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Contrast-induced acute kidney injury (CIAKI) is a common complication with limited treatments. Intermedin (IMD), a peptide belonging to the calcitonin gene-related peptide family, promotes vasodilation and endothelial stability, but its role in mitigating CIAKI remains unexplored. This study investigates the protective effects of IMD in CIAKI, focusing on its mechanisms, particularly the cAMP/Rac1 signaling pathway. Human umbilical vein endothelial cells (HUVECs) were treated with iohexol to simulate kidney injury in vitro. The protective effects of IMD were assessed using CCK8 assay, flow cytometry, ELISA, and Western blotting. A CIAKI rat model was utilized to evaluate renal peritubular capillary endothelial cell injury and renal function through histopathology, immunohistochemistry, immunofluorescence, Western blotting, and transmission electron microscopy. In vitro, IMD significantly enhanced HUVEC viability and mitigated iohexol-induced toxicity by preserving intercellular adhesion junctions and activating the cAMP/Rac1 pathway, with Rac1 inhibition attenuating these protective effects. In vivo, CIAKI caused severe damage to peritubular capillary endothelial cell junctions, impairing renal function. IMD treatment markedly improved renal function, an effect negated by Rac1 inhibition. IMD protects against renal injury in CIAKI by activating the cAMP/Rac1 pathway, preserving peritubular capillary endothelial integrity and alleviating acute renal injury from contrast media. These findings suggest that IMD has therapeutic potential in CIAKI and highlight the cAMP/Rac1 pathway as a promising target for preventing contrast-induced acute kidney injury in at-risk patients, ultimately improving clinical outcomes. [ABSTRACT FROM AUTHOR]

Disulfidptosis is a novel discovered form of programmed cell death (PCD) that diverges from apoptosis, necroptosis, ferroptosis, and cuproptosis, stemming from disulfide stress-induced cytoskeletal collapse. In cancer cells exhibiting heightened expression of the solute carrier family 7 member 11 (SLC7A11), excessive cystine importation and reduction will deplete nicotinamide adenine dinucleotide phosphate (NADPH) under glucose deprivation, followed by an increase in intracellular disulfide stress and aberrant disulfide bond formation within actin networks, ultimately culminating in cytoskeletal collapse and disulfidptosis. Disulfidptosis involves crucial physiological processes in eukaryotic cells, such as cystine and glucose uptake, NADPH metabolism, and actin dynamics. The Rac1-WRC pathway-mediated actin polymerization is also implicated in this cell death due to its contribution to disulfide bond formation. However, the precise mechanisms underlying disulfidptosis and its role in tumors are not well understood. This is probably due to the multifaceted functionalities of SLC7A11 within cells and the complexities of the downstream pathways driving disulfidptosis. This review describes the critical roles of SLC7A11 in cells and summarizes recent research advancements in the potential pathways of disulfidptosis. Moreover, the less-studied aspects of this newly discovered cell death process are highlighted to stimulate further investigations in this field. [ABSTRACT FROM AUTHOR]

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen‐harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two‐cell stage after cell division, the cell doublet typically displays “inverted” polarity, with apical and basolateral proteins being located to the ECM‐facing and cell–cell‐contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM‐abutting periphery and subsequent apical vesicle delivery to a cell–cell contact site for lumen formation. Here, we show that downstream of the ECM‐signal‐transducer β1‐integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin‐Darby canine kidney (MDCK) cells at a later two‐cell stage in three‐dimensional culture. Rac1–GTP facilitates IQGAP1 interaction with the Rac‐specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1–GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin‐mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two‐cell stage via its interaction with IQGAP1. [ABSTRACT FROM AUTHOR]

Objective: To investigate the correlation between serum Rac1 enzyme (Rac1) level with asthma control, airway inflammatory response and lung function in asthmatic children. Methods: A retrospective analysis was performed on 79 children with asthma who were diagnosed and treated in our hospital from June 2020 to January 2023. According to the severity of the disease, the children were divided into mild group (25 cases), moderate group (30 cases) and severe group (24 cases). 36 healthy children who underwent physical examination at the same period in our hospital were selected as the control group. The state of an illness, control level, serum mRNA Rac1, inflammatory factors, and lung function of the children in two groups were compared between the control group and the observation group. Results: The Rac1 mRNA levels, forced vital capacity (FVC), forced expiratory volume in one second/FVC (FEV1/FVC), peak expiratory flow (PEF), and maximum mid-expiratory flow (MMEF) in the observation group were significantly lower than these in the control group (P < 0.05). The tumor necrosis factor-alpha (TNF-α), interleukin-5 (IL-5), IL-6, and IL-33 in the observation group were markedly higher than these in the control group (P < 0.05). As the state of an illness worsened, the Rac1 mRNA levels, FVC, FEV1/FVC, PEF, and MMEF gradually reduced (P < 0.05), while the levels of TNF-α, IL-5, IL-6, and IL-33 increased (P < 0.05). As the degree of disease control improved, the Rac1 mRNA levels, FVC, FEV1/FVC, PEF, and MMEF gradually elevated (P < 0.05), and the levels of TNF- α, IL-5, IL-6, and IL-33 showed the opposite trend (P < 0.05). Rac1 was negatively related to the levels of TNF-α, IL-5, IL-6 and IL-33 (P < 0.05), and positively to the levels of FVC, FEV1/FVC, PEF and MMEF (P < 0.001). Rac1 mRNA levels, FVC, FEV1/FVC, PEF and MMEF were protective factors, while TNF-α, IL-5, IL-6 and IL-33 were risk factors for the prognosis of children with asthma (P < 0.05). Conclusion: Children with asthma have obviously lower serum Rac1 mRNA levels, higher inflammatory factor levels and lower lung function. Serum Rac1 mRNA level may be associated with better asthma control, lower airway inflammatory response, better lung function and lower disease severity. It has important reference value for the evaluation of the state of an illness, efficacy and prognosis of children with bronchial asthma. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (LIHC) has severe consequences due to late diagnosis and the lack of effective therapies. Currently, potential biomarkers for the diagnosis and prognosis of LIHC have not been systematically evaluated. Previous studies have reported that RAC1 is associated with the B cell receptor signaling pathway in various tumor microenvironments, but its relationship with LIHC remains unclear. We investigated the relationship between RAC1 and the prognosis and immune infiltration microenvironment of LIHC, exploring its potential as a prognostic biomarker for this type of cancer. Methods: In this study, we analyzed data from The Cancer Genome Atlas (TCGA) using the Wilcoxon signed-rank test and logistic regression to assess the association between RAC1 expression and clinical characteristics in LIHC patients. Additionally, Kaplan-Meier and Cox regression methods were employed to confirm the impact of RAC1 expression levels on overall survival. Immunohistochemistry was used to validate RAC1 protein expression in LIHC. We constructed RAC1 knockdown LIHC cells and studied the effects of RAC1 protein on cell proliferation and migration at both cellular and animal levels. Results: RAC1 expression levels were significantly elevated in LIHC tissues compared to normal tissues. High RAC1 expression was strongly associated with advanced pathological stages and was identified as an independent factor negatively affecting overall survival. At both cellular and animal levels, RAC1 knockdown significantly inhibited the proliferation and migration of LIHC cells. Furthermore, RAC1 expression was positively correlated with the infiltration of Th2 cells and macrophages in the tumor microenvironment, suggesting that RAC1 may contribute to the deterioration of the tumor immunosuppressive microenvironment and potentially lead to reduced patient survival. Conclusion: These findings indicate that RAC1 expression promotes LIHC proliferation and migration and influences the landscape of immune cell infiltration in the tumor microenvironment. Based on these results, RAC1 is proposed as a potential prognostic biomarker for LIHC, associated with both cancer progression and tumor immune cell infiltration. [ABSTRACT FROM AUTHOR]

Pyroptosis may play an important role in the resistance of ovarian cancer (OC) to chemotherapy. However, the mechanism by which pyroptosis modulation can attenuate chemotherapy resistance has not been comprehensively studied in OC. Here, we demonstrated that RAS‐associated C3 botulinum toxin substrate 1 (RAC1) is highly expressed in OC and is negatively correlated with patient outcomes. Through cell function tests and in vivo tumor formation tests, we found that RAC1 can promote tumor growth by mediating paclitaxel (PTX) resistance. RAC1 can mediate OC progression by inhibiting pyroptosis, as evidenced by high‐throughput automated confocal imaging, the release of lactate dehydrogenase (LDH), the expression of the inflammatory cytokines IL‐1β/IL‐18 and the nucleotide oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome. Mechanically, RNA‐seq, gene set enrichment analysis (GSEA), coimmunoprecipitation (Co‐IP), mass spectrometry (MS), and ubiquitination tests further confirmed that RAC1 inhibits caspase‐1/gasdermin D (GSDMD)‐mediated canonical pyroptosis through the P21‐activated kinase 4 (PAK4)/mitogen‐activated protein kinase (MAPK) pathway, thereby promoting PTX resistance in OC cells. Finally, the whole molecular pathway was verified by the results of in vivo drug combination tests, clinical specimen detection and the prognosis. In summary, our results suggest that the combination of RAC1 inhibitors with PTX can reverse PTX resistance by inducing pyroptosis through the PAK4/MAPK pathway. [ABSTRACT FROM AUTHOR]

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

Abstract Metastatic breast-cancer is one of the major causes of death, due to remaining dormant cancer cells for several years. Rac1 is upregulated with cancer and stay elevated throughout the metastatic pathway to regulate the formation of lamellipodia and filopodia. This work developed peptide FGDWS as novel inhibitor targeting Rac1-Tiam1 binding site by in-silico as it was found to be the strongest interacting peptide with Rac1 at the Tiam binding site. The binding and inhibition study of peptide with Rac1 was performed by Surface plasmon resonance and MTT assay, respectively. Cell-migration, apoptotic assay and western-blot in breast-cancer cells were performed with FGDWS and in combination with Doxorubicin (Dox). Tumor regression experiment was done with mice model. The strong binding of FGDWS with Rac1 and reduction of cell-viability were observed in breast-cancer cell-lines. The cell-migration was suppressed, and higher regression were obtained in synergy group. The apoptotic effect of FGDWS alone and with Dox were detected by annexin-V via activating caspase3/7. The tumor size was reduced by the treatment of FGDWS and more reduced in combinatorial effect. The combinatorial effect of FGDWS-Dox may enhance the treatment efficacy without side-effects.

Abstract Disulfidptosis is a novel discovered form of programmed cell death (PCD) that diverges from apoptosis, necroptosis, ferroptosis, and cuproptosis, stemming from disulfide stress-induced cytoskeletal collapse. In cancer cells exhibiting heightened expression of the solute carrier family 7 member 11 (SLC7A11), excessive cystine importation and reduction will deplete nicotinamide adenine dinucleotide phosphate (NADPH) under glucose deprivation, followed by an increase in intracellular disulfide stress and aberrant disulfide bond formation within actin networks, ultimately culminating in cytoskeletal collapse and disulfidptosis. Disulfidptosis involves crucial physiological processes in eukaryotic cells, such as cystine and glucose uptake, NADPH metabolism, and actin dynamics. The Rac1-WRC pathway-mediated actin polymerization is also implicated in this cell death due to its contribution to disulfide bond formation. However, the precise mechanisms underlying disulfidptosis and its role in tumors are not well understood. This is probably due to the multifaceted functionalities of SLC7A11 within cells and the complexities of the downstream pathways driving disulfidptosis. This review describes the critical roles of SLC7A11 in cells and summarizes recent research advancements in the potential pathways of disulfidptosis. Moreover, the less-studied aspects of this newly discovered cell death process are highlighted to stimulate further investigations in this field. Graphical Abstract

Abstract Objective To investigate the correlation between serum Rac1 enzyme (Rac1) level with asthma control, airway inflammatory response and lung function in asthmatic children. Methods A retrospective analysis was performed on 79 children with asthma who were diagnosed and treated in our hospital from June 2020 to January 2023. According to the severity of the disease, the children were divided into mild group (25 cases), moderate group (30 cases) and severe group (24 cases). 36 healthy children who underwent physical examination at the same period in our hospital were selected as the control group. The state of an illness, control level, serum mRNA Rac1, inflammatory factors, and lung function of the children in two groups were compared between the control group and the observation group. Results The Rac1 mRNA levels, forced vital capacity (FVC), forced expiratory volume in one second/FVC (FEV1/FVC), peak expiratory flow (PEF), and maximum mid-expiratory flow (MMEF) in the observation group were significantly lower than these in the control group (P

Abstract 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.

Abstract 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.

The mevalonate pathway plays an important role in breast cancer and other tumor types. However, many issues remain obscure as yet regarding its mechanism of regulation and action. In the present study, we report that the expression of mevalonate pathway enzymes is mediated by the RHO guanosine nucleotide exchange factors VAV2 and VAV3 in a RAC1‐ and sterol regulatory element‐binding factor (SREBF)‐dependent manner in breast cancer cells. Furthermore, in vivo tumorigenesis experiments indicated that the two most upstream steps of this metabolic pathway [3‐hydroxy‐3‐methylglutaryl‐coenzyme A synthase 1 (HMGCS1) and 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR)] are important for primary tumorigenesis, angiogenesis, and cell survival in breast cancer cells. HMGCR, but not HMGCS1, is also important for the extravasation and subsequent fitness of breast cancer cells in the lung parenchyma. Genome‐wide expression analyses revealed that HMGCR influences the expression of gene signatures linked to proliferation, metabolism, and immune responses. The HMGCR‐regulated gene signature predicts long‐term tumor recurrence but not metastasis in cohorts of nonsegregated and chemotherapy‐resistant breast cancer patients. These results reveal a hitherto unknown, VAV‐catalysis‐dependent mechanism involved in the regulation of the mevalonate pathway in breast cancer cells. They also identify specific mevalonate‐pathway‐dependent processes that contribute to the malignant features of breast cancer cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Activation of Rac1 in DG engrams destabilizes fear memory. • Activation of Rac1 upregulates ATG7 expression in the engrams. • ATG7 overexpression or Rac1 activation in the engrams upregulate microglial Tlr2/4. • Rac1 and ATG7 in the engrams destabilizes fear memory via microglia Tlr2/4. Rac1 is a key regulator of the cytoskeleton and neuronal plasticity, and is known to play a critical role in psychological and cognitive brain disorders. To elucidate the engram specific Rac1 signaling in fear memory, a doxycycline (Dox)-dependent robust activity marking (RAM) system was used to label dorsal dentate gyrus (DG) engram cells in mice during contextual fear conditioning. Rac1 mRNA and protein levels in DG engram cells were peaked at 24 h (day 1) after fear conditioning and were more abundant in the fear engram cells than in the non-engram cells. Optogenetic activation of Rac1 in a temporal manner in DG engram cells before memory retrieval decreased the freezing level in the fear context. Optogenetic activation of Rac1 increased autophagy protein 7 (ATG7) expression in the DG engram cells and activated DG microglia. Microglia-specific transcriptomics and fluorescence in situ hybridization revealed that overexpression of ATG7 in the fear engram cells upregulated the mRNA of Toll-like receptor TLR2/4 in DG microglia. Knockdown of microglial TLR2/4 rescued fear memory destabilization induced by ATG7 overexpression or Rac1 activation in DG engram cells. These results indicate that Rac1-driven communications between engram cells and microglia contributes to contextual fear memory destabilization, and is mediated by ATG7 and TLR2/4, and suggest a novel mechanistic framework for the cytoskeletal regulator in fear memory interference. [ABSTRACT FROM AUTHOR]

Simple Summary: Cetuximab is a treatment widely used to treat advanced, metastatic colorectal cancer (CRC), and it works by blocking epidermal growth factor receptor signaling. Unfortunately, patients inevitably develop resistance to cetuximab. The most common resistance mechanisms are well established, but there are still patients that become resistant to cetuximab due to unknown mechanisms. The small guanosine triphosphatases (GTPases) RAC1 and RAC1B have been shown to contribute to CRC progression, but their role in cetuximab resistance is unclear. This review highlights the known cetuximab resistance mechanisms and summarizes how RAC1 and RAC1B could contribute to these resistance mechanisms to propose RAC1 and RAC1B as additional therapeutic targets that could increase the efficacy of cetuximab. Colorectal cancer (CRC) is one of the most diagnosed cancers and a leading contributor to cancer-related deaths in the United States. Clinically, standard treatment regimens include surgery, radiation, and chemotherapy; however, there has been increasing development and clinical use of targeted therapies for CRC. Unfortunately, many patients develop resistance to these treatments. Cetuximab, the first targeted therapy approved to treat advanced CRC, is a monoclonal antibody that targets the epidermal growth factor receptor and inhibits downstream pathway activation to restrict tumor cell growth and proliferation. CRC resistance to cetuximab has been well studied, and common resistance mechanisms include constitutive signal transduction through downstream protein mutations and promotion of the epithelial-to-mesenchymal transition. While the most common resistance mechanisms are known, a proportion of patients develop resistance through unknown mechanisms. One protein predicted to contribute to therapy resistance is RAC1, a small GTPase that is involved in cytoskeleton rearrangement, cell migration, motility, and proliferation. RAC1 has also been shown to be overexpressed in CRC. Despite evidence that RAC1 and its alternative splice isoform RAC1B play important roles in CRC and the pathways known to contribute to cetuximab resistance, there is a need to directly study the relationship between RAC1 and RAC1B and cetuximab resistance. This review highlights the recent studies investigating RAC1 and RAC1B in the context of CRC and suggests that these proteins could play a role in resistance to cetuximab. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis. IMPORTANCE Though neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Neuroinflammation plays a crucial part in the initial onset and progression of Alzheimer's disease (AD). NLRP3 inflammasome was demonstrated to get involved in amyloid-β (Aβ)-induced neuroinflammation. However, the mechanism of Aβ-triggered activation of NLRP3 inflammasome remains poorly understood. Objective: Based on our previous data, the study aimed to identify the downstream signals that bridge the activation of TLR4 and NLRP3 inflammasome associated with Aβ. Methods: BV-2 cells were transfected with TLR4siRNA or pretreated with a CLI-095 or NSC23766, followed by Aβ1–42 treatment. APP/PS1 mice were injected intraperitoneally with CLI-095 or NSC23766. NLRP3 inflammasome and microglia activation was detected with immunostaining and western blot. G-LISA and Rac1 pull-down activation test were performed to investigate the activation of Rac1. Real-time PCR and ELISA were used to detect the inflammatory cytokines. Aβ plaques were assessed by western blotting and immunofluorescence staining. Morris water maze test was conducted to determine the spatial memory in mice. Results: Rac1 and NLRP3 inflammasome were activated by Aβ in both in vitro and in vivo experiments. Inhibition of TLR4 reduced the activity of Rac1 and NLRP3 inflammasome induced by Aβ1–42. Furthermore, inhibition of Rac1 blocked NLRP3 inflammasome activation mediated by TLR4. Blocking the pathway by CLI095 or NSC23766 suppressed Aβ1–42-triggered activation of microglia, reduced the expression of pro-inflammatory mediators and ameliorated the cognition deficits in APP/PS1 mice. Conclusions: Our study demonstrated that TLR4/Rac1/NLRP3 pathway mediated Aβ-induced neuroinflammation, which unveiled a novel pathway and key contributors underlying the pathogenic mechanism of Aβ. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a debilitating form of pain arising from injury or disease of the nervous system that affects millions of people worldwide. Despite its prevalence, the underlying mechanisms of neuropathic pain are still not fully understood. Dendritic spines are small protrusions on the surface of neurons that play an important role in synaptic transmission. Recent studies have shown that dendritic spines reorganize in the superficial and deeper laminae of the spinal cord dorsal horn with the development of neuropathic pain in multiple models of disease or injury. Given the importance of dendritic spines in synaptic transmission, it is possible that studying dendritic spines could lead to new therapeutic approaches for managing intractable pain. In this review article, we highlight the emergent role of dendritic spines in neuropathic pain, as well as discuss the potential for studying dendritic spines for the development of new therapeutics. [ABSTRACT FROM AUTHOR]

Circular RNA (circRNA) can be used as a potential target for cancer treatment. However, the biological function and potential molecular mechanism of circ_0058123 in the development of colorectal cancer (CRC) are still unclear. The expression levels of circ_0058123, microRNA-939-5p (miR-939-5p) and Rac family small GTPase 1 (RAC1) were measured by quantitative real-time polymerase chain reaction or western blot assay. 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay, transwell assay, tube formation assay and flow cytometry apoptosis assay were conducted to assess CRC cell functions. In addition, protein expression was measured with western blot assay. Dual-luciferase reporter assays and RNA immunoprecipitation assay were conducted to confirm the relationships between miR-939-5p and circ_0058123, and miR-939-5p and RAC1. In vivo CRC tumor growth experiment also were carried out to determine circ_0058123-mediatede effects on tumor formation. Our data showed that circ_0058123 and RAC1 expression were increased, but miR-939-5p was decreased in both of CRC tissues and cell lines. Circ_0058123 depletion repressed CRC cell proliferation, migration, invasion and tube formation but promoted cell apoptosis. Down-regulation of circ_0058123 could significantly suppress the CRC progression, while the addition of miR-939-5p inhibitor could reverse this effect. Circ_0058123 directly targeted miR-939-5p, and RAC1 was a target of miR-939-5p. Furthermore, RAC1 overexpression could rescue the effect of miR-939-5p on CRC development. Lastly, silence of circ_0058123 inhibited CRC tumor growth in vivo. In conclusion, circ_0058123 could promote CRC progression through regulating the miR-939-5p/RAC1 axis and may be a valuable biomarker for early diagnosis and prognosis of CRC. [ABSTRACT FROM AUTHOR]

ABSTRACT Phagocytosis of Mycobacterium tuberculosis (Mtb) followed by its integration into the matured lysosome is critical in the host defense against tuberculosis. How Mtb escapes this immune attack remains elusive. In this study, we unveiled a novel regulatory mechanism by which SIRT7 regulates cytoskeletal remodeling by modulating RAC1 activation. We discovered that SIRT7 expression was significantly reduced in CD14+ monocytes of TB patients. Mtb infection diminished SIRT7 expression by macrophages at both the mRNA and protein levels. SIRT7 deficiency impaired actin cytoskeleton-dependent macrophage phagocytosis, LC3II expression, and bactericidal activity. In a murine tuberculosis model, SIRT7 deficiency detrimentally impacted host resistance to Mtb, while Sirt7 overexpression significantly increased the host defense against Mtb, as determined by bacterial burden and inflammatory–histopathological damage in the lung. Mechanistically, we demonstrated that SIRT7 limits Mtb infection by directly interacting with and activating RAC1, through which cytoskeletal remodeling is modulated. Therefore, we concluded that SIRT7, in its role regulating cytoskeletal remodeling through RAC1, is critical for host responses during Mtb infection and proposes a potential target for tuberculosis treatment.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health issue. Critical to macrophages’ defense against Mtb is phagocytosis, governed by the actin cytoskeleton. Previous research has revealed that Mtb manipulates and disrupts the host’s actin network, though the specific mechanisms have been elusive. Our study identifies a pivotal role for SIRT7 in this context: Mtb infection leads to reduced SIRT7 expression, which, in turn, diminishes RAC1 activation and consequently impairs actin-dependent phagocytosis. The significance of our research is that SIRT7 directly engages with and activates Rac Family Small GTPase 1 (RAC1), thus promoting effective phagocytosis and the elimination of Mtb. This insight into the dynamic between host and pathogen in TB not only broadens our understanding but also opens new avenues for therapeutic development.

Abstract Pyroptosis may play an important role in the resistance of ovarian cancer (OC) to chemotherapy. However, the mechanism by which pyroptosis modulation can attenuate chemotherapy resistance has not been comprehensively studied in OC. Here, we demonstrated that RAS‐associated C3 botulinum toxin substrate 1 (RAC1) is highly expressed in OC and is negatively correlated with patient outcomes. Through cell function tests and in vivo tumor formation tests, we found that RAC1 can promote tumor growth by mediating paclitaxel (PTX) resistance. RAC1 can mediate OC progression by inhibiting pyroptosis, as evidenced by high‐throughput automated confocal imaging, the release of lactate dehydrogenase (LDH), the expression of the inflammatory cytokines IL‐1β/IL‐18 and the nucleotide oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome. Mechanically, RNA‐seq, gene set enrichment analysis (GSEA), coimmunoprecipitation (Co‐IP), mass spectrometry (MS), and ubiquitination tests further confirmed that RAC1 inhibits caspase‐1/gasdermin D (GSDMD)‐mediated canonical pyroptosis through the P21‐activated kinase 4 (PAK4)/mitogen‐activated protein kinase (MAPK) pathway, thereby promoting PTX resistance in OC cells. Finally, the whole molecular pathway was verified by the results of in vivo drug combination tests, clinical specimen detection and the prognosis. In summary, our results suggest that the combination of RAC1 inhibitors with PTX can reverse PTX resistance by inducing pyroptosis through the PAK4/MAPK pathway.

The small GTPase Rac1 (Ras‐related C3 botulinum toxin substrate 1) has been implicated in cancer progression and in the poor prognosis of various types of tumors. Rac1 SUMOylation occurs during epithelial‐mesenchymal transition (EMT), and it is required for tumor cell migration and invasion. Here we identify POTEE (POTE Ankyrin domain family member E) as a novel Rac1‐SUMO1 effector involved in breast cancer malignancy that controls invadopodium formation through the activation of Rac1‐SUMO1. POTEE activates Rac1 in the invadopodium by recruiting TRIO‐GEF (triple functional domain protein), and it induces tumor cell proliferation and metastasis in vitro and in vivo. We found that the co‐localization of POTEE with Rac1 is correlated with more aggressive breast cancer subtypes. Given its role in tumor dissemination, the leading cause of cancer‐related deaths, POTEE could represent a potential therapeutic target for these types of cancer.

Abstract Background Cingulin (CGN) is a pivotal cytoskeletal adaptor protein located at tight junctions. This study investigates the link between CGN mutation and increased cancer susceptibility through genetic and mechanistic analyses and proposes a potential targeted therapeutic approach. Methods In a high-cancer-density family without known pathogenic variants, we performed tumor-targeted and germline whole-genome sequencing to identify novel cancer-associated variants. Subsequently, these variants were validated in a 222 cancer patient cohort, and CGN c.3560C > T was identified as a potential cancer-risk allele. Both wild-type (WT) (c.3560C > C) and variant (c.3560C > T) were transfected into cancer cell lines and incorporated into orthotopic xenograft mice model for evaluating their effects on cancer progression. Western blot, immunofluorescence analysis, migration and invasion assays, two-dimensional gel electrophoresis with mass spectrometry, immunoprecipitation assays, and siRNA applications were used to explore the biological consequence of CGN c.3560C > T. Results In cancer cell lines and orthotopic animal models, CGN c.3560C > T enhanced tumor progression with reduced sensitivity to oxaliplatin compared to the CGN WT. The variant induced downregulation of epithelial marker, upregulation of mesenchymal marker and transcription factor, which converged to initiate epithelial-mesenchymal transition (EMT). Proteomic analysis was conducted to investigate the elements driving EMT in CGN c.3560C > T. This exploration unveiled overexpression of IQGAP1 induced by the variant, contrasting the levels observed in CGN WT. Immunoprecipitation assay confirmed a direct interaction between CGN and IQGAP1. IQGAP1 functions as a regulator of multiple GTPases, particularly the Rho family. This overexpressed IQGAP1 was consistently associated with the activation of Rac1, as evidenced by the analysis of the cancer cell line and clinical sample harboring CGN c.3560C > T. Notably, activated Rac1 was suppressed following the downregulation of IQGAP1 by siRNA. Treatment with NSC23766, a selective inhibitor for Rac1-GEF interaction, resulted in the inactivation of Rac1. This intervention mitigated the EMT program in cancer cells carrying CGN c.3560C > T. Consistently, xenograft tumors with WT CGN showed no sensitivity to NSC23766 treatment, but NSC23766 demonstrated the capacity to attenuate tumor growth harboring c.3560C > T. Conclusions CGN c.3560C > T leads to IQGAP1 overexpression, subsequently triggering Rac1-dependent EMT. Targeting activated Rac1 is a strategy to impede the advancement of cancers carrying this specific variant.

Abstract Background Beta‐defensins (BDs) are antimicrobial peptides secreted upon epithelial injury. Both chemotactic and antimicrobial properties of BDs function as initial steps in host defence and prime the adaptive immune system in the body. Psoriasis, a chronic immune‐mediated inflammatory disease, has both visible cutaneous manifestations as well as known associations with higher incidence of cardiometabolic complications and vascular inflammation. Objectives We aimed to investigate the circulating expression of beta‐defensin‐2 (BD2) in psoriasis at baseline compared to control subjects, along with changes in BD2 levels following biologic treatment at 1 year. The contribution of BD2 to subclinical atherosclerosis is also assessed. In addition, we have sought to unravel signalling mechanisms linking inflammation with BD2 expression. Methods Multimodality imaging, as well as inflammatory biomarker assays, were performed in biological naïve psoriasis (n = 71) and non‐psoriasis (n = 53) subjects. A subset of psoriasis patients were followed for 1 year after biological intervention (antitumour necrosis factor‐α [TNF‐α], n = 30; anti‐interleukin17A [IL17A], n = 21). Measurements of circulating BD2 were completed by enzyme‐linked immunosorbent assay (ELISA). Using HaCaT‐transformed keratinocytes, expression of BD2 upon cytokine treatment was assessed by quantitative polymerase chain reaction and ELISA. Results Herein, we confirm that human circulating BD2 levels are associated with psoriasis, which attenuate upon biological interventions (anti‐TNF‐α, anti‐IL‐17A). A link between circulating BD2 and subclinical atherosclerosis markers was not observed. Furthermore, we demonstrate that IL‐17A‐driven BD2 expression occurs in a Phosphatidylinositol 3‐kinase (PI3‐kinase) and Rac1 GTPase‐dependent manner. Conclusions Our findings expand on the potential role of BD2 as a tractable biomarker in psoriasis patients and describe the role of an IL‐17A‐PI3‐kinase/Rac signalling axis in regulating BD2 levels in keratinocytes.

Objectives The small GTPase Rac1 (RAC1) has been linked to podocyte disorders and steroid-sensitive nephrotic syndrome (SSNS). The aim of this study was to explore and validate the potential causal association between circulating RAC1 and SSNS.Methods The association between circulating RAC1 and SSNS at both gene expression and proteomic levels was investigated using Mendelian randomization analysis, and further validated by single-cell RNA-sequencing, proteomic analysis, and experimental studies. The genetic instruments comprised cis-expression quantitative trait loci (cis-eQTLs) associated with RAC1 gene expression and protein QTLs correlated with plasma RAC1 protein levels. Causal associations were estimated utilizing the inverse variance weighted and MR-PRESSO methods. Validation of RAC1 expression was conducted through single-cell RNA-sequencing of peripheral blood mononuclear cells from patients with SSNS and healthy controls. Proteomic analysis was performed among patients with minimal change nephrotic syndrome. Experimental validation was conducted using a puromycin aminonucleoside (PAN)-induced nephrosis model.Results Increased expression of RAC1 was associated with a higher risk of SSNS (gene expression level: odds ratio [OR], 1.53; 95% confidence interval [CI], 1.02–2.28; protein level: OR, 1.82; 95% CI, 1.05–3.17). The results of MR-PRESSO were consistent (gene expression level: OR, 1.49; 95% CI, 1.17–1.92; protein level: OR, 1.81; 95% CI, 1.16–2.85). Single-cell RNA sequencing and proteomic analysis confirmed elevated RAC1 expression in patients with SSNS compared to healthy controls. Experimental data further supported increased RAC1 expression in PAN-induced nephropathy.Conclusions Increased expression of RAC1 might be causally associated with SSNS, suggesting that targeting RAC1 might represent a potential therapeutic strategy for SSNS.

Background Irisin, as a myokine, plays a protective role against cardiovascular disease, including myocardial infarction, atherosclerosis and hypertension. However, whether irisin attenuates salt-sensitive hypertension and the related underlying mechanisms is unknown.Methods Male Dahl salt-resistant (DSR) and Dahl salt-sensitive (DSS) (12 weeks) rats were fed a high salt diet (8% NaCl) with or without irisin treatment by intraperitoneal injection for 8 weeks.Results Compared with DSR rats, DSS rats showed higher systolic blood pressure (SBP), impaired natriuresis and diuresis and renal dysfunction. In addition, it was accompanied by downregulation of renal p-AMPKα and upregulation of renal RAC1 and nuclear mineralocorticoid receptor (MR). Irisin intervention could significantly up-regulated renal p-AMPKα level and down-regulated renal RAC1-MR signal, thereby improving renal sodium excretion and renal function, and ultimately reducing blood pressure in DSS rats. Ex vivo treatment with irisin reduced the expression of RAC1 and nuclear MR in primary renal distal convoluted tubule cells from DSS rats and the effects of irisin were abolished by cotreatment of compound C (AMPK inhibitor), indicating that the regulation of RAC1-MR signals by irisin depended on the activation of AMPK.Conclusions Irisin administration lowered salt-sensitive hypertension through regulating RAC1-MR signaling via activation of AMPK, which may be a promising therapeutic approach for salt-sensitive hypertension.

Background: Recent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH. Methods: We analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression. Results: Forty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1's effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation. Conclusions: Our findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment. 1. Identification of Differentially Expressed microRNAs (DEmiRs) in ITGB1 overexpressed TCs. 2. Effects of Exo-ITGB1 or miR-429-3p on Hypoxia-Induced PASMCs in vitro. 3. Exo-ITGB1 inhibits the hyper-proliferation and migration of PASMCs through regulating miR-429-3p/Rac1 axis in vitro. 4. The therapeutic potential of Exo-ITGB1 in hypoxia-induced PAH model in vivo. [ABSTRACT FROM AUTHOR]

Macrophage-mediated inflammation plays a significant role in the development and progression of diabetic kidney disease (DKD). Studies have suggested that impaired macrophage efferocytosis aggravates the inflammatory response. However, its contribution to DKD progression remains unknown. Using single-cell RNA sequencing (scRNA-seq) data obtained from the GSE131882, GSE195460, GSE151302, GSE195460, and GSE131685 datasets, we successfully clustered 13 cell types. Through analysis of the ligand-receptor network, it was discovered that macrophages interact with other cells. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that macrophages exhibit a heightened presence of phagocytosis signaling. We discovered that RAC1 was closely related to macrophage efferocytosis through a Venn diagram and protein-protein interaction (PPI) analysis, which predicted the correlation with the clinical features of DKD using the NephroseqV5 tool. Furthermore, we verified that RAC1 exhibited decreased expression in macrophages cultured with lipopolysaccharide (LPS) and high glucose. Nevertheless, the overexpression of RAC1 promoted macrophage efferocytosis and inhibited the inflammatory response. In summary, our study focused on examining the presence and importance of efferocytosis-related molecules in DKD macrophages. Through a comprehensive analysis using scRNA-seq, we discovered that RAC1 plays a crucial role as an efferocytosis molecule in DKD. These findings enhance our current knowledge of the molecular mechanisms involved in the development of DKD and aid the exploration of new treatments. [ABSTRACT FROM AUTHOR]

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling. [ABSTRACT FROM AUTHOR]

Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs. [ABSTRACT FROM AUTHOR]

Background: Kinase D‐interacting substrate of 220 kDa (KIDINS220) is a multifunctional scaffolding protein essential for neuronal development. It has been implicated in neurological diseases with either autosomal dominant (AD) or autosomal recessive (AR) inheritance patterns. The molecular mechanisms underlying the AR/AD dual nature of KIDINS220 remain elusive, posing challenges to genetic interpretation and clinical interventions. Moreover, increased KIDINS220 exhibited neurotoxicity, but its role in neurodevelopment remains unclear. Objective: The aim was to investigate the genotype–phenotype correlations of KIDINS220 and elucidate its pathophysiological role in neuronal development. Methods: Whole‐exome sequencing was performed in a four‐generation family with cerebral palsy. CRISPR/Cas9 was used to generate KIDINS220 mutant cell lines. In utero electroporation was employed to investigate the effect of KIDINS220 variants on neurogenesis in vivo. Results: We identified in KIDINS220 a pathogenic nonsense variant (c.4177C > T, p.Q1393*) that associated with AD cerebral palsy. We demonstrated that the nonsense variants located in the terminal exon of KIDINS220 are gain‐of‐function (GoF) variants, which enable the mRNA to escape nonsense‐mediated decay and produce a truncated yet functional KIDINS220 protein. The truncated protein exhibited significant resistance to calpain and consequently accumulated within cells, resulting in the hyperactivation of Rac1 and defects in neuronal development. Conclusions: Our findings demonstrate that the location of variants within KIDINS220 plays a crucial role in determining inheritance patterns and corresponding clinical outcomes. The proposed interaction between Rac1 and KIDINS220 provides new insights into the pathogenesis of cerebral palsy, implying potential therapeutic perspectives. © 2023 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Background: Cingulin (CGN) is a pivotal cytoskeletal adaptor protein located at tight junctions. This study investigates the link between CGN mutation and increased cancer susceptibility through genetic and mechanistic analyses and proposes a potential targeted therapeutic approach. Methods: In a high-cancer-density family without known pathogenic variants, we performed tumor-targeted and germline whole-genome sequencing to identify novel cancer-associated variants. Subsequently, these variants were validated in a 222 cancer patient cohort, and CGN c.3560C > T was identified as a potential cancer-risk allele. Both wild-type (WT) (c.3560C > C) and variant (c.3560C > T) were transfected into cancer cell lines and incorporated into orthotopic xenograft mice model for evaluating their effects on cancer progression. Western blot, immunofluorescence analysis, migration and invasion assays, two-dimensional gel electrophoresis with mass spectrometry, immunoprecipitation assays, and siRNA applications were used to explore the biological consequence of CGN c.3560C > T. Results: In cancer cell lines and orthotopic animal models, CGN c.3560C > T enhanced tumor progression with reduced sensitivity to oxaliplatin compared to the CGN WT. The variant induced downregulation of epithelial marker, upregulation of mesenchymal marker and transcription factor, which converged to initiate epithelial-mesenchymal transition (EMT). Proteomic analysis was conducted to investigate the elements driving EMT in CGN c.3560C > T. This exploration unveiled overexpression of IQGAP1 induced by the variant, contrasting the levels observed in CGN WT. Immunoprecipitation assay confirmed a direct interaction between CGN and IQGAP1. IQGAP1 functions as a regulator of multiple GTPases, particularly the Rho family. This overexpressed IQGAP1 was consistently associated with the activation of Rac1, as evidenced by the analysis of the cancer cell line and clinical sample harboring CGN c.3560C > T. Notably, activated Rac1 was suppressed following the downregulation of IQGAP1 by siRNA. Treatment with NSC23766, a selective inhibitor for Rac1-GEF interaction, resulted in the inactivation of Rac1. This intervention mitigated the EMT program in cancer cells carrying CGN c.3560C > T. Consistently, xenograft tumors with WT CGN showed no sensitivity to NSC23766 treatment, but NSC23766 demonstrated the capacity to attenuate tumor growth harboring c.3560C > T. Conclusions: CGN c.3560C > T leads to IQGAP1 overexpression, subsequently triggering Rac1-dependent EMT. Targeting activated Rac1 is a strategy to impede the advancement of cancers carrying this specific variant. [ABSTRACT FROM AUTHOR]

The small GTPase Rac1 (Ras‐related C3 botulinum toxin substrate 1) has been implicated in cancer progression and in the poor prognosis of various types of tumors. Rac1 SUMOylation occurs during epithelial‐mesenchymal transition (EMT), and it is required for tumor cell migration and invasion. Here we identify POTEE (POTE Ankyrin domain family member E) as a novel Rac1‐SUMO1 effector involved in breast cancer malignancy that controls invadopodium formation through the activation of Rac1‐SUMO1. POTEE activates Rac1 in the invadopodium by recruiting TRIO‐GEF (triple functional domain protein), and it induces tumor cell proliferation and metastasis in vitro and in vivo. We found that the co‐localization of POTEE with Rac1 is correlated with more aggressive breast cancer subtypes. Given its role in tumor dissemination, the leading cause of cancer‐related deaths, POTEE could represent a potential therapeutic target for these types of cancer. [ABSTRACT FROM AUTHOR]

Osteoporosis is a prevalent metabolic bone disease. While drug therapy is essential to prevent bone loss in osteoporotic patients, current treatments are limited by side effects and high costs, necessitating the development of more effective and safer targeted therapies. Utilizing a zebrafish (Danio rerio) larval model of osteoporosis, we explored the influence of the metabolite spermine on bone homeostasis. Results showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone resorption. Spermine not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and cardiotoxicity. Notably, spermine showcased protective attributes in the nervous systems of both zebrafish embryos and larvae. At the molecular level, Rac1 was identified as playing a pivotal role in mediating the antiosteoporotic effects of spermine, with P53 potentially acting downstream of Rac1. These findings were confirmed using mouse (Mus musculus) models, in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions, suggesting strong potential as a bone strengthening agent. This study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development, highlighting pivotal molecular mediators. Given their efficacy and safety, human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents. [ABSTRACT FROM AUTHOR]

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase belonging to the Rho family. It acts as a binary molecular switch regulating several cellular functions, including cell adhesion and migration. Malfunctions due to the P29S mutation in Rac1 increase the stability of the activated form of Rac1. This sustained activation can drive aberrant cellular processes associated with cancer, such as cell proliferation, survival, and migration. Therefore, finding an inhibitor that can inhibit the mutant form of the protein is very important. Rhein, a natural compound with diverse pharmacological properties, has been studied in relation to Rac1. However, specific interactions between Rhein and Rac1 have not been examined. In this study, we investigated the potential of Rhein, a natural compound, as an inhibitor of two forms of Rac1: the wild type and the P29S mutation, using molecular dynamics simulations. Results indicated that the P29S mutation led to structural changes in the Rac1 protein, which resulted in greater accessibility of the Rhein to the active site. In addition, the binding energy of Rhein to mutant Rac1 was more negative than the native protein. Therefore, it seems that the Rhein has a better inhibitory effect on the P29S-mutated form of the Rac1 protein.

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 chaperone protein EROS (“Essential for Reactive Oxygen Species”) was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration in vivo.

Abstract VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV−) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles’ heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

Abstract Background Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. Methods NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. Results Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. Conclusions Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

Objective(s): The prognosis of endometrial cancer (EC) is significantly affected by tumor infiltration and metastasis. Cortactin (CTTN) regulates infiltration and metastasis in other tumors. Studies on the role and mechanism of CTTN in EC are limited and further studies are needed.Materials and Methods: Quantitative PCR and immunohistochemistry were used to detect Ras-associated C3 botulinum toxin substrate 1 (Rac1) and CTTN in EC and normal tissues. The relationship between the expression of these two genes and their prognostic factors was analyzed. A CTTN-RNAi lentiviral system was constructed and transfected into EC cells. Migration and invasion were evaluated by scratch assay, transwell migration, and invasion assays. Pseudopodia formation was observed by immunofluorescence staining. Western blotting was performed to detect the expression of Rac1.Results: The expression levels of Rac1 and CTTN in EC tissues were significantly higher than those in normal tissues. In the EC group, Rac1 and CTTN levels were correlated. The protein expression levels of Rac1 and CTTN were related to myometrial invasion and stage. After CTTN knockdown, the migration rate, invasiveness, and migratory ability of EC cells decreased significantly. Lamellipodia was observed to disappear with the appearance of blebs. Rac1 protein expression was decreased after CTTN knockdown.Conclusion: CTTN may promote the invasion and migration of EC by lamellipodia. This effect may be related to the regulation of Rac1 by CTTN.

Abstract Background Despite recent advances in locoregional, systemic, and novel checkpoint inhibitor treatment, hepatocellular carcinoma (HCC) is still associated with poor prognosis. The feasibility of potentially curative liver resection (LR) and transplantation (LT) is limited by the underlying liver disease and a shortage of organ donors. Especially after LR, high recurrence rates present a problem and circulating tumor cells are a major cause of extrahepatic recurrence. Tigecycline, a commonly used glycylcycline antibiotic, has been shown to have antitumorigenic effects and could be used as a perioperative and adjuvant therapeutic strategy to target circulating tumor cells. We aimed to investigate the effect of tigecycline on HCC cell lines and its mechanisms of action. Methods Huh7, HepG2, Hep3B, and immortalized hepatocytes underwent incubation with clinically relevant tigecycline concentrations, and the influence on proliferation, migration, and invasion was assessed in two- and three-dimensional in vitro assays, respectively. Bioinformatic analysis was used to identify specific targets of tigecycline. The expression of RAC1 was detected using western blot, RT-PCR and RNA sequencing. ELISA and flow cytometry were utilized to measure reactive oxygen species (ROS) generation upon tigecycline treatment and flow cytometry to detect alterations in cell cycle. Changes in mitochondrial function were detected via seahorse analysis. RNA sequencing was performed to examine involved pathways. Results Tigecycline treatment resulted in a significant reduction of mitochondrial function with concomitantly preserved mitochondrial size, which preceded the observed decrease in HCC cell viability. The sensitivity of HCC cells to tigecycline treatment was higher than that of immortalized non-cancerous THLE-2 hepatocytes. Tigecycline inhibited both migratory and invasive properties. Tigecycline application led to an increase of detected ROS and an S-phase cell cycle arrest. Bioinformatic analysis identified RAC1 as a likely target for tigecycline and the expression of this molecule was increased in HCC cells as a result of tigecycline treatment. Conclusion Our study provides evidence for the antiproliferative effect of tigecycline in HCC. We show for the first time that this effect, likely to be mediated by reduced mitochondrial function, is associated with increased expression of RAC1. The reported effects of tigecycline with clinically relevant and achievable doses on HCC cells lay the groundwork for a conceivable use of this agent in cancer treatment.