scholarly journals TFEB phosphorylation on Serine 211 is induced by autophagy in human synovial fibroblasts and by p62/SQSTM1 overexpression in HEK293 cells

2021 ◽  
Author(s):  
Biserka Relic ◽  
Celine Deroyer ◽  
Olivier Malaise ◽  
Zelda Plener ◽  
Philippe Gillet ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Kinase Inhibitor ◽  
Synovial Fibroblasts ◽  
Hek293 Cells ◽  
Autophagic Flux ◽  
Nuclear Fraction ◽  
Akt Phosphorylation ◽  
Coordinated Expression ◽  
A Cell ◽  
Lysosomal System

Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II upregulation, Akt phosphorylation inhibition on Serine 473 but downregulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was downregulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349.

scholarly journals Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization

2021 ◽  
Vol 9 ◽  
Author(s):  
Naila Umer ◽  
Lena Arévalo ◽  
Sharang Phadke ◽  
Keerthika Lohanadan ◽  
Gregor Kirfel ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Regulatory Proteins ◽  
Autophagic Flux ◽  
Nuclear Fraction ◽  
Rna Seq ◽  
Head Shape ◽  
Male Factor ◽  
Protein Levels ◽  
Acrosome Formation ◽  
Actin Regulatory Proteins

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

scholarly journals Axitinib and HDAC Inhibitors Interact to Kill Sarcoma Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Jane L. Roberts ◽  
Laurence Booth ◽  
Andrew Poklepovic ◽  
Paul Dent
Keyword(s):  
Hdac Inhibitor ◽  
Kinase Inhibitor ◽  
Hdac Inhibitors ◽  
Autophagic Flux ◽  
Drug Induced ◽  
Ampk Signaling ◽  
Knock Down ◽  
Reduce Tumor Growth ◽  
Sarcoma Cells

We have extended our analyses of HDAC inhibitor biology in sarcoma. The multi-kinase inhibitor axitinib interacted with multiple HDAC inhibitors to kill sarcoma cells. Axitinib and HDAC inhibitors interacted in a greater than additive fashion to inactivate AKT, mTORC1 and mTORC2, and to increase Raptor S722/S792 phosphorylation. Individually, all drugs increased phosphorylation of ATM S1981, AMPKα T172, ULK1 S317 and ATG13 S318 and reduced ULK1 S757 phosphorylation; this correlated with enhanced autophagic flux. Increased phosphorylation of ULK1 S317 and of Raptor S722/S792 required ATM-AMPK signaling. ULK1 S757 is a recognized site for mTORC1 and knock down of either ATM or AMPKα reduced the drug-induced dephosphorylation of this site. Combined exposure of cells to axitinib and an HDAC inhibitor significantly reduced the expression of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6 and HDAC7. No response was observed for HDACs 10 and 11. Knock down of ULK1, Beclin1 or ATG5 prevented the decline in HDAC expression, as did expression of a constitutively active mTOR protein. Axitinib combined with HDAC inhibitors enhanced expression of Class I MHCA and reduced expression of PD-L1 which was recapitulated via knock down studies, particularly of HDACs 1 and 3. In vivo, axitinib and the HDAC inhibitor entinostat interacted to significantly reduce tumor growth. Collectively our findings support the exploration of axitinib and HDAC inhibitors being developed as a novel sarcoma therapy.

scholarly journals oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading

2017 ◽  
Vol 313 (3) ◽  
pp. C340-C351 ◽  
Author(s):  
Chongxu Zhang ◽  
Crystal Adamos ◽  
Myung-Jin Oh ◽  
Jugajyoti Baruah ◽  
Manuela A. A. Ayee ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Kinase Inhibitor ◽  
Oxidized Ldl ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Aortic Endothelial Cells ◽  
Akt Phosphorylation ◽  
Downstream Target ◽  
Underlying Mechanisms

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs’ proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1. Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

scholarly journals Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K

2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Giulia Petroni ◽  
Giacomo Bagni ◽  
Jessica Iorio ◽  
Claudia Duranti ◽  
Tiziano Lottini ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Therapeutic Option ◽  
Autophagic Flux ◽  
Macromolecular Complex ◽  
Human Colorectal Cancer ◽  
Akt Phosphorylation ◽  
Channel Interaction ◽  
Dependent Cell ◽  
Resistance To Chemotherapy

AbstractWe have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K+ channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.

scholarly journals Volume-regulated Cl− current: contributions of distinct Cl− channels and localized Ca2+ signals

2019 ◽  
Vol 317 (3) ◽  
pp. C466-C480 ◽  
Author(s):  
Yani Liu ◽  
Huiran Zhang ◽  
Hongchao Men ◽  
Yuwei Du ◽  
Ziqian Xiao ◽  
...  
Keyword(s):  
Anion Channel ◽  
Fluorescent Imaging ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Anoctamin 1 ◽  
Component Cell ◽  
Intracellular Ca ◽  
Cl Channels ◽  
A Cell ◽  
Necessary And Sufficient

The swelling-activated chloride current ( ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl− channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

scholarly journals Serotonin transporter interacts with the PDGFβ receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells

2011 ◽  
Vol 300 (3) ◽  
pp. L486-L497 ◽  
Author(s):  
Wenying Ren ◽  
Stephanie W. Watts ◽  
Barry L. Fanburg
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Serotonin Transporter ◽  
Growth Factor Receptor ◽  
Hek293 Cells ◽  
Comparable Efficacy ◽  
Akt Phosphorylation ◽  
Genetic Ablation ◽  
Pdgf Signaling ◽  
Pulmonary Artery Smooth Muscle

The serotonin transporter (SERT) and the platelet-derived growth factor receptor (PDGFR) have been implicated in both clinical and experimental pulmonary hypertension (PH) and the facilitation of pulmonary artery smooth muscle cell (PASMC) growth. To gain a better understanding of the possible relationship of these two cell surface molecules we have explored interactions between SERT and PDGFR. We have previously demonstrated that SERT transactivates PDGFRβ in serotonin-stimulated PASMC proliferation. We now provide evidence for a role for SERT in PDGF-BB signaling and PASMC proliferation by using pharmacological inhibitors, genetic ablation, and construct overexpression of SERT. The results show that four tested SERT blockers dose dependently inhibit PDGF-stimulated human and bovine PASMC proliferation with comparable efficacy to that of PDGFR inhibitors, whereas 5-HT1B or 5-HT2A receptor inhibitors had no effect. Combinations of the SERT and PDGFR inhibitors led to synergistic/additive inhibition. Similarly, PDGF-induced PASMC proliferation was attenuated by small interfering RNA downregulation of SERT. Inhibition of SERT in PASMCs attenuated PDGF-induced phosphorylation of PDGFRβ, Akt, and p38 but not Erk. Overexpression of SERT in HEK293 cells led to enhanced Akt phosphorylation by PDGF, which was blunted by a SERT PDZ motif mutant, indicating the mechanistic need for the PDZ motif of SERT in PDGF signaling. Furthermore, coimmunoprecipitation experiments showed that SERT and PDGFRβ become physically associated upon PDGF stimulation. In total, the data show for the first time an important interactive relationship between SERT and the PDGFRβ in the production of PASMC proliferation triggered by PDGF that may be important in PH.

scholarly journals Regulation of Cloned Atp–Sensitive K Channels by Phosphorylation, Mgadp, and Phosphatidylinositol Bisphosphate (Pip2)

2000 ◽  
Vol 116 (3) ◽  
pp. 391-410 ◽  
Author(s):  
Bernard Ribalet ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Adenine Nucleotides ◽  
Katp Channels ◽  
Hek293 Cells ◽  
Slow Phase ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
Phosphatidylinositol Bisphosphate

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6.2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of KATP channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP2), and phosphorylation. Upon excision of inside-out patches into a Ca2+- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6.2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca2+ accelerated this process, suggesting a role for PIP2 hydrolysis mediated by a Ca2+-dependent phospholipase C. PIP2 could reactivate channel activity after a brief exposure to Ca2+, but not after prolonged exposure. However, in both cases, PIP2 reversed the increase in ATP sensitivity, indicating that PIP2 lowers the ATP sensitivity by increasing Po as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca2+ facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5–15 min, increased ATP sensitivity and loss of reactivation by PIP2 and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP2 responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP2 and phosphorylation.

scholarly journals Antibodies against nodo-paranodal proteins are not present in genetic neuropathies

Neurology ◽  
2020 ◽  
Vol 95 (4) ◽  
pp. e427-e433 ◽  
Author(s):  
Lorena Martín-Aguilar ◽  
Elba Pascual-Goñi ◽  
Cinta Lleixà ◽  
Marina Frasquet ◽  
Herminia Argente ◽  
...  
Keyword(s):  
Staining Pattern ◽  
Nerve Fibers ◽  
Immunoglobulin M ◽  
Hek293 Cells ◽  
Igg Antibodies ◽  
Routine Testing ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
A Cell ◽  
Tooth Disease

ObjectiveTo study the presence of nodal and paranodal immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies in patients with genetic neuropathies.MethodsA total of 108 patients with genetic neuropathies from 3 different centers were included. The presence of IgG and IgM antibodies against neurofascin-155 (NF155), nodal neurofascin (NF186 and NF140), and contactin-1 (CNTN1) were investigated with a cell-based assay (CBA) using immunocytochemistry in transfected HEK293 cells. Sera with positive or uncertain results were further tested by ELISA and immunohistochemistry in pig teased-nerve fibers.ResultsSix patients with Charcot-Marie-Tooth disease (CMT) had an uncertain staining pattern for IgM against nodal neurofascin that was not confirmed by ELISA. Two patients with CMT had an uncertain staining pattern for IgG against nodal neurofascin that was not confirmed by ELISA or immunohistochemistry. One patient with CMT with a confirmed GJB1 mutation tested positive for IgG against NF155 by CBA and ELISA (1/900), but was not confirmed by immunohistochemistry and was ultimately classified as negative.ConclusionsAntibodies against nodal or paranodal antigens were not detected in our cohort of patients with CMT, as previously reported. Some patients may falsely test positive for any of the techniques; confirmatory techniques should be incorporated into the routine testing.

scholarly journals Seizure-related 6 homolog like 2 autoimmunity

2020 ◽  
Vol 8 (1) ◽  
pp. e916
Author(s):  
Jon Landa ◽  
Mar Guasp ◽  
Mar Petit-Pedrol ◽  
Eugenia Martínez-Hernández ◽  
Jesús Planagumà ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Brain Mri ◽  
Index Patient ◽  
Clinical Syndrome ◽  
Extrapyramidal Symptoms ◽  
Hek293 Cells ◽  
Neuronal Cultures ◽  
Neurologic Disorders ◽  
A Cell ◽  
Csf Pleocytosis

ObjectiveTo describe the clinical syndrome of 4 new patients with seizure-related 6 homolog like 2 antibodies (SEZ6L2-abs), study the antibody characteristics, and evaluate their effects on neuronal cultures.MethodsSEZ6L2-abs were initially identified in serum and CSF of a patient with cerebellar ataxia by immunohistochemistry on rat brain sections and immunoprecipitation from rat cerebellar neurons. We used a cell-based assay (CBA) of HEK293 cells transfected with SEZ6L2 to test the serum of 95 patients with unclassified neuropil antibodies, 331 with different neurologic disorders, and 10 healthy subjects. Additional studies included characterization of immunoglobulin G (IgG) subclasses and the effects of SEZ6L2-abs on cultures of rat hippocampal neurons.ResultsIn addition to the index patient, SEZ6L2-abs were identified by CBA in 3/95 patients with unclassified neuropil antibodies but in none of the 341 controls. The median age of the 4 patients was 62 years (range: 54–69 years), and 2 were female. Patients presented with subacute gait ataxia, dysarthria, and mild extrapyramidal symptoms. Initial brain MRI was normal, and CSF pleocytosis was found in only 1 patient. None improved with immunotherapy. SEZ6L2-abs recognized conformational epitopes. IgG4 SEZ6L2-abs were found in all 4 patients, and it was the predominant subclass in 2. SEZ6L2-abs did not alter the number of total or synaptic SEZ6L2 or the AMPA glutamate receptor 1 (GluA1) clusters on the surface of hippocampal neurons.ConclusionsSEZ6L2-abs associate with a subacute cerebellar syndrome with frequent extrapyramidal symptoms. The potential pathogenic effect of the antibodies is not mediated by internalization of the antigen.

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