Spontaneous rotations in epithelia as an interplay between cell polarity and RhoA activity at boundaries.

Directed flows of cells in vivo are essential in morphogenesis. They shape living matter in phenomena involving cell mechanics and regulations of the acto-myosin cytoskeleton. However the onset of coherent motion during collective cell migration is still poorly understood. Here we show that coherence is set by spontaneous alignments of cell polarity by designing cellular rings of controlled dimensions. A tug-of-war between opposite polarities dictates the onset of coherence, as assessed by tracking live cellular shapes and motions in various experimental conditions. In addition, we identify an internally driven constraint by cellular acto-myosin cables at boundaries as essential to ensure coherence and active force is generated as evaluated by the high RhoA activity. Its contribution is required to trigger coherence as shown by our numerical simulations based on a novel Vicsek-type model including free active boundaries. Altogether, spontaneous coherent motion results from basic interplay between cell orientations and active cables at boundaries.

Structure-Activity Relationship Analysis of Analogs of Rhosin, a RhoA Inhibitor, Reveals a New Generation of Improved Antiplatelet Agents

Platelet activation and aggregation play a key role in mediating hemostasis and thrombosis. The antiplatelet therapies currently available in the market are associated with a high risk of hemorrhage and are mostly irreversible in suppressing platelet activity; hence, there is a need to develop better therapeutic agents. Previous genetic and pharmacological studies have implicated the small GTPase RhoA in multiple platelet signaling pathways. We devised a lead RhoA activity-specific inhibitor, Rhosin/G04, based on the structure-function relationship of RhoA interaction with its activator, guanine nucleotide exchange factor (GEF) (Figure 1A). Rhosin/G04 binds to RhoA directly with micromolar affinity at a surface groove that is essential for GEF recognition and blocks GEF-mediated GTP loading to RhoA. Rhosin/G04 inhibits platelet spreading on fibrinogen and thrombin-induced platelet aggregation, mimicking effects of RhoA gene targeting. In the current work, we have utilized the inhibitory activity of G04 for platelet activation and its biochemical activity to define its structure-activity relationship (SAR) and to understand its mechanism of action in an effort to improve efficacy and druggability. The structure of G04 in a groove of RhoA interaction was hypothesized based on the docking studies using Molsoft ICM-Pro. Cincinnati Children's Hospital Medical Center's compound library of over 360,000 chemicals was scanned for G04 analogs by similarity and substructure searches. In the initial screen, a human platelet aggregation assay was performed at both a low concentration (1 µg/ml) and a high concentration (5 µg/ml) of collagen. The first round similarity search resulted in a set of 7 compounds (Set-1), from which, compound 177629 showed significantly enhanced potency relative to G04 (Figure 1B). The second round of similarity searches for compounds more closely related to 177629 (Set-2) identified 14 compounds. The third-round search for other related compounds (Set-3) led to 9 additional compounds that add to the understanding of the SAR. The compounds that showed enhanced antiplatelet activity were examined for their potency and selectivity in in vitro biochemical binding assays and in suppressing RhoA-GTP formation and downstream phosphorylation of myosin light chain (p-MLC) signaling in platelets. The active compounds were further examined for their anti-platelet activities under diverse stimuli including thrombin, ADP, U46619 (a stable thromboxane receptor agonist), and arachidonic acid. The most active compounds from Set-1, Set-2, and Set-3 inhibited platelet aggregation by at least 70% and showed IC 50 values below 6 µM. Of these compounds, 12 showed significantly greater potency than the initial compound, G04. The most active compounds were 177618, 177619, 177628, 177629, 177633, and 177634. These compounds specifically inhibited RhoA activity and blocked p-MLC. SAR analyses led us to believe that the quinoline is optimally attached to the hydrazine at the 4-position. The halogen (choloro- or trifluoromethyl-) substitution at the 7- or 8- position improved activity, and the 7- position may be slightly favored. The aryl group is considerably variable with similar potency between the indole, methylphenyl, and dichlorophenyl- groups. Rhosin/G04 is the R enantiomer (i.e. Rhosin is R-G04), so its S enantiomer, S-G04 was also evaluated (Figure 1C). S-G04 is significantly more potent than R-G04 in inhibiting collagen-stimulated RhoA-GTP formation and aggregation of platelets, and its effect is completely reversible by washing the platelets. Finally, R-G04 and S-G04 showed differential inhibition of arachidonic acid and U46619 stimulated primary and secondary aggregation, highlighting the potential utilities of the inhibitors in dissecting different platelet activation mechanisms. S-G04 is active in inhibiting thrombin, ADP, U46619, and arachidonic acid-mediated platelet activation at submicromolar concentration, suggesting a broad role of RhoA signaling in integrating platelet signal cross talk. In summary, evaluation of Rhosin/R-G04 analogs in a platelet activity screen identified a new generation of improved small-molecule RhoA inhibitors, including an enantiomer with significantly improved efficacy. These analog studies of novel anti-platelet agents provide a new approach to effectively and reversibly manipulate platelet activities. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway

Objective Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. Materials and methods HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. Results HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. Conclusions Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

N6-Isopentenyladenosine Hinders the Vasculogenic Mimicry in Human Glioblastoma Cells through Src-120 Catenin Pathway Modulation and RhoA Activity Inhibition

Background: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. Methods: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. Results: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. Conclusions: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Ursodeoxycholic acid suppresses the malignant progression of colorectal cancer through TGR5-YAP axis

The Hippo/YAP pathway plays an important role in the development of cancers. Previous studies have reported that bile acids can activate YAP (Yes Associated Protein) to promote tumorigenesis and tumor progression. Ursodeoxycholic acid (UDCA) is a long-established old drug used for cholestasis treatment. So far, the effect of UDCA on YAP signaling in colorectal cancer (CRC) is not well defined. This study means to explore relationship of UDCA and YAP in CRC. UDCA suppressed YAP signaling by activating the membrane G-protein-coupled bile acid receptor (TGR5). TGR5 mainly regulated cAMP/PKA signaling pathway to inhibit RhoA activity, thereby suppressing YAP signaling. Moreover, the restoration of YAP expression alleviated the inhibitory effect of UDCA on CRC cell proliferation. In AOM/DSS-induced CRC model, UDCA inhibited tumor growth in a concentration-dependent manner and decreased expression of YAP and Ki67. UDCA plays a distinguished role in regulating YAP signaling and CRC growth from the primary bile acids and partial secondary bile acids, demonstrating the importance of maintaining normal intestinal bile acid metabolism in cancer patients. It also presents a potential therapeutic intervention for CRC.

Acute RhoA/Rho Kinase Inhibition is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

High-throughput sequencing identified circular RNA circUBE2K mediating RhoA associated bladder cancer phenotype via regulation of miR-516b-5p/ARHGAP5 axis

Bladder cancer (BC) is known as a common and lethal urinary malignancy worldwide. Circular RNAs (circRNAs), an emerging non-coding RNA, participate in carcinogenesis process of several cancers including BC. In this study, high-throughput sequencing and RT-qPCR were applied to discover and validate abnormal high expression of circUBE2K in BC tissues. Fluorescence in situ hybridization (FISH) was used to detect hsa_circ_0009154 (circUBE2K) expression and subcellular localization in BC tissues. High circUBE2K predicted unfavorable prognoses in BCs, as well as correlated with clinical features. CCK8, transwell, EdU and wound healing assays demonstrated down-regulating circUBE2K decreased BC cell phenotype as proliferation, invasion, and migration, respectively. Further studies showed that circUBE2K promoted BC progression via sponging miR-516b-5p and enhancing ARHGAP5 expression through regulating RhoA activity. Dual-luciferase reporter, FISH and RNA pulldown assays were employed to verify the relationships among circUBE2K/miR-516b-5p/ARHGAP5/RhoA axis. Down-regulating miR-516b-5p or overexpressing ARHGAP5 restored RhoA activity mediated BC cell properties after silencing circUBE2K. Subcutaneous xenograft and metastasis model identified circUBE2K significantly increased BC cell metastasis and proliferation in-vivo. Taken together, we found that circUBE2K is a tumor-promoting circRNA in BC that functions as a ceRNA to regulate ARHGAP5 expression via sponging miR-516b-5p.

S-Nitrosylation of RhoGAP Myosin9A Is Altered in Advanced Diabetic Kidney Disease

The molecular pathogenesis of diabetic kidney disease progression is complex and remains unresolved. Rho-GAP MYO9A was recently identified as a novel podocyte protein and a candidate gene for monogenic FSGS. Myo9A involvement in diabetic kidney disease has been suggested. Here, we examined the effect of diabetic milieu on Myo9A expression in vivo and in vitro. We determined that Myo9A undergoes S-nitrosylation, a post-translational modification dependent on nitric oxide (NO) availability. Diabetic mice with nodular glomerulosclerosis and severe proteinuria associated with doxycycline-induced, podocyte-specific VEGF164 gain-of-function showed markedly decreased glomerular Myo9A expression and S-nitrosylation, as compared to uninduced diabetic mice. Immortalized mouse podocytes exposed to high glucose revealed decreased Myo9A expression, assessed by qPCR, immunoblot and immunocytochemistry, and reduced Myo9A S-nitrosylation (SNO-Myo9A), assessed by proximity link assay and biotin switch test, functionally resulting in abnormal podocyte migration. These defects were abrogated by exposure to a NO donor and were not due to hyperosmolarity. Our data demonstrate that high-glucose induced decrease of both Myo9A expression and SNO-Myo9A is regulated by NO availability. We detected S-nitrosylation of Myo9A interacting proteins RhoA and actin, which was also altered by high glucose and NO dependent. RhoA activity inversely related to SNO-RhoA. Collectively, data suggest that dysregulation of SNO-Myo9A, SNO-RhoA and SNO-actin may contribute to the pathogenesis of advanced diabetic kidney disease and may be amenable to therapeutic targeting.