed VE-cadherin-mediated adhesion and perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction resulting in RhoGEF1-mediated RhoA activation. VE-PTP inhibitor AKB9778 and Rho-kinase inhibitor Y27632 blunted all sVE-cadherinEC1-5-induced effects, which uncovers a pathophysiological role of sVE-cadherin via dysbalanced VE-PTP/RhoA signaling.

Hypoxia-induced TMTC3 expression in esophageal squamous cell carcinoma potentiates tumor angiogenesis through Rho GTPase/STAT3/VEGFA pathway

BackgroundHypoxia is one of most typical features in the tumor microenvironment of solid tumor and an inducer of endoplasmic reticulum (ER) stress, and HIF-1α functions as a key transcription factor regulator to promote tumor angiogenesis in the adaptive response to hypoxia. Increasing evidence has suggested that hypoxia plays an important regulatory role of ER homeostasis. We previously identified TMTC3 as an ER stress mediator under nutrient-deficiency condition in esophageal squamous cell carcinoma (ESCC), but the molecular mechanism in hypoxia is still unclear.MethodsRNA sequencing data of TMTC3 knockdown cells and TCGA database were analyzed to determine the association of TMTC3 and hypoxia. Moreover, ChIP assay and dual-luciferase reporter assay were performed to detect the interaction of HIF-1α and TMTC3 promoter. In vitro and in vivo assays were used to investigate the function of TMTC3 in tumor angiogenesis. The molecular mechanism was determined using co-immunoprecipitation assays, immunofluorescence assays and western blot. The TMTC3 inhibitor was identified by high-throughput screening of FDA-approved drugs. The combination of TMTC3 inhibitor and cisplatin was conducted to confirm the efficiency in vitro and in vivo.ResultsThe expression of TMTC3 was remarkably increased under hypoxia and regulated by HIF-1α. Knockdown of TMTC3 inhibited the capability of tumor angiogenesis and ROS production in ESCC. Mechanistically, TMTC3 promoted the production of GTP through interacting with IMPDH2 Bateman domain. The activity of Rho GTPase/STAT3, regulated by cellular GTP levels, decreased in TMTC3 knockdown cells, whereas reversed by IMPDH2 overexpression. Additionally, TMTC3 regulated the expression of VEGFA through Rho GTPase/STAT3 pathway. Allopurinol inhibited the expression of TMTC3 and further reduced the phosphorylation and activation of STAT3 signaling pathway in a dose-dependent manner in ESCC. Additionally, the combination of allopurinol and cisplatin significantly inhibited the cell viability in vitro and tumor growth in vivo, comparing with single drug treatment, respectively.ConclusionsCollectively, our study clarified the molecular mechanism of TMTC3 in regulating tumor angiogenesis and highlighted the potential therapeutic combination of TMTC3 inhibitor and cisplatin, which proposed a promising strategy for the treatment of ESCC.

Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants.

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and –LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and –LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO–K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and –LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors.

TIPE2 ameliorates neuroinflammation and cognitive impairment in sepsis-associated encephalopathy through regulating RhoA/ROCK2–NF-κB signaling pathway

Sepsis-associated encephalopathy (SAE) is an acute brain dysfunction induced by systemic inflammation caused by sepsis and is one of the most common types of encephalopathy in intensive care units. Deteriorative neuroinflammation is closely related to the development of brain injury, which often transforms into common pathological manifestations in patients with severe sepsis. Therefore, taking necessary preventive and protective measures for potential brain injury and promptly reducing neuroinflammatory injury is necessary to improve the long-term prognoses of patients. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) can play a significant protective role in septic lung injury, but studies on its expression and role in neurological diseases are rare. In the present study, we found that TIPE2 can expressed in microglia and ameliorate brain injury caused by SAE by suppressing neuroinflammation. The RhoA/ROCK2 pathway is the central coordinator of tissue injury response, and the activation of RhoA participates in the lipopolysaccharide-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway. The activation of RhoA and phosphorylation of NF-κB was enhanced after TIPE2 deficiency. Importantly, TIPE2 negatively regulates inflammatory responses in vivo and in vitro and plays a protective role in SAE by inhibiting the activation of RhoA/ROCK2–NF-κB signaling pathways. The ultimate aim of our proposed project is to provide a theoretical basis for the development of a novel strategy for the early prevention and therapy of SAE.

Genome-Wide CRISPR Screens Identify Multiple Synthetic Lethal Targets That Enhance KRASG12C Inhibitor Efficacy.

Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005.

Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells.

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

CTNNAL1 deficiency suppresses CFTR expression in HDM-induced asthma mouse model through ROCK1-CAL signaling pathway.

The downregulation of adhesion molecule catenin alpha-like 1 (CTNNAL1) in airway epithelial cells of asthma patients and house dust mite (HDM)-induced asthma animal models was illustrated in our previous study. It is assumed to contribute to airway inflammation and mucus hypersecretion. In this work, we further explore the underlying mechanism of CTNNAL1 in asthma. CTNNAL1-silenced female mice exhibit a decreased level of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated and ATP-gated Cl - channel that correlates with mucus hypersecretion. Our previous study demonstrated that ROCK1 expression decreases but ROCK2 expression increases in the lungs of a CTNNAL1-silenced mouse model. Inhibition of ROCK1 leads to a reduction in CFTR expression in CTNNAL1-overexpressing and CTNNAL1-silenced human bronchial epithelial (HBE) cells. It has been reported that ROCK1 is a downstream target of RhoA and that activation of RhoA increases CFTR expression after CTNNAL1 deficiency in vitro and in vivo. The above results indicate that CTNNAL1 regulates CFTR expression through the ROCK1 pathway. In addition, the expression of CFTR-associated ligand (CAL) is increased after CTNNAL1 silencing, and immunoprecipitation results confirm the interaction between ROCK1 and CAL. Inhibition of CAL does not influence ROCK1 expression but increases CFTR expression in CTNNAL1-silenced HBE cells. These data suggest that CTNNAL1 deficiency decreases CFTR expression in the HDM-induced asthma mouse model through the ROCK1-CAL signaling pathway.

Ouabain Induces Transcript Changes and Activation of RhoA/ROCK Signaling in Cultured Epithelial Cells (MDCK).

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Cingulin regulates hair cell cuticular plate morphology and is required for hearing in human and mouse.

Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.

Intracellular signaling pathways of muscarinic acetylcholine receptor-mediated detrusor muscle contractions.

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gαi-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gαi and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are of utmost importance in physiological regulation of micturition and also in the development of voiding disorders. We demonstrate that the RhoA-Rho-associated kinase (ROCK) pathway plays a crucial role in contractions induced by cholinergic stimulation in detrusor muscle. Activation of RhoA is mediated by both M2 and M3 receptors as well as by Gi but not Gq/11 proteins. The Gi-RhoA-ROCK pathway may provide a novel therapeutic target for overactive voiding disorders.

Role of Rho/MRTF in Aggressive Vemurafenib-Resistant Murine Melanomas and Immune Checkpoint Upregulation.

Cutaneous melanoma is the deadliest skin cancer. Most have Ras-MAPK pathway (BRAFV600E or NRAS) mutations and highly effective targeted therapies exist; however, they and immune therapies are limited by resistance, in part driven by small GTPase (Rho and Rac) activation. To facilitate preclinical studies of combination therapies to provide durable responses, we describe the first mouse melanoma lines resistant to BRAF inhibitors. Treatment of mouse lines, YUMM1.7 and YUMMER, with vemurafenib (Vem), the BRAFV600E-selective inhibitor, resulted in high-level resistance (IC50 shifts 20-30-fold). Resistant cells showed enhanced activation of Rho and the downstream transcriptional coactivator, myocardin-related transcription factor (MRTF). Resistant cells exhibited increased stress fibers, nuclear translocation of MRTF-A, and an increased MRTF-A gene signature. Pharmacological inhibition of the Rho/MRTF pathway using CCG-257081 reduced viability of resistant lines and enhanced sensitivity to Vem. Remarkably, co-treatment of parental lines with Vem and CCG-257081 eliminated resistant colony development. Resistant cells grew more slowly in vitro, but they developed highly aggressive tumors with a shortened survival of tumor-bearing mice. Increased expression of immune checkpoint inhibitor proteins (ICIs) in resistant lines may contribute to aggressive in vivo behavior. Here, we introduce the first drug-resistant mouse melanoma models for assessing combinations of targeted and immune therapies.

Tensin-3 is involved in osteogenic versus adipogenic fate of human bone marrow stromal cells

BackgroundThe tightly controlled balance between osteogenic and adipogenic differentiation of human bone marrow-derived stromal cells (BMSCs) is critical to maintain bone homeostasis. Age-related osteoporosis is characterized by low bone mass with excessive infiltration of adipose tissue in the bone marrow compartment. The shift of BMSC differentiation from osteoblasts to adipocytes could result in bone loss and adiposity.MethodsTNS3 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of TNS3 was used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining at multiple time points. The role of TNS3 and its domain function in osteogenic differentiation were evaluated by ALP activity, calcium assay, and Alizarin Red S staining. The expression of Rho-GTP was determined using the RhoA pull-down activation assay.ResultsLoss of TNS3 impaired osteogenic differentiation of BMSCs but promoted adipogenic differentiation. Conversely, TNS3 overexpression hampered adipogenesis while enhancing osteogenesis. The expression level of TNS3 determined cell shape and cytoskeletal reorganization during osteogenic differentiation. TNS3 truncation experiments revealed that for optimal osteogenesis to occur, all domains proved essential. Pull-down and immunocytochemical experiments suggested that TNS3 mediates osteogenic differentiation through RhoA.ConclusionsHere, we identify TNS3 to be involved in BMSC fate decision. Our study links the domain structure in TNS3 to RhoA activity via actin dynamics and implicates an important role for TNS3 in regulating osteogenesis and adipogenesis from BMSCs. Furthermore, it supports the critical involvement of cytoskeletal reorganization in BMSC differentiation.

Mechanosensitive accumulation of non-muscle myosin IIB during mitosis requires its translocation activity

Non-muscle myosin II (NMII) is a force-generating mechanosensitive enzyme that responds to mechanical forces. NMIIs mechanoaccumulate at the cell cortex in response to mechanical forces. It is essential for cells to mechanically adapt to the physical environment, failure of which results in mitotic defects when dividing in confined environment. Much less is known about how NMII mechanoaccumulation is regulated during mitosis. We show that mitotic cells respond to compressive stress by promoting accumulation of active RhoA at the cell cortex as in interphase cells. RhoA mechanoresponse during mitosis activates and stabilizes NMIIB via ROCK signaling, leading to NMIIB mechanoaccumulation at the cell cortex. Using disease-related myosin II mutations, we found that NMIIB mechanoaccumulation requires its motor activity that translocates actin filaments, but not just its actin-binding function. Thus, the motor activity coordinates structural movement and nucleotide state changes to fine-tune actin-binding affinity optimal for NMIIs to generate and respond to forces.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs invitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI invivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Yes-associated protein regulates cortical actin architecture and dynamics through intracellular translocation of Rho GTPase-activating protein 18.

Yes-associated protein (YAP) is a transcriptional co-activator that controls the transcription of target genes and modulates the structures of various cytoskeletal architecture as mechanical responses. Although it has been known that YAP regulates actin-regulatory proteins, the detailed molecular mechanism of how they control and coordinate intracellular actin architecture remains elusive. Herein, we aimed to examine the structure and dynamics of intracellular actin architecture from molecular to cellular scales in normal and YAP-knockout (YAP-KO) cells utilizing high-speed atomic force microscopy (HS-AFM) for live-cell imaging and other microscope-based mechanical manipulation and measurement techniques. YAP-KO Madin-Darby canine kidney cells had a higher density and turnover of actin filaments in the cell cortex and a higher elastic modulus. Laser aberration assay demonstrated that YAP-KO cells were more resistant to damage than normal cells. We also found that Rho GTPase-activating protein 18 (ARHGAP18), a downstream factor of YAP, translocated from the cortex to the edge of sparsely cultured YAP-KO cells. It resulted in high RhoA activity and promotion of actin polymerization in the cell cortex and their reductions at the edge. HS-AFM imaging of live cell edge and a cell-migration assay demonstrated lower membrane dynamics and motility of YAP-KO cells than those of normal cells, suggesting lower actin dynamics at the edge. Together, these results demonstrate that a YAP-dependent pathway changes the intracellular distribution of RhoGAP and modulates actin dynamics in different parts of the cell, providing a mechanistic insight into how a mechano-sensitive transcription cofactor regulates multiple intracellular actin architecture and coordinates mechano-responses.

Integrin β3 directly inhibits the Gα13-p115RhoGEF interaction to regulate G protein signaling and platelet exocytosis

The integrins and G protein-coupled receptors are both fundamental in cell biology. The cross talk between these two, however, is unclear. Here we show that β3 integrins negatively regulate G protein-coupled signaling by directly inhibiting the Gα13-p115RhoGEF interaction. Furthermore, whereas β3 deficiency or integrin antagonists inhibit integrin-dependent platelet aggregation and exocytosis (granule secretion), they enhance G protein-coupled RhoA activation and integrin-independent secretion. In contrast, a β3-derived Gα13-binding peptide or Gα13 knockout inhibits G protein-coupled RhoA activation and both integrin-independent and dependent platelet secretion without affecting primary platelet aggregation. In a mouse model of myocardial ischemia/reperfusion injury in vivo, the β3-derived Gα13-binding peptide inhibits platelet secretion of granule constituents, which exacerbates inflammation and ischemia/reperfusion injury. These data establish crucial integrin-G protein crosstalk, providing a rationale for therapeutic approaches that inhibit exocytosis in platelets and possibly other cells without adverse effects associated with loss of cell adhesion.