scholarly journals The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

Genetics ◽  
2003 ◽  
Vol 165 (1) ◽  
pp. 159-169
Author(s):  
Benjamin Boettner ◽  
Phoebe Harjes ◽  
Satoshi Ishimaru ◽  
Michael Heke ◽  
Hong Qing Fan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Interaction ◽  
Critical Role ◽  
Adherens Junction ◽  
Small Gtpases ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Cell Sheets ◽  
Jnk Pathway

Abstract Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process.

scholarly journals The potential renal toxicity of silver nanoparticles after repeated oral exposure and its underlying mechanisms

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hamed Nosrati ◽  
Manijeh Hamzepoor ◽  
Maryam Sohrabi ◽  
Massoud Saidijam ◽  
Mohammad Javad Assari ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Molecular Mechanisms ◽  
Renal Toxicity ◽  
Periodic Acid ◽  
Critical Role ◽  
Oral Exposure ◽  
Control Group ◽  
Rt Pcr ◽  
Periodic Acid Schiff

Abstract Background Silver nanoparticles (AgNPs) can accumulate in various organs after oral exposure. The main objective of the current study is to evaluate the renal toxicity induced by AgNPs after repeated oral exposure and to determine the relevant molecular mechanisms. Methods In this study, 40 male Wistar rats were treated with solutions containing 30, 125, 300, and 700 mg/kg of AgNPs. After 28 days of exposure, histopathological changes were assessed using hematoxylin-eosin (H&E), Masson’s trichrome, and periodic acid-Schiff (PAS) staining. Apoptosis was quantified by TUNEL and immunohistochemistry of caspase-3, and the level of expression of the mRNAs of growth factors was determined using RT-PCR. Results Histopathologic examination revealed degenerative changes in the glomeruli, loss of tubular architecture, loss of brush border, and interrupted tubular basal laminae. These changes were more noticeable in groups treated with 30 and 125 mg/kg. The collagen intensity increased in the group treated with 30 mg/kg in both the cortex and the medulla. Apoptosis was much more evident in middle-dose groups (i.e., 125 and 300 mg/kg). The results of RT-PCR indicated that Bcl-2 and Bax mRNAs upregulated in the treated groups (p < 0.05). Moreover, the data related to EGF, TNF-α, and TGF-β1 revealed that AgNPs induced significant changes in gene expression in the groups treated with 30 and 700 mg/kg compared to the control group. Conclusion Our observations showed that AgNPs played a critical role in in vivo renal toxicity.

scholarly journals Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy

2021 ◽  
Author(s):  
Xin Peng ◽  
Shaolu Zhang ◽  
Wenhui Jiao ◽  
Zhenxing Zhong ◽  
Yuqi Yang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Single Agent ◽  
Antitumor Efficacy ◽  
Antitumor Effects ◽  
Autophagy Inhibitor

Abstract Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

FC 006PP2A PHOSPHATASE INHIBITION IS ANTI-FIBROTIC THROUGH SER77 PHOSPHORYLATION-MEDIATED ARNT/ARNT HOMODIMER FORMATION

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Gunsmaa Nyamsuren ◽  
Gregor Christof Rapp ◽  
Björn Tampe ◽  
Michael Zeisberg
Keyword(s):  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Receptor Expression ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Aryl Hydrocarbon ◽  
Ureter Obstruction

Abstract Background and Aims Aryl hydrocarbon receptor nuclear translocator (ARNT) mediates anti-fibrotic activity in kidney and liver through induction of ALK3-receptor expression and subsequently increased Smad1/5/8 signaling. While expression of ARNT can be pharmacologically induced by sub-immunosuppressive doses of FK506 or by GPI1046, its anti-fibrotic activity is only realized when ARNT-ARNT homodimers form, as opposed to formation of ARNT-AHR or ARNT-HIF1α heterodimers. Mechanisms underlying ARNTs dimerization decision to specifically form ARNT-ARNT homodimers and possible cues to specifically induce ARNT homodimerization have been previously unknown. We here aimed to elucidate the molecular mechanisms underlying control of ARNT dimerization decision and to explore its therapeutic potential. Method We analyzed dimerization of recombinant and native ARNT by immunoprecipitation, MALDI-TOF mass spectrometry, and LS-MS/MS analysis and proximity ligation assay. Phosphorylation sites were mapped through generation of phosphorylation site mutants and through pharmacological inhibition. For in vivo analysis we challenged mice with model of unilateral ureter obstruction and carbon tetrachloride to induce fibrosis in kidney and liver. Results Here we report that inhibition of PP2A phosphatase activity increases intracellular accumulation of ARNT-ARNT homodimers. This effect is dependent on enhanced ARNT-ARNT homodimerization and decreased ARNT proteolytic degradation, but independent of ARNT transcription (which remains unchanged upon PP2A inhibition). We further identify that Ser77 phosphorylation plays a critical role in ARNT homodimerization, as ARNT-ARNT homodimers do not form with Ser77/Asp-mutant ARNT proteins. In light of previous studies which identified anti-fibrotic activity upon increased ARNT expression, we further demonstrate attenuated fibrosis upon monotherapy with the PP2A inhibitor LB100, and additive anti-fibrotic activities upon combination with pharmacological inducers of ARNT expression FK506 or GPI1046 in murine models of kidney and liver fibrosis. Conclusion Our study provides additional evidence for the anti-fibrotic activity of ARNT and reveals Ser77 phosphorylation as a novel pharmacological target to realize the therapeutic potential of increased ARNT transactivation activity.

Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Ryan A. Wilcox ◽  
Koji Tamada ◽  
Dallas B. Flies ◽  
Gefeng Zhu ◽  
Andrei I. Chapoval ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Mechanisms ◽  
Transplant Rejection ◽  
Critical Role ◽  
Tumor Necrosis Factor Receptor ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Induced Tolerance

Abstract T-cell anergy is a tolerance mechanism defined as a hyporesponsive status of antigen-specific T cells upon prior antigen encounter and is believed to play a critical role in the evasion of tumor immunity and the amelioration of allogeneic transplant rejection. Molecular mechanisms in controlling T-cell anergy are less known. We show here that administration of an agonistic monoclonal antibody (mAb) to CD137, a member of the tumor necrosis factor receptor superfamily, prevents the induction of CD8+ cytolytic T-lymphocyte (CTL) anergy by soluble antigens. More importantly, CD137 mAb restores the functions of established anergic CTLs upon reencountering their cognate antigen. As a result, infusion of CD137 mAb inhibits progressive tumor growth that is caused by soluble tumor antigen-induced tolerance in a P815R model. CD137 mAb also restores proliferation and effector functions of anergic alloreactive 2C T cells in a bone marrow transplantation model. Our results indicate that ligation of CD137 receptor delivers a regulatory signal for T-cell anergy and implicate manipulation of the CD137 pathway as a new approach to break T-cell tolerance.

scholarly journals Discrete Functions of TRAF1 and TRAF2 in Drosophila melanogaster Mediated by c-Jun N-Terminal Kinase and NF-κB-Dependent Signaling Pathways

2003 ◽  
Vol 23 (22) ◽  
pp. 7982-7991 ◽  
Author(s):  
Guang-Ho Cha ◽  
Kyoung Sang Cho ◽  
Jun Hee Lee ◽  
Myungjin Kim ◽  
Euysoo Kim ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Signaling Pathways ◽  
Nuclear Translocation ◽  
Genetic Interaction ◽  
Ectopic Expression ◽  
Tumor Necrosis Factor Receptor ◽  
Null Mutant ◽  
Microbial Infection ◽  
Jnk Pathway

ABSTRACT Two Drosophila tumor necrosis factor receptor-associated factors (TRAF), DTRAF1 and DTRAF2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, their in vivo functions and related signaling pathways are not fully understood yet. Here, we show that DTRAF1 is an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila melanogaster. Ectopic expression of DTRAF1 in the developing eye induced apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses revealed that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by DTRAF1 are dependent on JNK and its upstream kinases, Hep and DTAK1. In support of these results, DTRAF1-null mutant showed a remarkable reduction in JNK activity with an impaired development of imaginal discs and a defective formation of photosensory neuron arrays. In contrast, DTRAF2 was demonstrated as an upstream activator of nuclear factor-κB (NF-κB). Ectopic expression of DTRAF2 induced nuclear translocation of two Drosophila NF-κBs, DIF and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of DTRAF2 showed immune deficiencies in which NF-κB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, our findings demonstrate that DTRAF1 and DTRAF2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-κB-dependent signaling pathway, respectively.

ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1767-1775 ◽  
Author(s):  
Markus Bender ◽  
Anita Eckly ◽  
John H. Hartwig ◽  
Margitta Elvers ◽  
Irina Pleines ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Actin Dynamics ◽  
Complex Process ◽  
First Time ◽  
Late Stages ◽  
Platelet Formation

Abstract The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

scholarly journals C. elegans PVD Neurons: A Platform for Functionally Validating and Characterizing Neuropsychiatric Risk Genes

2016 ◽  
Author(s):  
Cristina Aguirre-Chen ◽  
Nuri Kim ◽  
Olivia Mendivil Ramos ◽  
Melissa Kramer ◽  
W. Richard McCombie ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
De Novo ◽  
Genetic Interaction ◽  
Cost Effective ◽  
Chromatin Remodelers ◽  
C Elegans ◽  
Effective Manner

AbstractOne of the primary challenges in the field of psychiatric genetics is the lack of an in vivo model system in which to functionally validate candidate neuropsychiatric risk genes (NRGs) in a rapid and cost-effective manner1−3. To overcome this obstacle, we performed a candidate-based RNAi screen in which C. elegans orthologs of human NRGs were assayed for dendritic arborization and cell specification defects using C. elegans PVD neurons. Of 66 NRGs, identified via exome sequencing of autism (ASD)4 or schizophrenia (SCZ)5−9 probands and whose mutations are de novo and predicted to result in a complete or partial loss of protein function, the C. elegans orthologs of 7 NRGs were found to be required for proper neuronal development and represent a variety of functional classes, including transcriptional regulators and chromatin remodelers, molecular chaperones, and cytoskeleton-related proteins. Notably, the positive hit rate, when selectively assaying C. elegans orthologs of ASD and SCZ NRGs, is enriched >14-fold as compared to unbiased RNAi screening10. Furthermore, we find that RNAi phenotypes associated with the depletion of NRG orthologs is recapitulated in genetic mutant animals, and, via genetic interaction studies, we show that the NRG ortholog of ANK2, unc-44, is required for SAX-7/MNR-1/DMA-1 signaling. Collectively, our studies demonstrate that C. elegans PVD neurons are a tractable model in which to discover and dissect the fundamental molecular mechanisms underlying neuropsychiatric disease pathogenesis.

scholarly journals Role of proinflammatory cytokines and growth factors in skeletal muscle regeneration

2016 ◽  
Vol 72 (8) ◽  
pp. 472-478
Author(s):  
Marta Milewska ◽  
Katarzyna Grzelkowska-Kowalczyk
Keyword(s):  
Skeletal Muscle ◽  
Growth Factors ◽  
Proinflammatory Cytokines ◽  
Muscle Regeneration ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Muscle Fibers ◽  
Skeletal Muscle Regeneration ◽  
Functional Efficiency

Skeletal muscle healing after injury can be divided into three distinct but overlapping phases. The destruction phase is characterized by rupture followed by necrosis of muscle fibers, formation of hematoma and inflammatory reaction. During the repair phase a necrotic tissue is phagocyted by macrophages, muscle fibers are regenerating and connective tissue scars are formed. The remodeling phase concerns the period when regenerating muscle fibers mature, scar contraction and reorganization occurs and the muscle recovers its functional efficiency. Proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and growth factors (FGF, IGF, TGF-β, HGF) play a critical role in all phases of muscle repair. Moreover, chemokines expressed at early stages of myogenesis can regulate the survival and proliferation of myoblasts. Chemokines expressed in vivo in muscle cells can directly influence myogenesis, but can also act in a paracrine manner by recruiting the immune cells (macrophages) to injured skeletal muscles, which is crucial for the regeneration process. Identification of molecules regulating myogenesis, like cytokines, chemokines and growth factors, contributes to the exploration of molecular mechanisms that can improve muscle regeneration after injury, diseases, surgery and increase the effectiveness of cell transplantation.

scholarly journals Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

2022 ◽  
Author(s):  
Jaclyn M Camuglia ◽  
Soline Chanet ◽  
Adam C Martin
Keyword(s):  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Drosophila Embryo ◽  
Spindle Orientation ◽  
Planar Polarity ◽  
Ventral Furrow ◽  
Adherens Junction Protein ◽  
Early Drosophila Embryo ◽  
Junction Protein

Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

scholarly journals Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3

2020 ◽  
Author(s):  
Marion Lenoir ◽  
Rebeca Martin ◽  
Edgar Torres-Maravilla ◽  
Sead Chadi ◽  
Pamela González-Dávila ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Commensal Bacterium ◽  
Jnk Pathway ◽  
Faecalibacterium Prausnitzii ◽  
Anti Inflammatory

Abstract BackgroundThe commensal bacterium Faecalibacterium prausnitzii plays a key role in inflammatory bowel disease (IBD) pathogenesis and serves as a general health biomarker in humans. However, the host molecular mechanisms that underlie its anti-inflammatory effects remain unknown.MethodsA transcriptomic approach on human intestinal epithelial cells (HT-29) that were stimulated with TNF-α and exposed to F. prausnitzii culture supernatant (SN) was used. Modulation of the most upregulated gene after F. prausnitzii SN contact was validated both in vitro and in vivo.ResultsF. prausnitzii SN upregulates the expression of Dact3, a gene linked to the Wnt/JNK pathway. Interestingly, when we silenced Dact3 expression, the effect of F. prausnitzii SN was lost. Butyrate was identified as the F. prausnitzii effector responsible for Dact3 modulation. Dact3 upregulation was also validated in vivo in both healthy and inflamed mice treated with either F. prausnitzii SN or the live bacteria, respectively. Finally, we demonstrated by colon transcriptomics that gut microbiota directly influences Dact3 expression.ConclusionsOur results provide new clues about the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium F. prausnitzii.*Contributed equally to this work

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