bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2021‒06‒06
33 papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

  1. J Cutan Pathol. 2021 Jun 03.
      BACKGROUND: BRCA1-associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1-inactivated melanocytic tumors (BIMTs).METHODS: We evaluated the cilia and centrosomes in eleven BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha-tubulin and gamma tubulin.
    RESULTS: We found that, compared to nevi, BIMTs demonstrate loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs.
    CONCLUSIONS: Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1-inactivated tumors. This article is protected by copyright. All rights reserved.
    Keywords:  BAP1; centrosomes; cilia; melanocytic nevi; melanocytic tumors
  2. Clin Exp Nephrol. 2021 May 31.
      BACKGROUND: Renal bilateral fluid filled-cyst in polycystic kidney disease (PKD) is associated with abnormal epithelial cell proliferation and transepithelial fluid secretion which leads to end-stage renal disease (ESRD). A chalcone derivative, isoliquiritigenin (ISLQ), has been shown to have various pharmacological properties. Since several studies have shown that ISLQ could inhibit CFTR channel activity, it is interesting to see whether it can inhibit renal cyst enlargement. The present study was aimed to determine an inhibitory effect and the mechanism of chalcone derivatives on MDCK cyst progression and Pkd1 mutant cells.METHODS: MDCK cyst growth and cyst formation experiments, MTT assay, Ussing chamber experiment, BrdU cell proliferation assay and western blot analysis were performed in this study.
    RESULTS: Among four compounds of chalcone derivatives tested, CHAL-005 (100 µM) was found to inhibit MDCK cyst growth in a dose-dependent manner without cytotoxicity. It inhibited short-circuit current of chloride secretion as well as CFTR protein expression in MDCK cells. CHAL-005 significantly suppressed cell proliferation. In addition, CHAL-005 strongly reduced phosphorylation ERK1/2 and phosphorylation S6 kinase in MDCK and Pkd1 mutant cells. Interestingly, CHAL-005 activated phosphorylation of AMP kinase protein expression in MDCK and Pkd1 mutant cells.
    CONCLUSION: CHAL-005 slowed MDCK cyst progression by inhibiting CFTR expression and reducing ERK1/2 and mTOR/S6K signaling pathways as well as activating AMPK expression. Therefore, a chalcone derivative could represent as a promising drug candidate for polycystic kidney disease intervention.
    Keywords:  AMPK; CFTR; Chalcone; ERK1/2; MDCK cyst enlargement; mTOR/S6K
  3. Nat Commun. 2021 06 02. 12(1): 3292
      Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.
  4. Methods Mol Biol. 2021 ;2329 291-309
      The cell and cilia cycles are inextricably linked through the dual functions of the centrioles at both the basal body of cilia and at mitotic centrosomes. How cilia assembly and disassembly, either through slow resorption or rapid deciliation, are coordinated with cell cycle progression remains unclear in many cell types and developmental paradigms. Moreover, little is known about how additional cilia parameters including changes in ciliary length or frequency of distal tip shedding change with cell cycle stage. In order to explore these questions, we have developed the Arl13bCerulean-Fucci2a tricistronic cilia and cell cycle biosensor (Ford et al., Dev Cell 47:509-523.e7, 2018). This reporter allowed us to document the heterogeneity in ciliary behaviors during the cell cycle at a population level. Without the need for external stimuli, it revealed that in several cell types and in the developing embryo cilia persist beyond the G1/S checkpoint. Here, we describe the generation of stable cell lines expressing Arl13bCerulean-Fucci2a and open-source software to aid morphometric profiling of the primary cilium with cell cycle phases, including changes in cilium length. This resource will allow the investigation of multiple morphometric questions relating to cilia and cell cycle biology.
    Keywords:  Biosensor; Cell cycle; Cell division; Cilia; Ciliogenesis; Fucci2A; Image analysis; ImageJ; Live cell imaging; Morphometrics
  5. Autophagy. 2021 May 31. 1-3
      The primary cilium (PC), a plasma membrane microtubule-based structure, is a sensor of extracellular chemical and mechanical stress stimuli. Upon ciliogenesis, the autophagy protein ATG16L1 and the ciliary protein IFT20 are co-transported to the PC. We demonstrated in a recent study that IFT20 and ATG16L1 interact in a multiprotein complex. This interaction is mediated by the ATG16L1 WD40 domain and an ATG16L1-binding motif newly identified in IFT20. ATG16L1-deficient cells are decorated by giant ciliary structures hallmarked by defects in PC-associated signaling. These structures uncommonly accumulate phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) while phosphatidylinositol-4-phosphate (PtdIns4P), a lipid normally concentrated in the PC, is excluded. We show that INPP5E, a phosphoinositide-associated phosphatase responsible for PtdIns4P generation, is a partner of ATG16L1 in this context. Perturbation of the ATG16L1-IFT20 complex alters INPP5E trafficking and proper function at the ciliary membrane. Altogether, these results reveal a novel autophagy-independent function of ATG16L1 that contributes to proper PC dynamics and function.
    Keywords:  ATG; IFT; INPP5E; macroautophagy; phosphoinositides; primary cilium
  6. Methods Mol Biol. 2021 ;2329 249-263
      Expansion microscopy is an imaging method based on isotropic physical expansion of biological samples, which improves optical resolution and allows imaging of subresolutional cellular components by conventional microscopes. Centrioles are small microtubule-based cylindrical structures that build centrosomes and cilia, two organelles essential for vertebrates. Due to a centriole's small size, electron microscopy has traditionally been used to study centriole length and ultrastructural features. Recently, expansion microscopy has been successfully used as an affordable and accessible alternative to electron microscopy in the analysis of centriole and cilia length and structural features. Here, we describe an expansion microscopy approach for the analysis of centrioles and cilia in large populations of mammalian adherent and nonadherent cells and multiciliated cultures.
    Keywords:  Centriole; Centriole length; Centrosome; Cilia; Expansion microscopy
  7. Curr Top Dev Biol. 2021 ;pii: S0070-2153(20)30105-8. [Epub ahead of print]145 3-39
      The epidermis of the Xenopus embryo has emerged as a powerful tool for studying the development of a ciliated epithelium. Interspersed throughout the epithelium are multiciliated cells (MCCs) with 100+ motile cilia that beat in a coordinated manner to generate fluid flow over the surface of the cell. MCCs are essential for various developmental processes and, furthermore, ciliary dysfunction is associated with numerous pathologies. Therefore, understanding the cellular mechanisms involved in establishing a ciliated epithelium are of particular interest. MCCs originate in the inner epithelial layer of Xenopus skin, where Notch signaling plays a critical role in determining which progenitors will adopt a ciliated cell fate. Then, activation of various transcriptional regulators, such as GemC1 and MCIDAS, initiate the MCC transcriptional program, resulting in centriole amplification and the formation of motile cilia. Following specification and differentiation, MCCs undergo the process of radial intercalation, where cells apically migrate from the inner layer to the outer epithelial layer. This process involves the cooperation of various cytoskeletal networks, activation of various signaling molecules, and changes in cell-ECM and cell-cell adhesion. Coordination of these cellular processes is required for complete incorporation into the outer epithelial layer and generation of a functional ciliated epithelium. Here, we highlight recent advances made in understanding the transcriptional cascades required for MCC specification and differentiation and the coordination of cellular processes that facilitate radial intercalation. Proper regulation of these signaling pathways and processes are the foundation for developing a ciliated epithelium.
    Keywords:  Actin; Ciliated epithelium; Ciliogenesis; Cytoskeleton; Differentiation; Microtubules; Migration; Multiciliated cells; Radial intercalation; Xenopus
  8. J Vis Exp. 2021 May 14.
      Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.
  9. Sci Rep. 2021 Jun 03. 11(1): 11760
      Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.
  10. Int J Mol Sci. 2021 May 01. pii: 4821. [Epub ahead of print]22(9):
      Schwann cells (SCs) are a highly plastic cell type capable of undergoing phenotypic changes following injury or disease. SCs are able to upregulate genes associated with nerve regeneration and ultimately achieve functional recovery. During the regeneration process, the extracellular matrix (ECM) and cell morphology play a cooperative, critical role in regulating SCs, and therefore highly impact nerve regeneration outcomes. However, the roles of the ECM and mechanotransduction relating to SC phenotype are largely unknown. Here, we describe the role that matrix stiffness and cell morphology play in SC phenotype specification via known mechanotransducers YAP/TAZ and RhoA. Using engineered microenvironments to precisely control ECM stiffness, cell shape, and cell spreading, we show that ECM stiffness and SC spreading downregulated SC regenerative associated proteins by the activation of RhoA and YAP/TAZ. Additionally, cell elongation promoted a distinct SC regenerative capacity by the upregulation of Rac1/MKK7/JNK, both necessary for the ECM and morphology changes found during nerve regeneration. These results confirm the role of ECM signaling in peripheral nerve regeneration as well as provide insight to the design of future biomaterials and cellular therapies for peripheral nerve regeneration.
    Keywords:  Rho GTPase; Schwann cell; YAP/TAZ; extracellular matrix; mechanobiology; peripheral nerve
  11. Int J Mol Sci. 2021 May 07. pii: 4923. [Epub ahead of print]22(9):
      Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.
    Keywords:  STOP suppression; aminoglycosides; premature termination codon; primary ciliary dyskinesia; rare disease; readthrough
  12. J Toxicol Sci. 2021 ;46(6): 255-262
      Fasudil is an inhibitor of Rhoa/ROCK signaling, which is involved in anti-inflammatory and anti-injury effects. The purpose of this study was to explore the effects of Fasudil on acetaminophen (APAP)-induced liver injury and reveal its potential molecular mechanism. In this study, C57BL/6 J mice were divided into different groups and treated with APAP and specified dose of Fasudil. HE staining was used to detect the changes of liver pathological tissues induced by APAP. ELISA assay was performed to detected the level of related factors. Western blot was used to detect the expressions of Rhoa, ROCK1, ROCK2. CD86 and CD6 were determined by RT-PCR and immunohistochemical staining detected the difference in CD86 expression. Rhoa/ROCK expression was increased in APAP-induced liver injury, and Fasudil targeted the expression of Rhoa/ROCK. Fasudil inhibits APAP-induced hepatic pathological changes and liver function injury. Fasudil inhibits the release of APAP-induced systemic inflammatory factors in liver tissue. Fasudil inhibits the activity of antioxidant enzymes, lipid peroxidation and macrophage infiltration induced by APAP in liver tissues. Fasudil alleviates APAP-induced liver injury via targeting Rhoa/ROCK signal pathway, indicating the possibility for clinical use of Fasudil in APAP-induced liver injury.
    Keywords:  Acetaminophen; Fasudil; Liver injury; Rhoa/ROCK
  13. Cell Death Discov. 2021 Jun 04. 7(1): 132
      Inhibition of RhoA-ROCK pathway is involved in the H2S-induced cerebral vasodilatation and H2S-mediated protection on endothelial cells against oxygen-glucose deprivation/reoxygenation injury. However, the inhibitory mechanism of H2S on RhoA-ROCK pathway is still unclear. The aim of this study was to investigate the target and mechanism of H2S in inhibition of RhoA/ROCK. GST-RhoAwild and GST-RhoAS188A proteins were constructed and expressed, and were used for phosphorylation assay in vitro. Recombinant RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids were constructed and transfected into primary hippocampal nerve cells (HNCs) to evaluate the neuroprotective mechanism of endothelial H2S by using transwell co-culture system with endothelial cells from cystathionine-γ-lyase knockout (CSE-/-) mice and 3-mercaptopyruvate sulfurtransferase knockout (3-MST-/-) rats, respectively. We found that NaHS, exogenous H2S donor, promoted RhoA phosphorylation at Ser188 in the presence of cGMP-dependent protein kinase 1 (PKG1) in vitro. Besides, both exogenous and endothelial H2S facilitated the RhoA phosphorylation at Ser188 in HNCs, which induced the reduction of RhoA activity and membrane transposition, as well as ROCK2 activity and expression. To further investigate the role of endothelial H2S on RhoA phosphorylation, we detected H2S release from ECs of CSE+/+ and CSE-/- mice, and 3-MST+/+ and 3-MST-/- rats, respectively, and found that H2S produced by ECs in the culture medium is mainly catalyzed by CSE synthase. Moreover, we revealed that both endothelial H2S, mainly catalyzed by CSE, and exogenous H2S protected the HNCs against hypoxia-reoxygenation injury via phosphorylating RhoA at Ser188.
  14. Front Cell Dev Biol. 2021 ;9 672625
      Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.
    Keywords:  IFT20; corneal inflammation; lymphangiogenesis; lymphatic; lymphatic development; primary cilia; vascular patterning
  15. Molecules. 2021 May 01. pii: 2658. [Epub ahead of print]26(9):
      Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.
    Keywords:  ROCK inhibitor; dissociation-induced cell death; salivary gland stem cells (SGSCs)
  16. Int J Mol Sci. 2021 May 04. pii: 4868. [Epub ahead of print]22(9):
      Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.
    Keywords:  PKD1; osteogenic differentiation; stemness; umbilical cord blood-derived mesenchymal stem cell (UCB-MSC)
  17. Front Cell Dev Biol. 2021 ;9 675562
      Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis.
    Keywords:  PP2A; VEGF; YAP; angiogenesis; matrix stiffness
  18. J Clin Med. 2021 May 01. pii: 1953. [Epub ahead of print]10(9):
      Rho-associated coiled-coil kinase (ROCK) signaling can affect glaucoma risk by regulating trabecular meshwork outflow. We investigated the effect of ROCK gene polymorphism on the risks of primary open-angle glaucoma (POAG) and POAG-related phenotypes including intraocular pressure (IOP) in a Korean population. A total of 24 single-nucleotide polymorphisms (SNPs) from ROCK1 and ROCK2 were selected and genotyped for 363 POAG patients and 213 healthy controls. Among the 363 POAG patients, 282 were normal-tension glaucoma (NTG, baseline IOP ≤ 21 mmHg) and 81 were high-tension glaucoma (HTG, baseline IOP > 21 mmHg). The SNPs rs288979, rs1006881, rs35996865, rs10083915, and rs11873284 in ROCK1 (tagged to each other, r2 = 1) were nominally associated with risk of HTG (OR = 0.52, p = 0.045). However, there were no SNPs that were significantly associated with the risk of NTG. In the genotype-phenotype correlation analysis, the SNPs rs2230773 and rs3771106 in ROCK2 were significantly correlated with central corneal thickness (CCT)-adjusted IOP (p = 0.024) and axial length (AXL; p = 0.024), respectively. The present data implicated the role of ROCK in POAG development, and as such, can serve as a good reference for upcoming Rho/ROCK-pathway-related studies on POAG.
    Keywords:  primary open-angle glaucoma; rho-associated coiled-coil kinase (ROCK); single nucleotide polymorphism
  19. Development. 2021 Jun 01. pii: dev199091. [Epub ahead of print]148(11):
      During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.
    Keywords:  Cochlea; Epithelial-mesenchymal transition; Mouse; Wnt pathway; β-catenin
  20. Genes (Basel). 2021 May 24. pii: 800. [Epub ahead of print]12(6):
      Many different regulatory mechanisms of renal fibrosis are known to date, and those related to transforming growth factor-β1 (TGF-β1)-induced signaling have been studied in greater depth. However, in recent years, other signaling pathways have been identified, which contribute to the regulation of these pathological processes. Several studies by our team and others have revealed the involvement of small Ras GTPases in the regulation of the cellular processes that occur in renal fibrosis, such as the activation and proliferation of myofibroblasts or the accumulation of extracellular matrix (ECM) proteins. Intracellular signaling mediated by TGF-β1 and Ras GTPases are closely related, and this interaction also occurs during the development of renal fibrosis. In this review, we update the available in vitro and in vivo knowledge on the role of Ras and its main effectors, such as Erk and Akt, in the cellular mechanisms that occur during the regulation of kidney fibrosis (ECM synthesis, accumulation and activation of myofibroblasts, apoptosis and survival of tubular epithelial cells), as well as the therapeutic strategies for targeting the Ras pathway to intervene on the development of renal fibrosis.
    Keywords:  Akt; MAP kinases; Ras; extracellular matrix; fibroblasts; kidney fibrosis
  21. Int J Mol Sci. 2021 May 31. pii: 5915. [Epub ahead of print]22(11):
      The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.
    Keywords:  CDX2; LPA; YAP; bovine; lineage segregation; trophectoderm
  22. Cells Dev. 2021 May 28. pii: S2667-2901(21)00034-6. [Epub ahead of print] 203687
      Bone marrow mesenchymal stem cells (BMSCs) have strong proliferative ability and multi-directional differentiation potential. Osteoarthritis is a degenerative joint disease that is closely related to the loss of osteogenic differentiation function of BMSCs. Autophagy, plays a crucial role in the maintenance of cellular functions, but its regulatory mechanism during the osteogenic differentiation of BMSCs remains unclear. In this study, we analyzed the differential gene networks and pathways during BMSC osteogenesis using bioinformatics, and further validated the regulatory roles of autophagy during the osteogenic differentiation of BMSCs in inflammatory condition in vitro. We found that Tumor necrosis factor alpha (TNF-α) treatment led to actin cytoskeleton rearrangements and inhibited osteogenic differentiation in BMSCs. In addition, TNF-α enhanced Rho-associated protein kinase 1 (ROCK1) expression and decreased autophagy activation. ROCK1 knockdown reduced Endoplasmic Reticulum stress (ER stress) and promoted autophagy, resulting reversion of osteogenic differentiation in BMSCs under inflammatory condition. Rapamycin reversed the TNF-α-induced decrease in osteogenesis of BMSCs, assessed by alkaline phosphatase (ALP) activity and Alizarin staining. Autophagy treated with inhibitor 3-Methyladenine (3-MA) further increased TNF-α-induced osteogenesis inhibition of BMSCs. Collectively, these results indicate that ER stress and dysfunction of autophagy promote inflammation-induced bone loss through the activation of ROCK1 signaling in BMSCs.
    Keywords:  BMSCs; ER stress/autophagy; Osteogenic differentiation; ROCK 1; TNF-α
  23. Mol Biol Rep. 2021 Jun 04.
      Cells translate the mechanosensing of extracellular matrix component dysregulation and stiffness into the signal transduction including Osteopontin (OPN) through the Hippo pathway. But how extracellular matrix (ECM) component dysregulation and stiffness are ultimately linked to transitional cell carcinoma (TCC) development remains poorly understood. This study was aimed to evaluate the possible links between ECM component alteration after cancer surgery and OPN and Yes-associated protein (YAP) expression in TCC and adjacent tissues. In this study, we used 50 TCC (25 newly diagnosed and 25 recurrent) and 50 adjacent tissues to determine the tissue stiffness using atomic force microscopy. The mRNA expression of SPP1, Indian hedgehog (IHH), and YAP was also determined using qRT-PCR. Western blotting and ELISA were performed to assess the tissue and serum levels of OPN, respectively. To assess the glycoproteins and elastic fibers content, Periodic Acid Schiff, and Verhoeff-Van Gieson Staining were performed, respectively. Matrix stiffness was markedly higher in TCCs than adjacent tissues (p < 0.05). Gene expression analysis showed that YAP, SPP1, and IHH genes were upregulated in TCC tissues (p < 0.05). Additionally, the OPN protein overexpression was observed in the tissue and the serum of TCC patients (p < 0.05). We also found that glycoproteins, elastic fibers content of recurrent TCC tissues was remarkably higher as compared to adjacent tissues (p < 0.05). Our results suggest that glycoproteins and elastic fibers content modulation and ECM stiffness may upregulates the expression of YAP, SPP1 and IHH genes, and possibly contribute to the TCC development and relapse.
    Keywords:  Cancer; Indian hedgehog (IHH); Osteopontin (OPN); Tissue stiffness; Transitional cell carcinoma (TCC); Yes-associated protein (YAP)
  24. Front Pharmacol. 2021 ;12 676817
      Background: One of the important pathogenesis of acute respiratory distress syndrome (ARDS) is the dysfunction of pulmonary microvascular endothelial barrier induced by a hyperinflammatory immune response. However, the potential mechanisms of such an imbalance in pulmonary microvascular endothelial cells (PMVECs) are not yet understood. Purpose: Explore the molecular mechanism of endothelial barrier dysfunction induced by inflammatory immune cytokines in ARDS, and find a therapeutic target for this syndrome. Methods: Rat PMVECs were cultured to form a monolayer. Immunofluorescence, flow cytometry, and Western blotting were selected to detect the distribution and the expression level of phosphorylated Ezrin protein and Ezrin protein. Transendothelial electrical resistance (TER) and transendothelial fluxes of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) were utilized to measure the permeability of the cell monolayer. Ezrin short hairpin RNA (shRNA) and Ezrin 567-site threonine mutant (EzrinT567A) were used to examine the role of Ezrin protein and phosphorylated Ezrin protein in endothelial response induced by tumor necrosis factor-alpha (TNF-α), respectively. The function of focal adhesion kinase (FAK) and Ras homolog gene family, member A (RhoA) signaling pathways were estimated by inhibitors and RhoA/FAK shRNA in TNF-α-stimulated rat PMVECs. The activation of FAK and RhoA was assessed by Western blotting or pull-down assay plus Western blotting. Results: The TER was decreased after TNF-α treatment, while the Ezrin protein phosphorylation was increased in a time- and dose-dependent manner. The phosphorylated Ezrin protein was localized primarily at the cell periphery, resulting in filamentous actin (F-actin) rearrangement, followed by a significant decrease in TER and increase in fluxes of FITC-BSA. Moreover, FAK and RhoA signaling pathways were required in the phosphorylation of Ezrin protein, and the former positively regulated the latter. Conclusion: The phosphorylated Ezrin protein was induced by TNF-α via the FAK/RhoA signaling pathway leading to endothelial hyperpermeability in PMVECs.
    Keywords:  Ezrin protein; acute respiratory distress syndrome; endothelial permeability; inflammatory immune response; molecular mechanism
  25. Oncol Rep. 2021 Jul;pii: 142. [Epub ahead of print]46(1):
      Aberrant expression of circular RNAs (circRNAs) has been demonstrated to be related to the development of colorectal cancer (CRC), the third most common cancer worldwide. However, the mechanism of the effect of circRNA NOP2/Sun domain family, member 2 (circNSUN2) on the malignant biological behavior of CRC remains unclear. In the present study, the expression of circNSUN2 and microRNA (miR)‑181a‑5p was detected by RT‑qPCR. The expression of Rho‑associated coiled‑coil‑containing protein kinase 2 (ROCK2) was measured by western blotting. Cell proliferation was detected by CCK‑8 assay. The cell apoptosis rate was measured by flow cytometry. Cell migration ability was evaluated by Transwell assay. The interactions between circNSUN2, miR‑181a‑5p and ROCK2 were verified by dual‑luciferase reporter assay. The results revealed that circNSUN2 was highly expressed in CRC tissues and cell lines. Knockdown of circNSUN2 inhibited the malignant biological behavior of CRC in vivo and in vitro. Moreover, miR‑181a‑5p was revealed to be a target gene of circNSUN2, and the expression of ROCK2 was negatively regulated by miR‑181a‑5p. Knockdown of circNSUN2 inhibited proliferation and migration, and induced apoptosis of CRC cells and suppressed tumor growth by targeting miR‑181a‑5p to decrease ROCK2 expression. In conclusion, circNSUN2 promoted the progression of CRC by sponging miR‑181a‑5p to increase the expression of ROCK2.
    Keywords:  Rho‑associated coiled‑coil‑containing protein kinase 2; apoptosis; circRNA NOP2/Sun domain family; colorectal cancer; member 2; microRNA‑181a‑5p; migration; proliferation
  26. J Cell Mol Med. 2021 Jun 01.
      Gastric cancer (GC) is the most frequent digestive system malignant tumour and the second most common cause of cancer death globally. Cancer stem cell (CSC) is a small percentage of cancer cells in solid tumours that have differentiation, self-renewal and tumorigenic capabilities. They have an active participation in the initiation, development, metastasis, recurrence and resistance of tumours to chemotherapy and radiotherapy. Gastric cancer stem cells (GCSCs) have been shown to be correlated with GC initiation and metastasis. In this study, we found that TAK1 expression level in GC tissues was significantly increased compared to the adjacent non-cancerous tissues by RT-qPCR, Western blot and immunohistochemistry. TAK1 has been identified as a critical molecule that promoted a variety of malignant GC phenotypes both in vivo and in vitro and promoted the self-renewal of GCSCs. Mechanistically, TAK1 was up-regulated by IL-6 and prevented the degradation of yes-associated protein (YAP) in the cytoplasm by binding to YAP. Thus, TAK1 promoted the SOX2 and SOX9 transcription and the self-renewal and oncogenesis of GCSCs. Our findings provide insights into the mechanism of self-renewal and tumorigenesis of TAK1 in GCSCs and have broad implications for clinical therapies.
    Keywords:  TGFβ-activated kinase 1; YAP; cancer stem cells; gastric cancer
  27. J Biol Chem. 2021 May 28. pii: S0021-9258(21)00646-3. [Epub ahead of print] 100848
      Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells (IECs) results in increased epithelial proliferation and nuclear accumulation of Yes-Associated Protein (YAP), a key Hippo-signaling transducer that senses cell-cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, β-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP-β-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo-YAP signaling pathway for rapid reparative response after tissue injury.
    Keywords:  Colitis; Colonic regeneration; Endosomes; Epithelial Junction; Hippo; Merlin; RAB11A; YAP
  28. Physiol Rep. 2021 Jun;9(11): e14881
      INTRODUCTION: (Pro)renin receptor has emerged as a new member of the renin-angiotensin system implicated in the pathogenesis of chronic kidney disease (CKD). Herein we report characterization of the therapeutic potential of (pro)renin receptor (PRR) antagonist PRO20 in 5/6 nephrectomy (5/6Nx) rats.METHODS: Male Wistar rats underwent 5/6Nx followed by treatment with vehicle or received daily injections of a PRR inhibitor PRO20 (700 μg/kg) via the 3 s.c. Sham group served as a control.
    RESULTS: As compared with the sham control, the 5/6Nx rats exhibited significant increases in proteinuria, glomerulosclerosis, tubular injury, and interstitial inflammation in the remnant kidneys. Treatment with PRO20 significantly attenuated these abnormalities, as evidenced by reduced expression of fibronectin, α-SMA, collagen 1, TGF-β1, IL-6, IL-8, IL-1β, MCP-1 and increased expression of E-cadherin. Increased urinary/renal levels of renin activity, angiotensinogen (AGT), and Angiotensin II (Ang II) by 5/6Nx, which were all ameliorated by PRO20. Renal PRR, the secreted proteolytic fragment of PRR (sPRR) in renal and urinary, were all elevated in 5/6Nx rats. Moreover, our results revealed that renal Wnt3A and β-catenin expression were upregulated during 5/6Nx, which were all attenuated by PRO20.
    CONCLUSIONS: Overall we conclude that in vivo antagonism of PRR with PRO20 will improve 5/6Nx-induced CKD mainly through inhibition of intrarenal RAS and Wnt/β-catenin signaling pathway.
    Keywords:  (Pro)renin receptor; 5/6 nephrectomy; renin-angiotensin system
  29. Antioxidants (Basel). 2021 May 30. pii: 879. [Epub ahead of print]10(6):
      Mesenchymal stem cells (MSCs) are self-renewal and capable of differentiating to various functional cell types, including osteocytes, adipocytes, myoblasts, and chondrocytes. They are, therefore, regarded as a potential source for stem cell therapy. Fisetin is a bioactive flavonoid known as an active antioxidant molecule that has been reported to inhibit cell growth in various cell types. Fisetin was shown to play a role in regulating osteogenic differentiation in animal-derived MSCs; however, its molecular mechanism is not well understood. We, therefore, studied the effect of fisetin on the biological properties of human MSCs derived from chorion tissue and its role in human osteogenesis using MSCs and osteoblast-like cells (SaOs-2) as a model. We found that fisetin inhibited proliferation, migration, and osteogenic differentiation of MSCs as well as human SaOs-2 cells. Fisetin could reduce Yes-associated protein (YAP) activity, which results in downregulation of osteogenic genes and upregulation of fibroblast genes. Further analysis using molecular docking and molecular dynamics simulations suggests that fisetin occupied the hydrophobic TEAD pocket preventing YAP from associating with TEA domain (TEAD). This finding supports the potential application of flavonoids like fisetin as a protein-protein interaction disruptor and also suggesting an implication of fisetin in regulating human osteogenesis.
    Keywords:  YAP; fisetin; flavonoid; inhibition; mesenchymal stem cells; osteogenic differentiation
  30. Methods Mol Biol. 2021 ;2329 1-18
      The cell cycle is the sequence of events through which a cell duplicates its genome, grows, and divides. Key cell cycle transitions are driven by oscillators comprising of protein kinases and their regulators. Different cell cycle oscillators are inextricably linked to ensure orderly activation of oscillators. A recurring theme in their regulation is the abundance of autoamplifying loops that ensure switch-like and unidirectional cell cycle transitions. The periodicity of many cell cycle oscillators is choreographed by inherent mechanisms that promote automatic inactivation, often involving dephosphorylation and ubiquitin-mediated protein degradation. These inhibitory signals are subsequently suppressed to enable the next cell cycle to occur. Although the activation and inactivation of cell cycle oscillators are in essence autonomous during the unperturbed cell cycle, a number of checkpoint mechanisms are able to halt the cell cycle until preconditions or defects are addressed. Together, these mechanisms orchestrate orderly progression of the cell cycle to produce more cells and to safeguard genome stability.
    Keywords:  Anaphase-promoting complex; Cell cycle; Cell division; Cell growth; Checkpoints; Cyclin; Cyclin-dependent kinases; DNA replication; Mitosis; Phosphorylation; Proteolysis; Ubiquitin-mediated degradation; pRb
  31. Front Cell Dev Biol. 2021 ;9 639111
      Metabolic reprogramming is a vital factor in the development of many types of cancer, including colon cancer. Serine metabolic reprogramming is a major feature of tumor metabolism. Yes-associated protein (YAP) participates in organ size control and tumorigenesis. However, the relationship between YAP and serine metabolism in colon cancer is unclear. In this study, RNA sequencing and metabolomics analyses indicated significant enrichment of the glycine, serine, and threonine metabolism pathways in serine starvation-resistant cells. Short-term serine deficiency inhibited YAP activation, whereas a prolonged response dephosphorylated YAP and promoted its activity. Mechanistically, USP7 increases YAP stability under increased serine conditions by regulating deubiquitination. Verteporfin (VP) effectively inhibited the proliferation of colon cancer cells and organoids and could even modulate serine metabolism by inhibiting USP7 expression. Clinically, YAP was significantly activated in colon tumor tissues and positively correlated with the expression of phosphoglycerate dehydrogenase (PHGDH) and USP7. Generally, our study uncovered the mechanism by which serine metabolism regulates YAP via USP7 and identified the crucial role of YAP in the regulation of cell proliferation and tumor growth; thus, VP may be a new treatment for colon cancer.
    Keywords:  USP7; YAP; colon cancer; organoid; serine metabolism
  32. Pediatr Rep. 2021 May 10. 13(2): 241-244
      We report a Japanese 5-year-old boy with primary ciliary dyskinesia (PCD) which was diagnosed owing to Clostridium difficile (CD) infection caused by prolonged antibiotic exposure. He had intractable otitis media with effusion (OME) and had abdominal pain and diarrhea for 4 months after starting antibiotics administration. His stool contained CD toxin. After vancomycin treatment, his symptoms improved and his stools did not contain CD toxin. His past medical history included frequent pneumonia. We, therefore, performed electron microscopy of the biopsy specimen from his nasal mucosa and genetic testing, and he was diagnosed with PCD. PCD is a rare inherited genetic disease causing ciliary dysfunction, which is very difficult to diagnose because some children without PCD also develop the same symptoms. Therefore, children who have intractable OME, rhinosinusitis, frequent pneumonia, or bronchitis and are taking antibiotics for long periods of time should be checked for underlying diseases, such as PCD.
    Keywords:  NODAL; OME; antibiotic exposure; ciliary dysfunction
  33. Genes (Basel). 2021 May 27. pii: 819. [Epub ahead of print]12(6):
      Ras and Rho proteins are GTP-regulated molecular switches that control multiple signaling pathways in eukaryotic cells. Ras was among the first identified oncogenes, and it appears mutated in many forms of human cancer. It mainly promotes proliferation and survival through the MAPK pathway and the PI3K/AKT pathways, respectively. However, the myriad proteins close to the plasma membrane that activate or inhibit Ras make it a major regulator of many apparently unrelated pathways. On the other hand, Rho is weakly oncogenic by itself, but it critically regulates microfilament dynamics; that is, actin polymerization, disassembly and contraction. Polymerization is driven mainly by the Arp2/3 complex and formins, whereas contraction depends on myosin mini-filament assembly and activity. These two pathways intersect at numerous points: from Ras-dependent triggering of Rho activators, some of which act through PI3K, to mechanical feedback driven by actomyosin action. Here, we describe the main points of connection between the Ras and Rho pathways as they coordinately drive oncogenic transformation. We emphasize the biochemical crosstalk that drives actomyosin contraction driven by Ras in a Rho-dependent manner. We also describe possible routes of mechanical feedback through which myosin II activation may control Ras/Rho activation.
    Keywords:  Ras; Rho; myosin II