Expression Of Cdc42 Research Articles

Cdc42 improve SARS-CoV-2 spike protein-induced cellular senescence through activating of Wnt/β-Catenin signaling pathway.

SARS-CoV-2 infection drove senescent cells and the senescence-associated phenotypes were reported playing roles in disease progression, which contributes to severe COVID-19 and related sequelae. Cdc42 is involved in the regulation of cellular senescence. This study, aimed to investigate the mechanism of the SARS-CoV-2 spike protein regulating cellular senescence through Cdc42. K18-hACE2 mice were infected with SARS-CoV-2 Omicron BA.4 or stimulated with spike protein through the airway, the senescent cells and Cdc42 expression in lung tissue were detected. Overexpression of spike protein or exogenous incubation of spike protein was used to simulate the induction of cellular senescence by spike protein. Mechanistic insights into the role of Cdc42 were mainly explored using Western Blot and qRT-PCR. Spike protein, SARS-CoV-2 infection related, accelerates cell aging by upregulating Cdc42 expression, which furtherly activated the Wnt/β-catenin signaling pathway. Conversely, treatment with ML141 in animal models, a Cdc42 inhibitor, reduced cellular senescence and ameliorated lung injury and inflammation. These results suggest that the upregulation of Cdc42 by the SARS-CoV-2 spike protein induces cellular senescence and enhances β-catenin nuclear translocation. This study provides insights into the mechanisms underlying cellular senescence induced by the SARS-CoV-2 spike protein, offering potential strategies to mitigate the inflammatory response and complications associated with COVID-19 in both the acute and long-term phases.

Overexpression of CDC42 causes accumulation of DNA damage leading to failure of oogenesis in triploid Pacific oyster Crassostrea gigas

Triploid Pacific oyster Crassostrea gigas exhibits notable differences in fecundity, with the majority being sterile individuals, referred to as female β, which produce few oocytes, while a minority are fertile individuals, referred to as female α, which produce abundant oocytes. However, the molecular mechanisms underlying these differences in triploid fecundity remain poorly understood. CDC42 has been implicated in processes related to increased DNA damage and genomic instability. Here, we investigate the crucial role of CDC42 in DNA damage repair during oogenesis in triploid C. gigas. Immunofluorescence analysis of γH2AX, a marker for DNA double-stranded breaks, showed significantly higher levels of DNA damage in gonadal cells of triploids compared to diploids, particularly in female β. Histological and ultrastructural analyses revealed abnormal germ cells, termed β gonia, characterized by giant nuclei condensed into irregular chromosome-like chromatin, present in triploid gonadal follicles. Analysis of mRNA and protein expression revealed significantly elevated CDC42 expression in triploid gonads compared to the diploids. Inhibition of CDC42 activity in triploids using ZCL278, a CDC42-specific inhibitor, resulted in a significant reduction in DNA damage, increased oocyte numbers, and a decrease in β gonia count. Transcriptome profiling revealed that CDC42 inhibition upregulated the PI3K-AKT signaling pathway along with DNA repair activation. Overall, our findings suggest that overexpression of CDC42 during oogenesis in triploid C. gigas impedes DNA repair, leading to the accumulation of DNA damage, and consequently, oogenesis blockade and abnormal germ cell differentiation. Conversely, inhibition of CDC42 activity activates the PI3K-AKT signaling pathway and promotes DNA repair, thereby mitigating DNA damage and facilitating oogenesis in triploids. This study provides new insights into the molecular mechanisms of sterility in female triploid C. gigas.

Key Genes Involved in the Beneficial Mechanism of Hyperbaric Oxygen for Glioblastoma and Predictive Indicators of Hyperbaric Oxygen Prolonging Survival in Glioblastoma Patients.

The prognosis of glioblastoma is poor, and therapy-resistance is largely attributed to intratumor hypoxia. Hyperbaric oxygen (HBO) effectively alleviates hypoxia. However, the sole role of HBO in glioblastoma remains controversial. We previously reported that HBO can promote apoptosis, shorten protrusions, and delay growth of glioblastoma, but the molecular mechanism is unclear. We aimed to test candidate genes in HBO-exposed glioblastoma cells and to analyze their correlation with the survival of glioblastoma patients. Glioblastoma cell lines exposed to repetitive HBO or normobaric air (NBA) were collected for RNA isolation and microarray data analysis. GO analysis, KEGG pathway analysis and survival analysis of the differentially expressed genes (DEGs) were performed. HBO not only inhibited hypoxia-inducing genes including CA9, FGF11, PPFIA4, TCAF2 and SLC2A12, but also regulated vascularization by downregulating the expression of COL1A1, COL8A1, COL12A1, RHOJ and FILIP1L, ultimately attenuated hypoxic microenvironment of glioblastoma. HBO attenuated inflammatory microenvironment by reducing the expression of NLRP2, CARD8, MYD88 and CD180. HBO prevented metastasis by downregulating the expression of NTM, CXCL12, CXCL13, CXCR4, CXCR5, CDC42, IGFBP3, IGFBP5, GPC6, MMP19, ADAMTS1, EFEMP1, PTBP3, NF1 and PDCD1. HBO upregulated the expression of BAK1, PPIF, DDIT3, TP53I11 and FAS, whereas downregulated the expression of MDM4 and SIVA1, thus promoting apoptosis. HBO upregulated the expression of CDC25A, MCM2, PCNA, RFC33, DSCC1 and CDC14A, whereas downregulated the expression of ASNS, CDK6, CDKN1B, PTBP3 and MAD2L1, thus inhibiting cell cycle progression. Among these DEGs, 17 indicator-genes of HBO prolonging survival were detected. HBO is beneficial for glioblastoma. Glioblastoma patients with these predictive indicators may prolong survival with HBO therapy. These potential therapeutic targets especially COL1A1, ADAMTS1 and PTBP3 deserve further validation.

Chungtaejeon (CTJ) inhibits adhesion and migration of VSMC through cytoskeletal remodeling pathway

IntroductionVascular remodeling is crucial for the progression of vascular disease such as atherosclerosis. We utilize the in vitro experimental model of atherosclerosis to elucidate the activity of Chungtaejeon (CTJ), a Korean fermented tea on adhesion and migration of human aortic vascular smooth muscle cells (HASMC). Materials and methodsVarious in vitro assays such as cell viability, cell adhesion, Western blot, immunofluorescence, were carried out on HASMC to explore pathway associated with cytoskeletal remodeling during the progression of atherosclerosis. ResultsIn result, CTJ significantly inhibited adhesion of HASMC as revealed by collagen assay. Similarly, CTJ inhibited the β1-integrin protein expression as well as FAK phosphorylation. Treatment of CTJ also inhibited stress fiber formation. Likewise, adherence of cells on collagen optimally increased the expression of both RhoA and Cdc42, however, treatment of CTJ dose dependently decreased their expression. The lysophosphatidic acid stimulation of HASMC rapidly increased the level of phosphorylated forms of MLC20 within 15 min, followed by an extended level of MLC20 phosphorylation. The treatment of CTJ at a dose of 50, 100 and 250 μg/ml remarkably reduced the diphosphorylated form while decreased the level of monophosphorylated form of MLC20. ConclusionsOur results suggests that, with further validation CTJ could be a promising herbal resource for prevention of atherosclerosis.

Harmine promotes megakaryocyte differentiation and thrombopoiesis by activating the Rac1/Cdc42/JNK pathway through a potential target of 5-HTR2A.

Harmine (HM), a β-carboline alkaloid extracted from plants, is a crucial component of traditional Chinese medicine (TCM) known for its diverse pharmacological activities. Thrombocytopenia, a common and challenging hematological disorder, often coexists with serious illnesses. Previous research has shown a correlation between HM and thrombocytopenia, but the mechanism needs further elucidation. The aim of this study was to clarify the mechanisms underlying the effects of HM on thrombocytopenia and to develop new therapeutic strategies. Flow cytometry, Giemsa staining, and Phalloidin staining were used to assess HM's impact on Meg-01 and HEL cell differentiation and maturation in vitro. A radiation-induced thrombocytopenic mouse model was employed to evaluate HM's effect on platelet production in vivo. Network pharmacology, molecular docking, and protein blotting were utilized to investigate HM's targets and mechanisms. The results demonstrated that HM dose-dependently promoted Meg-01 and HEL cell differentiation and maturation in vitro and restored platelet levels in irradiated mice in vivo. Subsequently, HM was found to be involved in the biological process of platelet production by upregulating the expressions of Rac1, Cdc42, JNK, and 5-HTR2A. Furthermore, the targeting of HM to 5-HTR2A and its correlation with downstream Rac1/Cdc42/JNK were also confirmed. In conclusion, HM regulates megakaryocyte differentiation and thrombopoiesis through the 5-HTR2A and Rac1/Cdc42/JNK pathways, providing a potential treatment strategy for thrombocytopenia.

Increased activity of epithelial Cdc42 Rho GTPase and tight junction permeability in the Cftr knockout intestine.

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/β-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pulldown assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled. With the use of a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3 kDa dextran relative to WT. Leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/β-catenin signaling with endo-IWR1 in Cftr KO relative to WT enteroids. Increased proliferation or inhibition of Cdc42 activity with ML141 in WT enteroids had no effect on permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3 kDa dextran and tight junction impermeant 500 kDa dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.NEW & NOTEWORTHY Increased tight junction localization and GTP-bound activity of the Rho GTPase Cdc42 was identified in small intestinal crypts and enteroids of cystic fibrosis (CF) transmembrane conductance regulator knockout (Cftr KO) mice. The increase in epithelial Cdc42 activity was associated with increased Wnt signaling. Paracellular flux of an uncharged solute (3 kDa dextran) in Cftr KO enteroids indicated a moderate leak permeability under basal conditions that was strongly exacerbated by Cdc42 inhibition. These findings suggest increased activity of Cdc42 in the Cftr KO intestine underlies alterations in intestinal permeability.

LncRNA MALAT1 facilitates BM-MSCs differentiation into endothelial cells and ameliorates erectile dysfunction via the miR-206/CDC42/PAK1/paxillin signalling axis

BackgroundErectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective.MethodsVascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining.ResultsMALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3’-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups.ConclusionsMALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.

Germacrone, isolated from Curcuma wenyujin, inhibits melanin synthesis through the regulation of the MAPK signaling pathway.

Abnormal melanin synthesis causes hyperpigmentation disorders, such as chloasma, freckles, and melanoma, which are highly multiple and prevalent. There were few reports on the anti-melanogenic effect of Curcuma wenyujin Y.H. Chen et C. Ling, and the bioactive compound has not been elucidated as well. The study aims to investigate the anti-melanogenic effect of C. wenyujin, and identify the bioactive compound, and further explore its underlying mechanism. Our results showed that the Petroleum ether fraction extracted from C. wenyujin rhizome had a significant anti-melanogenic effect, and germacrone isolated from it was confirmed as the major bioactive compound. To our data, germacrone significantly inhibited tyrosinase (TYR) activity, reduced melanosome synthesis, reduced dendrites formation of B16F10 cells, and melanosome transport to keratinocytes. Moreover, germacrone effectively decreased the hyperpigmentation in zebrafish and the skin of guinea pigs in vivo. Western-blot analysis showed that germacrone down-regulated the expression of TYR, TRP-1, TRP-2, Rab27a, Cdc42, and MITF proteins via the activation of the MAPK signaling pathway. Taken together, germacrone is an effective bioactive compound for melanogenesis inhibition. Our studies suggest that germacrone may be considered a potential candidate for skin whitening.

Cdc42 Rho GTPase Activity Sustains Epithelial Barrier Function in the Cystic Fibrosis Intestine

Increased paracellular permeability is a common manifestation of cystic fibrosis (CF) intestinal disease in people with CF and in CF mouse models. Increased paracellular permeability has been found in CF airway epithelium cultured in the absence of inflammatory factors, suggesting an inherent defect in CF epithelial barrier function (Weiser et al., Cell Physiol Biochem. 2011;28(2):289-96). CF transmembrane conductance regulator knockout ( Cftr KO) mouse intestinal crypts demonstrate an increase in epithelial cell proliferation, which correlates with an alkaline intracellular pH (pHi), a pHi-dependent increase in lateral cell membrane localization of the Wnt transducer Disheveled (Dvl), and increased Wnt/β-catenin signaling (Strubberg et al., Cell Mol Gastroenterol Hepatol. 2017 Dec 7;5(3):253-271). We hypothesized that the alkaline pHi of Cftr KO crypts also facilitates Dvl-dependent noncanonical Wnt signaling to increase Rho GTPase activity and thereby tight junction remodeling during increased proliferation. In Dvl2-eGFP rescue mice, Dvl2-eGFP intensity near the apical membrane of Cftr KO enteroid crypts was greater versus wild-type (WT) at both the crypt base and the transit-amplifying zone. Immunofluorescence studies of both freshly isolated crypts and early passage enteroids also found increased accumulation of the Rho GTPase Cdc42 at the tight junctions of Cftr KO versus WT crypts. Live imaging revealed a small increase in leak pathway permeability (3kD dextran) in Cftr KO as compared to WT murine enteroids. Increased leak permeability and Cdc42 tight junction localization were normalized in Cftr KO enteroids by suppressing proliferation through inhibition of Wnt/β-catenin signaling. There was no difference in Cdc42 expression in Cftr KO fresh crypts or enteroids, but initial studies show increased Cdc42 GTP-loaded activity in Cftr KO enteroids relative to WT. Similarly, CFTR KO human intestinal epithelial Caco-2 cells, which also show an alkaline pHi, increased Wnt/β-catenin signaling and a hyperproliferative phenotype, demonstrated a significant increase in GTP-loaded Cdc42 activity compared to WT cells. Inhibition of Cdc42 activity with ML141 (R&D Systems) resulted in a significant disruption of the Cftr KO epithelial barrier, whereas WT enteroid permeability was not affected by Cdc42 inhibition, Wnt inhibition or Wnt3a-induced proliferation. We conclude that increased Cdc42 Rho GTPase activity is cell-autonomous and is necessary for maintaining barrier integrity of the Cftr KO crypt epithelium. This work was supported by the Cystic Fibrosis Foundation grant CLARKE20G0 and the Crohn’s and Colitis Foundation grant #693938. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oleate Promotes Triple-Negative Breast Cancer Cell Migration by Enhancing Filopodia Formation through a PLD/Cdc42-Dependent Pathway.

Breast cancer, particularly triple-negative breast cancer (TNBC), poses a global health challenge. Emerging evidence has established a positive association between elevated levels of stearoyl-CoA desaturase 1 (SCD1) and its product oleate (OA) with cancer development and metastasis. SCD1/OA leads to alterations in migration speed, direction, and cell morphology in TNBC cells, yet the underlying molecular mechanisms remain elusive. To address this gap, we aim to investigate the impact of OA on remodeling the actin structure in TNBC cell lines, and the underlying signaling. Using TNBC cell lines and bioinformatics tools, we show that OA stimulation induces rapid cell membrane ruffling and enhances filopodia formation. OA treatment triggers the subcellular translocation of Arp2/3 complex and Cdc42. Inhibiting Cdc42, not the Arp2/3 complex, effectively abolishes OA-induced filopodia formation and cell migration. Additionally, our findings suggest that phospholipase D is involved in Cdc42-dependent filopodia formation and cell migration. Lastly, the elevated expression of Cdc42 in breast tumor tissues is associated with a lower survival rate in TNBC patients. Our study outlines a new signaling pathway in the OA-induced migration of TNBC cells, via the promotion of Cdc42-dependent filopodia formation, providing a novel insight for therapeutic strategies in TNBC treatment.

Motility and tumor infiltration are key aspects of invariant natural killer T cell anti-tumor function

Dysfunction of invariant natural killer T (iNKT) cells contributes to immune resistance of tumors. Most mechanistic studies focus on their static functional status before or after activation, not considering motility as an important characteristic for antigen scanning and thus anti-tumor capability. Here we show via intravital imaging, that impaired motility of iNKT cells and their exclusion from tumors both contribute to the diminished anti-tumor iNKT cell response. Mechanistically, CD1d, expressed on macrophages, interferes with tumor infiltration of iNKT cells and iNKT-DC interactions but does not influence their intratumoral motility. VCAM1, expressed by cancer cells, restricts iNKT cell motility and inhibits their antigen scanning and activation by DCs via reducing CDC42 expression. Blocking VCAM1-CD49d signaling improves motility and activation of intratumoral iNKT cells, and consequently augments their anti-tumor function. Interference with macrophage-iNKT cell interactions further enhances the anti-tumor capability of iNKT cells. Thus, our findings provide a direction to enhance the efficacy of iNKT cell-based immunotherapy via motility regulation.

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

ANGI-09. UNRAVELING THE INVOLVEMENT OF BAIAP2 AND CDC42 IN MEDULLOBLASTOMA PATHOGENESIS

Abstract The most common malignant pediatric brain tumor is medulloblastoma. Despite advancements in treatment, medulloblastoma survivors have significant long-term medical side effects and increased all-cause mortality. That is why novel, more effective, and safe treatments are direly necessary. One treatment challenge is that medulloblastoma tumors can disseminate via the bloodstream and cerebrospinal fluid leading to a poorer prognosis. The underlying mechanisms that promote metastasis and invasion in medulloblastoma are not clearly understood. One method we used to identify potential key regulators in the pathogenesis of medulloblastoma is by analyzing microarray and single-cell RNA sequencing data. We mined publicly-available datasets along with our data from genetically engineered models of medulloblastoma to identify differentially expressed genes compared to non-tumor brain cells. We identified BAIAP2 and CDC42 to be differentially expressed and have higher expression in medulloblastoma compared to other brain cancers. BAIAP2 and CDC42 are known to be implicated in migration and invasion. Extensive literature demonstrates an interaction between BAIAP2 and CDC42 in multiple cell types. However, this interaction has yet to be thoroughly studied in medulloblastoma. We hypothesize that CDC42 interacts with BAIAP2 in medulloblastoma cells to facilitate tumor pathogenesis. To further investigate this, we conducted siRNA-mediated gene knockdown of BAIAP2 and CDC42 in ONS-76 cells. We determined that BAIAP2 and CDC42 are highly expressed in medulloblastoma, and knockdown of BAIAP2 modulates CDC42 expression. Further, we determined that cell proliferation and migration were reduced upon BAIAP2 and CDC42 knockdown. Our findings suggest that BAIAP2 and CDC42 have a role in the migration and proliferation of medulloblastoma ONS-76 cells. Altogether, these findings indicate BAIAP2 and CDC42 as possible targets for future studies.

M4IDP stimulates ROS elevation through inhibition of mevalonate pathway and pentose phosphate pathway to inhibit colon cancer cells

Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.

Monotropein Induced Apoptosis and Suppressed Cell Cycle Progression in Colorectal Cancer Cells.

To determine whether monotropein has an anticancer effect and explore its potential mechanisms against colorectal cancer (CRC) through network pharmacology and molecular docking combined with experimental verification. Network pharmacology and molecular docking were used to predict potential targets of monotropein against CRC. Cell counting kit assay, plate monoclonal assay and microscopic observation were used to investigate the antiproliferative effects of monotropein on CRC cells HCT116, HT29 and LoVo. Flow cytometry and scratch assay were used to analyze apoptosis and cell cycle, as well as cell migration, respectively in HCT116, HT29, and LoVo cells. Western blotting was used to detect the expression of proteins related to apoptosis, cell cycle, and cell migration, and the expression of proteins key to the Akt pathway. The Gene Ontology and Reactome enrichment analyses indicated that the anticancer potential of monotropein against CRC might be involved in multiple cancer-related signaling pathways. Among these pathways, RAC-beta serine/threonine-protein kinase (Akt1, Akt2), cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase-9 (MMP9), epidermal growth factor receptor (EGFR), cell division control protein 42 homolog (CDC42) were shown as the potential anticancer targets of monotropein against CRC. Molecular docking suggested that monotropein may interact with the 6 targets (Akt1, Akt2, CDK6, MMP9, EGFR, CDC42). Subsequently, cell activity of HCT116, HT29 and LoVo cell lines were significantly suppressed by monotropein (P<0.05). Furthermore, our research revealed that monotropein induced cell apoptosis by inhibiting Bcl-2 and increasing Bax, induced G1-S cycle arrest in colorectal cancer by decreasing the expressions of CyclinD1, CDK4 and CDK6, inhibited cell migration by suppressing the expressions of CDC42 and MMP9 (P<0.05), and might play an anticancer role through Akt signaling pathway. Monotropein exerts its antitumor effects primarily by arresting the cell cycle, causing cell apoptosis, and inhibiting cell migration. This indicates a high potential for developing novel medication for treating CRC.

CDC42-mediated Wnt signaling facilitates odontogenic differentiation of DPCs during tooth root elongation

BackgroundCDC42 is a member of Rho GTPase family, acting as a molecular switch to regulate cytoskeleton organization and junction maturation of epithelium in organ development. Tooth root pattern is a highly complicated and dynamic process that dependens on interaction of epithelium and mesenchyme. However, there is a lack of understanding of the role of CDC42 during tooth root elongation.MethodsThe dynamic expression of CDC42 was traced during tooth development through immunofluorescence staining. Then we constructed a model of lentivirus or inhibitor mediated Cdc42 knockdown in Herwig’s epithelial root sheath (HERS) cells and dental papilla cells (DPCs), respectively. Long-term influence of CDC42 abnormality was assessed via renal capsule transplantation and in situ injection of alveolar socket.ResultsCDC42 displayed a dynamic spatiotemporal pattern, with abundant expression in HERS cells and apical DPCs in developing root. Lentivirus-mediated Cdc42 knockdown in HERS cells didn’t disrupt cell junctions as well as epithelium-mesenchyme transition. However, inhibition of CDC42 in DPCs undermined cell proliferation, migration and odontogenic differentiation. Wnt/β-catenin signaling as the downstream target of CDC42 modulated DPCs’ odontogenic differentiation. The transplantation and in situ injection experiments verified that loss of CDC42 impeded root extension via inhibiting the proliferation and differentiation of DPCs.ConclusionsWe innovatively revealed that CDC42 was responsible for guiding root elongation in a mesenchyme-specific manner. Furthermore, CDC42-mediated canonical Wnt signaling regulated odontogenic differentiation of DPCs during root formation.

TMT-Based Proteomics Analysis of Senescent Nucleus Pulposus from Patients with Intervertebral Disc Degeneration.

Lower back pain, a leading cause of disability worldwide, is associated with intervertebral disc degeneration (IDD) in approximately 40% of cases. Although nucleus pulposus (NP) cell senescence is a major contributor to IDD, the underlying mechanisms remain unclear. We collected NP samples from IDD patients who had undergone spinal surgery. Healthy and senescent NP tissues (n = 3) were screened using the Pfirrmann grading system combined with immunohistochemistry, as well as hematoxylin and eosin, Safranin O, Alcian blue, and Masson staining. Differentially expressed proteins (DEPs) were identified using quantitative TMT-based proteomics technology. Bioinformatics analyses included gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) analyses. In addition, immunofluorescence was used to verify protein expression. In total, 301 DEPs were identified in senescent NP tissues, including 92 upregulated and 209 downregulated proteins. In GO, DEPs were primarily associated with NF-kappaB transcription factor, extracellular regions, cellular protein metabolic processes, and post-translational protein modification. The enriched KEGG pathways included TGF-β, Wnt, RAP1, interleukin-17, extracellular matrix-receptor adhesion, and PI3K/Akt signaling pathways. PPI analysis demonstrated interactions between multiple proteins. Finally, immunofluorescence verified the expressions of MMP3, LUM, TIMP1, and CDC42 in senescent NP cells. Our study provides valuable insights into the mechanisms underlying senescent NP tissues in IDD patients. DEPs provide a basis for further investigation of the effects of senescent factors on IDD.

Epithelial-specific deletion of FAM20A leads to short root defects

Short Root Defects defined by a reduced ratio of root to crown, may culminate in root resorption and subsequent tooth loss, in spite of the absence of apparent symptoms. Such defects present considerable impediments to orthodontic treatment and restoration. Recent identification of Fam20a, an emergent pseudokinase, has been associated with enamel development and tooth eruption, yet its definitive role in root formation and eruption remains ambiguous. In this research, we initially ascertained that the targeted knockout of Fam20a within the epithelium led to truncated tooth roots, irregular breaks in the epithelial root sheath initiation of the WNT signaling pathway, and decreased expression of the cell polarity-related transcription factor Cdc42 in murine models. This was concomitant with the participation of the associated epithelial root sheath developmental pathways BMP2, Gli1, and Nfic. Furthermore, we observed that Fam20a predominantly affects the intraosseous eruption phase of tooth emergence. During this phase, the osteoclast peak around the mandibular first molar in cKO mice is delayed, leading to a slower formation of the eruption pathway, ultimately resulting in delayed tooth eruption in mice. The findings of this study enrich the extant knowledge regarding the role of Fam20a, suggesting its potential regulatory function in tooth root development through the WNT/β-catenin/Cdc42 pathway.

Silencing of CDC42 inhibits contraction and growth-related functions in prostate stromal cells, which is mimicked by ML141

BackgroundProstate smooth muscle contraction and stromal growth may contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia, but are incompletely understood. A role of the monomeric GTPase CDC42 for smooth muscle contraction and proliferation appears possible, but is unknown for the prostate. Here, we silenced CDC42 expression in prostate stromal cells (WPMY-1), and examined contractility, growth-related functions and responses to the presumed CDC42 inhibitor, ML141. MethodsWPMY-1 cells were transfected with scrambled or CDC42-specific siRNA, and characterized for GTPase activities, contraction, proliferation, colony formation, apoptosis, cell death and viability. Effects of ML141 were examined in cells with and without silencing. ResultsCDC42 silencing was confirmed by reduced mRNA and protein expression, and reduced CDC42 activity. Silencing impaired contraction (23–47 %), actin organization (25 %), proliferation (17–63 %), colony formation and viability (64–89 %), and increased the percentage of dead cells (2.6-fold). ML141 mimicked the phenotype of silencing in scrambled siRNA-transfected controls, and in non-transfected WPMY-1 cells, including inhibition of contraction, proliferation, colony formation and viability, breakdown of actin organization and increased cell death. In CDC42-silenced cells, ML141 still affected phalloiding organization, proliferation and cell death, with effect sizes resembling controls without silencing. ML141 inhibited RhoA activity in CDC42-silenced cells, but not in cells without silencing. ConclusionsCDC42 promotes contraction of prostate stromal cells, and drives stromal growth by CDC42-mediated proliferation and suppression of apoptosis-independent cell death. ML141 mimicks all effects of CDC42 silencing, but its specificity may be limited and depends on GTPase phenotypes of cells.