Search

Cryptococcus neoformans (Cn) is an opportunistic fungus that causes severe central nervous system (CNS) disease in immunocompromised individuals. Brain parenchyma invasion requires fungal traversal of the blood-brain barrier. In this study, we describe that Cn alters the brain endothelium by activating small GTPase RhoA, causing reorganization of the actin cytoskeleton and tight junction modulation to regulate endothelial barrier permeability. We confirm that the main fungal capsule polysaccharide glucuronoxylomannan is responsible for these alterations. We reveal a therapeutic benefit of RhoA inhibition by CCG-1423 in vivo. RhoA inhibition prolonged survival and reduced fungal burden in a murine model of disseminated cryptococcosis, supporting the therapeutic potential of targeting RhoA in the context of cryptococcal infection. We examine the complex virulence of Cn in establishing CNS disease, describing cellular components of the brain endothelium that may serve as molecular targets for future antifungal therapies to alleviate the burden of life-threatening cryptococcal CNS infection. [ABSTRACT FROM AUTHOR]

The Rho GTPase signaling pathway is responsible for cell-specific processes, including actin cytoskeleton organization, cell motility, cell division, and the transcription of specific genes. The implications of RhoA and the downstream effector ROCK2 in cancer epithelial-mesenchymal transition, migration, invasion, and therapy resistance associated with stem cells highlight the potential of targeting RhoA/ROCK2 signaling in therapy. Tumor relapse can occur due to cancer cells that do not fully respond to adjuvant chemoradiotherapy, targeted therapy, or immunotherapy. Rho signaling-mediated mitotic defects and cytokinesis failure lead to asymmetric cell division, allowing cells to form polyploids to escape cytotoxicity and promote tumor recurrence and metastasis. In this review, we elucidate the significance of RhoA/ROCK2 in the mechanisms of cancer progression and summarize their inhibitors that may improve treatment strategies. [ABSTRACT FROM AUTHOR]

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoAmediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endodermC&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis. [ABSTRACT FROM AUTHOR]

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors. [ABSTRACT FROM AUTHOR]

Ultraviolet-B (UV-B) radiation overexposure causes function impairment of epidermal stem cells (ESCs). We explored the mechanism of Annexin A1 (ANXA1) ameliorating UV-B-induced ESC mitochondrial dysfunction/cell injury. ESCs were cultured in vitro and irradiated with different doses of UV-B. Cell viability/ANXA1 protein level were assessed. After oe-ANXA1 transfection, ESCs were treated with oe-ANXA1/UV-B irradiation/CCCP/CCG-1423/3-methyladenine for 12 h. Cell viability/death, and adenosine triphosphate (ATP)/reactive oxygen species (ROS) levels were determined. Mitochondrial membrane potential (MMP) changes/DNA (mtDNA) content/oxygen consumption and RhoA activation were assessed. ROCK1/p-MYPT1/MYPT1/(LC3BII/I)/Beclin-1/p62 protein levels were determined. Mitochondrial morphology was observed. Mito-Tracker Green (MTG) and LC3B levels were determined. UV-B irradiation decreased cell viability/ANXA1 expression in a dose-dependent manner. UV-B-treated ESCs exhibited reduced cell viability/ATP content/MMP level/mitochondrial respiratory control ratio/mtDNA number/RhoA activity/MYPT1 phosphorylation/MTG+LC3B+ cells/(LC3BII/I) and Beclin-1 proteins, increased cell death/ROS/p62/IL-1β/IL-6/TNF-α expression, contracted mitochondrial, disappeared mitochondrial cristae, and increased vacuolar mitochondria, which were averted by ANXA1 overexpression, suggesting that UV-B induced ESC mitochondrial dysfunction/cell injury/inflammation by repressing mitophagy, but ANXA1 promoted mitophagy by activating the RhoA/ROCK1 pathway, thus repressing UV-B’s effects. Mitophagy activation ameliorated UV-B-caused ESC mitochondrial dysfunction/cell injury/inflammation. Mitophagy inhibition partly diminished ANXA1-ameliorated UV-B’s effects. Conjointly, ANXA1 promoted mitophagy by activating the RhoA/ROCK1 pathway, thereby improving UV-B-induced ESC mitochondrial dysfunction/cell injury. [ABSTRACT FROM AUTHOR]

Background: The adhesive properties of vitiligo melanocytes have decreased under oxidative stress., cytoskeleton proteins can control cell adhesion. Paeoniflorin (PF) was proved to resist hydrogen peroxide (H2O2)-induced oxidative stress in melanocytes via nuclear factorE2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Objectives: This study was to investigate whether PF exerts anti-oxidative effect through influencing cytoskeleton markers or potential signaling pathway. Methods: Human Oxidative Stress Plus array was used to identify the differentially expressed genes between H2O2 + PF group and H2O2 only group, in PIG1 and PIG3V melanocyte cell lines respectively. Western blotting was used to verify the PCR array results and to test the protein expression levels of cytoskeleton markers including Ras homolog family member A (RhoA), Rho-associated kinase 1 (ROCK1) and antioxidative marker Nrf2. Small interfering RNA was used to knock down PDZ and LIM domain 1 (PDLIM1). Results: PF increased the expressions of PDLIM1, RhoA and ROCK1 in H2O2-induced PIG1, in contrast, decreased the expressions of PDLIM1 and ROCK1 in H2O2-induced PIG3V. Knockdown of PDLIM1 increased the expressions of RhoA and Nrf2 in PF-pretreated H2O2-induced PIG1, and ROCK1 and Nrf2 in PF-pretreated H2O2-induced PIG3V. Conclusions: PF regulates RhoA/ROCK1 and Nrf2 pathways in PDLIM1-dependent or independent manners in H2O2-induced melanocytes. In PIG1, PF promotes PDLIM1 to inhibit RhoA/ROCK1 pathway or activates Nrf2/HO-1 pathway, separately. In PIG3V, PF directly downregulates ROCK1 in PDLIM1-independent manner or upregulates Nrf2 dependent of PDLIM1. [ABSTRACT FROM AUTHOR]

Shortening of airway smooth muscle and bronchoconstriction are pathognomonic for asthma. Airway shortening occurs through calcium-dependent activation of myosin light chain kinase, and RhoA-dependent calcium sensitization, which inhibits myosin light chain phosphatase. The mechanism through which pro-contractile stimuli activate calcium sensitization is poorly understood. Our review of the literature suggests that pro-contractile G protein coupled receptors likely signal through G12/13 to activate RhoA and mediate calcium sensitization. This hypothesis is consistent with the effects of pro-contractile agonists on RhoA and Rho kinase activation, actin polymerization and myosin light chain phosphorylation. Recognizing the likely role of G12/13 signaling in the pathophysiology of asthma rationalizes the effects of pro-contractile stimuli on airway hyperresponsiveness, immune activation and airway remodeling, and suggests new approaches for asthma treatment. [ABSTRACT FROM AUTHOR]

Abstract The Rho GTPase signaling pathway is responsible for cell-specific processes, including actin cytoskeleton organization, cell motility, cell division, and the transcription of specific genes. The implications of RhoA and the downstream effector ROCK2 in cancer epithelial-mesenchymal transition, migration, invasion, and therapy resistance associated with stem cells highlight the potential of targeting RhoA/ROCK2 signaling in therapy. Tumor relapse can occur due to cancer cells that do not fully respond to adjuvant chemoradiotherapy, targeted therapy, or immunotherapy. Rho signaling-mediated mitotic defects and cytokinesis failure lead to asymmetric cell division, allowing cells to form polyploids to escape cytotoxicity and promote tumor recurrence and metastasis. In this review, we elucidate the significance of RhoA/ROCK2 in the mechanisms of cancer progression and summarize their inhibitors that may improve treatment strategies.

Central nervous system diseases pose a significant threat to human health. Clinical observations have demonstrated that celabenzine, the primary active component of the natural plant ginseng, can potentially alleviate cognitive dysfunction in patients with central nervous system disorders. However, the specific mechanisms of its action remain unclear. The PubMed database was used to identify the targets of celabenzine. Subsequently, a Venn diagram of intersecting targets was constructed by utilizing the identified central nervous system injury targets. Additionally, Cytoscape software (version 3.7.2) was used to construct active ingredient-target and Protein-protein interaction networks. The Metascape database was used to perform Gene Ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Molecular docking was performed on active ingredients and target genes. Network pharmacology analysis revealed that celabenzine shares 175 intersecting targets with central nervous system damage, manifesting its effects through PI3K-Akt and MAPK signaling pathways and proteoglycans in the cancer pathway. Celabenzine demonstrates a strong binding affinity with its target gene, RhoA, indicating stable binding. This study was accomplished using network pharmacology and bioinformatics. Our findings suggested that celabenzine shows its protective effects against central nervous system damage by modulating the RhoA gene. [ABSTRACT FROM AUTHOR]

Histone lactylation is a metabolic stress‐related histone modification. However, the role of histone lactylation in the development of sepsis‐associated acute kidney injury (SA‐AKI) remains unclear. Here, histone H3K18 lactylation (H3K18la) is elevated in SA‐AKI, which is reported in this study. Furthermore, this lactate‐dependent histone modification is enriched at the promoter of Ras homolog gene family member A (RhoA) and positively correlated with the transcription. Correction of abnormal lactate levels resulted in a reversal of abnormal histone lactylation at the promoter of RhoA. Examination of related mechanism revealed that histone lactylation promoted the RhoA/Rho‐associated protein kinase (ROCK) /Ezrin signaling, the activation of nuclear factor‐κB (NF‐κB), inflammation, cell apoptosis, and aggravated renal dysfunction. In addition, Ezrin can undergo lactylation modification. Multiple lactylation sites are identified in Ezrin and confirmed that lactylation mainly occurred at the K263 site. The role of histone lactylation is revealed in SA‐AKI and reportes a novel post‐translational modification in Ezrin. Its potential role in regulating inflammatory metabolic adaptation of renal proximal tubule epithelial cells is also elucidated. The results provide novel insights into the epigenetic regulation of the onset of SA‐AKI. [ABSTRACT FROM AUTHOR]

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA–Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA–mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA–ROCK signaling, which restrained growth cone microtubule assembly and protrusion. [ABSTRACT FROM AUTHOR]

Introduction: Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma characterized by a T follicular helper cell phenotype expressing PD-1 (programmed cell death-1). AITL exhibits a poor response to conventional chemotherapy, with a median 5-year overall survival of 44% and a progressionfree survival of 32%. Relapse is common, resulting in a median overall survival of 6 months. Recurrent mutations are detected in genes regulating DNA methylation, including TET2, DNMT3A, and IDH2 variants, along with the prevalent RHOA G17V mutation. In this context, patients treated with the hypomethylating agent 5-azacytidine achieved overall response and complete response rates of 75% and 41%, respectively. We hypothesized that targeted therapies combining anti-PD-1 checkpoint blockers with hypomethylating agents could be efficient in AITL patients and less toxic than standard chemotherapy. Methods: Here, we report the efficacy of a regimen combining 5-azacytidine and nivolumab in nine relapsed or refractory AITL patients. Results: This regimen was well-tolerated, especially in elderly patients. The overall response rate was 78%, including four partial responses (44%) and three complete responses (33%). Allogeneic hematopoietic stem cell transplantation was performed in two patients who reached complete response. Discussion: These preliminary favorable results may serve as a basis for further investigation in prospective studies. [ABSTRACT FROM AUTHOR]

Plakophilin 4 (PKP4) is a component of cell–cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization. [ABSTRACT FROM AUTHOR]

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage. [ABSTRACT FROM AUTHOR]

Aminoglycoside nephrotoxicity stands as a primary contributor to the development of acute intrinsic renal failure. Distinctive characteristic associated with this nephrotoxicity is the occurrence of tubular necrosis, which is why it is commonly referred to as acute tubular necrosis. Studies have demonstrated that inhibiting rhoA/rho-kinase pathway is beneficial for kidney damage induced by diabetes and renal ischemia. Comparable pathological conditions can be observed in aminoglycoside nephrotoxicity, like those found in diabetes and renal ischemia. Gentamicin, an aminoglycoside, is known to activate Rho/Rho-kinase pathway. The primary goal of this study is to explore influence of oxidative stress on this pathway by concurrently administering gentamicin and alpha-linolenic acid (ALA) possessing known antioxidant properties. To achieve this, gentamicin (100 mg kg−1) and ALA (70 mg kg−1) were administered to mice for a period of 9 days, and Rho/Rho-kinase pathway was examined by using ELISA. Administration of gentamicin to mice led to an elevation in RhoA and rho-kinase II levels, along with the activity of rho-kinase in kidneys. However, ALA effectively reversed this heightened response. ALA, known for its antioxidant properties, inhibited activation of Rho/Rho-kinase pathway induced by gentamicin. This finding suggests that gentamicin induces nephrotoxicity through oxidative stress.

Mechanical stimulation is the key physical factor in cell environment. Mechanotransduction acts as a fundamental regulator of cell behavior, regulating cell proliferation, differentiation, apoptosis, and exhibiting specific signature alterations during the pathological process. As research continues, the role of epigenetic science in mechanotransduction is attracting attention. However, the molecular mechanism of the synergistic effect between mechanotransduction and epigenetics in physiological and pathological processes has not been clarified. We focus on how histone modifications, as important components of epigenetics, are coordinated with multiple signaling pathways to control cell fate and disease progression. Specifically, we propose that histone modifications can form regulatory feedback loops with signaling pathways, that is, histone modifications can not only serve as downstream regulators of signaling pathways for target gene transcription but also provide feedback to regulate signaling pathways. Mechanotransduction and epigenetic changes could be potential markers and therapeutic targets in clinical practice.

ABSTRACT Within the eukaryotic cell, the actin cytoskeleton is a crucial structural framework that maintains cellular form, regulates cell movement and division, and facilitates the internal transportation of proteins and organelles. External cues induce alterations in the actin cytoskeleton primarily through the activation of Rho GTPases, which then bind to a diverse array of effector proteins to promote the local assembly or disassembly of actin. We have harnessed the extensively studied functions of RhoA in the dynamics of the actin cytoskeleton to craft a practical series for Stage 2 Biology students. This series not only imparts essential tissue culture laboratory skills but also reinforces them through repetition. These activities are presented in a scenario designed for students to explore the function of a hypothetical RhoA family member. Students produce slides from transfected cells, undertake fluorescence microscopy, process the images using ImageJ, and compile their findings in a comprehensive scientific report. The composition of the report requires independent acquisition of new knowledge and synoptic learning. According to student feedback, this early experience greatly aids in solidifying and honing the skills required to report on more extensive and intricate research projects, such as capstone projects.

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 Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration. Methods Immunoprecipitation, GST pull-down assay, and proximity ligation assay (PLA) were performed to analyze the interaction between RhoGDI1 and PLK1. In vitro kinase assay and immunoprecipitation were performed with Phospho-(Ser/Thr) antibody. We evaluated RhoA activation using RhoGTPases activity assay. Cell migration and invasion were analyzed by transwell assays. Results GST pull-down assays and PLA showed that PLK1 directly interacted with RhoGDI1 in vitro and in vivo. Truncation mutagenesis revealed that aa 90-111 of RhoGDI1 are critical for interacting with PLK1. We also showed that PLK1 phosphorylated RhoGDI1 at Thr7 and Thr91, which induces cell motility. Overexpression of the GFP-tagged RhoGDI1 truncated mutant (aa 90-111) inhibited the interaction of PLK1 with RhoGDI1 and attenuated RhoA activation by PLK1. Furthermore, the overexpression of the RhoGDI1 truncated mutant reduced cancer cell migration and invasion in vitro and suppressed lung metastasis in vivo. Conclusions Collectively, we demonstrate that the phosphorylation of RhoGDI1 by PLK1 promotes cancer cell migration and invasion through RhoA activation. This study connects the interaction between PLK1 and RhoGDI1 to the promotion of cancer cell behavior associated with malignant progression, thereby providing opportunities for cancer therapeutic interventions.

Our previous research revealed that androgen receptor (AR) activation reduces endothelial cell proliferation via non-genomic pathways. We hypothesized that AR activation might also affect endothelial cell migration, a critical step in angiogenesis. Our data demonstrates that treatment of human umbilical vein endothelial cells (HUVECs) with AR agonists, metribolone (R1881) or dihydrotestosterone (DHT), results in a dose-dependent reduction in migration, which can be reversed by AR antagonists or AR knockdown. Mechanistically, R1881 inhibits HUVEC migration by suppressing RhoA activity through the cSrc/FAK/paxillin pathway and promoting RhoA degradation via RhoA-p27 complex formation, ultimately resulting in RhoA ubiquitination. Transfection with constitutively active RhoA-V14 rescues the inhibitory effect of R1881 on HUVEC migration. Furthermore, R1881 elevates intracellular vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels but reduces VEGF secretion from HUVECs. This reduction is attributed to the formation of VEGF-CTGF complexes in the cytosol induced by R1881. Transfection with RhoA-V14 reduces CTGF levels and VEGF-CTGF complex formation, leading to enhanced VEGF secretion. Pre-treatment with WP631, a CTGF inhibitor, mitigates the R1881-induced reduction in VEGF secretion and HUVECs migration. In vivo assessments using zebrafish angiogenesis and mouse matrigel plug assays validate the anti-angiogenic effects of R1881. These findings provide insight into the molecular mechanisms through which AR activation modulates endothelial cell migration and angiogenesis.

Forsythia suspensa tea is a popular traditional Chinese medicine decoction for its healthy and therapeutic benefits. However, its effects in bone metabolism were not clear. In recent study, we uncovered anti‐osteoclastogenesis property of Phillygenin (Phi), a compound abundant in Forsythia suspensa leaves, and aimed to investigate the effect and mechanism of Phi on bone metabolism in vivo and in vitro. Lipopolysaccharides‐induced murine calvaria osteolysis and ovariectomy‐induced bone loss animal models were used to identify the bone‐protective effect of Phi in vivo and micro‐CT, pQCT, and TRAP staining were applied. We used CCK8, TUNEL, BrdU, and TRAP staining to evaluate the efficacy of Phi on the proliferation and formation of OCs in primary mBMMs. RNA sequence, activity‐based protein profiling, molecular docking, G‐LISA, and WB were used to inspect the target and underlying mechanism of Phi's actions in mBMMs. We found Phi significantly inhibited bone resorption in vivo and inhibited mBMMs osteoclastogenesis in vitro. Ras homolog gene family member A (RhoA) was identified as the direct target of Phi. It counteracted the effects of RhoA activator and acted as a RhoA inhibitor. By targeting RhoA, Phi modulated Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) activity and regulated its downstream NF‐κB/NFATc1/c‐fos pathway. Furthermore, Phi depressed the disassembling of F‐actin ring through cofilin and myosin1a. Our findings provided Phi as a potential option for treating bone loss diseases by targeting RhoA and highlighted the importance of F. suspensa as a preventive approach in bone disorders. [ABSTRACT FROM AUTHOR]

Background Triple-negative breast cancer (TNBC) lacks effective therapeutic targets. Scutellaria barbata D.Don (SB) has been revealed to have anti-breast cancer (BC) effect, but the effect of SB extract in TNBC is still unclear. Herein, this research delves into the underlying mechanism. Methods SB was extracted by solvent extraction, and the main components were identified using an Agilent 6,520 HPLC-Chip/Q-TOF (Chip/Q-TOF) MS system. In vitro cell experiments were conducted. The effects of SB extract alone, SB extract plus EGF, GSK alone, GSK plus Ezrin overexpression, or SB extract plus Ezrin overexpression on cell viability, invasion, migration, and apoptosis were examined by cell function experiments. The apoptosis- and RhoA/ROCK1 pathway-related protein levels were analyzed by western blot assay. Results Mass spectrometry analysis exhibited that SB extract mainly contains long-chain fatty acids and ursolic acid. SB extract mitigated TNBC cell biological phenotypes, apoptosis- and RhoA/ROCK1 pathway-related marker expressions, which were reversed by EGF. The further results found that GSK obviously weakens TNBC cell biological behaviors, apoptosis- and RhoA/ROCK1 signaling-related protein levels, while oe-Ezrin treatment reverses the effect of GSK on TNBC cells. Moreover, SB extract regulated Ezrin-mediated function of TNBC cells by impeding the RhoA/ROCK1 pathway. Conclusion Our findings demonstrated that SB extract regulated Ezrin-mediated proliferation, migration, invasion, and apoptosis of TNBC cells via suppressing the RhoA /ROCK1 signaling. Our results offer the experimental foundation for further investigation of the anti-cancer role of SB in TNBC cells. Highlights SB extract inhibits the biological phenotypes of TNBC cells. SB extract inhibits the biological behaviors of TNBC cells through the RhoA/ROCK1 pathway. SB extract modulates Ezrin-mediated TNBC cell proliferation, migration, invasion, and apoptosis via restraining the RhoA/ROCK1 signaling. [ABSTRACT FROM AUTHOR]

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells. [ABSTRACT FROM AUTHOR]

How receptors juggle their interactions with multiple downstream effectors remains poorly understood. Here we show that the outcome of death receptor p75NTR signaling is determined through competition of effectors for interaction with its intracellular domain, in turn dictated by the nature of the ligand. While NGF induces release of RhoGDI through recruitment of RIP2, thus decreasing RhoA activity in favor of NFkB signaling, MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, disengaging RIP2, and enhancing RhoA activity in detriment of NF-kB. This results in stunted neurite outgrowth and apoptosis in cerebellar granule neurons. If presented simultaneously, MAG prevails over NGF. The NMR solution structure of the complex between the RhoGDI N-terminus and p75NTR juxtamembrane domain reveals previously unknown structures of these proteins and clarifies the mechanism of p75NTR activation. These results show how ligand-directed competition between RIP2 and RhoGDI for p75NTR engagement determine axon growth and neuron survival. Similar principles are likely at work in other receptors engaging multiple effectors and signaling pathways. Synopsis: MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, locking p75NTR in a configuration that promotes RhoA activity, growth cone collapse, stunted neurite outgrowth, and neuronal apoptosis. MAG induces PKC-mediated phosphorylation of RhoGDI at Ser34, leading to phosphorylation at Ser96, which results in the release and activation of RhoA. Phosphorylated Ser34 interacts in trans with Lys303 of the p75NTR juxtamembrane domain, which becomes structured by wrapping around RhoGDI N-terminal domain. Fully phosphorylated RhoGDI outcompetes RIP2 and locks p75NTR in a configuration that promotes growth cone collapse, stunted neurite outgrowth, and apoptosis in cerebellar granule neurons. MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, locking p75NTR in a configuration that promotes RhoA activity, growth cone collapse, stunted neurite outgrowth, and neuronal apoptosis. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Background: Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration. Methods: Immunoprecipitation, GST pull-down assay, and proximity ligation assay (PLA) were performed to analyze the interaction between RhoGDI1 and PLK1. In vitro kinase assay and immunoprecipitation were performed with Phospho-(Ser/Thr) antibody. We evaluated RhoA activation using RhoGTPases activity assay. Cell migration and invasion were analyzed by transwell assays. Results: GST pull-down assays and PLA showed that PLK1 directly interacted with RhoGDI1 in vitro and in vivo. Truncation mutagenesis revealed that aa 90-111 of RhoGDI1 are critical for interacting with PLK1. We also showed that PLK1 phosphorylated RhoGDI1 at Thr7 and Thr91, which induces cell motility. Overexpression of the GFP-tagged RhoGDI1 truncated mutant (aa 90-111) inhibited the interaction of PLK1 with RhoGDI1 and attenuated RhoA activation by PLK1. Furthermore, the overexpression of the RhoGDI1 truncated mutant reduced cancer cell migration and invasion in vitro and suppressed lung metastasis in vivo. Conclusions: Collectively, we demonstrate that the phosphorylation of RhoGDI1 by PLK1 promotes cancer cell migration and invasion through RhoA activation. This study connects the interaction between PLK1 and RhoGDI1 to the promotion of cancer cell behavior associated with malignant progression, thereby providing opportunities for cancer therapeutic interventions. [ABSTRACT FROM AUTHOR]

Hepatoblastoma (HB) is a common primary liver malignant tumor in children. Long non-coding RNAs (lncRNAs) are closely engaged in HB progression. The role and regulatory molecule mechanism of lncRNA small nucleolar RNA host gene 1 (SNHG1) in HB remain unclear. Through qRT-PCR or western blot, we found that SNHG1 and proviral integration site for moloney murine leukemia virus 3 (PIM3) were elevated but miR-6838-5p was decreased in HB cells. Cell biology experiments revealed that SNHG1 depletion or miR-6838-5p upregulation suppressed cell proliferation, migration, and invasion of HB cells. Mechanistically, luciferase activity assay validated that miR-6838-5p could interact with SNHG1 or PIM3. SNHG1 up-regulated PIM3 expression via sponging miR-6838-5p. Moreover, miR-6838-5p inhibitor abolished SNHG1 depletion-mediated suppression of malignant behaviors in HB cells. PIM3 overexpression neutralized miR-6838-5p mimics-mediated repression of malignant phenotypes in HB cells. Furthermore, miR-6838-5p overexpression suppressed RhoA activation, which was restored by PIM3 upregulation. What's more, the results at the cellular level were further verified by nude mice tumor formation experiment. In conclusion, SNHG1 regulated miR-6838-5p/PIM3/RhoA axis to promote malignant phenotypes of HB, which might provide novel therapeutic target for HB treatment. [ABSTRACT FROM AUTHOR]

Background: Glioma, a type of malignant brain tumor, has become a challenging health issue globally in recent years. Methods: In this study, we investigated the potential therapeutic role of scoparone in glioma and the underlying mechanism. Initially, transcriptome sequencing was conducted to identify genes that exhibited differential expression in glioma cells treated with scoparone compared to untreated cells. Subsequently, the impact of scoparone on the proliferation, migration, and invasion of glioma cells was assessed in vitro using a range of assays including cell viability, colony formation, wound healing, and transwell assays. Moreover, the apoptotic effects of scoparone on glioma cells were evaluated through flow cytometry and western blot analysis. Furthermore, we established a glioma xenograft mouse model to assess the in vivo antitumor activity of scoparone. Lastly, by integrating transcriptome analysis, we endeavored to unravel the molecular mechanisms underlying the observed antitumor effects of scoparone by examining the expression levels of RhoA/ROCK1 signaling pathway components using western blot analysis and qRT‐PCR. Results: Our transcriptome sequencing results revealed that scoparone significantly downregulated RhoA/ROCK1 signaling in glioma cells. Furthermore, scoparone treatment inhibited glioma cell proliferation, migration, and invasion, and promoted cell apoptosis in vitro. Moreover, scoparone reduced tumor growth and prolonged survival in a glioma xenograft mouse model, and improved the toxicity of temozolomide. Finally, our results showed that the antitumor effects of scoparone were mediated by the suppression of RhoA/ROCK1 signaling. Conclusion: Scoparone could be a promising therapeutic agent for glioma by suppressing RhoA/ROCK1 signaling. These findings pave the way for future research endeavors aimed at the development and optimization of scoparone‐based therapeutic strategies. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by aggregation of amyloid beta (Aβ) plaque. RhoA may serve as a potential target for prevention against AD given its role in the amyloidogenic pathway. The recent emergence of the gut-brain axis has linked lactic acid bacteria (LAB) to neuroprotection against AD. This study assessed the importance of RhoA inhibition in mediating the neuroprotective potential of LAB. To this end, de Man, Rogosa and Sharpe (MRS) broth fermented by lactobacilli or pediococci were tested against SK-N-SH (a human neuroblastoma cell line) in the presence of RhoA activator II for 24 h after which the RhoA activity was measured using the G-LISA Kit. Fluorescence staining of f-actin stress fibres was performed to validate RhoA inhibition. SK-N-SH was transfected with plasmid expressing amyloid precursor protein (APP) gene. The Aβ concentration in transfected cells exposed to LAB-derived cell free supernatant (CFS) in the presence of RhoA activator II was measured using the ELISA kit. Furthermore, this study measured organic acids in LAB-derived CFS using the gas chromatography. It was found that LAB-derived CFS yielded strain-dependent inhibition of RhoA, with LAB6- and LAB12-derived CFS being the most potent Pediococcal- and Lactiplantibacillus-based RhoA inhibitor, respectively. Lesser stress fibres were formed under treatment with LAB-derived CFS. The LAB-derived CFS also significantly inhibited Aβ in SK-N-SH transfected with APP gene in the presence of RhoA activator II. The LAB-derived CFS was presented with increased lactic acid, acetic acid, butyric acid and propionic acid. The present findings warrant in-depth study using animal models. [ABSTRACT FROM AUTHOR]

Abstract Histone lactylation is a metabolic stress‐related histone modification. However, the role of histone lactylation in the development of sepsis‐associated acute kidney injury (SA‐AKI) remains unclear. Here, histone H3K18 lactylation (H3K18la) is elevated in SA‐AKI, which is reported in this study. Furthermore, this lactate‐dependent histone modification is enriched at the promoter of Ras homolog gene family member A (RhoA) and positively correlated with the transcription. Correction of abnormal lactate levels resulted in a reversal of abnormal histone lactylation at the promoter of RhoA. Examination of related mechanism revealed that histone lactylation promoted the RhoA/Rho‐associated protein kinase (ROCK) /Ezrin signaling, the activation of nuclear factor‐κB (NF‐κB), inflammation, cell apoptosis, and aggravated renal dysfunction. In addition, Ezrin can undergo lactylation modification. Multiple lactylation sites are identified in Ezrin and confirmed that lactylation mainly occurred at the K263 site. The role of histone lactylation is revealed in SA‐AKI and reportes a novel post‐translational modification in Ezrin. Its potential role in regulating inflammatory metabolic adaptation of renal proximal tubule epithelial cells is also elucidated. The results provide novel insights into the epigenetic regulation of the onset of SA‐AKI.

IntroductionAngioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma characterized by a T follicular helper cell phenotype expressing PD-1 (programmed cell death-1). AITL exhibits a poor response to conventional chemotherapy, with a median 5-year overall survival of 44% and a progression-free survival of 32%. Relapse is common, resulting in a median overall survival of 6 months. Recurrent mutations are detected in genes regulating DNA methylation, including TET2, DNMT3A, and IDH2 variants, along with the prevalent RHOA G17V mutation. In this context, patients treated with the hypomethylating agent 5-azacytidine achieved overall response and complete response rates of 75% and 41%, respectively. We hypothesized that targeted therapies combining anti-PD-1 checkpoint blockers with hypomethylating agents could be efficient in AITL patients and less toxic than standard chemotherapy.MethodsHere, we report the efficacy of a regimen combining 5-azacytidine and nivolumab in nine relapsed or refractory AITL patients.ResultsThis regimen was well-tolerated, especially in elderly patients. The overall response rate was 78%, including four partial responses (44%) and three complete responses (33%). Allogeneic hematopoietic stem cell transplantation was performed in two patients who reached complete response.DiscussionThese preliminary favorable results may serve as a basis for further investigation in prospective studies.

Osteocytes are terminally differentiated cells derived from osteoblasts and are deeply embedded within the bone matrix. They play a critical role in bone remodeling by generating a lacuno-canalicular network (LCN) and controlling the transport of nutrients. Due to the absence of blood vessels within the bone matrix, it is widely believed that osteocytes develop in a hypoxic environment. However, the mechanisms of osteocytogenesis and the role of oxygen sensing in this process remain unclear. Hypoxia-inducible factors (HIFs) are major transcriptional factors involved in oxygen sensing. Previous studies have shown that accumulation of HIFs in osteoblasts leads to abnormal bone remodeling, potentially linked with the alterations of the LCN network. Specifically, HIF-1α is hypothesized to play a more significant role in regulating bone remodeling compared to HIF-2α. Therefore, we investigated the functions of HIF-1α in dendrite formation and the establishment of the LCN network during osteocytogenesis. Immunostaining and scanning electron microscopy revealed that the E11 protein aggregates to form a ring structure that defines the site for dendrite initiation. This process is followed by activation of the ERM/RhoA pathway and recruitment of matrix metalloproteinase 14 (MMP14) to facilitate extracellular matrix degradation, enabling dendrite elongation. However, both hypoxic treatment and overexpression of HIF-1α impair ring formation, resulting in reduced ERM/RhoA activity and decreased matrix degradation capability. These findings suggest that abnormal HIF-1α activity in local areas could contribute to impaired LCN network formation and abnormal bone remodeling observed in bone diseases such as osteopenia and aging.

Abstract Objective Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by abnormal activation of the type I interferon (IFN) pathway, which results in tissue inflammation and organ damage. We explored the role of the RhoA GTPase in the type I IFN activation pathway to provide a potential basis for targeting GTPase signaling for the treatment of SLE. Methods Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls, and the mRNA expression levels of RhoA and IFN-stimulated genes were measured by SYBR Green quantitative reverse transcriptase-polymerase chain reaction. IFN-a-stimulated response element (ISRE)-luciferase reporter gene assays and Western blotting were conducted to assess the biologic function of RhoA. An enzyme-linked immunoassay (ELISA) measured C-X-C motif chemokine ligand 10 (CXCL10) protein expression. Results Our studies demonstrate that the expression of RhoA in the PBMCs of SLE subjects was significantly higher than in healthy controls and positively correlated with type I IFN scores and type I IFN-stimulated gene (ISGs) expression levels. SiRNA-mediated knockdown of RhoA and the RhoA/ROCK inhibitor Y27632 reduced the activity of the type I IFN-induced ISRE, the signal transducer and activator of transcription 1 (STAT-1) phosphorylation, and the expression of CXCL10 and 2′-5′-oligoadenylate synthetase 1 (OAS1). Finally, we verified that Y27632 could significantly down-regulate the OAS1 and CXCL10 expression levels in the PBMCs of SLE patients. Conclusion Our study shows that RhoA positively regulates the activation of the type I IFN response pathway. Reducing the expression level of RhoA inhibits the abnormal activation of the type I IFN system, and the RhoA/ROCK inhibitor Y27632 decreases aberrant type I IFN signaling in SLE PBMCs, suggesting the possibility of targeting the RhoA GTPase for the treatment of SLE.

Abstract The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.

Kuo-Feng Hua,1,2,* Lan-Hui Li,3,* Hsin-Chiao Yu,4 Wei-Ting Wong,1 Hsien-Ta Hsu4,5 1Department of Biotechnology and Animal Science, National Ilan University, Yilan, 26047, Taiwan; 2Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 404333, Taiwan; 3Department of Laboratory Medicine, Linsen, Chinese Medicine and Kunming Branch, Taipei City Hospital, Taipei, 108, Taiwan; 4Division of Neurosurgery, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, 231, Taiwan; 5School of Medicine, Buddhist Tzu Chi University, Hualien, 970, Taiwan*These authors contributed equally to this workCorrespondence: Hsien-Ta Hsu, Division of Neurosurgery, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, No. 289, Jianguo Road, Xindian Dist, New Taipei City, 231016, Taiwan, Tel +866-2-6628-9779, Email j1208192@ms45.hinet.netPurpose: Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin’s influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells.Methods: The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors.Results: Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation.Conclusion: These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration. Keywords: intervertebral disc degeneration, leptin, matrix metalloproteinase, intervertebral disc cartilage endplate-derived stem cells, RhoA, ERK1/2

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-b (TGF-b) is one of the most established drivers of fibrotic processes. TGF-b promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-b robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle a-actin (SMA)-positive myofibroblasts. TGF-b-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-b signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-b treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing “with-no-lysine (K)” kinase (WNK1). WNK1 siRNA significantly attenuates TGF-b-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-b-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-b; and 2) ANO1 mediates TGF-b-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity. NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-b (TGF-b) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

This article discusses the process of replicative senescence in human embryonic bone marrow mesenchymal stem cells (MSCs). The study focuses on changes in cell motility and the organization of the actin cytoskeleton during replicative senescence. The researchers found that there are alterations in the localization of certain proteins and changes in cell morphology and motility during this process. The study suggests that Rho signaling pathways may be involved in replicative senescence, but further research is needed to understand the underlying mechanisms. The article also provides a list of references for further reading on related topics in cell biology and biochemistry. [Extracted from the article]

Objective: Inflammation is a complex reaction present in numerous disorders. Indomethacin, a compound possessing an indoline core, is a Nonsteroidal Anti-Inflammatory Drug (NSAID) that is commonly prescribed for inflammation and pain. The actin network, plays a major role in cellular activities and it's regulated by by Rho GTPases has important implications for cellular dynamics and orientation. In this research, we explore the effects of indomethacin on the inflammatory response as mediated via RhoA and pyrin inflammatory complexes using an inflammatory disease model with relation actin cytoskeleton. Materials and Methods: This study used Western blotting to examine the impact of indomethacin on the assembly processes related to the pyrin inflammasome complex and the RhoA signaling pathway in Lipopolysaccharide-stimulated THP-1 cells. Actin-indomethacin interaction was analyzed by Differential Scanning Fluorimetry (DSF). Results: We found that while the expression levels of pyrin decreased, phosphorylated-RhoA increased but overall RhoA levels did not change. The equilibrium dissociation constant (KD) for the G-actin-indomethacin complex was calculated to be 9.591± 1.608 ng/mL (R² = 0.8582) using ΔTm measurements of indomethacin by DSF. Conclusion: Moreover, the effects of indomethacin on inflammation pathways may provide insight into the molecular mechanisms of pyrin inflammasome formation in various autoimmune diseases. [ABSTRACT FROM AUTHOR]

The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs. [ABSTRACT FROM AUTHOR]

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gai-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gai and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of isoprene cysteine carboxymethyltransferase (ICMT) gene on the migration and invasion of salivary adenoid cystic cancer cells (SACC) and the related mechanism, to provide experimental evidence for molecular targeted therapy of SACC. Methods Adenoid cystic cancer cells SACC-LM and SACC-83 were cultured in vitro, and siRNA was transfected into human SACC-LM and SACC-83 cells (experimental group) by transient transfection of a liposome vector. A blank control group and negative control group were set up respectively (transfected NC-siRNA). qRT-PCR was peformed to measure the mRNA expression of ICMT and RhoA in each group after transfection and to determine the silencing efficiency. The expression of ICMT, membrane RhoA, total RhoA, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and Rho associated with coiled helical binding protein kinase 1 (ROCK1) in each group was detected by Western blot. The proliferation abilityies of SACC cells was detected by CCK-8 assay. The migration and invasion ability of SACC cells were detected by comparing the relative healing area of cell scratch assay and the number of Transwell assay cells. Results After transfection of ICMT-siRNA into SACC-LM and SACC-83 cells, the expression of ICMT gene and protein in the experimental group was significantly decreased compared with the negative control group and blank control group (P0.05). The expression of RhoA membrane proteins, ROCK1, MMP-2, MMP-9 in the experimental group was significantly decreased compared with that in the negative control group and blank control group (P

Objective: The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Methods: The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. Results: The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. Conclusion: The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. The translational potential of this article: Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Abstract Background Diabetic cardiomyopathy results in cardiac structural and functional abnormalities. Previous studies have demonstrated that inhibiting the RhoA/ROCK signalling pathway increases the injury resistance of cardiomyocytes. The early detection of cardiac structural and functional alterations may facilitate an improved understanding of the pathophysiologic progress and guide therapy. This study aimed to identify the optimal diagnostic measures for the subtle early alterations of cardiac dysfunction in type 2 diabetes mellitus (T2DM) rats. Methods Twenty-four rat models were divided into four groups and received treatments for 4 weeks: the CON group (control rats), the DM group (T2DM rats), the DMF group (T2DM rats receiving fasudil) and the CONF group (control rats receiving fasudil) group. Left ventricular (LV) structure was quantified by histological staining and transmission electron microscopy. LV function and myocardial deformation were assessed by high-frequency echocardiography. Results Treatment with fasudil, a ROCK inhibitor, significantly protected against diabetes-induced myocardial hypertrophy, fibrosis and mitochondrial dysfunction. Impaired LV performance was found in T2DM rats, as evidenced by significant reductions in the ejection fraction (EF), fractional shortening (FS) and the mitral valve (MV) E/A ratio (which decreased 26%, 34% and 20%, respectively). Fasudil failed to improve the conventional ultrasonic parameters in T2DM rats, but the myocardial deformation measured by speckle-tracking echocardiography (STE) were significantly improved (global circumferential strain, GCS: P = 0.003; GCS rate, GCSR: P = 0.021). When receiver operating characteristic (ROC) curves were used in combination with linear regression analysis, STE parameters were found to be characterized by both optimal prediction of cardiac damage [AUC (95% CI): fractional area change, FAC: 0.927 (0.744, 0.993); GCS: 0.819 (0.610, 0.945); GCSR: 0.899 (0.707, 0.984)] and stronger correlations with cardiac fibrosis (FAC: r = -0.825; GCS: r = 0.772; GCSR: r = 0.829) than conventional parameters. Conclusion The results suggest that STE parameters are more sensitive and specific than conventional parameters in predicting the subtle cardiac functional changes that occur in the early stage, providing new insight into the management of diabetic cardiomyopathy.

Folic acid (FA) plays multiple roles in many biological processes. Myoblast intakes FA through endocytosis by folate receptor 1 (Folr1), which has vital role in regulating muscle development. However, the mechanism remains unclear. In this study, FA supplement experiment showed that FA promoted C2C12 cell differentiation through Folr1. Further exploration found that FA could promote C2C12 cell migration through Folr1 interacting with RhoA. Then stimulated RhoA/ROCK/LIMK/cofilin signaling pathway, and affected the distribution of microfilaments to improve C2C12 cell migration. Meanwhile, mice experiments also proved that FA could activate the expression of RhoA/ROCK/LIMK/cofilin signaling pathway in vivo and accelerate the process of skeletal muscle regeneration in mice. This discovery is helpful to further understand the molecular mechanism of FA regulating muscle development and provide new ideas for the treatment of muscle diseases.