Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.

DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

Unveiling the role of RAC3 in the growth and invasion of cisplatin-resistant bladder cancer cells.

Bladder cancer is one of the most prevalent cancers worldwide, and its morbidity and mortality rates have been increasing over the years. However, how RAC family small GTPase 3 (RAC3) affects the proliferation, migration and invasion of cisplatin-resistant bladder cancer cells remains unclear. Bioinformatics techniques were used to investigate the expression of RAC3 in bladder cancer tissues. Influences of RAC3 in the grade, stage, distant metastasis, and survival rate of bladder cancer were also examined. Analysis of the relationship between RAC3 expression and the immune microenvironment (TIME), genomic mutations, and stemness index. In normal bladder cancer cells (T24, 5637, and BIU-87) and cisplatin-resistant bladder cancer cells (BIU-87-DDP), the expression of RAC3 was detected separately with Western blotting. Plasmid transfection was used to overexpress or silence the expression of RAC3 in bladder cancer cells resistant to cisplatin (BIU-87-DDP). By adding activators and inhibitors, the activities of the JNK/MAPK signalling pathway were altered. Cell viability, invasion, and its level of apoptosis were measured in vitro using CCK-8, transwell, and flow cytometry. The bioinformatics analyses found RAC3 levels were elevated in bladder cancer tissues and were associated with a poor prognosis in bladder cancer. RAC3 in BIU-87-DDP cells expressed a higher level than normal bladder cancer cells. RAC3 overexpression promoted BIU-87-DDP proliferation. The growth of BIU-87-DDP cells slowed after the knockdown of RAC3, and RAC3 may have had an impact on the activation of the JNK/MAPK pathway.

The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis.

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

DOCK1 regulates the malignant biological behavior of endometrial cancer through c-Raf/ERK pathway.

BACKGROUND: The effect of DOCK1 gene on the biological behavior of endometrial carcinoma cells and its related pathway has not been reported. METHODS: The immunohistochemical method and western blot were utilized to analyze DOCK1 protein expression in endometrial tissues and cells, respectively. CCK-8, BrdU, transwell and flow cytometry were performed to analyze the effect of DOCK1 expression changes on the viability, proliferation, invasion, migration and apoptosis of endometrial cancer cells, respectively. The effects of DOCK1 gene on Bcl-2, MMP9, Ezrin, E-cadherin and c-RAF/ERK1/2 signaling pathway were evaluated by western blot. The xenograft models were constructed to analyze the effect of DOCK1 in vivo. RESULTS: DOCK1 expression was increased in endometrial cancer tissues and cells compared with those in normal adjacent tissues and cells. DOCK1 knockout could inhibit the malignant biological behavior of endometrial cancer cells, while DOCK1 overexpression played the opposite effect. The expression of E-cadherin was upregulated and those of MMP9, Ezrin, Bcl-2, p-c-RAF (S338) and p-ERK1/2 (T202/Y204) were downregulated after DOCK1 knockout, while DOCK1 overexpression played the opposite effect. Additionally, Raf inhibitor LY3009120 reversed the function of DOCK1 on malignant biological behavior. In vivo experiment results showed that the growth and weight of transplanted tumors in nude mice were inhibited after DOCK1 knockout. The changes of E-cadherin, MMP9, Ezrin and Bcl-2 expressions in the transplanted tumors were consistent with those in vitro. CONCLUSION: DOCK1 could enhance the malignant biological behavior of endometrial cancer cells, which might be through c-RAF/ERK1/2 signaling pathways in vitro and in vivo.

Pulsatilla saponin D regulates ras-related C3 botulinum toxin substrate 3 (RAC3) to overcome resistance to paclitaxel in lung adenocarcinoma cells.

BACKGROUND: Paclitaxel, a tubulin-binding agent, is a Food and Drug Administration-approved first-line drug for the treatment of non-small cell lung cancer (NSCLC), for both squamous and non-squamous cell lung carcinoma, with paclitaxel/carboplatin + bevacizumab a common chemotherapy regimen for stage IV non-squamous NSCLC; however, primary or acquired resistance to paclitaxel is gradually increasing, leading to treatment failure. METHODS: Our results show that Ras-related C3 botulinum toxin substrate 3 (RAC3) is overexpressed in cultured paclitaxel-resistant cells and that RAC3 expression levels are negatively correlated with sensitivity of lung adenocarcinoma cells to paclitaxel. Pulsatilla saponin D could inhibit RAC3 expression, and we hypothesize that it may block paclitaxel resistance. Further, we found that treatment with paclitaxel combined with Pulsatilla saponin D, can overcome lung adenocarcinoma cell resistance to paclitaxel alone in cell culture and mouse xenograft models.

Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

An unusual presentation of de novo RAC3 variation in prenatal diagnosis.

Pathogenic variants in RAC3 cause a neurodevelopmental disorder with brain malformations and craniofacial dysmorphism, called NEDBAF. This gene encodes a small GTPase, which plays a critical role in neurogenesis and neuronal migration. We report a 31 weeks of gestation fetus with triventricular dilatation, and temporal and perisylvian polymicrogyria, without cerebellar, brainstem, or callosal anomalies. Trio whole exome sequencing identified a RAC3 (NM_005052.3, GRCh38) probably pathogenic de novo variant c.276 T>A p.(Asn92Lys). Eighteen patients harboring 13 different and essentially de novo missense RAC3 variants were previously reported. All the patients presented with corpus callosum malformations. Gyration disorders, ventriculomegaly (VM), and brainstem and cerebellar malformations have frequently been described. The only previous prenatal case associated with RAC3 variant presented with complex brain malformations, mainly consisting of midline and posterior fossa anomalies. We report the second prenatal case of NEDBAF presenting an undescribed pattern of cerebral anomalies, including VM and polymicrogyria, without callosal, cerebellar, or brainstem malformations. All neuroimaging data were reviewed to clarify the spectrum of cerebral malformations.

Macrophages inhibit extracellular hyphal growth of A. fumigatus through Rac2 GTPase signaling.

Macrophages act as a first line of defense against pathogens. Against Aspergillus fumigatus, a fungus with pathogenic potential in immunocompromised patients, macrophages can phagocytose fungal spores and inhibit spore germination to prevent the development of tissue-invasive hyphae. However, the cellular pathways that macrophages use to accomplish these tasks and any roles macrophages have later in infection against invasive forms of fungi are still not fully known. Rac-family Rho GTPases are signaling hubs for multiple cellular functions in leukocytes, including cell migration, phagocytosis, reactive oxygen species (ROS) generation, and transcriptional activation. We therefore aimed to further characterize the function of macrophages against A. fumigatus in an in vivo vertebrate infection model by live imaging of the macrophage behavior in A. fumigatus-infected rac2 mutant zebrafish larvae. While Rac2-deficient zebrafish larvae are susceptible to A. fumigatus infection, Rac2 deficiency does not impair macrophage migration to the infection site, interaction with and phagocytosis of spores, spore trafficking to acidified compartments, or spore killing. However, we reveal a role for Rac2 in macrophage-mediated inhibition of spore germination and control of invasive hyphae. Re-expression of Rac2 under a macrophage-specific promoter rescues the survival of A. fumigatus-infected rac2 mutant larvae through increased control of germination and hyphal growth. Altogether, we describe a new role for macrophages against extracellular hyphal growth of A. fumigatus and report that the function of the Rac2 Rho GTPase in macrophages is required for this function.

Establishment of a prognostic model to predict chemotherapy response and identification of RAC3 as a chemotherapeutic target in bladder cancer.

Cisplatin-based chemotherapy is considered the primary treatment option for patients with advanced bladder cancer (BCa). However, the objective response rate to chemotherapy is often unsatisfactory, leading to a poor 5-year survival rate. Furthermore, current strategies for evaluating chemotherapy response and prognosis are limited and inefficient. In this study, we aimed to address these challenges by establishing a chemotherapy response type gene (CRTG) signature consisting of 9 genes and verified the prognostic value of this signature using TCGA and GEO BCa cohorts. The risk scores based on the CRTG signature were found to be associated with advanced clinicopathological status and demonstrated favorable predictive power for chemotherapy response in the TCGA cohort. Meanwhile, tumors with high risk scores exhibited a tendency toward a "cold tumor" phenotype. These tumors showed a low abundance of T cells, CD8+ T cells and cytotoxic lymphocytes, along with a high abundance of cancer-associated fibroblasts. Moreover, they displayed higher mRNA levels of these immune checkpoints: CD200, CD276, CD44, NRP1, PDCD1LG2 (PD-L2), and TNFSF9. Furthermore, we developed a nomogram that integrated the CRTG signature with clinicopathologic risk factors. This nomogram proved to be a more effective tool for predicting the prognosis of BCa patients. Additionally, we identified Rac family small GTPase 3 (RAC3) as a biomarker in our model. RAC3 was found to be overexpressed in chemoresistant BCa tissues and enhance the chemotherapeutic resistance of BCa cells in vitro and in vivo by regulating the PAK1-ERK1/2 pathway. In conclusion, our study presents a novel CRTG model for predicting chemotherapy response and prognosis in BCa. We also highlight the potential of combining chemotherapy with immunotherapy as a promising strategy for chemoresistant BCa and that RAC3 might be a latent target for therapeutic intervention.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

Early diagnosis and prognostic potential of RAC3 in bladder tumor.

BACKGROUND AND PURPOSE: Bladder tumors are among the most prevalent malignancies in the urinary system, and RAC3 has been linked to various types of cancer. This article seeks to explore the potential of RAC3 as both an early diagnostic marker for bladder tumors and a novel therapeutic target. METHODS/PATIENTS: The expression of RAC3 in bladder tissue was detected using immunohistochemical staining. Additionally, the protein expression of RAC3 was measured and quantified through enzyme-linked immunosorbent assay (ELISA). Subsequently, the correlation between the expression level of RAC3 and bladder tumors was investigated through multifactorial analysis and survival analysis. RESULTS: Our findings revealed that RAC3 expression was upregulated in bladder tumor tissues. Moreover, we observed higher levels of RAC3 expression in the serum and urine of patients with bladder tumors compared to those with non-bladder tumors. Additionally, we identified a significant positive correlation between RAC3 expression levels and the stage, degree of differentiation, and infiltration of bladder tumors. Importantly, high RAC3 expression emerged as an influential factor in the poor prognosis of bladder tumors, as patients with high RAC3 expression exhibited a lower overall survival rate than those with low RAC3 expression. CONCLUSION: Based on our results, RAC3 shows promise as both a marker for early diagnosis of bladder tumors and a potential therapeutic target.

DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway.

Abnormal trophoblast function is associated with diseases such as recurrent spontaneous abortion, pre-eclampsia, and preterm birth, and endangers maternal and fetal health. However, the underlying regulatory mechanisms remain unclear. In this study, we found DOCK1 expression is decreased in the placental villi of patients with recurrent spontaneous abortion, and that its expression determined the invasive properties of extravillous trophoblasts (EVTs), highlighting a previously unknown role of DOCK1 in regulating EVT function. Furthermore, DOCK1 deficiency disturbed the ubiquitinated degradation of DUSP4, leading to its accumulation. This caused inactivation of the ERK signaling pathway, resulting in inadequate EVT migration and invasion. DOCK1 was implicated in regulating the ubiquitin levels of DUSP4, possibly by modulating the E3 ligase enzyme HUWE1. The results of our in vivo experiments confirmed that the DOCK1 inhibitor TBOPP caused miscarriage in mice by inactivating the DUSP4/ERK pathway. Collectively, our results revealed the crucial role of DOCK1 in the regulation of EVT function via the DUSP4-ERK pathway and a basis for the development of novel treatments for adverse pregnancy outcomes caused by trophoblast dysfunction.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating ß2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and ß2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing.

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

Expression Analysis of the Small GTP-Binding Protein Rac in Pterygium

Objectives: To determine the roles of small GTP-binding proteins Rac1, Rac2, and Rac3 expression in pterygial tissue and to compare these expressions with normal conjunctival tissue. Materials and Methods: Seventy-eight patients with primary pterygium were enrolled. Healthy conjunctival graft specimens obtained during pterygium surgery were used as control tissue. The real-time polymerase chain reaction method on the BioMark HD dynamic array system was utilized in genomic mRNA for the gene expression analysis. Protein expressions were analyzed using western blot and immunohistochemical methods. Results: RAC1, RAC2, and RAC3 gene expressions in pterygial tissues were not markedly elevated when compared to the control specimens (p>0.05). As a very low level of RAC1 gene expression was observed, further protein expression analysis was performed for the Rac2 and Rac3 proteins. Western blot and immunohistochemical analysis of Rac2 and Rac3 protein expression revealed no significant differences between pterygial and healthy tissues (p>0.05). Conclusion: This is the first study to identify the contribution of Rac proteins in pterygium. Our results indicate that the small GTP-binding protein Rac may not be involved in pterygium pathogenesis.

Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3.

The Rho family GTPases Rac and Rho play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well-described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac was dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified activating transcription factor 3 (ATF3) as a major target of stiffness- and Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.

Integrated analysis identifies RAC3 as an immune-related prognostic biomarker associated with chemotherapy sensitivity in endometrial cancer.

Endometrial cancer (EC) is one of the most common gynaecological malignant tumours with a high incidence, leading to urgent demands for exploring novel carcinogenic mechanisms and developing rational therapeutic strategies. The rac family of small GTPase 3 (RAC3) functions as an oncogene in various human malignant tumours and plays an important role in tumour development. However, the critical roles of RAC3 in the progression of EC need further investigation. Based on TCGA, single-cell RNA-Seq, CCLE and clinical specimens, we revealed that the RAC3 was specifically distributed in EC tumour cells compared to normal tissues and functioned as an independent diagnostic marker with a high area under curve (AUC) score. Meanwhile, the RAC3 expression in EC tissues was also correlated with a poor prognosis. In detail, the high levels of RAC3 in EC tissues were reversely associated with CD8+ T cell infiltration and orchestrated an immunosuppressive microenvironment. Furthermore, RAC3 accelerated tumour cell proliferation and inhibited its apoptosis, without impacting cell cycle stages. Importantly, silencing RAC3 improved the sensitivity of EC cells to chemotherapeutic drugs. In this paper, we revealed that RAC3 was predominantly expressed in EC and significantly correlated with the progression of EC via inducing immunosuppression and regulating tumour cell viability, providing a novel diagnostic biomarker and a promising strategy for sensitizing chemotherapy to EC.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Dispensable role of Rac1 and Rac3 after cochlear hair cell specification.

Rac small GTPases play important roles during embryonic development of the inner ear; however, little is known regarding their function in cochlear hair cells (HCs) after specification. Here, we revealed the localization and activation of Racs in cochlear HCs using GFP-tagged Rac plasmids and transgenic mice expressing a Rac1-fluorescence resonance energy transfer (FRET) biosensor. Furthermore, we employed Rac1-knockout (Rac1-KO, Atoh1-Cre;Rac1flox/flox) and Rac1 and Rac3 double KO (Rac1/Rac3-DKO, Atoh1-Cre;Rac1flox/flox;Rac3-/-) mice, under the control of the Atoh1 promoter. However, both Rac1-KO and Rac1/Rac3-DKO mice exhibited normal cochlear HC morphology at 13 weeks of age and normal hearing function at 24 weeks of age. No hearing vulnerability was observed in young adult (6-week-old) Rac1/Rac3-DKO mice even after intense noise exposure. Consistent with prior reports, the results from Atoh1-Cre;tdTomato mice confirmed that the Atoh1 promoter became functional only after embryonic day 14 when the sensory HC precursors exit the cell cycle. Taken together, these findings indicate that although Rac1 and Rac3 contribute to the early development of sensory epithelia in cochleae, as previously shown, they are dispensable for the maturation of cochlear HCs in the postmitotic state or for hearing maintenance following HC maturation. KEY MESSAGES: Mice with Rac1 and Rac3 deletion were generated after HC specification. Knockout mice exhibit normal cochlear hair cell morphology and hearing. Racs are dispensable for hair cells in the postmitotic state after specification. Racs are dispensable for hearing maintenance after HC maturation.

Cancer-associated fibroblasts promote migration and invasion of non-small cell lung cancer cells via METTL3-mediated RAC3 m6A modification.

Cancer progression depends on the communication between tumor cells and tumor microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of stromal cells. CAFs promote cancer metastasis; however, it has not been evaluated whether N6-methyladenosine (m6A) modification is responsible for CAFs' role in metastasis. In the present study, we found that CAFs promoted migration and invasion of non-small cell lung cancer (NSCLC) cells by elevating m6A modification in NSCLC cells. Methyltransferase-like 3 (METTL3) in NSCLC cells mediated CAFs' effect on m6A modification, and was regulated by CAFs-secreted vascular endothelial growth factor A (VEGFA). METTL3 knockdown in NSCLC cells dramatically inhibited cell migration and invasion, and suppressed tumor growth in vivo. Database analysis revealed that METTL3 was associated with poor prognosis of lung cancer. The mechanism study showed that METTL3 increased m6A level of RAC3 mRNA, resulting in increased stability and translation of RAC3 mRNA. RAC3 was responsible for the CAFs' promoting effect on cell migration via the AKT/NF-κB pathway. This study established a CAF-METTL3-RAC3 m6A modification-dependent regulation system in NSCLC metastasis, suggesting potential candidates for metastasis treatment.