Search Results (49)

Susac Syndrome (SuS) is a rare autoimmune neurovascular disorder characterized by sudden visual loss, hearing disturbances, and encephalopathy. Pathology affects the small vessels of the brain, retina, and inner ear. Diagnosing SuS is challenging due to its rarity, complexity, and nonspecific symptoms. This single-case study presents a proteomic analysis of tear fluid from a patient with SuS, revealing upregulated proteins involved in immune dysregulation, cytoskeletal remodeling, and cellular repair. The activation of inflammatory proteins (e.g., S100), cytoskeletal and motility-related proteins (e.g., ezrin, radixin), and membrane transport proteins (e.g., aquaporin-5, chloride intracellular channel protein), together with activation of MAPK and NF-κB signaling pathways, highlights immune dysregulation and neurovascular damage in SuS. Hyperactivation of MAPK and NF-κB pathways leads to chronic neuroinflammation and decreased expression of neutrophil defensin 1, indicating a shift from a protective to a chronic inflammatory response. These findings from the personalized proteomic pattern of SuS support the potential of tear fluid proteomics for diagnosing SuS and offer valuable insights into its underlying molecular mechanisms. Full article

Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer’s disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired. Full article

Background: Over the last years, the phenomenon of entosis, a form of cell-in-cell structure, has been highlighted in various tumors, including poorly treatable breast or pancreatic cancers. Nevertheless, not only the biological properties, but also the molecular drivers of entosis remain unclear. Here, we evaluated SRC tyrosine kinase, a key proto-oncogene, and podoplanin (PDPN), a membrane glycoprotein, as potential regulators of entotic cell formation. Methods: In the study, two entosis-competent cell lines, BxPC-3 and MFC-7, originating from pancreatic and breast cancers, respectively, were used. SRC or PDPN genes were silenced using dedicated siRNA and the frequency of entotic structure formation was assessed using fluorescent staining and confocal imaging. Results: It was found that BxPC-3 cells deficient in PDPN are more prone to form entotic structures and that over 90% of all entotic figures formed by mixed PDPN⁺ and PDPN^- BxPC-3 cells involved PDPN-silenced cells. The SRC data supports this observation, as the suppressed entotic formation ability presented by SRC-deficient cells was linked with increased expression of PDPN. Even though the observed effects were mainly limited to BxPC-3 cells, as PDPN expression in MCF-7 cells is restricted, overall, the obtained data suggest a strong anti-entotic function of PDPN. Additionally, the performed Western blotting indicated the activation of ezrin-radixin-moesin (ERM) proteins in PDPN-deficient cells. Conclusions: Taken together, these data suggest that the negatively controlled PDPN-ERM axis may act as a molecular factor controlling the development of entotic structures and cells with naturally low PDPN expression may be more liable to form entoses. Full article

Organic anion transporting polypeptide 1B1 (OATP1B1) is selectively expressed at the basolateral membrane of human hepatocytes and plays a crucial role in the absorption of various xenobiotic compounds, including many important clinical drugs. Oligomerization with regulatory proteins is a common mechanism for regulating membrane protein functions. In the present study, we found that knocking down the scaffold protein radixin, which is the major member of the ERM family expressed in the liver, significantly enhanced the uptake function of OATP1B1. On the other hand, the overexpression of the phospho-mimic form of radixin (radixin-D) reduced the uptake function and cell surface level of OATP1B1, while the wild-type and phospho-dormant form of radixin (radixin-A) did not exhibit the same effect. Further investigation revealed that radixin interacts with OATP1B1. Activation of protein kinase C (PKC), which our previous study showed accelerates the internalization of OATP1B1, was found to increase the phosphorylation level of radixin associated with OATP1B1. The knockdown of radixin significantly diminished the suppressive effect of PKC on the function and cell surface levels of OATP1B1. These results suggested that OATP1B1 forms complexes with radixin, which may be phosphorylated by PKC, leading to reduced cell surface expression and activity of the transporter. Full article

Background: Radixin is an ERM family protein that includes radixin, moesin, and ezrin. The importance of ERM family proteins has been attracting more attention, and studies on the roles of ERM in biological function and the pathogenesis of some diseases are accumulating. In particular, we have found that radixin is the most dramatically changed ERM protein in elevated glucose-treated Schwann cells. Method: We systemically review the literature on ERM, radixin in focus, and update the roles of radixin in regulating cell morphology, interaction, and cell signaling pathways. The potential of radixin as a therapeutic target in neurodegenerative diseases and cancer was also discussed. Results: Radixin research has focused on its cell functions, activation, and pathogenic roles in some diseases. Radixin and other ERM proteins maintain cell shape, growth, and motility. In the nervous system, radixin has been shown to prevent neurodegeneration and axonal growth. The activation of radixin is through phosphorylation of its conserved threonine residues. Radixin functions in cell signaling pathways by binding to membrane proteins and relaying the cell signals into the cells. Deficiency of radixin has been involved in the pathogenic process of diseases in the central nervous system and diabetic peripheral nerve injury. Moreover, radixin also plays a role in cell growth and drug resistance in multiple cancers. The trials of therapeutic potential through radixin modulation have been accumulating. However, the exact mechanisms underlying the roles of radixin are far from clarification. Conclusions: Radixin plays various roles in cells and is involved in developing neurodegenerative diseases and many types of cancers. Therefore, radixin may be considered a potential target for developing therapeutic strategies for its related diseases. Further elucidation of the function and the cell signaling pathways that are linked to radixin may open the avenue to finding novel therapeutic strategies for diseases in the nervous system and other body systems. Full article

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico—an ESE-15-one and an ESE-16 one—with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class. Full article

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners. Full article

Ezin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that interacts with several partner molecules including β-catenin. Here, we examined the crosstalk between EBP50 and nuclear catenin during colorectal carcinoma (CRC) progression. In clinical samples, there were no correlations between the subcellular location of EBP50 and any clinicopathological factors. However, EBP50 expression was significantly lower specifically in the outer areas of tumor lesions, in regions where tumor budding (BD) was observed. Low EBP50 expression was also significantly associated with several unfavorable prognostic factors, suggesting that EBP50 depletion rather than its overexpression or subcellular distribution plays an important role in CRC progression. In CRC cell lines, knockout of EBP50 induced epithelial–mesenchymal transition (EMT)-like features, decreased proliferation, accelerated migration capability, and stabilized nuclear β-catenin due to disruption of the interaction between EBP50 and β-catenin at the plasma membrane. In addition, Slug expression was significantly higher in outer lesions, particularly in BD areas, and was positively correlated with nuclear β-catenin status, consistent with β-catenin-driven transactivation of the Slug promoter. Together, our data suggest that EBP50 depletion releases β-catenin from the plasma membrane in outer tumor lesions, allowing β-catenin to accumulate and translocate to the nucleus, where it transactivates the Slug gene to promote EMT. This in turn triggers tumor budding and contributes to the progression of CRC to a more aggressive phase. Full article

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family produces the critical lipid regulator phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM). Here, we investigated the potential role of PIP5Kγ, a PIP5K isoform, in the Hippo pathway. The ectopic expression of PIP5Kγ87 or PIP5Kγ90, two major PIP5Kγ splice variants, activated large tumor suppressor kinase 1 (LATS1) and inhibited Yes-associated protein (YAP), whereas PIP5Kγ knockdown yielded opposite effects. The regulatory effects of PIP5Kγ were dependent on its catalytic activity and the presence of Merlin and LATS1. PIP5Kγ knockdown weakened the restoration of YAP phosphorylation upon stimulation with epidermal growth factor or lysophosphatidic acid. We further found that PIP5Kγ90 bound to the Merlin’s band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PI(4,5)P2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, potentiated Merlin–LATS1 interaction and recruited LATS1 to the PM. Consistently, PIP5Kγ knockdown or inhibitor (UNC3230) enhanced colony formation in carcinoma cell lines YAP-dependently. In addition, PIP5Kγ90 interacted with heat shock cognate 71-kDa protein (Hsc70), which also contributed to Hippo pathway activation. Collectively, our results suggest that PIP5Kγ regulates the Hippo–YAP pathway by forming a functional complex with Merlin and LATS1 at the PI(4,5)P2-rich PM and via interplay with Hsc70. Full article

NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron–glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABA_A receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron–glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABA_A receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity. Full article

Mast cells have existed for millions of years in species that never suffer from allergic reactions. Hence, in addition to allergies, mast cells can play a critical role in homeostasis and inflammation via secretion of numerous vasoactive, pro-inflammatory and neuro-sensitizing mediators. Secretion may utilize different modes that involve the cytoskeleton, but our understanding of the molecular mechanisms regulating secretion is still not well understood. The Ezrin/Radixin/Moesin (ERM) family of proteins is involved in linking cell surface-initiated signaling to the actin cytoskeleton. However, how ERMs may regulate secretion from mast cells is still poorly understood. ERMs contain two functional domains connected through a long α-helix region, the N-terminal FERM (band 4.1 protein-ERM) domain and the C-terminal ERM association domain (C-ERMAD). The FERM domain and the C-ERMAD can bind to each other in a head-to-tail manner, leading to a closed/inactive conformation. Typically, phosphorylation on the C-terminus Thr has been associated with the activation of ERMs, including secretion from macrophages and platelets. It has previously been shown that the ability of the so-called mast cell “stabilizer” disodium cromoglycate (cromolyn) to inhibit secretion from rat mast cells closely paralleled the phosphorylation of a 78 kDa protein, which was subsequently shown to be moesin, a member of ERMs. Interestingly, the phosphorylation of moesin during the inhibition of mast cell secretion was on the N-terminal Ser56/74 and Thr66 residues. This phosphorylation pattern could lock moesin in its inactive state and render it inaccessible to binding to the Soluble NSF attachment protein receptors (SNAREs) and synaptosomal-associated proteins (SNAPs) critical for exocytosis. Using confocal microscopic imaging, we showed moesin was found to colocalize with actin and cluster around secretory granules during inhibition of secretion. In conclusion, the phosphorylation pattern and localization of moesin may be important in the regulation of mast cell secretion and could be targeted for the development of effective inhibitors of secretion of allergic and inflammatory mediators from mast cells. Full article

CRC is the second leading cause of cancer-related death. The complex mechanisms of metastatic CRC limit available therapeutic choice. Thus, identifying new CRC therapeutic targets is essential. Moesin (MSN), a member of the ezrin–radixin–moesin family, connects the cell membrane to the actin-based cytoskeleton and regulates cell morphology. We investigated the role of MSN in the progression of CRC. GENT2 and oncomine were used to study MSN expression and CRC patient outcomes. MSN-specific shRNAs or MSN-overexpressed plasmid were used to establish MSN-KD and MSN overexpressed cell lines, respectively. SRB, migration, wound healing, and flow cytometry were used to test cell survival and migration. Propidium iodide and annexin V stain were used to analyze the cell cycle and apoptosis. MSN expression was found to be higher in CRC tissues than in normal tissues. Higher MSN expression is associated with poor overall survival, disease-free survival, and relapse-free survival rates in CRC patients. MSN silencing inhibits cell proliferation, adhesion, migration, and invasion in vitro, whereas MSN overexpression accelerates cell proliferation, adhesion, migration, and invasion. RNA sequencing was used to investigate differentially expressed genes, and RUNX2 was discovered as a possible downstream target for MSN. In CRC patients, RUNX2 expression was significantly correlated with MSN expression. We also found that MSN silencing decreased cytoplasmic and nuclear β-catenin levels. Additionally, pharmacological inhibition of β-catenin in MSN-overexpressed cells led to a reduction of RUNX2, and activating β-catenin signaling by inhibiting GSK3β rescued the RUNX2 downregulation in MSN-KD cells. This confirms that MSN regulates RUNX2 expression via activation of β-catenin signaling. Finally, our result further determined that RUNX2 silencing reduced the ability of MSN overexpression cells to proliferate and migrate. MSN accelerated CRC progression via the β-catenin-RUNX2 axis. As a result, MSN holds the potential to become a new target for CRC treatment. Full article

Pancreatic cancer is one of the most aggressive forms of cancer and is the seventh leading cause of cancer deaths worldwide. Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of pancreatic cancers. Most pancreatic cancers are recalcitrant to radiation, chemotherapy, and immunotherapy, highlighting the urgent need for novel treatment options for this deadly disease. To this end, we screened a library of kinase inhibitors in the PDAC cell lines PANC-1 and BxPC-3 and identified two highly potent molecules: Aurora kinase inhibitor AT 9283 (AT) and EGFR kinase inhibitor WZ 3146 (WZ). Both AT and WZ exhibited a dose-dependent inhibition of viability in both cell lines. Thus, we conducted an in-depth multilevel (cellular, molecular, and proteomic) analysis with AT and WZ in PANC-1 cells, which harbor KRAS mutation and exhibit quasimesenchymal properties representing pancreatic cancer cells as having intrinsic chemoresistance and the potential for differential response to therapy. Elucidation of the molecular mechanism of action of AT and WZ revealed an impact on the programmed cell death pathway with an increase in apoptotic, multicaspase, and caspase 3/7 positive cells. Additionally, the key survival molecule Bcl-2 was impacted. Moreover, cell cycle arrest was observed with both kinase inhibitors. Additionally, an increase in superoxide radicals was observed in the AT-treated group. Importantly, proteomic profiling revealed differentially regulated key entities with multifaceted effects, which could have a deleterious impact on PDAC. These findings suggest potential targets for efficacious treatment, including a possible increase in the efficacy of immunotherapy using PD-L1 antibody due to the upregulation of lactoferrin and radixin. Furthermore, combination therapy outcomes with gemcitabine/platinum drugs may also be more effective due to an increase in the NADH dehydrogenase complex. Notably, protein–protein interaction analysis (STRING) revealed possible enrichment of reactome pathway entities. Additionally, novel therapy options, such as vimentin-antibody--drug conjugates, could be explored. Therefore, future studies with the two kinases as monotherapy/combination therapy are warranted. Full article

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin β2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer. Full article

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent “do not eat me” signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells. Full article