Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer.

Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.

PI3K couples long-term synaptic potentiation with cofilin recruitment and actin polymerization in dendritic spines via its regulatory subunit p85α.

Long-term synaptic plasticity is typically associated with morphological changes in synaptic connections. However, the molecular mechanisms coupling functional and structural aspects of synaptic plasticity are still poorly defined. The catalytic activity of type I phosphoinositide-3-kinase (PI3K) is required for specific forms of synaptic plasticity, such as NMDA receptor-dependent long-term potentiation (LTP) and mGluR-dependent long-term depression (LTD). On the other hand, PI3K signaling has been linked to neuronal growth and synapse formation. Consequently, PI3Ks are promising candidates to coordinate changes in synaptic strength with structural remodeling of synapses. To investigate this issue, we targeted individual regulatory subunits of type I PI3Ks in hippocampal neurons and employed a combination of electrophysiological, biochemical and imaging techniques to assess their role in synaptic plasticity. We found that a particular regulatory isoform, p85α, is selectively required for LTP. This specificity is based on its BH domain, which engages the small GTPases Rac1 and Cdc42, critical regulators of the actin cytoskeleton. Moreover, cofilin, a key regulator of actin dynamics that accumulates in dendritic spines after LTP induction, failed to do so in the absence of p85α or when its BH domain was overexpressed as a dominant negative construct. Finally, in agreement with this convergence on actin regulatory mechanisms, the presence of p85α in the PI3K complex determined the extent of actin polymerization in dendritic spines during LTP. Therefore, this study reveals a molecular mechanism linking structural and functional synaptic plasticity through the coordinate action of PI3K catalytic activity and a specific isoform of the regulatory subunits.

CED-5/CED-12 (DOCK/ELMO) can promote and inhibit F-actin formation via distinct motifs that may target different GTPases.

Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. We interfered with GEF function by interfering with CED-5's ability to bind Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies strongly support that the GAP function likely acts on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.

Dermatan Sulfate Affects the Activation of the Necroptotic Effector MLKL in Breast Cancer Cell Lines via the NFκB Pathway and Rac-Mediated Oxidative Stress.

Dermatan sulfate (DS) is a glycosaminoglycan characterized by having a variable structure and wide distribution in animal tissues. We previously demonstrated that some structural variants of DS were able to rapidly induce moderate necroptosis in luminal breast cancer cells when used at a high concentration. We have now investigated the mechanisms underlying the DS-mediated activation of the necroptotic executor MLKL using immunofluorescence, Western blotting and pharmacological inhibition. The two main processes, by which DS influences the phosphorylation of MLKL, are the activation of NFκB, which demonstrates a suppressive impact, and the induction of oxidative stress, which has a stimulatory effect. Moreover, the triggering of the redox imbalance by DS occurs via the modulatory influence of this glycosaminoglycan on the rearrangement of the actin cytoskeleton, requiring alterations in the activity of small Rho GTP-ase Rac1. All of these processes that were elicited by DS in luminal breast cancer cells showed a dependence on the structure of this glycan and the type of cancer cells. Furthermore, our results suggest that a major mechanism that is involved in the stimulation of necroptosis in luminal breast cancer cells by high doses of DS is mediated via the effect of this glycan on the activity of adhesion molecules.

Salicylate induces epithelial actin reorganization via activation of the AMP-activated protein kinase and promotes wound healing and contraction in mice.

Wounds that occur in adults form scars due to fibrosis, whereas those in embryos regenerate. If wound healing in embryos is mimicked in adults, scarring can be reduced. We found that mouse fetuses could regenerate tissues up to embryonic day (E) 13, but visible scars remained thereafter. This regeneration pattern requires actin cable formation at the epithelial wound margin via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). Here, we investigated whether the AMPK-activating effect of salicylate, an anti-inflammatory drug, promotes regenerative wound healing. Salicylate administration resulted in actin cable formation and complete wound regeneration in E14 fetuses, in which scarring should have normally occurred, and promoted contraction of the panniculus carnosus muscle, resulting in complete wound regeneration. In vitro, salicylate further induced actin remodeling in mouse epidermal keratinocytes in a manner dependent on cell and substrate target-specific AMPK activation and subsequent regulation of Rac1 signaling. Furthermore, salicylate promoted epithelialization, enhanced panniculus carnosus muscle contraction, and inhibited scar formation in adult mice. Administration of salicylates to wounds immediately after injury may be a novel method for preventing scarring by promoting a wound healing pattern similar to that of embryonic wounds.

Identification and characterization of RAC1-related immune and prognostic subtypes of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant tumor with significant variability in prognosis among patients. Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key focus in the area of cancer research. However, the molecular mechanisms of RAC1 in HCC remain incompletely elucidated. MATERIALS AND METHODS: In this study, bioinformatics analysis was used, and public databases were used to obtain information about HCC cases. The samples were categorized into two groups of high and low expression based on the expression level of RAC1 gene. The limma package was used to calculate the differentially expressed genes between the two groups, and univariate Cox regression analysis was used to screen the prognostic related factors. Consensus clustering analysis was performed using the ConsensusClusterPlus package to identify molecular subtypes of HCC patients. Immune cell infiltration and ESTIMATE scores were assessed using the single sample gene set enrichment analysis and ESTIMATE algorithms. The sensitivity of different isoforms to chemotherapeutic agents was predicted by the oncoPredict package. Finally, we also performed cell function experiments to validate the biological role of RAC1 in vitro. Initially, we classified patients into high and low expression groups based on RAC1 gene expression levels and identified 195 up-regulated genes and 107 down-regulated genes. Through univariate Cox regression analysis, we screened out 169 prognosis-related factors. Furthermore, HCC patients were categorized into two subtypes. Subsequently, Kaplan-Meier survival curves showed that there was a significant difference in prognosis between the two molecular subtypes. Further analysis indicated substantial differences in gene expression levels and TIDE scores between two molecular subtypes. Moreover, these two subtypes exhibited varying sensitivity to chemotherapy drugs, as evidenced by differences in IC50 values. In addition, we found that the silence of RAC1 could effectively inhibit the migration and invasion of HCC cells in vitro. CONCLUSION: This study sheds light on the molecular intricacies of RAC1 in HCC and identifies patient populations that may benefit from immunotherapeutic interventions, with potential implications for tailored treatment strategies.

Glucose-oxygen deprivation constrains HMGCR function and Rac1 prenylation and activates the NLRP3 inflammasome in human monocytes.

Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.

Injective hydrogel loaded with liposomes-encapsulated MY-1 promotes wound healing and increases tensile strength by accelerating fibroblast migration via the PI3K/AKT-Rac1 signaling pathway.

Failed skin wound healing, through delayed wound healing or wound dehiscence, is a global public health issue that imposes significant burdens on individuals and society. Although the application of growth factor is an effective method to improve the pace and quality of wound healing, the clinically approved factors are limited. Parathyroid hormone (PTH) demonstrates promising results in wound healing by promoting collagen deposition and cell migration, but its application is limited by potentially inhibitory effects when administered continuously and locally. Through partially replacing and repeating the amino acid domains of PTH(1-34), we previously designed a novel PTH analog, PTH(3-34)(29-34) or MY-1, and found that it avoided the inhibitory effects of PTH while retaining its positive functions. To evaluate its role in wound healing, MY-1 was encapsulated in liposomes and incorporated into the methacryloyl gelatin (GelMA) hydrogel, through which an injectable nanocomposite hydrogel (GelMA-MY@Lipo, or GML) was developed. In vitro studies revealed that the GML had similar properties in terms of the appearance, microstructure, functional groups, swelling, and degradation capacities as the GelMA hydrogel. In vitro drug release testing showed a relatively more sustainable release of MY-1, which was still detectable in vivo 9 days post-application. When the GML was topically applied to the wound areas of rat models, wound closure as well as tensile strength were improved. Further studies showed that the effects of GML on wound repair and tensile strength were closely related to the promotion of fibroblast migration to the wound area through the controlled release of MY-1. Mechanically, MY-1 enhanced fibroblast migration by activating PI3K/AKT signaling and its downstream molecule, Rac1, by which it increased fibroblast aggregation in the early stage and resulting in denser collagen deposition at a later time. Overall, these findings demonstrated that the nanocomposite hydrogel system promoted skin wound healing and increased tensile strength, thus offering new potential in the treatment of wound healing.

Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

The S-Nitrosylation of Septin2 (SNO-Septin2) axis: A novel potential therapeutic target for treating aneurysms and dissection.

Aortic aneurysm and aortic dissection (AAD) are severe life-threatening cardiovascular disorders for which no approved pharmaceutical therapies are currently available. Protein S-nitrosylation (SNO) is a typical redox-dependent posttranslational modification whose role in AAD has yet to be described. Recently, Zhang et al. revealed for the first time that SNO modification of macrophage cytoskeletal protein septin2 promotes vascular inflammation and extracellular matrix degradation in aortic aneurysm. Mechanically, the TIAM1-RAC1(T lymphoma invasion and metastasis-inducing protein 1-Ras-related C3 botulinum toxin substrate 1) axis participates in the progression of AAD induced with S-nitrosylated septin2. More importantly, developing R-ketorolac and NSC23766 compounds that specifically target the TIAM1-RAC1 pathway may be new a potential strategy for alleviating AAD.

Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5ß1 integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

Activation of the Mevalonate Pathway in Response to Anti-cancer Treatments Drives Glioblastoma Recurrences Through Activation of Rac-1.

Glioblastoma (GBM) is the deadliest adult brain cancer. Under the current standard of care, almost all patients succumb to the disease and novel treatments are urgently needed. Recognizing that GBMs are addicted to cholesterol, past clinical trials have repurposed statins against GBM but failed. The purpose of this study was to test whether treatments that upregulate the cholesterol biosynthesis pathway in GBM would generate a metabolic vulnerability that can be exploited using statins and to determine the underlying mechanisms.Effects of radiotherapy and temozolomide or dopamine receptor antagonists on the mevalonate pathway in GBM were assessed in vitro and in vivo. The impact of statins on self-renewal of glioma stem cells and median survival was studied. Branches of the mevalonate pathway were probed to identify relevant effector proteins.Cells surviving combination treatments that converge in activating the immediate early response, universally upregulated the mevalonate pathway and increased stemness of GBM cells through activation of the Rho-GTPase Rac-1. Activation of the mevalonate pathway and Rac-1 was inhibited by statins, which led to improved survival in mouse models of glioblastoma when combined with radiation and drugs that target the glioma stem cell pool and plasticity of glioma cells.We conclude that a combination of dopamine receptor antagonists and statins could potentially improve radiotherapy outcome and warrants further investigation. SIGNIFICANCE: Combination therapies that activate the mevalonate pathway in GBM cells after sublethal treatment enhance self-renewal and migratory capacity through Rac-1 activation, which creates a metabolic vulnerability that can be further potentially exploited using statins.

Novel mechanisms of Anshen Dingzhi prescription against PTSD: Inhibiting DCC to modulate synaptic function and inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Anshen Dingzhi prescription (ADP), documented in "Yi Xue Xin Wu", is a famous prescription for treating panic-related mental disorders such as post-traumatic stress disorder (PTSD). However, the underlying mechanism remains unclear. AIM OF THE STUDY: This study aimed to investigate the mechanisms by which ADP intervened in PTSD-like behaviors. METHODS: A mouse model of single prolonged stress (SPS) was established to evaluate the ameliorative effects and mechanisms of ADP on PTSD. Behavioral tests were used to assess PTSD-like behaviors in mice; transmission electron microscopy was used to observe changes in the ultrastructure of hippocampal synapses, and western blot, immunofluorescence, and ELISA were used to detect the expression of hippocampal deleted in colorectal cancer (DCC) and downstream Ras-related C3 botulinum toxin substrate 1 (Rac1) - P21-activated kinase 1 (PAK1) signal, as well as levels of synaptic proteins and inflammatory factors. Molecular docking technology simulated the binding of potential brain-penetrating components of ADP to DCC. RESULTS: SPS induced PTSD-like behaviors in mice and increased expression of hippocampal netrin-1 (NT-1) and DCC on the 14th day post-modeling, with concurrent elevation in serum NT-1 levels. Simultaneously, SPS also decreased p-Rac1 level and increased p-PAK1 level, the down-stream molecules of DCC. Lentiviral overexpression of DCC induced or exacerbated PTSD-like behaviors in control and SPS mice, respectively, whereas neutralization antibody against NT-1 reduced DCC activation and ameliorated PTSD-like behaviors in SPS mice. Interestingly, downstream Rac1-PAK1 signal was altered according to DCC expression. Moreover, DCC overexpression down-regulated N-methyl-d-aspartate (NMDA) receptor 2A (GluN2A) and postsynaptic density 95 (PSD95), up-regulated NMDA receptor 2B (GluN2B) and increased neuroinflammatory responses. Administration of ADP (36.8 mg/kg) improved PTSD-like behaviors in the SPS mice, suppressed hippocampal DCC, and downstream Rac1-PAK1 signal, upregulated GluN2A and PSD95, downregulated GluN2B, and reduced levels of inflammatory factors NOD-like receptor protein 3 (NLRP3), nuclear factor kappa-B (NF-κB) and interleukin-6 (IL-6). Importantly, DCC overexpression could also reduce the ameliorative effect of ADP on PTSD. Additionally, DCC demonstrated a favorable molecular docking pattern with the potential brain-penetrating components of ADP, further suggesting DCC as a potential target of ADP. CONCLUSION: Our data indicate that DCC is a key target for the regulation of synaptic function and inflammatory response in the onset of PTSD, and ADP likely reduces DCC to prevent PTSD via modulating downstream Rac1-PAK1 pathway. This study provides a novel mechanism for the onset of PTSD and warrants the clinical application of ADP.

Rac1 plays a crucial role in MCP-1-induced monocyte adhesion and migration.

Monocyte migration is an important process in inflammation and atherogenesis. Identification of the key signalling pathways that regulate monocyte migration can provide prospective targets for prophylactic treatments in inflammatory diseases. Previous research showed that the focal adhesion kinase Pyk2, Src kinase and MAP kinases play an important role in MCP-1-induced monocyte migration. In this study, we demonstrate that MCP-1 induces iPLA2 activity, which is regulated by PKCß and affects downstream activation of Rac1 and Pyk2. Rac1 interacts directly with iPLA2 and Pyk2, and plays a crucial role in MCP-1-mediated monocyte migration by modulating downstream Pyk2 and p38 MAPK activation. Furthermore, Rac1 is necessary for cell spreading and F-actin polymerization during monocyte adhesion to fibronectin. Finally, we provide evidence that Rac1 controls the secretion of inflammatory mediator vimentin from MCP-1-stimulated monocytes. Altogether, this study demonstrates that the PKCß/iPLA2/Rac1/Pyk2/p38 MAPK signalling cascade is essential for MCP-1-induced monocyte adhesion and migration.

The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice.

The small G-protein Ras-related C3 botulinum toxin substrate 1 (Rac1) promotes the formation of filamentous actin (F-actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an adeno-associated virus (AAV) expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning.

Potential Involvement of M1 Macrophage and VLA4/VCAM-1 Pathway in the Valvular Damage Due to Rheumatic Heart Disease.

BACKGROUND: Rheumatic heart disease (RHD) is caused by inflammatory cells mistakenly attacking the heart valve due to Group A Streptococcus (GAS) infection, but it is still unclear which cells or genes are involved in the process of inflammatory cells infiltrating the valve. Inflammatory infiltration into the target tissue requires an increase in the expression of phosphorylated vascular endothelial-cadherin (p-VE-cad), p-VE-cad can increase the endothelial permeability and promote the migration of inflammatory cells across the endothelium. P-VE-cad is potentially regulated by RAS-related C3 botulinum substrate 1 (RAC1), together with phosphorylated proline-rich tyrosine kinase 2 (p-PYK2). While RAC1/p-PYK2/p-VE-cad is triggered by the activation of vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is related to M1 macrophages adhering to the endothelium via very late antigen 4 (VLA4). Inflammatory infiltration into the valve is extremely important in the early pathogenesis of RHD. However, there is no relevant research on whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved in RHD; therefore, what we explored in this study was whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved. METHODS: We established a rat model of RHD and a cell model of M1 macrophage and endothelial cell cocultivation. Subsequently, we measured the degree of inflammatory cell infiltration, the levels of IL-6/IL-17, the degree of fibrosis (COL3/1), and the expression levels of fibrosis markers (FSP1, COL1A1 and COL3A1) in the heart valves of RHD rats. Additionally, we detected the expression of M1/M2 macrophage biomarkers in rat model and cell model, as well as the expression of M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad. We also tested the changes in endothelial permeability after coculturing M1 macrophages and endothelial cells. RESULTS: Compared to those in the control group, the levels of inflammatory cell infiltration and fibrotic factors in the heart valves of RHD rats were significantly higher; the expression of M1 macrophage biomarkers (iNOS, CD86 and TNF-α) in RHD rats was significantly higher; and significantly higher than the expression of M2 macrophage biomarkers (Arg1 and TGF-ß). And the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad in the hearts of RHD rats were significantly higher. At the cellular level, after coculturing M1 macrophages with endothelial cells, the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad were significantly higher, and the permeability of the endothelium was significantly greater due to cocultivation with M1 macrophages. CONCLUSIONS: All the results suggested that M1 macrophages and the VLA4/VCAM-1 pathway are potentially involved in the process of inflammatory infiltration in RHD.

FLNA regulates neuronal maturation by modulating RAC1-Cofilin activity in the developing cortex.

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.

Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA.

Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.