TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation.
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ABSTRACT:
SUBMITTER: Kolliopoulos C
PROVIDER: S-EPMC7957764 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a "stellate"-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.
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Project description:Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co-receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F-actin in chemokine-treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN-depleted NK cells, chemokine-mediated or target cell-mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.
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Project description:This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).
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Project description:Aims and Hypothesis: NEDD9 is highly expressed in gastric cancer and has a significant involvement in its pathogenesis. However, the mechanism behind hypoxia-promoted cancer cell migration and its regulation because of NEDD9 is still unknown. The aim of this study is to investigate the involvement of NEDD9 in gastric cancer cell migration under hypoxia and explore the underlying potential molecular mechanisms.MethodsCell motility was measured by wound healing and transwell assay. NEDD9 and MICAL1 expressions were examined by western blot analysis. Interaction between NEDD9 and MICAL1 was assessed by immunohistochemistry and co-immunoprecipitation assay, respectively. Cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of NEDD9 and MICAL1. Rac1, Cdc42, and RhoA activation was assessed by pulldown assay.ResultsThe mRNA and protein level of NEDD9 increased as a result of hypoxia in gastric cancer cell lines BGC-823 and SGC-7901 while decreased levels of NEDD9 caused reduced cell migratory potential in response to hypoxia. Hypoxia also caused the enhancement of MICAL1 expression. Furthermore, it was revealed that there is a positive correlation between NEDD9 and MICAL1 protein while hypoxia played role in increasing their interaction. Under hypoxic conditions, silencing of NEDD9 caused reduction in the stability of MICAL1 protein, while depletion of MICAL1 also inhibited the migration of NEDD9-overexpressing gastric cancer cells. In addition, silencing of NEDD9 or MICAL1 expression reversed the increased GTP forms of Rac1 and Cdc42 in hypoxic cells. However, only the upregulation of Rac1-GTP level was observed in gastric cancer cells that were already overexpressed by MICAL1.ConclusionIn all, it is concluded that MICAL1 is regulated by NEDD9 that facilitates hypoxia-induced gastric cancer cell migration via Rac1-dependent manner.
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Project description:Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.
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Project description:In the developing cortex, excitatory neurons migrate along the radial fibers to their final destinations and build up synaptic connection with each other to form functional circuitry. The shaping of neuronal morphologies by actin cytoskeleton dynamics is crucial for neuronal migration. However, it is largely unknown how the distribution and assembly of the F-actin cytoskeleton are coordinated. In the present study, we found that an actin regulatory protein, coronin 2B, is indispensable for the transition from a multipolar to bipolar morphology during neuronal migration in ICR mice of either sex. Loss of coronin 2B led to heterotopic accumulation of migrating neurons in the intermediate zone along with reduced dendritic complexity and aberrant neuronal activity in the cortical plate. This was accompanied by increased seizure susceptibility, suggesting the malfunction of cortical development in coronin 2B-deficient brains. Coronin 2B knockdown disrupted the distribution of the F-actin cytoskeleton at the leading processes, while the migration defect in coronin 2B-deficient neurons was partially rescued by overexpression of Rac1 and its downstream actin-severing protein, cofilin. Our results collectively reveal the physiological function of coronin 2B during neuronal migration whereby it maintains the proper distribution of activated Rac1 and the F-actin cytoskeleton.SIGNIFICANCE STATEMENT Deficits in neuronal migration during cortical development result in various neurodevelopmental disorders (e.g., focal cortical dysplasia, periventricular heterotopia, epilepsy, etc.). Most signaling pathways that control neuronal migration process converge to regulate actin cytoskeleton dynamics. Therefore, it is important to understand how actin dynamics is coordinated in the critical processes of neuronal migration. Herein, we report that coronin 2B is a key protein that regulates neuronal migration through its ability to control the distribution of the actin cytoskeleton and its regulatory signaling protein Rac1 during the multipolar-bipolar transition in the intermediate zone, providing insights into the molecular machinery that drives the migration process of newborn neurons.
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Project description:Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.
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Project description:The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.
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Project description:P66Shc and Rac1 proteins are responsible for tumor-associated inflammation, particularly in brain tumors characterized by elevated oxidative stress and increased reactive oxygen species (ROS) production. Quercetin, a natural polyphenolic flavonoid, is a well-known redox modulator with anticancer properties. It has the capacity to cross the blood-brain barrier and, thus, could be a possible drug against brain tumors. In this study, we explored the effect of quercetin on Rac1/p66Shc-mediated tumor cell inflammation, which is the principal pathway for the generation of ROS in brain cells. Glioma cells transfected with Rac1, p66Shc, or both were treated with varying concentrations of quercetin for different time points. Quercetin significantly reduced the viability and migration of cells in an ROS-dependent manner with the concomitant inhibition of Rac1/p66Shc expression and ROS production in naïve and Rac1/p66Shc-transfected cell lines, suggestive of preventing Rac1 activation. Through molecular docking simulations, we observed that quercetin showed the best binding compared to other known Rac1 inhibitors and specifically blocked the GTP-binding site in the A-loop of Rac1 to prevent GTP binding and, thus, Rac1 activation. We conclude that quercetin exerts its anticancer effects via the modulation of Rac1-p66Shc signaling by specifically inhibiting Rac1 activation, thus restraining the production of ROS and tumor growth.
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Project description:Background:Macrophage-derived foam cells play a central role in atherosclerosis, and their ultimate fate includes apoptosis, promotion of vascular inflammation, or migration to other tissues. N?-Carboxymethyl-lysine (CML), the key active component of advanced glycation end products, induced foam cell formation and apoptosis. Previous studies have shown that the Vav1/Rac1 pathway affects the macrophage cytoskeleton and cell migration, but its role in the pathogenesis of diabetic atherosclerosis is unknown. Methods and Results:In this study, we used anterior tibiofibular vascular samples from diabetic foot amputation patients and accident amputation patients, and histological and cytological tests were performed using a diabetic ApoE-/- mouse model and primary peritoneal macrophages, respectively. The results showed that the atherosclerotic plaques of diabetic foot amputation patients and diabetic ApoE-/- mice were larger than those of the control group. Inhibition of the Vav1/Rac1 pathway reduced vascular plaques and promoted the migration of macrophages to lymph nodes. Transwell and wound healing assays showed that the migratory ability of macrophage-derived foam cells was inhibited by CML. Cytoskeletal staining showed that advanced glycation end products inhibited the formation of lamellipodia in foam cells, and inhibition of the Vav1/Rac1 pathway restored the formation of lamellipodia. Conclusion:CML inhibits the migration of foam cells from blood vessels via the Vav1/Rac1 pathway, and this process affects the formation of lamellipodia.
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