Rac1 Activation Research Articles

Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2− levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2−. l-NAME promoted Rac1 activation and enhanced Rac1–PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2− mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1–PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2− and NO as positive regulators of Rac1–PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.

Spontaneous recovery of reward memory through active forgetting of extinction memory

Learned behavior can be suppressed by the extinction procedure. Such extinguished memory often returns spontaneously over time, making it difficult to treat diseases such as addiction. However, the biological mechanisms underlying such spontaneous recovery remain unclear. Here, we report that the extinguished reward memory in Drosophila recovers spontaneously because extinction training forms an aversive memory that can be actively forgotten via the Rac1/Dia pathway. Manipulating Rac1 activity does not affect sugar-reward memory and its immediate extinction effect but bidirectionally regulates spontaneous recovery-the decay process of extinction. Experiments using thermogenetic inhibition and functional imaging support that such extinction appears to be coded as an aversive experience. Genetic and pharmacological inhibition of formin Dia, a downstream effector of Rac1, specifically prevents spontaneous recovery after extinction in both behavioral performance and corresponding physiological traces. Together, ourdata suggest that spontaneous recovery is caused by active forgetting of the opposing extinction memory.

Pathogenic TRIO variants associated with neurodevelopmental disorders perturb the molecular regulation of TRIO and axon pathfinding in vivo.

The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations.

Deficiency of filamin A in smooth muscle cells protects against hypoxia‑mediated pulmonary hypertension in mice.

Filamin A (FLNA) is a high molecular weight cytoskeleton protein important for cell locomotion. A relationship between FLNA mutations and pulmonary arterial hypertension (PAH) has previously been reported; however, the detailed mechanism remains unclear. The present study aimed to explore the role of FLNA in vascular smooth muscle cells during the development of PAH. Smooth muscle cell (SMC)‑specific FLNA‑deficient mice were generated and the mice were then exposed to hypoxia for 28days to build the mouse model of PAH. Human pulmonary arterial smooth muscle cells (PASMCs) were also cultured and transfected with FLNA small interfering RNA or overexpression plasmids to investigate the effects of FLNA on PASMC proliferation and migration. Notably, compared with control individuals, the expression levels of FLNA were increased in lung tissues from patients with PAH, and it was obviously expressed in the PASMCs of pulmonary arterioles. FLNA deficiency in SMCs attenuated hypoxia‑induced pulmonary hypertension and pulmonary vascular remodeling. Invitro studies suggested that absence of FLNA impaired PASMC proliferation and migration, and produced lower levels of phosphorylated (p)‑PAK‑1 and RAC1 activity. However, FLNA overexpression promoted PASMC proliferation and migration, and increased the expression levels of p‑PAK‑1 and RAC1 activity. The present study highlights the role of FLNA in pulmonary vascular remodeling; therefore, it could serve as a potential target for the treatment of PAH.

Wdr4 promotes cerebellar development and locomotion through Arhgap17-mediated Rac1 activation

Patients with mutations of WDR4, a substrate adaptor of the CUL4 E3 ligase complex, develop cerebellar atrophy and gait phenotypes. However, the underlying mechanisms remain unexplored. Here, we identify a crucial role of Wdr4 in cerebellar development. Wdr4 deficiency in granule neuron progenitors (GNPs) not only reduces foliation and the sizes of external and internal granular layers but also compromises Purkinje neuron organization and the size of the molecular layer, leading to locomotion defects. Mechanistically, Wdr4 supports the proliferation of GNPs by preventing their cell cycle exit. This effect is mediated by Wdr4-induced ubiquitination and degradation of Arhgap17, thereby activating Rac1 to facilitate cell cycle progression. Disease-associated Wdr4 variants, however, cannot provide GNP cell cycle maintenance. Our study identifies Wdr4 as a previously unappreciated participant in cerebellar development and locomotion, providing potential insights into treatment strategies for diseases with WDR4 mutations, such as primordial dwarfism and Galloway-Mowat syndrome.

The variant senescence-associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia-inducible factor-1α.

The senescence-associated secretory phenotype (SASP) can promote paracrine invasion while suppressing tumour growth, thus generating complex phenotypic outcomes. Likewise, centrosome amplification can induce proliferation arrest yet also facilitate tumour invasion. However, the eventual fate of cells with centrosome amplification remains elusive. Here, we report that centrosome amplification induces a variant of SASP, which constitutes a pathway activating paracrine invasion. The centrosome amplification-induced SASP is non-canonical as it lacks the archetypal detectable DNA damage and prominent NF-κB activation, but involves Rac activation and production of reactive oxygen species. Consequently, it induces hypoxia-inducible factor 1α and associated genes, including pro-migratory factors such as ANGPTL4. Of note, cellular senescence can either induce tumourigenesis through paracrine signalling or conversely suppress tumourigenesis through p53 induction. By analogy, centrosome amplification-induced SASP may therefore be one reason why extra centrosomes promote malignancy in some experimental models but are neutral in others.

Effects of Different Irradiance Conditions on Photosynthetic Activity, Photosystem II, Rubisco Enzyme Activity, Chloroplast Ultrastructure, and Chloroplast-Related Gene Expression in Clematis tientaiensis Leaves

Clematis is a perennial ornamental vine known as the “Vine Queen” for its gorgeous floral color and rich flower shape. Clematis tientaiensis, an endangered plant, is a key protected wild plant and a rare breeding parent of Clematis because of its extremely high ornamental value. Light environment is one of the important environmental factors affecting the space distribution and the size of C. tientaiensis population. One-year-old homogenous and healthy potted C. tientaiensis plants were grown under four different light intensities (T1: 1800 ± 30/0 µmol m−2 s−1; T2: 1500 ± 30/0 µmol m−2 s−1; T3: 1200 ± 30/0 µmol m−2 s−1; T4: 900 ± 30/0 µmol m−2 s−1). This study analyzed the potential adaptive mechanism of C. tientaiensis in response to irradiance by investigating the photosynthesis, rapid light curve, chloroplast ultrastructure, Rubisco activase enzyme (RAC), Rubisco enzyme, amino acids, and gene expression under four irradiance treatments. High light caused the leaves chlorosis and yellowing, reduced the net photosynthetic rate (Pn), stomatal conductance (Gs), RAC and Rubisco enzyme activity; the quantum yield of unregulated energy dissipation [Y(NO)], and increased the content of eight amino acids content. The expression of psbA, psbB, psbC, and Psb(OEC) were down-regulated with decreasing irradiance. The results showed that C. tientaiensis plants grown under T1 (1800 ± 30 µmol m−2 s−1) irradiance were in danger of absorbing more light energy than they could use for photosynthesis, while they exhibited good adaptability to the T3 (1200 ± 30 µmol m−2 s−1) irradiance, and the PSII reaction center and Rubisco and RCA enzymes could be the key points in response to high light stress, which also emphasized the importance of appropriate light management practices for promoting the growth and population expansion of C. tientaiensis.

Abstract B011: Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis

Abstract Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion, modulation of the tumor microenvironment (TME), plasticity, and ability to colonize secondary tissues. Therefore, a viable anti-metastasis strategy needs to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, the purpose of this study was to test the hypothesis that Rac and Cdc42 inhibitors target both migratory cancer cells and immunosuppressive cells in the TME. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Since the targets of these inhibitors may also affect immune cell behavior, we first tested their potential to target Rac/Cdc42 function in human and rat macrophage cell lines and show that EHop-016 and MBQ-167 inhibit Rac activation (at 5X higher concentration than the IC50 in breast cancer cells), actin cytoskeletal extensions, and migration, without affecting cell viability. EHop-016 and MBQ-167 significantly decrease the proinflammatory cytokine Interleukin-6 (Il-6) from plasma and the TME. Next, we tested the in vivo effect of MBQ-167 on immune cells from breast cancer mouse models. Tumors from immunodeficient mice bearing mammary tumors that were treated with vehicle or 50mg/kg MBQ-167 5x a week for 5 weeks, were analyzed by flow cytometry to quantify different immune cell types. MBQ-167 treated mice demonstrated elevated CD11+F4/80+CD86+ M1 macrophages and reduced MDSCs in mammary tumors. In the immunocompetent 4T-1 breast cancer model, spleens contained a lower frequency of macrophages and neutrophils, accompanied by an increase in activated cytotoxic CD69+, CD8+T cells. Moreover, when metastasis to the lungs was reduced by ~90% following MBQ-167 treatment, granzyme B+ CD8+ T cells were elevated in the lung extracts, without affecting the immunosuppressive CD4+ regulatory T cell counts. In vitro, we also demonstrated that increasing doses of MBQ-167 induced T cell activation and interferon-gamma secretion, a cytokine known to have antitumor properties. Taken together, Rac/Cdc42 inhibitors induce anti-metastatic cancer effects via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells, while promoting the activation of cytotoxic T cells, in the tumor and the metastasis microenvironment. Therefore, MBQ-167 promises to be an effective antimetastatic cancer therapeutic that is poised to enter Phase I clinical trials. Citation Format: Stephanie Dorta-Estremera, Anamaris Torres, Michael Rivera-Robles, Ailed Cruz-Collazo, Luis Borrero-Garcia, Suranganie Dharmawardhane. Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B011.

Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of β1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and β1 integrins. Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of β1 integrins. These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells.

Extracellular matrix (ECM) has a well-recognized impact on the progression of solid tumors, including hepatocellular carcinoma (HCC). Laminin 332 (Ln332) is a ECM molecule of epithelial basal lamina, composed of three polypeptide chains (α3, β3, and γ2), that is usually poorly expressed in the normal liver but is detected at high levels in HCC. This macromolecule was shown to promote the proliferation, epithelial-to-mesenchymal transition (EMT), and drug resistance of HCC cells. The monomeric γ2 chain is up-regulated and localized preferentially at the invasive edge of metastatic intrahepatic HCC nodules, suggesting its potential involvement in the acquisition of invasive properties of HCC cells. HCC cells were tested in in vitro adhesion, scattering, and transwell migration assays in response to fibronectin and the Ln332 and Ln332 γ2 chains, and the activation status of major signaling pathways involved was evaluated. Here, we show that the Ln332 γ2 chain promotes HCC the cell adhesion, migration, and scattering of HCC cells that express the Ln332 receptor α3β1 integrin, proving to be a causal factor of the EMT program achievement. Moreover, we found that efficient HCC cell adhesion and migration on γ2 require the activation of the small cytosolic GTPase Rac1 and ERKs signaling. These data suggest that the γ2 chain, independently from the full-length Ln332, can contribute to the pro-invasive potential of aggressive HCC cell subpopulations.

The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

S-38-1: EPIGENETIC MECHANISM OF SALT-SENSITIVE HYPERTENSION

Nutritional and environmental factors contribute to the development of salt sensitive hypertension. DNA methylation, the most extensively studied epigenetic marker, may have more stable, longer lasting effects than transient changes in gene expression. Protein malnutrition during pregnancy causes prenatal programmed salt sensitive hypertension via aberrant DNA methylation of the angiotensin receptor, which is induced by exposure to excessive glucocorticoid in the fetal hypothalamus. In adults, salt sensitive hypertension induced by obesity is attributable to increased renal activity of Rac1 and mineralocorticoid receptor (MR) pathway, and adverse prenatal exposure induces transgenerational hypertension through activation of Rac1 and MR pathway by an epigenetic regulated mechanism. Finally, DNA methylation of Klotho in aged kidneys leads to a reduction in the circulating levels of soluble Klotho, and in turn, resulting in the activation of noncanonical Wnt5a and RhoA signaling in the vasculature and the development of aging associated salt sensitive hypertension. These findings indicate that epigenetic modulation has an important role in the development of salt sensitive hypertension throughout life.

Sigma-1 receptor-regulated efferocytosis by infiltrating circulating macrophages/microglial cells protects against neuronal impairments and promotes functional recovery in cerebral ischemic stroke.

Background: Efferocytosis of apoptotic neurons by macrophages is essential for the resolution of inflammation and for neuronal protection from secondary damage. It is known that alteration of the Sigma-1 receptor (Sig-1R) is involved in the pathological development of some neurological diseases, including ischemic stroke. The present study aimed to investigate whether and how Sig-1R regulates the phagocytic activity of macrophages/microglia and its significance in neuroprotection and neurological function in stroke. Methods: The roles of Sig-1R in the efferocytosis activity of microglia/macrophages using bone marrow-derived macrophages (BMDMs) or using Sig-1R knockout mice subjected to transient middle artery occlusion (tMCAO)-induced stroke were investigated. The molecular mechanism of Sig-1R in the regulation of efferocytosis was also explored. Adoptive transfer of Sig-1R intact macrophages to recipient Sig-1R knockout mice with tMCAO was developed to observe its effect on apoptotic neuron clearance and stroke outcomes. Results: Depletion of Sig-1R greatly impaired the phagocytic activity of macrophages/microglia, accordingly with worsened brain damage and neurological defects in Sig-1R knockout mice subjected to tMCAO. Adoptive transfer of Sig-1R intact bone marrow-derived macrophages (BMDMs) to Sig-1R knockout mice restored the clearance activity of dead/dying neurons, reduced infarct area and neuroinflammation, and improved long-term functional recovery after cerebral ischemia. Mechanistically, Sig-1R-mediated efferocytosis was dependent on Rac1 activation in macrophages, and a few key sites of Rac1 in its binding pocket responsible for the interaction with Sig-1R were identified. Conclusion: Our data provide the first evidence of the pivotal role of Sig-1R in macrophage/microglia-mediated efferocytosis and elucidate a novel mechanism for the neuroprotection of Sig-1R in ischemic stroke.

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition

Background and aimsThiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear.MethodsTo model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl– and HCO3– secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization.ResultsWe demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3– and Cl– secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR.ConclusionsAZA impaired the ductal HCO3– and Cl– secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

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 β2 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 β2 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.

Serum-Derived Exosomes of Patients with Depression with Increased miR-96-5p Inhibit Neuronal Differentiation by Targeting RAC1

Exosomes are associated with neuronal differentiation in mental disorders, such as depression. This study explored the effects of exosomes on neuronal differentiation and their underlying molecular mechanisms. We isolated exosomes from the sera of patients with depression. These characteristics were authenticated by TEM, NTA and western blotting. A differentiated cell model was established using all-trans retinoic acid (RA) to treat SH-SY5Y cells. Exosomes from depressed serum co-cultured with miR-96-5p mimic-transfected RA-treated cells, and PMA was used to activate RAC1. Neuronal differentiation indices were analyzed by microscopy, quantitative PCR, immunofluorescence assay, and western blotting. The results illustrated that exosomes inhibited RA-induced differentiation, downregulated SYP, upregulated nestin, and decreased SYP-positive cell number. miR-96-5p was elevated in serum-released exosomes from depressed patients, which impeded RA-induced neuronal differentiation. RAC1 is an miR-96-5p target. Activation of RAC1 partly counteracted the effects on neuronal differentiation induced by enhanced miR-96-5p levels. Additionally, decreasing miR-96-5p attenuated depression-like behaviors and promoted hippocampal neuron differentiation induced by CUMS. Summarily, serum-derived exosomes from patients with depression suppress neuronal differentiation via the miR-96-5p/RAC1 axis. Moreover, decreased miR-96-5p levels suppresses CUMS-induced depression. These consequences suggest that regulating exosomes secretion and exosomal miR-96-5p expression will be a new approach for therapy of depression.

SH3BP1 Regulates Melanoma Progression Through Race1/Wace2 Signaling Pathway.

SH3-domain binding protein-1 (SH3BP1), which specifically inactivates Rac1 and its target protein Wave2, has been shown to be an important regulator of cancer metastasis. However, the effects of SH3BP1 in melanoma progression remain unclear. The current study aimed to explore the function of SH3BP1 in melanoma and its possible molecular mechanism. TCGA database was used to analyze the expression of SH3BP1 in melanoma. Then, reverse transcription-quantitative polymerase chain reaction was performed to detect the expression of SH3BP1 in melanoma tissues and cells. Next, genes related to SH3BP1 were analyzed by LinkedOmics database, and protein interactions were analyzed by STRING database. These genes were further subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. In addition, the signaling pathway of SH3BP1 action was screened by bioinformatics analysis. Finally, the function of SH3BP1 and its mediated signaling pathway in melanoma progression were investigated in vitro and in vivo. SH3BP1 was significantly upregulated in melanoma tissues and cells. The pathways regulated by SH3BP1 are closely related to the occurrence and development of tumors. And we found that overexpression of SH3BP1 promoted the proliferation, migration, and invasion of melanoma cells by increasing Rac1 activity and Wave2 protein levels in vitro. Similarly, overexpression of SH3BP1 facilitated melanoma progression by upregulating Wave2 protein expression in vivo. In summary, this study revealed for the first time that SH3BP1 promoted melanoma progression through Rac1/Wave2 signaling pathway, providing a new therapeutic target for melanoma.

Characterization of Novel Derivatives of MBQ-167, an inhibitor of the GTP-binding proteins Rac/Cdc42.

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

High MICAL1 expression correlates with cancer progression and immune infiltration in renal clear cell carcinoma

BackgroundMolecule interacting with CasL 1 (MICAL1), a multidomain flavoprotein monooxygenase, is strongly involved in the biological processes related to cancer cell proliferation and metastasis. However, there were few reports on the clinical significance of MICAL1 in renal clear cell carcinoma.MethodsThe expression and prognostic value of MICAL1 in renal clear cell carcinoma were explored using immunohistochemical assays, public TCGA-KIRC databases and multiple analysis methods, including survival analysis, univariate and multivariate analyses, KEGG and GSEA. Wound healing and Transwell assays were performed to check the 786-O cell and Caki-1 cell migration abilities after knockdown of MICAL1. Western blotting was used to assess the regulatory effect of MICAL1 on the Rac1 activation. Additionally, the function of MICAL1 and the correlations between MICAL1 and immune infiltration levels in KIRC were investigated using TIMER and TISIDB.ResultsMICAL1 expression was significantly higher in carcinoma tissue compared with non-cancerous tissue. A survival analysis revealed that patients with high MICAL1 expression had shorter overall survival (OS) and disease-specific survival (DSS) compared with patients with low MICAL1 expression. ROC analysis also confirmed that MICAL1 has a high diagnostic value in KIRC. Importantly, the univariate and multivariate Cox analysis further confirmed that high MICAL1 expression was an independent risk factor for OS in patients with KIRC. In accordance with this, knockdown of MICAL1 expression decreased Rac1 activation and cell migration. KEGG and GSEA analysis revealed that the immune infiltration and Ras signaling pathways were significantly upregulated in the high MICAL1 expression group. In terms of immune infiltrating levels, MICAL1 expression was positively associated with CD8+/Treg cell infiltration levels. Specifically, bioinformatic analysis showed that MICAL1 expression had strong relationships with various T cell exhaustion markers.ConclusionsMICAL1 expression may act as a prognostic biomarker for determining the prognosis in renal clear cell carcinoma and plays an important role in regulating tumor immune microenvironment and cell migratory capacity.