Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes.

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity.

Tumor-intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in breast cancer.

Immune checkpoint blockade has been used to treat breast cancer, but the clinical responses remain relatively poor. We have used the CRISPR-Cas9 kinome knockout library consisting of 763 kinase genes to identify tumor-intrinsic kinases conferring resistance to anti-PD-1 immune checkpoint blockade. We have identified the CDC42BPB kinase as a potential target to overcome the resistance to anti-PD-1 immune checkpoint blockade immunotherapy. We found that CDC42BPB is highly expressed in breast cancer patients who are non-responsive to immunotherapy. Furthermore, a small-molecule pharmacological inhibitor, BDP5290, which targets CDC42BPB, synergized with anti-PD-1 and enhanced tumor cell killing by promoting T cell proliferation in both in vitro and in vivo assays. Moreover, anti-PD-1-resistant breast cancer cells showed higher expression of CDC42BPB, and its inhibition rendered the resistant cells more susceptible to T cell killing in the presence of anti-PD-1. We also found that CDC42BPB phosphorylated AURKA, which in turn upregulated PD-L1 through cMYC. Our results have revealed a robust link between tumor-intrinsic kinase and immunotherapy resistance and have provided a rationale for a unique combination therapy of CDC42BPB inhibition and anti-PD-1 immunotherapy for breast cancer.

[Autoinflammatory diseases associated with IL-18]. / Maladies auto-inflammatoires associées à l'IL-18.

Autoinflammatory diseases (AIDs) are conditions characterized by dysfunction of innate immunity, causing systemic inflammation and various clinical symptoms. The field of AIDs has expanded due to improved comprehension of pathophysiological mechanisms and advancements in genomics techniques. A new emerging category of AIDs is characterized by a significant increase in interleukin 18 (IL-18), a pro-inflammatory cytokine synthesized in macrophages and activated by caspase 1 via various inflammasomes. IL-18 plays a role in the regulation of innate and adaptive immunity. IL-18 is involved in various functions, such as the proliferation, survival, and differentiation of immune cells, tissue infiltration of immune cells, polarization of immune responses, and production of other pro-inflammatory cytokines. This review analyzes the literature on IL-18 regarding its functions and its implications in the diagnosis and treatment of AIDs. IL-18-associated AIDs comprise Still's disease and diseases associated with mutations in NLRC4, XIAP, CDC42, and PSTPIP1, as well as IL-18BP deficiencies. With the exception of PSTPIP1-associated diseases, these conditions all carry a risk of macrophagic activation syndrome. Measuring IL-18 levels in serum can aid in the diagnosis, prognosis, and monitoring of these diseases. Therapies targeting IL-18 and its signaling pathways are currently under investigation.

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

Harmine (HM), a ß-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/ß-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/ß-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.

Pharmacokinetic profiling of ZCL-278, a cdc42 inhibitor, and its effectiveness against chronic kidney disease.

ZCL-278 is a selective small molecule specifically inhibiting the Cdc42-intersectin interaction, yet its in-vivo pharmacokinetic and pharmacodynamic properties against renal diseases had not been determined. Thus, our study explored the absorption, distribution and excretion of ZCL-278 as well as its pharmacological efficacy against chronic kidney disease (CKD). With the optimized detection method, absolute oral bioavailability of ZCL-278 was determined as 10.99 % in male and 17.34 % in female rats. ZCL-278 was rapidly and abundantly distributed in various tissues, especially the kidney and heart, while few excreted through urine and feces. In the adenine-induced CKD mice, the increased plasma creatinine and urea, the decreased body weight as well as the renal pathological alterations, including vacuolization of renal tubular epithelial cells, granular degeneration, cell flattening, luminal dilation, and cylindruria, were significantly ameliorated after ZCL-278 administration. Moreover, ZCL-278 could also reverse the increased intensities of renal inflammation and fibrosis in the CKD mice. These results clarified the pharmacokinetics of ZCL-278 in rats and preliminarily indicated that ZCL-278 has favorable pharmacodynamic properties for CKD primed for lead development and optimization, warranting further drug development.

PLEKHG1: New Potential Candidate Gene for Periventricular White Matter Abnormalities.

Hypoxic-ischemic brain damage presents a significant neurological challenge, often manifesting during the perinatal period. Specifically, periventricular leukomalacia (PVL) is emerging as a notable contributor to cerebral palsy and intellectual disabilities. It compromises cerebral microcirculation, resulting in insufficient oxygen or blood flow to the periventricular region of the brain. As widely documented, these pathological conditions can be caused by several factors encompassing preterm birth (4-5% of the total cases), as well single cotwin abortion and genetic variants such as those associated with GTPase pathways. Whole exome sequencing (WES) analysis identified a de novo causative variant within the pleckstrin homology domain-containing family G member 1 (PLEKHG1) gene in a patient presenting with PVL. The PLEKHG1 gene is ubiquitously expressed, showing high expression patterns in brain tissues. PLEKHG1 is part of a family of Rho guanine nucleotide exchange factors, and the protein is essential for cell division control protein 42 (CDC42) activation in the GTPase pathway. CDC42 is a key small GTPase of the Rho-subfamily, regulating various cellular functions such as cell morphology, migration, endocytosis, and cell cycle progression. The molecular mechanism involving PLEKHG1 and CDC42 has an intriguing role in the reorientation of cells in the vascular endothelium, thus suggesting that disruption responses to mechanical stress in endothelial cells may be involved in the formation of white matter lesions. Significantly, CDC42 association with white matter abnormalities is underscored by its MIM phenotype number. In contrast, although PLEKHG1 has been recently associated with patients showing white matter hyperintensities, it currently lacks a MIM phenotype number. Additionally, in silico analyses classified the identified variant as pathogenic. Although the patient was born prematurely and subsequently to dichorionic gestation, during which its cotwin died, we suggest that the variant described can strongly contribute to PVL. The aim of the current study is to establish a plausible association between the PLEKHG1 gene and PVL.

Multiscale Modeling of Bistability in the Yeast Polarity Circuit.

Cell polarity refers to the asymmetric distribution of proteins and other molecules along a specified axis within a cell. Polarity establishment is the first step in many cellular processes. For example, directed growth or migration requires the formation of a cell front and back. In many cases, polarity occurs in the absence of spatial cues. That is, the cell undergoes symmetry breaking. Understanding the molecular mechanisms that allow cells to break symmetry and polarize requires computational models that span multiple spatial and temporal scales. Here, we apply a multiscale modeling approach to examine the polarity circuit of yeast. In addition to symmetry breaking, experiments revealed two key features of the yeast polarity circuit: bistability and rapid dismantling of the polarity site following a loss of signal. We used modeling based on ordinary differential equations (ODEs) to investigate mechanisms that generate these behaviors. Our analysis revealed that a model involving positive and negative feedback acting on different time scales captured both features. We then extend our ODE model into a coarse-grained reaction-diffusion equation (RDE) model to capture the spatial profiles of polarity factors. After establishing that the coarse-grained RDE model qualitatively captures key features of the polarity circuit, we expand it to more accurately capture the biochemical reactions involved in the system. We convert the expanded model to a particle-based model that resolves individual molecules and captures fluctuations that arise from the stochastic nature of biochemical reactions. Our models assume that negative regulation results from negative feedback. However, experimental observations do not rule out the possibility that negative regulation occurs through an incoherent feedforward loop. Therefore, we conclude by using our RDE model to suggest how negative feedback might be distinguished from incoherent feedforward regulation.

Paeoniflorin alleviates DSS-induced ulcerative colitis by suppressing inflammation, oxidative stress, and apoptosis via regulating serum metabolites and inhibiting CDC42/JNK signaling pathway.

Ulcerative colitis (UC) poses a threat to human health. The present study attempts to unravel the efficacy and potential mechanisms of paeoniflorin (PF), a naturally sourced active ingredient, for the management of UC. By establishing a DSS (dextran sulphate sodium)-induced experimental rat model of UC, this study found that PF was effective in ameliorating UC symptoms, inhibiting oxidative stress, inflammation and apoptosis, and repairing colonic epithelial damage. In addition, metabolomics revealed that PF may alleviate UC by primarily improving linoleic acid metabolism. Mechanistically, PF inhibited the CDC42/JNK signaling pathway by targeting CDC42. In particular, HuProtTM20K proteomics, molecular docking and MST revealed that PF is a novel CDC42 inhibitor. In LPS-treated Caco-2 cells, PF similarly inhibited oxidative stress, inflammation, and apoptosis and down-regulated the CDC42/JNK signaling pathway. Overall, PF inhibits oxidative stress, inflammation and apoptosis and repairs colonic epithelial damage through modulation of serum metabolites and inhibition of the CDC42/JNK signaling pathway, leading to alleviation of UC.

Emu oil alleviates atopic dermatitis-like responses by inhibiting Cdc42 signaling of keratinocyte.

Emu oil is the oil extracted from the body fat of the Australian bird emu. Although previous studies have reported that emu oil has anti-inflammatory effects, the effect and mechanism of emu oil on the treatment of atopic dermatitis have not been reported. Here, 2, 4-dinitrofluorobenzene was used to induce atopic dermatitis-like appearance on the back skin of C57BL/6 mice. And then, the effect of emu oil in the atopic dermatitis treatment was evaluated. We found that emu oil reduced the transdermal water loss in the atopic dermatitis model. Additionally, the epidermal thickness treated with emu oil was significantly thinner. The number of mast cells and inflammatory cells were significantly decreased. The thymic stromal lymphopoietin (TSLP), which is secreted by keratinocyte, was decreased significantly after treatment. Moreover, the serum levels of cytokines TSLP, interleukin-4, interleukin-13, and immunoglobulin (Ig) E were decreased after emu oil treatment. Surprisingly, we found that the high level of Cdc42 expression in the atopic dermatitis, which was decreased after emu oil treatment. To detect the role of Cdc42 in atopic dermatitis, we constructed atopic dermatitis model in mice with sustained activation of Cdc42 signaling. Furthermore, we have confirmed that emu oil demonstrates anti-inflammatory effects in atopic dermatitis by inhibiting the expression of Cdc42 signaling in keratinocytes. In conclusion, we discovered a new role of Cdc42 in the development of atopic dermatitis, which mediated the therapeutic effect of emu oil on atopic dermatitis.

Cdc42 deletion yielded enamel defects by disrupting mitochondria and producing reactive oxygen species in dental epithelium.

Developmental defects of enamel are common due to genetic and environmental factors before and after birth. Cdc42, a Rho family small GTPase, regulates prenatal tooth development in mice. However, its role in postnatal tooth development, especially enamel formation, remains elusive. Here, we investigated Cdc42 functions in mouse enamel development and tooth repair after birth. Cdc42 showed highly dynamic temporospatial patterns in the developing incisors, with robust expression in ameloblast and odontoblast layers. Strikingly, epithelium-specific Cdc42 deletion resulted in enamel defects in incisors. Ameloblast differentiation was inhibited, and hypomineralization of enamel was observed upon epithelial Cdc42 deletion. Proteomic analysis showed that abnormal mitochondrial components, phosphotransferase activity, and ion channel regulator activity occurred in the Cdc42 mutant dental epithelium. Reactive oxygen species accumulation was detected in the mutant mice, suggesting that abnormal oxidative stress occurred after Cdc42 depletion. Moreover, Cdc42 mutant mice showed delayed tooth repair and generated less calcified enamel. Mitochondrial dysfunction and abnormal oxygen consumption were evidenced by reduced Apool and Timm8a1 expression, increased Atp5j2 levels, and reactive oxygen species overproduction in the mutant repair epithelium. Epithelium-specific Cdc42 deletion attenuated ERK1/2 signaling in the labial cervical loop. Aberrant Sox2 expression in the mutant labial cervical loop after clipping might lead to delayed tooth repair. These findings suggested that mitochondrial dysfunction, up-regulated oxidative stress, and abnormal ion channel activity may be among multiple factors responsible for the observed enamel defects in Cdc42 mutant incisors. Overall, Cdc42 exerts multidimensional and pivotal roles in enamel development and is particularly required for ameloblast differentiation and enamel matrix formation.

Gut commensal metabolite rhamnose promotes macrophages phagocytosis by activating SLC12A4 and protects against sepsis in mice.

Sepsis progression is significantly associated with the disruption of gut eubiosis. However, the modulatory mechanisms of gut microbiota operating during sepsis are still unclear. Herein, we investigated how gut commensals impact sepsis development in a pre-clinical model. Cecal ligation and puncture (CLP) surgery was used to establish polymicrobial sepsis in mice. Mice depleted of gut microbiota by an antibiotic cocktail (ABX) exhibited a significantly higher level of mortality than controls. As determined by metabolomics analysis, ABX treatment has depleted many metabolites, and subsequent supplementation with l-rhamnose (rhamnose, Rha), a bacterial carbohydrate metabolite, exerted profound immunomodulatory properties with a significant enhancement in macrophage phagocytosis, which in turn improved organ damage and mortality. Mechanistically, rhamnose binds directly to and activates the solute carrier family 12 (potassium-chloride symporter), member 4 (SLC12A4) in macrophages and promotes phagocytosis by activating the small G-proteins, Ras-related C3 botulinum toxin substrate1 (Rac1) and cell division control protein 42 homolog (Cdc42). Interestingly, rhamnose has enhanced the phagocytosis capacity of macrophages from sepsis patients. In conclusion, by identifying SLC12A4 as the host interacting protein, we disclosed that the gut commensal metabolite rhamnose is a functional molecular that could promote the phagocytosis capacity of macrophages and protect the host against sepsis.

LncRNA ZFPM2-AS1 promotes phyllodes tumor progression by binding to CDC42 and inhibiting STAT1 activation.

Breast phyllodes tumor (PT) is a rare fibroepithelial neoplasm with potential malignant behavior. Long non-coding RNAs (lncRNAs) play multifaceted roles in various cancers, but their involvement in breast PT remains largely unexplored. In this study, microarray was leveraged for the first time to investigate the role of lncRNA in PT. We identified lncRNA ZFPM2-AS1 was significantly upregulated in malignant PT, and its overexpression endowed PT with high tumor grade and adverse prognosis. Furthermore, we elucidated that ZFPM2-AS1 promotes the proliferation, migration, and invasion of malignant PT in vitro. Targeting ZFPM2-AS1 through nanomaterial-mediated siRNA delivery in patient-derived xenograft (PDX) model could effectively inhibit tumor progression in vivo. Mechanistically, our findings showed that ZFPM2-AS1 is competitively bound to CDC42, inhibiting ACK1 and STAT1 activation, thereby launching the transcription of TNFRSF19. In conclusion, our study provides evidence that ZFPM2-AS1 plays a pivotal role in the pathogenesis of breast PT, and suggests that ZFPM2-AS1 could serve as a prognostic indicator for patients with PT as well as a promising novel therapeutic target.

Inflammatory gene regulation by Cdc42 in airway epithelial cells.

Cytokine release from airway epithelial cells is a key immunological process that coordinates an immune response in the lungs. We propose that the Rho GTPase, Cdc42, regulates both transcription and trafficking of cytokines, ultimately affecting the essential process of cytokine release and subsequent inflammation in the lungs. Here, we examined the pro-inflammatory transcriptional profile that occurs in bronchial epithelial cells (BEAS-2B) in response to TNF-α using RNA-Seq and differential gene expression analysis. To interrogate the role of Cdc42 in inflammatory gene expression, we used a pharmacological inhibitor of Cdc42, ML141, and determined changes in the transcriptomic profile induced by Cdc42 inhibition. Our results indicated that Cdc42 inhibition with ML141 resulted in a unique inflammatory phenotype concomitant with increased gene expression of ER stress genes, Golgi membrane and vesicle transport genes. To further interrogate the inflammatory pathways regulated by Cdc42, we made BEAS-2B knockdown strains for the signaling targets TRIB3, DUSP5, SESN2 and BMP4, which showed high differential expression in response to Cdc42 inhibition. Depletion of DUSP5 and TRIB3 reduced the pro-inflammatory response triggered by Cdc42 inhibition as shown by a reduction in cytokine transcript levels. Depletion of SESN2 and BMP4 did not affect cytokine transcript level, however, Golgi fragmentation was reduced. These results provide further evidence that in airway epithelial cells, Cdc42 is part of a signaling network that controls inflammatory gene expression and secretion by regulating Golgi integrity. Summary sentence:We define the Cdc42-regulated gene networks for inflammatory signaling in airway epithelial cells which includes regulation of ER stress response and vesicle trafficking pathways.

The small GTPase Cdc42 regulates shell field morphogenesis in a gastropod mollusk.

In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes.

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

Proangiogenic effect and underlying mechanism of holmium oxide nanoparticles: a new biomaterial for tissue engineering.

BACKGROUND: Early angiogenesis provides nutrient supply for bone tissue repair, and insufficient angiogenesis will lead tissue engineering failure. Lanthanide metal nanoparticles (LM NPs) are the preferred materials for tissue engineering and can effectively promote angiogenesis. Holmium oxide nanoparticles (HNPs) are LM NPs with the function of bone tissue "tracking" labelling. Preliminary studies have shown that HNPs has potential of promote angiogenesis, but the specific role and mechanism remain unclear. This limits the biological application of HNPs. RESULTS: In this study, we confirmed that HNPs promoted early vessel formation, especially that of H-type vessels in vivo, thereby accelerating bone tissue repair. Moreover, HNPs promoted angiogenesis by increasing cell migration, which was mediated by filopodia extension in vitro. At the molecular level, HNPs interact with the membrane protein EphrinB2 in human umbilical vein endothelial cells (HUVECs), and phosphorylated EphrinB2 can bind and activate VAV2, which is an activator of the filopodia regulatory protein CDC42. When these three molecules were inhibited separately, angiogenesis was reduced. CONCLUSION: Overall, our study confirmed that HNPs increased cell migration to promote angiogenesis for the first time, which is beneficial for bone repair. The EphrinB2/VAV2/CDC42 signalling pathway regulates cell migration, which is an important target of angiogenesis. Thus, HNPs are a new candidate biomaterial for tissue engineering, providing new insights into their biological application.

Apigenin inhibits tumor angiogenesis by hindering microvesicle biogenesis via ARHGEF1.

Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin.

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

BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular 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. RESULTS: MALAT1 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. CONCLUSIONS: MALAT1 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.

NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.