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Abstract The intricate involvement of Rho GTPases in a multitude of human malignancies and their diverse array of biological functions has garnered substantial attention within the scientific community. However, their expression pattern and potential role in gastric cancer (GC) remain unclear. In this study, we successfully identified two distinct subtypes associated with Rho GTPase-related gene (RGG) through consensus clustering analysis, which exhibited significant disparities in overall survival and the tumor microenvironment. Subsequently, an extensively validated risk model termed RGGscore was meticulously constructed to prognosticate the outcomes of GC patients. This model was further assessed and validated using an external cohort. Notably, the high RGGscore group was indicative of a poorer prognosis. Univariate and multivariate Cox regression analyses unveiled the RGGscore as an autonomous prognostic indicator for GC patients. Subsequent external validation, utilizing two cohorts of patients who underwent immunotherapy, demonstrated a significant correlation between a low RGGscore and improved response to immunotherapy. Additionally, the expression levels of three genes associated with RGGscore were examined using qRT-PCR. Taken together, a pioneering RGGscore model has been successfully established, showcasing its potential efficacy in offering valuable therapeutic guidance for GC.

Vascular permeability is mediated by Cortactin (Cttn) and regulated by several molecules including cyclic-adenosine-monophosphate, small Rho family GTPases and the actin cytoskeleton. However, it is unclear whether Cttn directly interacts with any of the junctional components or if Cttn intervenes with signaling pathways affecting the intercellular contacts and the cytoskeleton. To address these questions, we employed immortalized microvascular myocardial endothelial cells derived from wild-type and Cttn-knock-out mice. We found that lack of Cttn compromised barrier integrity due to fragmented membrane distribution of different junctional proteins. Moreover, immunoprecipitations revealed that Cttn is within the VE-cadherin-based adherens junction complex. In addition, lack of Cttn slowed-down barrier recovery after Ca2+ repletion. The role of Cttn for cAMP-mediated endothelial barrier regulation was analyzed using Forskolin/Rolipram. In contrast to Cttn-KO, WT cells reacted with increased transendothelial electrical resistance. Absence of Cttn disturbed Rap1 and Rac1 activation in Cttn-depleted cells. Surprisingly, despite the absence of Cttn, direct activation of Rac1/Cdc42/RhoA by CN04 increased barrier resistance and induced well-defined cortical actin and intracellular actin bundles. In summary, our data show that Cttn is required for basal barrier integrity by allowing proper membrane distribution of junctional proteins and for cAMP–mediated activation of the Rap1/Rac1 signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

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.

Regulation of Rho GTPases remains a topic of active investigation as they are essential participants in cell biology and the pathophysiology of many human diseases. Non-degrading ubiquitination (NDU) is a critical regulator of the Ras superfamily, but its relevance to Rho proteins remains unknown. We show that RhoC, but not RhoA, is a target of NDU by E3 ubiquitin ligase, LNX1. Furthermore, LNX1 ubiquitination of RhoC is negatively regulated by LIS1 (aka, PAFAH1B1). Despite multiple reports of functional interaction between LIS1 and activity of Rho proteins, a robust mechanism linking the two has been lacking. Here, LIS1 inhibition of LNX1 effects on RhoGDI-RhoC interaction provides a molecular mechanism underpinning the enhanced activity of Rho proteins observed upon reduction in LIS1 protein levels. Since LNX1 and RhoC are only found in vertebrates, the LIS1-LNX1-RhoC module represents an evolutionarily acquired function of the highly conserved LIS1. While these nearly identical proteins have several distinct RhoA and RhoC downstream effectors, our data provide a rare example of Rho-isoform specific, upstream regulation that opens new therapeutic opportunities. [ABSTRACT FROM AUTHOR]

Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1flox/flox; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1flox/flox; Rac3+/−; Cre mice showed intermediate phenotype between Rac1flox/flox; Cre and Rac1flox/flox; Rac3−/−; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1+/flox; Rac3−/−; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons. [ABSTRACT FROM AUTHOR]

Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury. [ABSTRACT FROM AUTHOR]

Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular structures such as lamellipodia and contractile actomyosin rings. Wiskott-Aldrich Syndrome (WAS) family proteins are downstream effectors of Rho family GTPases that usually function in a one-to-one correspondence to regulate branched actin nucleation. In particular, the WAS protein Scar/WAVE has been shown to exhibit one-to-one correspondence with Rac GTPase. Here we show that Rac and SCAR are recruited to cell wounds in the Drosophila repair model and are required for the proper formation and maintenance of the dynamic actomyosin ring formed at the wound periphery. Interestingly, we find that SCAR is recruited to wounds earlier than Rac and is still recruited to the wound periphery in the presence of a potent Rac inhibitor. We also show that while Rac is important for actin recruitment to the actomyosin ring, SCAR serves to organize the actomyosin ring and facilitate its anchoring to the overlying plasma membrane. These differing spatiotemporal recruitment patterns and wound repair phenotypes highlight the Rac-independent functions of SCAR and provide an exciting new context in which to investigate these newly uncovered SCAR functions. [ABSTRACT FROM AUTHOR]

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

Biological therapies have dramatically improved the therapeutic landscape of psoriatic arthritis (PsA); however, 40–50% of patients are primary non-responders with response rates declining significantly with each successive biological therapy. Therefore, there is a pressing need to develop a coherent strategy for effective initial and subsequent selection of biologic agents. We interrogated 40 PsA patients initiating either tumour necrosis factor inhibitors (TNFi) or interleukin-17A inhibitors (17Ai) for active PsA. Patients achieving low disease activity according to the Disease Activity Index for PsA (DAPSA) at 3 months were classified as responders. Baseline and 3-month CD4+ transcript profiling were performed, and novel signaling pathways were identified using a multi-omics profiling and integrative computational analysis approach. Using transcriptomic data at initiation of therapy, we identified over 100 differentially expressed genes (DEGs) that differentiated IL-17Ai response from non-response and TNFi response from non-response. Integration of cell-type-specific DEGs with protein–protein interactions and further comprehensive pathway enrichment analysis revealed several pathways. Rho GTPase signaling pathway exhibited a strong signal specific to IL-17Ai response and the genes, RAC1 and ROCKs, are supported by results from prior research. Our detailed network and pathway analyses have identified the rewiring of Rho GTPase pathways as potential markers of response to IL17Ai but not TNFi. These results need further verification. [ABSTRACT FROM AUTHOR]

Heterogeneity of cell phenotypes remains a barrier in progressing cell research and a challenge in conquering cancer-related drug resistance. Cell morphology, the most direct property of cell phenotype, evolves along the progression of the cell cycle; meanwhile, cell motility, the dynamic property of cell phenotype, also alters over the cell cycle. However, a quantifiable research understanding the relationship between the cell cycle and cell migration is missing. Herein, we coordinate the migratory behaviours of NIH 3T3 fibroblasts to their corresponding phases of the cell cycle, the G1, the S, and the G2 phases, and explain the relationship through the spatiotemporal arrangements between the Rho GTPases' signals and cyclin-dependent kinase inhibitors, p21Cip1, and p27Kip1. Taken together, we demonstrate that both cell morphology and the dynamic subcellular behaviour are homogenous within each stage of the cell cycle phases but heterogenous between phases through quantitative cell analyses and an interactive molecular mechanism between the cell cycle and cell migration, posing potential implications in countering drug resistance. [ABSTRACT FROM AUTHOR]

Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20–30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/β-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/β-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC. [ABSTRACT FROM AUTHOR]

GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient’s iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy. [ABSTRACT FROM AUTHOR]

The Rho GTPase family consists of 20 genes encoding intracellular signalling proteins that influence cytoskeletal dynamics, cell migration and cell cycle progression. They are implicated in breast cancer progression but their role in breast cancer aetiology is unknown. As aberrant Rho GTPase activity could be associated with breast cancer, we aimed to determine the potential for a causal role of Rho GTPase gene expression in breast cancer risk, using two-sample Mendelian randomization (MR). MR was undertaken in 122,977 breast cancer cases and 105,974 controls, including 69,501 estrogen receptor positive (ER+) cases and 105,974 controls, and 21,468 ER negative (ER−) cases and 105,974 controls. Single nucleotide polymorphisms (SNPs) underlying expression quantitative trait loci (eQTLs) obtained from normal breast tissue, breast cancer tissue and blood were used as genetic instruments for Rho GTPase expression. As a sensitivity analysis, we undertook co-localisation to examine whether findings reflected shared causal variants or genomic confounding. We identified genetic instruments for 14 of the 20 human Rho GTPases. Using eQTLs obtained from normal breast tissue and normal blood, we identified evidence of a causal role of RHOD in overall and ER+ breast cancers (overall breast cancer: odds ratio (OR) per standard deviation (SD) increase in expression level 1.06; (95% confidence interval (CI) 1.03, 1.09; P = 5.65 × 10–5) and OR 1.22 (95% CI 1.11, 1.35; P = 5.22 × 10–5) in normal breast tissue and blood respectively). There was a consistent direction of association for ER− breast cancer, although the effect-estimate was imprecisely estimated. Using eQTLs from breast cancer tissue and normal blood there was some evidence that CDC42 was negatively associated with overall and ER + breast cancer risk. The evidence from colocalization analyses strongly supported our MR results particularly for RHOD. Our study suggests a potential causal role of increased RHOD gene expression, and, although the evidence is weaker, a potential protective role for CDC42 gene expression, in overall and ER+ breast cancers. These finding warrant validation in independent samples and further biological investigation to assess whether they may be suitable targets for drug targeting. [ABSTRACT FROM AUTHOR]

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. The disease has an acute and a chronic phase in which approximately 30% of the chronic patients suffer from heart disease and/or gastrointestinal symptoms. The pathogenesis of the disease is multifactorial and involves the virulence of the strains, immunological factors and extracellular vesicles (EV) shed by the parasite which participate in cell–cell communication and evasion of the immune response. In this work, we present a transcriptomic analysis of cells stimulated with EV of the trypomastigote stage of T. cruzi. Results after EV-cell incubation revealed 322 differentially expressed genes (168 were upregulated and 154 were downregulated). In this regard, the overexpression of genes related to ubiquitin-related processes (Ube2C, SUMO1 and SUMO2) is highlighted. Moreover, the expression of Rho-GTPases (RhoA, Rac1 and Cdc42) after the interaction was analyzed, revealing a downregulation of the analyzed genes after 4 h of interaction. Finally, a protective role of EV over apoptosis is suggested, as relative values of cells in early and late apoptosis were significantly lower in EV-treated cells, which also showed increased CSNK1G1 expression. These results contribute to a better understanding of the EV-cell interaction and support the role of EV as virulence factors. [ABSTRACT FROM AUTHOR]

Rho GTPases are important regulators of many cellular functions like cell migration, adhesion and polarity. The molecular switches are often dysregulated in cancer. We detected Rho-dependent upregulation of the orphan seven-transmembrane receptor G-protein-coupled receptor family C group 5 member A (GPRC5A). GPRC5A is highly expressed in breast cancer whereas in lung cancer, it is often downregulated. Here, we analyzed the function of GPRC5A in breast epithelial and breast cancer cells. Activation or expression of RhoA/C led to GPRC5A-dependent inhibition of proliferation and reduction of the colony forming capacity of benign breast epithelial cells. This effect is based on an inhibition of EGFR signalling. Knockout of retinoic acid induced 3 (RAI3, the gene for GPRC5A) in breast cancer cells increased cell division, whereas Rho activation had no effect on proliferation. Knockout of RAI3 in benign breast epithelial cells led to decrease of EGFR expression and diminished proliferation. [ABSTRACT FROM AUTHOR]

Mechanotransduction has been divided into mechanotransmission, mechanosensing, and mechanoresponse, although how a cell performs all three functions using the same set of structural components is still highly debated. Here, we bridge the gap between emerging molecular and systems-level understandings of mechanotransduction through a multiscale model linking these three phases. Our model incorporates a discrete network of actin filaments and associated proteins that responds to stretching through geometric relaxation. We assess three potential activating mechanisms at mechanosensitive crosslinks as inputs to a mixture model of molecular release and benchmark each using experimental data of mechanically-induced Rho GTPase FilGAP release from actin-filamin crosslinks. Our results suggest that filamin-FilGAP mechanotransduction response is best explained by a bandpass mechanism favoring release when crosslinking angles fall outside of a specific range. Our model further investigates the difference between ordered versus disordered networks and finds that a more disordered actin network may allow a cell to more finely tune control of molecular release enabling a more robust response. [ABSTRACT FROM AUTHOR]

Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1. [ABSTRACT FROM AUTHOR]

Abstract Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20–30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac’s impact on metastasis by influencing Rac-1/HIF-1α/DDX3/β-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/β-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.

Raf-1 kinase inhibitor protein was initially discovered as a physiological kinase inhibitor of the MAPK signaling pathway and was later shown to suppress cancer cell invasion and metastasis. Yet, the molecular mechanism through which RKIP executes its effects is not completely defined. RhoA has both a pro- and anti-metastatic cell-context dependent functions. Given that Rho GTPases primarily function on actin cytoskeleton dynamics and cell movement regulation, it is possible that one way RKIP hinders cancer cell invasion/metastasis is by targeting these proteins. Here we show that RKIP inhibits cancer cell invasion and metastasis by stimulating RhoA anti-tumorigenic functions. Mechanistically, RKIP activates RhoA in an Erk2 and GEF-H1 dependent manner to enhance E-cadherin membrane localization and inhibit CCL5 expression. [ABSTRACT FROM AUTHOR]

Actin-dependent mechanisms drive the nuclear translocation of Yap1 to enable its co-activation of transcription factors that induce pro-growth and survival programs. While Rho GTPases are necessary for the nuclear import of YAP1, the relevant Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) that connect this process to upstream signaling are not well defined. To this end, we measured the impact of expressing sixty-seven RhoGEFs and RhoGAPs on the YAP1 dependent activity of a TEAD element transcriptional reporter. Robust effects by all three members of the regulator of G-protein signaling (RGS) domain containing RhoGEFs (ArhGEF1, ArhGEF11 and ArhGEF12) prompted studies relating their known roles in serum signaling onto the regulation of Yap1. Under all conditions examined, ArhGEF12 preferentially mediated the activation of YAP1/TEAD by serum versus ArhGEF1 or ArhGEF11. Conversely, ArhGEF1 in multiple contexts inhibited both basal and serum elevated YAP1 activity through its GAP activity for Gα13. The sensitivity of such inhibition to cellular density and to low states of serum signaling supports that ArhGEF1 is a context dependent regulator of YAP1. Taken together, the relative activities of the RGS-RhoGEFs were found to dictate the degree to which serum signaling promotes YAP1 activity. [ABSTRACT FROM AUTHOR]

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of 'sub-states' such as state 1 &state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound 'OFF' state have very similar conformations, whereas the GTP bound 'ON' state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors. [ABSTRACT FROM AUTHOR]

Abstract Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1 flox/flox ; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1 flox/flox ; Rac3 +/−; Cre mice showed intermediate phenotype between Rac1 flox/flox ; Cre and Rac1 flox/flox ; Rac3 −/−; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1 +/flox ; Rac3 −/−; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons.

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder whose major hallmark is the deposition of mutated transthyretin (TTR) in the form of amyloid fibrils in the peripheral nervous system (PNS). The exposure of PNS axons to extracellular TTR deposits leads to an axonopathy that culminates in neuronal death. However, the molecular mechanisms underlying TTR-induced neurodegeneration are still unclear, despite the extensive studies in vertebrate models. In this work we used a Drosophila FAP model, based on the expression of the amyloidogenic TTR (V30M) in the fly retina, to uncover genetic interactions with cytoskeleton regulators. We show that TTR interacts with actin regulators and induces cytoskeleton alterations, leading to axonal defects. Moreover, our study pinpoints an interaction between TTRV30M and members of Rho GTPase signaling pathways, the major actin regulators. Based on these findings we propose that actin cytoskeleton alterations may mediate the axonopathy observed in FAP patients, and highlight a molecular pathway, mediated by Rho GTPases, underlying TTR-induced neurodegeneration. We expect this work to prompt novel studies and approaches towards FAP therapy. [ABSTRACT FROM AUTHOR]

Vav1 regulates Rac activation as a hematopoietic-specific Rho/Rac-family guanine nucleotide exchange factor. Rac is a subfamily of Rho GTPases that regulates the bone-resorbing capacity of osteoclasts (OCs). In this study, we show that hematopoietic-specific Rac2 and Vav1 play opposing roles by enhancing or attenuating OC differentiation, respectively. This was demonstrated by higher and lower bone density in the femurs from Rac2-deficient (Rac2−/−) and Vav1-deficient (Vav1−/−) mice, respectively, compared to the wild-type (WT) mice. Accordingly, Rac2−/− cells displayed low numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (41%) compared to WT cells, whereas, Vav1−/− cells showed high TRAP-positive cell numbers (150%), and the double-knockout Rac2−/−Vav1−/− mice nullified the effects on OC numbers achieved by the individual knockouts. These reciprocal roles of Rac2 and Vav1 in OC differentiation were confirmed by reduced and increased levels of OC-specific markers, such as TRAP, calcitonin receptor, cathepsin K, and DC-STAMP in the Rac2−/− and Vav1−/− OCs, respectively. Our findings of decrease and increase in actin ring formation and αvβ3 integrin-mediated adhesion in Rac2−/− and Vav1−/− mice, respectively, suggest that Vav1 and its downstream GTPase, Rac2, may counteract to fine-tune OC differentiation and bone resorption. [ABSTRACT FROM AUTHOR]

The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk. [ABSTRACT FROM AUTHOR]

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology. [ABSTRACT FROM AUTHOR]

AMPylation is a biologically significant yet understudied post-translational modification where an adenosine monophosphate (AMP) group is added to Tyrosine and Threonine residues primarily. While recent work has illuminated the prevalence and functional impacts of AMPylation, experimental identification of AMPylation sites remains challenging. Computational prediction techniques provide a faster alternative approach. The predictive performance of machine learning models is highly dependent on the features used to represent the raw amino acid sequences. In this work, we introduce a novel feature extraction pipeline to encode the key properties relevant to AMPylation site prediction. We utilize a recently published dataset of curated AMPylation sites to develop our feature generation framework. We demonstrate the utility of our extracted features by training various machine learning classifiers, on various numerical representations of the raw sequences extracted with the help of our framework. Tenfold cross-validation is used to evaluate the model's capability to distinguish between AMPylated and non-AMPylated sites. The top-performing set of features extracted achieved MCC score of 0.58, Accuracy of 0.8, AUC-ROC of 0.85 and F1 score of 0.73. Further, we elucidate the behaviour of the model on the set of features consisting of monogram and bigram counts for various representations using SHapley Additive exPlanations. [ABSTRACT FROM AUTHOR]

ARHGAP25, a crucial molecule in immunological processes, serves as a Rac-specific GTPase-activating protein. Its role in cell migration and phagocyte functions, affecting the outcome of complex immunological diseases such as rheumatoid arthritis, renders it a promising target for drug research. Despite its importance, our knowledge of its intracellular interactions is still limited. This study employed proteomic analysis of glutathione S-transferase (GST)-tag pulldowns and co-immunoprecipitation from neutrophilic granulocyte cell lysate, revealing 76 candidates for potential physical interactions that complement ARHGAP25's known profile. Notably, four small GTPases (RAC2, RHOG, ARF4, and RAB27A) exhibited high affinity for ARHGAP25. The ARHGAP25–RAC2 and ARHGAP25–RHOG interactions appeared to be affected by the activation state of the small GTPases, suggesting a GTP–GDP cycle-dependent interaction. In silico dimer prediction pinpointed ARHGAP25's GAP domain as a credible binding interface, suggesting its suitability for GTP hydrolysis. Additionally, a list of Fc receptor-related kinases, phosphatases, and three of the 14-3-3 members were identified as potential partners, with in silico predictions highlighting eight binding sites, presenting novel insight on a potential regulatory mechanism for ARHGAP25. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competitive kinase inhibitors with known target profiles from two Published Kinase Inhibitor Sets (PKIS1 and PKIS2) using intracellular infection models for Stm and Mtb, based on human cell lines and primary macrophages. Additionally, the in vivo safety and efficacy of the compounds were assessed using zebrafish embryo infection models. Our screen identified 11 hit compounds for Stm and 17 hit compounds for Mtb that were effective against intracellular bacteria and non-toxic for host cells. Further experiments were conducted to prioritize Stm hit compounds that were able to clear the intracellular infection in primary human macrophages. From these, two structurally related Stm hit compounds, GSK1379738A and GSK1379760A, exhibited significant activity against Stm in infected zebrafish embryos. In addition, we identified compounds that were active against intracellular Mtb, including morpholino-imidazo/triazolo-pyrimidinones that target PIK3CB, as well as 2-aminobenzimidazoles targeting ABL1. Overall, this study provided insights into kinase targets acting at the host–pathogen interface and identified several kinase inhibitors as potential HDTs. [ABSTRACT FROM AUTHOR]

In a prospective cohort of subjects who subsequently developed preeclampsia (PE, n = 14) versus remaining healthy (NORM, n = 12), early gestation circulating extracellular vesicles (EVs) containing a panel of microRNA signatures were characterized and their biological networks of targets deciphered. Multiple microRNAs of which some arose from the placenta (19MC and 14MC) demonstrated changes in association with advancing gestation, while others expressed were pathognomonic of the subsequent development of characteristic clinical features of PE which set in as a late-onset subtype. This panel of miRNAs demonstrated a predictability with an area under the curve of 0.96 using leave-one-out cross-validation training in a logistic regression model with elastic-net regularization and precautions against overfitting. In addition, this panel of miRNAs, some of which were previously detected in either placental tissue or as maternal cell-free non-coding transcripts, lent further validation to our EV studies and the observed association with PE. Further, the identified biological networks of targets of these detected miRNAs revealed biological functions related to vascular remodeling, cellular proliferation, growth, VEGF, EGF and the PIP3/Akt signaling pathways, all mediating key cellular functions. We conclude that we have demonstrated a proof-of-principle by detecting a panel of EV packaged miRNAs in the maternal circulation early in gestation with possibilities of biological function in the placenta and other maternal tissues, along with the probability of predicting the subsequent clinical appearance of PE, particularly the late-onset subtype. [ABSTRACT FROM AUTHOR]

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration—an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial–mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior. [ABSTRACT FROM AUTHOR]

Lichens are symbiotic organisms that produce various secondary metabolites. Here, different lichen extracts were examined to identify secondary metabolites with anti-migratory activity against human lung cancer cells. Everniastrum vexans had the most potent inhibitory activity, and atranorin was identified as an active subcomponent of this extract. Atranorin suppressed β-catenin-mediated TOPFLASH activity by inhibiting the nuclear import of β-catenin and downregulating β-catenin/LEF and c-jun/AP-1 downstream target genes such as CD44, cyclin-D1 and c-myc. Atranorin decreased KAI1 C-terminal interacting tetraspanin (KITENIN)-mediated AP-1 activity and the activity of the KITENIN 3′-untranslated region. The nuclear distribution of the AP-1 transcriptional factor, including c-jun and c-fos, was suppressed in atranorin-treated cells, and atranorin inhibited the activity of Rho GTPases including Rac1, Cdc42, and RhoA, whereas it had no effect on epithelial-mesenchymal transition markers. STAT-luciferase activity and nuclear STAT levels were decreased, whereas total STAT levels were moderately reduced. The human cell motility and lung cancer RT² Profiler PCR Arrays identified additional atranorin target genes. Atranorin significantly inhibited tumorigenesis in vitro and in vivo. Taken together, our results indicated that E. vexans and its subcomponent atranorin may inhibit lung cancer cell motility and tumorigenesis by affecting AP-1, Wnt, and STAT signaling and suppressing RhoGTPase activity. [ABSTRACT FROM AUTHOR]

Diabetic corneal neuropathy (DCN) is a common diabetic ocular complication with limited treatment options. In this study, we investigated the effects of topical and oral fenofibrate, a peroxisome proliferator-activated receptor-α agonist, on the amelioration of DCN using diabetic mice (n = 120). Ocular surface assessments, corneal nerve and cell imaging analysis, tear proteomics and its associated biological pathways, immuno-histochemistry and western blot on PPARα expression, were studied before and 12 weeks after treatment. At 12 weeks, PPARα expression markedly restored after topical and oral fenofibrate. Topical fenofibrate significantly improved corneal nerve fibre density (CNFD) and tortuosity coefficient. Likewise, oral fenofibrate significantly improved CNFD. Both topical and oral forms significantly improved corneal sensitivity. Additionally, topical and oral fenofibrate significantly alleviated diabetic keratopathy, with fenofibrate eye drops demonstrating earlier therapeutic effects. Both topical and oral fenofibrate significantly increased corneal β-III tubulin expression. Topical fenofibrate reduced neuroinflammation by significantly increasing the levels of nerve growth factor and substance P. It also significantly increased β-III-tubulin and reduced CDC42 mRNA expression in trigeminal ganglions. Proteomic analysis showed that neurotrophin signalling and anti-inflammation reactions were significantly up-regulated after fenofibrate treatment, whether applied topically or orally. This study concluded that both topical and oral fenofibrate ameliorate DCN, while topical fenofibrate significantly reduces neuroinflammation. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) have essential roles in cardiac tissue repair after myocardial infarction (MI). To establish stage-specific and long-term effects of the ischemic injury on cardiac ECs, we analyzed their transcriptome at landmark time points after MI in mice. We found that early EC response at Day 2 post-MI centered on metabolic changes, acquisition of proinflammatory phenotypes, initiation of the S phase of cell cycle, and activation of stress-response pathways, followed by progression to mitosis (M/G2 phase) and acquisition of proangiogenic and mesenchymal properties during scar formation at Day 7. In contrast, genes involved in vascular physiology and maintenance of vascular tone were suppressed. Importantly, ECs did not return to pre-injury phenotypes after repair has been completed but maintained inflammatory, fibrotic and thrombotic characteristics and lost circadian rhythmicity. We discovered that the highest induced transcript is the mammalian-specific Sh2d5 gene that promoted migration and invasion of ECs through Rac1 GTPase. Our results revealed a synchronized, temporal activation of disease phenotypes, metabolic pathways, and proliferation in quiescent ECs after MI, indicating that precisely-timed interventions are necessary to optimize cardiac tissue repair and improve outcomes. Furthermore, long-term effects of acute ischemic injury on ECs may contribute to vascular dysfunction and development of heart failure. [ABSTRACT FROM AUTHOR]

ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1's physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging. [ABSTRACT FROM AUTHOR]

Intervertebral Disc (IVD) degeneration has been associated with a chronic inflammatory response, but knowledge on the contribution of distinct IVD cells, namely CD44, to the progression of IVD degeneration remains elusive. Here, bovine nucleus pulposus (NP) CD44 cells were sorted and compared by gene expression and proteomics with the negative counterpart. NP cells were then stimulated with IL-1b (10 ng/ml) and dynamics of CD44 gene and protein expression was analyzed upon pro-inflammatory treatment. The results emphasize that CD44 has a multidimensional functional role in IVD metabolism, ECM synthesis and production of neuropermissive factors. CD44 widespread expression in NP was partially associated with CD14 and CD45, resulting in the identification of distinct cell subsets. In conclusion, this study points out CD44 and CD44-based cell subsets as relevant targets in the modulation of the IVD pro-inflammatory/degenerative cascade. [ABSTRACT FROM AUTHOR]

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts. [ABSTRACT FROM AUTHOR]

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents. [ABSTRACT FROM AUTHOR]

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules. [ABSTRACT FROM AUTHOR]

Fibromyalgia (FM) is a chronic pain syndrome characterized by widespread pain. The pathophysiology of fibromyalgia is not clearly understood and there are no specific biomarkers available for accurate diagnosis. Here we define genomic signatures using high throughput RNA sequencing on 96 fibromyalgia and 93 control cases. Our findings revealed three major fibromyalgia-associated expression signatures. The first group included 43 patients with a signature enriched for gene expression associated with extracellular matrix and downregulation of RhoGDI signaling pathway. The second group included 30 patients and showed a profound reduction in the expression of inflammatory mediators with an increased expression of genes involved in the CLEAR signaling pathway. These results suggest defective tissue homeostasis associated with the extra-cellular matrix and cellular program that regulates lysosomal biogenesis and participates in macromolecule clearance in fibromyalgia. The third group of 17 FM patients showed overexpression of pathways that control acute inflammation and dysfunction of the global transcriptional process. The result of this study indicates that FM is a heterogeneous and complex disease. Further elucidation of these pathways will lead to the development of accurate diagnostic markers, and effective therapeutic options for fibromyalgia. [ABSTRACT FROM AUTHOR]

KPNA1 is a mediator of nucleocytoplasmic transport that is abundantly expressed in the mammalian brain and regulates neuronal differentiation and synaptic function. De novo mutations in Kpna1 have been identified using genome-wide association studies in humans with schizophrenia; however, it remains unclear how KPNA1 contributes to schizophrenia pathogenesis. Recent studies have suggested a complex combination of genetic and environmental factors that are closely related to psychiatric disorders. Here, we found that subchronic administration of phencyclidine, a psychotropic drug, induced vulnerability and behavioral abnormalities consistent with the symptoms of schizophrenia in Kpna1-deficient mice. Microarray assessment revealed that the expression levels of dopamine d1/d2 receptors, an RNA editing enzyme, and a cytoplasmic dynein component were significantly altered in the nucleus accumbens brain region in a gene-environment (G × E) interaction-dependent manner. Our findings demonstrate that Kpna1-deficient mice may be useful as a G × E interaction mouse model for psychiatric disorders and for further investigation into the pathogenesis of such diseases and disorders. [ABSTRACT FROM AUTHOR]

Podocytes form the kidney filtration barrier and continuously adjust to external stimuli to preserve their integrity even in the presence of inflammation. It was suggested that canonical toll-like receptor signaling, mediated by the adaptor protein MYD88, plays a crucial role in initiating inflammatory responses in glomerulonephritis (GN). We explored the influence of podocyte-intrinsic MYD88 by challenging wild-type (WT) and podocyte-specific Myd88 knockout (MyD88pko) mice, with a model of experimental GN (nephrotoxic nephritis, NTN). Next-generation sequencing revealed a robust upregulation of inflammatory pathways and changes in cytoskeletal and cell adhesion proteins in sorted podocytes from WT mice during disease. Unchallenged MyD88pko mice were healthy and showed no proteinuria, normal kidney function and lacked morphological changes. During NTN, MyD88pko exhibited a transient increase in proteinuria in comparison to littermates, while histological damage, podocyte ultrastructure in STED imaging and frequencies of infiltrating immune cells by flow cytometry were unchanged. MYD88-deficiency led to subtle changes in the podocyte transcriptome, without a significant impact on the overall podocyte response to inflammation, presumably through MYD88-independent signaling pathways. In conclusion, our study reveals a comprehensive analysis of podocyte adaptation to an inflammatory environment on the transcriptome level, while MYD88-deficiency had only limited impact on the course of GN suggesting additional signaling through MYD88-independent signaling. [ABSTRACT FROM AUTHOR]

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention. [ABSTRACT FROM AUTHOR]