TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation

ABSTRACT DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies—chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo. This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

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Acid and neutral sphingomyelinases: roles and mechanisms of regulation

Biochemistry and Cell Biology ◽

10.1139/o03-091 ◽

2004 ◽

Vol 82 (1) ◽

pp. 27-44 ◽

Cited By ~ 224

Author(s):

Norma Marchesini ◽

Yusuf A Hannun

Keyword(s):

Nitric Oxide ◽

Molecular Mechanisms ◽

Caveolin 1 ◽

Niemann Pick Disease ◽

Extracellular Stimuli ◽

Niemann Pick ◽

Hydrolysis Of ◽

Pick Disease

Ceramide, an emerging bioactive lipid and second messenger, is mainly generated by hydrolysis of sphingomyelin through the action of sphingomyelinases. At least two sphingomyelinases, neutral and acid sphingo myelinases, are activated in response to many extracellular stimuli. Despite extensive studies, the precise cellular function of each of these sphingomyelinases in sphingomyelin turnover and in the regulation of ceramide-mediated responses is not well understood. Therefore, it is essential to elucidate the factors and mechanisms that control the activation of acid and neutral sphingomyelinases to understand their the roles in cell regulation. This review will focus on the molecular mechanisms that regulate these enzymes in vivo and in vitro, especially the roles of oxidants (glu ta thi one, peroxide, nitric oxide), proteins (saposin, caveolin 1, caspases), and lipids (diacylglycerol, arachidonic acid, and ceramide).Key words: sphingomyelinase, ceramide, apoptosis, Niemann-Pick disease, FAN (factor associated with N-SMase activation).

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Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.401-411.1991 ◽

1991 ◽

Vol 11 (1) ◽

pp. 401-411

Author(s):

S Cuthill ◽

A Wilhelmsson ◽

L Poellinger

Keyword(s):

Dna Binding ◽

Cellular Response ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Rank Order ◽

Receptor Ligands ◽

Free System ◽

Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

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Increased nuclear translocation of catalytically active PKC-ζ during mouse colonocyte hyperproliferation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.1.g223 ◽

2000 ◽

Vol 279 (1) ◽

pp. G223-G237 ◽

Cited By ~ 16

Author(s):

Shahid Umar ◽

Joseph H. Sellin ◽

Andrew P. Morris

Keyword(s):

Kinase Activity ◽

Nuclear Translocation ◽

Physiological Role ◽

Subcellular Fractionation ◽

Nuclear Antigen ◽

Nuclear Staining ◽

Catalytically Active ◽

Pkc Ζ

Protein kinase (PK) C-ζ is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-ζ, 3.2-fold), 48-kDa catalytic subunit (PKM-ζ, 3- to 9-fold), and 24-kDa membrane-bound fragment (Mf-ζ, >10-fold) expression. Both PKC-ζ and PKM-ζ exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-ι/PKM-ι expression occurred. The subcellular distribution of immunoreactive PKC-ζ changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-ζ and PKM-ζ expression and activity within nuclei, which preferentially accumulated PKM-ζ. These results suggest separate cellular and nuclear roles, respectively, for PKC-ζ in quiescent and mitotically active colonocytes. PKM-ζ may specifically act as a modulator of proliferation during TMCH.

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Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽

10.1093/brain/awz202 ◽

2019 ◽

Vol 142 (8) ◽

pp. 2352-2366 ◽

Cited By ~ 15

Author(s):

Guo-zhong Yi ◽

Guanglong Huang ◽

Manlan Guo ◽

Xi’an Zhang ◽

Hai Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Progression Free Survival ◽

Recurrent Glioblastoma ◽

Dna Lesions ◽

Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

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CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

Neuro-Oncology ◽

10.1093/neuonc/noz175.185 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi47-vi47

Author(s):

Marilin Koch ◽

Stefan Czemmel ◽

Felix Lennartz ◽

Sarah Beyeler ◽

Justyna Przystal ◽

...

Keyword(s):

Molecular Mechanisms ◽

Nuclear Translocation ◽

Clonogenic Survival ◽

Dominant Negative ◽

Molecular Profile ◽

Transcriptional Networks ◽

Molecular Alterations ◽

Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

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Caveolin-1-Mediated Tumor Suppression Is Linked to Reduced HIF1α S-Nitrosylation and Transcriptional Activity in Hypoxia

Cancers ◽

10.3390/cancers12092349 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2349

Author(s):

Carlos Sanhueza ◽

Jimena Castillo Bennett ◽

Manuel Valenzuela-Valderrama ◽

Pamela Contreras ◽

Lorena Lobos-González ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Tumor Suppression ◽

Transcriptional Activity ◽

Nuclear Translocation ◽

Caveolin 1 ◽

Oxide Synthase ◽

E Cadherin

Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.

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β2-AR Activation promotes cleavage and nuclear translocation of Her2 and metastatic potential of cancer cells

10.21203/rs.3.rs-17687/v1 ◽

2020 ◽

Author(s):

Dan Liu ◽

Xiyue Xu ◽

Shuci Liu ◽

Xuan Zhao ◽

Anqun Tang ◽

...

Keyword(s):

Cancer Cells ◽

Signaling Pathways ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Biological Response ◽

Metastatic Potential ◽

Adrenergic Signaling ◽

Signaling Activation

Abstract Background The prolonged hypersecretion of catecholamine induced by chronic stress may correlate with various steps of malignant progression of cancer and β2-AR overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins such as Her2. Crosstalk of the cell signaling pathways mediated by β2-AR and Her2 may promote a stronger or more sustained biological response. However, the molecular mechanisms underlying cross-communication between β2-AR and Her2 mediated signaling pathways are not fully understood. Methods In this study, the effects of adrenergic signaling on Her2 cleavage were evaluated by various assays, such as western blot, immunofluorescence and immunohistochemistry. In order to reveal the mechanism about Her2 cleavage triggered by β2-AR activation, the molecular and pharmacological means were employed. By using in vitro and in vivo assay, the influences of the crosstalk between β2-AR and Her2 on the bio-behaviors of tumor cells were demonstrated. Results Our data demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR-199a-5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ-secretase, leading to shedding of Her2 ECD by ADAM10 and subsequent intramembranous cleavage of Her2 ICD by presenilin-dependent γ-secretase, nuclear translocation of Her2 ICD and enhanced transcription of tumor metastasis-associated gene COX-2 . Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, the nuclear localization of Her2 was significantly correlated with overexpression of β2-AR in human breast cancer tissues. Conclusion This study illustrates that adrenergic signaling activation triggers Her2 cleavage, resulting in enhanced invasive and metastasis activities of cancer cells. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by β2-AR activation controls a novel Her2-mediated signaling transduction under physiological and pathological conditions.

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High glucose promotes nascent nephron apoptosis via NF-κB and p53 pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00361.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. F147-F156 ◽

Cited By ~ 16

Author(s):

Yun-Wen Chen ◽

Isabelle Chenier ◽

Shiao-Ying Chang ◽

Stella Tran ◽

Julie R. Ingelfinger ◽

...

Keyword(s):

High Glucose ◽

Nuclear Translocation ◽

Dominant Negative ◽

In Vivo Studies ◽

Ros Generation ◽

Small Interfering Rnas ◽

Kidney Size ◽

P53 Signaling

A hyperglycemic environment in utero reduces kidney size and nephron number due to nascent nephron apoptosis. However, the underlying mechanisms are incompletely understood. The present study investigated whether the nascent nephron apoptosis promoted by high glucose is mediated via the transcription factor NF-κB and p53 signaling pathways. Neonatal mouse kidneys from the offspring of nondiabetic, diabetic, and insulin-treated diabetic dams were used for in vivo studies, and MK4 cells, an embryonic metanephric mesenchymal (MM) cell line, were used for in vitro studies. Neonatal kidneys of the offspring of diabetic mothers exhibited an increased number of apoptotic cells and reactive oxygen species (ROS) generation, enhanced NF-κB activation, and nuclear translocation of its subunits (p50 and p65 subunits) as well as phosphorylation (Ser 15) of p53 compared with kidneys of offspring of nondiabetic mothers. Insulin treatment of diabetic dams normalized these parameters in the offspring. In vitro, high-glucose (25 mM) induced ROS generation and significantly increased MK4 cell apoptosis and caspase-3 activity via activation of NF-κB pathway, with p53 phosphorylation and nuclear translocation compared with normal glucose (5 mM). These changes in a high-glucose milieu were prevented by transient transfection of small interfering RNAs for dominant negative IκBα or IKK or p53. Our data demonstrate that high glucose-induced nascent nephron apoptosis is mediated, at least in part, via ROS generation and the activation of NF-κB and p53 pathways.

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PRMT5 regulates cell pyroptosis by silencing CASP1 in multiple myeloma

Cell Death and Disease ◽

10.1038/s41419-021-04125-5 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Tian Xia ◽

Ming Liu ◽

Quan Zhao ◽

Jian Ouyang ◽

Bing Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Molecular Mechanisms ◽

Hematological Malignancies ◽

Bioinformatics Analysis ◽

Histone H4 ◽

Poor Overall Survival ◽

Arginine Methyltransferase ◽

Cell Progression

AbstractProtein arginine methyltransferase 5 (PRMT5), a histone methyltransferase responsible for the symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s), is an enzyme that participates in tumor cell progression in a variety of hematological malignancies. However, the biological functions of PRMT5 in multiple myeloma (MM) and the underlying molecular mechanisms remain unclear. In this study, we conducted a bioinformatics analysis and found that PRMT5 expression was significantly upregulated in MM. In vitro and in vivo phenotypic experiments revealed that knockdown of PRMT5 expression enhanced cell pyroptosis in MM. Moreover, we found that CASP1 expression was negatively correlated with PRMT5 expression, and repressing PRMT5 expression rescued both the phenotype and expression markers (N-GSDMD, IL-1b, and IL-18). Inhibition of PRMT5 activity increased CASP1 expression and promoted MM cell pyroptosis. Finally, high expression of PRMT5 or low expression of CASP1 was correlated with poor overall survival in MM. Collectively, our results provide a mechanism by which PRMT5 regulates cell pyroptosis by silencing CASP1 in MM.

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