Exome sequencing implicates ancestry-related Mendelian variation at SYNE1 in childhood-onset essential hypertension.

Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

RVG29 Peptide-Modified Exosomes Loaded with Mir-133b Mediate the RhoA-ROCK Pathway to Improve Motor and Neurological Symptoms in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Drug delivery to the brain through the blood-brain barrier (BBB) is a significant challenge in PD treatment. Exosomes, which can efficiently traverse the BBB, which many drugs cannot penetrate, are ideal natural carriers for drug delivery. In this study, the BBB shuttle peptide was modified on the exosome surfaces. Three types of exosomes were constructed, each modified with a distinct peptide (RVG29, TAT, or Ang2) and loaded with miR-133b. The safety and brain-targeting capabilities of these peptide-modified exosomes were then evaluated. Finally, the mechanism by which RVG29-Exo-133b regulates the RhoA-ROCK signaling pathway was investigated. The findings indicate that the three peptide-modified exosomes were adequately tolerated, safe, and effectively assimilated in vivo and ex vivo, with RVG29 exhibiting superior targeting to the brain. Furthermore, RVG29-Exo-133b decreased the phosphorylation level of the Tau protein by targeting the RhoA-ROCK signaling pathway. It also enhanced the motor function in mice with PD, thereby reducing the degree of depression, improving dopaminergic neuron function, and attenuating 6-OHDA-induced nerve damage. In this study, we developed a stable drug delivery mechanism that targets the intracerebral region using exosomes. Furthermore, a novel strategy was developed to manage PD and can potentially serve as a preclinical basis for utilizing exosomes in the diagnosis and treatment of neurodegenerative conditions.

Genomic and Pathologic Profiling of Very Well-Differentiated Gastric Adenocarcinoma of Intestinal Type: A Study With Emphasis on Diffuse-Type Transformation.

Very well-differentiated adenocarcinoma of intestinal type is a distinct subtype of gastric cancer characterized by anastomosing glands with a hand-in-hand pattern and low-grade cytologic atypia resembling intestinal metaplasia. This is a slow-growing neoplasm with an indolent clinical course; however, a subset demonstrates transformation into adenocarcinoma with higher-grade histology, typically diffuse-type carcinoma, and behaves aggressively. This study aimed to better characterize the genomic and pathologic features, with a focus on factors associated with diffuse-type transformation. A total of 58 cases with (n=31) and without (n=27) diffuse-type transformation were analyzed for molecular and pathologic features. First, comprehensive deep DNA sequencing was conducted in 18 cases (discovery cohort), followed by a digital droplet polymerase chain reaction of hot spot RHOA mutations in 40 cases (validation cohort). In total, RHOA mutations were the most common alteration (34%), followed by loss of ARID1A (12%), p53 alterations (10%), and CLDN18 :: ARHGAP26/6 fusions (3.4%). FGFR2 amplification was identified in an advanced case with a p53 alteration. Altered p53 expression was recognized only in higher-grade components and was significantly associated with advanced disease ( P =0.0015) and diffuse-type transformation ( P =0.026). A mixed mucin phenotype was also strongly correlated with advanced disease ( P <0.001) and diffuse-type transformation ( P <0.001). Decreased E-cadherin expression was frequently observed (74%) in poorly cohesive components. This study demonstrated that a subset of RHOA -mutant diffuse-type gastric cancers develops through the transformation of very well-differentiated adenocarcinoma of intestinal type. Our observations suggest a mixed mucin phenotype as a risk factor and alterations in p53 and E-cadherin as drivers of diffuse-type transformation.

Dynamic light-responsive RhoA activity regulates mechanosensitive stem cell fate decision in 3D matrices.

The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner.

LAMB3 Promotes Myofibrogenesis and Cytoskeletal Reorganization in Endometrial Stromal Cells via the RhoA/ROCK1/MYL9 Pathway.

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman's syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-ß-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS.

RhoA Is a Crucial Regulator of Myoblast Fusion.

Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here we show that RhoA in SCs is indispensable to have correct muscle regeneration and hypertrophy. In particular, the absence of RhoA in SCs prevents a correct SC fusion both to other RhoA-deleted SCs (regeneration context) and to growing control myofibers (hypertrophy context). We demonstrated that RhoA is dispensable for SCs proliferation and differentiation; however, RhoA-deleted SCs have an inefficient movement even if their cytoskeleton assembly is not altered. Proliferative myoblast and differentiated myotubes without RhoA display a decreased expression of Chordin, suggesting a crosstalk between these genes for myoblast fusion regulation. These findings demonstrate the importance of RhoA in SC fusion regulation and its requirement to achieve an efficient skeletal muscle homeostasis restoration.

RHOAL57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Genomic and Reverse Translational Analysis Discloses a Role for Small GTPase RhoA Signaling in the Pathogenesis of Schizophrenia: Rho-Kinase as a Novel Drug Target.

Schizophrenia is one of the most serious psychiatric disorders and is characterized by reductions in both brain volume and spine density in the frontal cortex. RhoA belongs to the RAS homolog (Rho) family and plays critical roles in neuronal development and structural plasticity via Rho-kinase. RhoA activity is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Several variants in GAPs and GEFs associated with RhoA have been reported to be significantly associated with schizophrenia. Moreover, several mouse models carrying schizophrenia-associated gene variants involved in RhoA/Rho-kinase signaling have been developed. In this review, we summarize clinical evidence showing that variants in genes regulating RhoA activity are associated with schizophrenia. In the last half of the review, we discuss preclinical evidence indicating that RhoA/Rho-kinase is a potential therapeutic target of schizophrenia. In particular, Rho-kinase inhibitors exhibit anti-psychotic-like effects not only in Arhgap10 S490P/NHEJ mice, but also in pharmacologic models of schizophrenia (methamphetamine- and MK-801-treated mice). Accordingly, we propose that Rho-kinase inhibitors may have antipsychotic effects and reduce cognitive deficits in schizophrenia despite the presence or absence of genetic variants in small GTPase signaling pathways.

Novel cardiovascular protective effects of RhoA signaling and its therapeutic implications.

Ras homolog gene family member A (RhoA) belongs to the Rho GTPase superfamily, which was first studied in cancers as one of the essential regulators controlling cellular function. RhoA has long attracted attention as a key molecule involved in cell signaling and gene transcription, through which it affects cellular processes. A series of studies have demonstrated that RhoA plays crucial roles under both physiological states and pathological conditions in cardiovascular diseases. RhoA has been identified as an important regulator in cardiac remodeling by regulating actin stress fiber dynamics and cytoskeleton formation. However, its underlying mechanisms remain poorly understood, preventing definitive conclusions being drawn about its protective role in the cardiovascular system. In this review, we outline the characteristics of RhoA and its related signaling molecules, and present an overview of RhoA classical function and the corresponding cellular responses of RhoA under physiological and pathological conditions. Overall, we provide an update on the novel signaling under RhoA in the cardiovascular system and its potential clinical and therapeutic targets in cardiovascular medicine.

Src stimulates Abl-dependent phosphorylation of the guanine exchange factor Net1A to promote its cytosolic localization and cell motility.

The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor-stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.

Septin11 promotes hepatocellular carcinoma cell motility by activating RhoA to regulate cytoskeleton and cell adhesion.

Septins as GTPases in the cytoskeleton, are linked to a broad spectrum of cellular functions, including cell migration and the progression of hepatocellular carcinoma (HCC). However, roles of SEPT11, the new member of septin, have been hardly understood in HCC. In the study, the clinical significance and biological function of SEPT11 in HCC was explored. SEPT11 was screened out by combining ATAC-seq with mRNA-seq. Role of SEPT11 in HCC was further investigated by using overexpression, shRNA and CRISPR/Cas9-mediated SEPT11-knockout cells or in vivo models. We found RNA-seq and ATAC-seq highlights LncRNA AY927503 (AY) induced SEPT11 transcription, resulting in Rho GTPase activation and cytoskeleton actin aggregation. The GTP-binding protein SEPT11 is thus considered, as a downstream factor of AY, highly expressed in various tumors, including HCC, and associated with poor prognosis of the patients. In vitro, SEPT11 overexpression promotes the migration and invasion of HCC cells, while SEPT11-knockout inhibits migration and invasion. In vivo, SEPT11-overexpressed HCC cells show high metastasis incidents but don't significantly affect proliferation. Meanwhile, we found SEPT11 targets RhoA, thereby regulating cytoskeleton rearrangement and abnormal cell adhesion through ROCK1/cofilin and FAK/paxillin signaling pathways, promoting invasion and migration of HCC. Further, we found SEPT11 facilitates the binding of GEF-H1 to RhoA, which enhances the activity of RhoA. Overall, our study confirmed function of SEPT11 in promoting metastasis in HCC, and preliminarily explored its related molecular mechanism. SEPT11 acts as an oncogene in HCC, also draws further interest regarding its clinical application as a potential therapeutic target.

MiR-490-3p Sponged by lncRNA NEAT1 Can Attenuate Lung Adenocarcinoma Progression by Suppressing the RhoA/ROCK Signaling Pathway.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are crucial regulators of lung adenocarcinoma (LUAD) progression. Herein, we explored the role of miR-490-3p and the underlying molecular mechanism involving key lncRNAs and pathways in LUAD. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect the expression of lncRNA NEAT1 and miR-490-3p in LUAD cells and tissues. Western blotting was used to determine protein expression levels of the Ras homologous gene family member A/Rho-related protein kinase (RhoA/ROCK) signal pathway marker. Considering cell functions, Cell Counting Kit-8 (CCK-8), Transwell, and xenograft experiments were employed to evaluate LUAD cell proliferation, migration, and tumor growth, respectively. The relationship between miR-490-3p and lncRNA NEAT1 was analyzed using a luciferase reporter assay. RESULTS: Herein, we found that miR-490-3p expression was significantly low in LUAD cells and tissues. MiR-490-3p overexpression markedly suppressed tumor growth, the RhoA/ROCK signaling pathway, migration, and proliferation of LUAD cells. Moreover, lncRNA NEAT1, which is highly expressed in LUAD, was detected upstream of miR-490-3p. Upregulation of lncRNA NEAT1 exacerbated the behavior of LUAD cells and offset the suppressive influence of miR-490-3p-mediated upregulation on malignant LUAD cell behavior. CONCLUSION: MiR-490-3p sponging by lncRNA NEAT1 could hamper LUAD progression by inhibiting the RhoA/ROCK signaling pathway. These findings provide new insights for LUAD diagnosis and treatment.

Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

GEF-H1/RhoA signaling pathway mediates pro-inflammatory effects of NF-κB on CD40L-induced pulmonary endothelial cells.

One of the key targets of the inflammatory response in acute lung injury (ALI) is the human pulmonary micro-vascular endothelial cells (HPMVECs). Owing to its role in the activation of endothelial cells (ECs), CD40L figures prominently in the pathogenesis of ALI. Increasing evidences have showed that CD40L mediates inflammatory effects on ECs, at least in part, by triggering NF-κB-dependent gene expression. However, the mechanisms of such signal transmission remain unknown. In this study, we found that CD40L stimulated the transactivation of NF-κB and expression of its downstream cytokines in a p38 MAPK-dependent mechanism in HPMVECs. In addition, CD40L-mediated inflammatory effects might be correlated with the activation of the IKK/IκB/NF-κB pathway and nuclear translocation of NF-κB, being accompanied by dynamic cytoskeletal changes. GEF-H1/RhoA signaling is best known for its role in regulating cytoskeletal rearrangements. An interesting finding was that CD40L induced the activation of p38 and IKK/IκB, and the subsequent transactivation of NF-κB via GEF-H1/RhoA signaling. The critical role of GEF-H1/RhoA in CD40L-induced inflammatory responses in the lung was further confirmed in GEF-H1 and RhoA knockout mouse models, both of which were established by adeno-associated virus (AAV)-mediated delivery of sgRNAs into mice with EC-specific Cas9 expression. These results taken together suggested that p38 and IKK/IκB-mediated signaling pathways, both of which lied downstream of GEF-H1/RhoA, may coordinately regulate the transactivation of NF-κB in CD40L-activated HPMVECs. These findings may help to determine key pharmacological targets of intervention for CD40L-activated inflammatory effects associated with ALI.

Fluoride exposure disrupts the cytoskeletal arrangement and ATP synthesis of HT-22 cell by activating the RhoA/ROCK signaling pathway.

BACKGROUND: Fluoride, an environmental contaminant, is ubiquitously present in air, water, and soil. It usually enters the body through drinking water and may cause structural and functional disorders in the central nervous system in humans and animals. Fluoride exposure affects cytoskeleton and neural function, but the mechanism is not clear. METHODS: The specific neurotoxic mechanism of fluoride was explored in HT-22 cells. Cellular proliferation and toxicity detection were investigated by CCK-8, CCK-F, and cytotoxicity detection kits. The development morphology of HT-22 cells was observed under a light microscope. Cell membrane permeability and neurotransmitter content were determined using lactate dehydrogenase (LDH) and glutamate content determination kits, respectively. The ultrastructural changes were detected by transmission electron microscopy, and actin homeostasis was observed by laser confocal microscopy. ATP enzyme and ATP activity were determined using the ATP content kit and ultramicro-total ATP enzyme content kit, respectively. The expression levels of GLUT1 and 3 were assessed by Western Blot assays and qRT-PCR. RESULTS: Our results showed that fluoride reduced the proliferation and survival rates of HT-22 cells. Cytomorphology showed that dendritic spines became shorter, cellular bodies became rounder, and adhesion decreased gradually after fluoride exposure. LDH results showed that fluoride exposure increased the membrane permeability of HT-22 cells. Transmission electron microscopy results showed that fluoride caused cells to swell, microvilli content decreased, cellular membrane integrity was damaged, chromatin was sparse, mitochondria ridge gap became wide, and microfilament and microtubule density decreased. Western Blot and qRT-PCR analyses showed that RhoA/ROCK/LIMK/Cofilin signaling pathway was activated by fluoride. F-actin/G-actin fluorescence intensity ratio remarkably increased in 0.125 and 0.5 mM NaF, and the mRNA expression of MAP2 was significantly decreased. Further studies showed that GLUT3 significantly increased in all fluoride groups, while GLUT1 decreased (p < 0.05). ATP contents remarkably increased, and ATP enzyme activity substantially decreased after NaF treatment with the control. CONCLUSION: Fluoride activates the RhoA/ROCK/LIMK/Cofilin signaling pathway, impairs the ultrastructure, and depresses the connection of synapses in HT-22 cells. Moreover, fluoride exposure affects the expression of glucose transporters (GLUT1 and 3) and ATP synthesis. Sum up fluoride exposure disrupts actin homeostasis, ultimately affecting structure, and function in HT-22 cells. These findings support our previous hypothesis and provide a new perspective on the neurotoxic mechanism of fluorosis.

Follicular Helper T-Cell-derived Nodal Lymphomas: Study of Histomorphologic, Immunophenotypic, Clinical, and RHOA G17V Mutational Profile.

The study was designed to review the demographic, clinical, and pathologic characteristics of follicular helper T cells (TFH)-derived nodal PTCL in India including angioimmunoblastic T-cell lymphoma (AITL), peripheral T-cell lymphoma (PTCL) with follicular helper T cell phenotype (P-TFH), and follicular T-cell lymphoma with additional immunohistochemistry (IHC) and RHOAG17V mutational analysis, as well as their impact on survival. This retrospective study included 88 cases of PTCL that were reclassified using IHC for TFH markers (PD1, ICOS, BCL6, and CD10) and dendritic-meshwork markers (CD21, CD23). Cases of TFH cell origin were evaluated for RHOAG17V mutation using Sanger sequencing and amplification-refractory mutation system-polymerase chain reaction (PCR) (validated using cloning and quantitative PCR) with detailed clinicopathologic correlation. Extensive re-evaluation with added IHC panel resulted in a total of 19 cases being reclassified, and the final subtypes were AITL (37 cases, 42%), PTCL-not otherwise specified (44, 50%), P-TFH (6, 7%), and follicular T-cell lymphoma (1, 1%). The presence of at least 2 TFH markers (>20% immunopositivity) determined the TFH origin. AITL patients tended to be male and showed increased presence of B-symptoms and hepatosplenomegaly. Histomorphology revealed that 92% of AITL cases had pattern 3 involvement. Sanger sequencing with conventional PCR did not yield any mutation, while RHOAG17V was detected by amplification-refractory mutation system-PCR in AITL (51%, P =0.027) and P-TFH (17%), which was validated with cloning followed by sequencing. Cases of RHOAG17V-mutant AITL had a worse Eastern Cooperative Oncology Group performance status initially but fared better in terms of overall outcome ( P =0.029). Although not specific for AITL, RHOAG17V mutation shows an association with diagnosis and requires sensitive methods for detection due to low-tumor burden. The mutant status of AITL could have prognostic implications and translational relevance.