In vitro Effects of the Specific Mitochondrial TSPO Ligand Ro5 4864 in Cultured Human Osteoblasts

2017 ◽  
Vol 126 (02) ◽  
pp. 77-84 ◽  
Author(s):  
Nahum Rosenberg ◽  
Orit Rosenberg ◽  
Abraham Weizman ◽  
Leo Veenman ◽  
Moshe Gavish
Keyword(s):  
Protein Expression ◽  
Cellular Proliferation ◽  
Protoporphyrin Ix ◽  
Anion Channel ◽  
Translocator Protein ◽  
Mitochondrial Activity ◽  
Voltage Dependent Anion Channel ◽  
Hexokinase 2 ◽  
Human Osteoblasts

AbstractThe 18 kDa mitochondrial translocator protein (TSPO) ligands (10 µM), e. g., protoporphyrin IX, PK 11195 and FGIN-1-27, have different effects on metabolism and protein expression in human osteoblasts. In this study, we investigated the archetypical TSPO specific ligand Ro5-4864 (10 µM) effect in primary osteoblasts in culture aiming to further understand the TSPO role in these mature metabolically active cells.We found that following exposure to Ro5-4864, cellular [18F]-FDG incorporation and ATP content were reduced by 48% (p<0.001) and 44% (p<0.001), respectively. The mitochondrial membrane potential (ΔΨm) increased by 50% (p<0.01), mRNA synthesis of TSPO and voltage dependent anion channel (VDAC1) decreased both by 70%, the TSPO and VDAC1 protein expression decreased by 80% and 68%, respectively (p<0.001). Ro5 4864 caused a decrease in the proportion of cells in the G1 phase (by 20%, p<0.05), shifting the cell cycle to the S and G2/M phases. Furthermore, 63% decrease in hexokinase 2 protein expression (p<0.001) was found. However, we found no significant effects on hexokinase 2 mRNA expression (by RT-PCR). We also did not see significant changes in mitochondrial mass (MitoTracker Green assay), apoptosis rate (TUNEL assay), overall cell death (LDH assay), cellular proliferation (BrdU assay), cell maturation (cellular alkaline phosphatase assay), and the number of cells in the culture.Therefore, an overall effect of Ro5-4864 exhorts is via pathways related to the mitochondrial activity, which is only partly like PK 11195, but not to the other TSPO ligands.

TSPO is a REDOX regulator of cell mitophagy

2015 ◽  
Vol 43 (4) ◽  
pp. 543-552 ◽  
Author(s):  
Jemma Gatliff ◽  
Michelangelo Campanella
Keyword(s):  
Cell Biology ◽  
Anion Channel ◽  
Translocator Protein ◽  
Control Mechanisms ◽  
Voltage Dependent Anion Channel ◽  
Core Element ◽  
Functional Link ◽  
Positron Emission

The mitochondrial 18-kDa translocator protein (TSPO) was originally discovered as a peripheral binding site of benzodiazepines to be later described as a core element of cholesterol trafficking between cytosol and mitochondria from which the current nomenclature originated. The high affinity it exhibits with chemicals (i.e. PK11195) has generated interest in the development of mitochondrial based TSPO-binding drugs for in vitro and in vivo analysis. Increased TSPO expression is observed in numerous pathologies such as cancer and inflammatory conditions of the central nervous system (CNS) that have been successfully exploited via protocols of positron emission tomography (PET) imaging. We endeavoured to dissect the molecular role of TSPO in mitochondrial cell biology and discovered a functional link with quality control mechanisms operated by selective autophagy. This review focuses on the current understanding of this pathway and focuses on the interplay with reactive oxygen species (ROS) and the voltage-dependent anion channel (VDAC), to which TSPO binds, in the regulation of cell mitophagy and hence homoeostasis of the mitochondrial network as a whole.

scholarly journals TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria

2016 ◽  
Vol 473 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Jemma Gatliff ◽  
Michelangelo Campanella
Keyword(s):  
Neurological Diseases ◽  
Anion Channel ◽  
Translocator Protein ◽  
Endocrine Regulation ◽  
Voltage Dependent Anion Channel ◽  
Steroid Synthesis ◽  
Cellular Processes ◽  
Cellular Events ◽  
Voltage Dependent

The 18-kDa translocator protein (TSPO) localizes in the outer mitochondrial membrane (OMM) of cells and is readily up-regulated under various pathological conditions such as cancer, inflammation, mechanical lesions and neurological diseases. Able to bind with high affinity synthetic and endogenous ligands, its core biochemical function resides in the translocation of cholesterol into the mitochondria influencing the subsequent steps of (neuro-)steroid synthesis and systemic endocrine regulation. Over the years, however, TSPO has also been linked to core cellular processes such as apoptosis and autophagy. It interacts and forms complexes with other mitochondrial proteins such as the voltage-dependent anion channel (VDAC) via which signalling and regulatory transduction of these core cellular events may be influenced. Despite nearly 40 years of study, the precise functional role of TSPO beyond cholesterol trafficking remains elusive even though the recent breakthroughs on its high-resolution crystal structure and contribution to quality-control signalling of mitochondria. All this along with a captivating pharmacological profile provides novel opportunities to investigate and understand the significance of this highly conserved protein as well as contribute the development of specific therapeutics as presented and discussed in the present review.

In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein

2013 ◽  
Vol 45 (4) ◽  
pp. 333-341 ◽  
Author(s):  
Nahum Rosenberg ◽  
Orit Rosenberg ◽  
Abraham Weizman ◽  
Leo Veenman ◽  
Moshe Gavish
Keyword(s):  
Protoporphyrin Ix ◽  
Translocator Protein ◽  
Human Osteoblast

scholarly journals Involvement of the IP3R-Grp75-VDAC1-MCU calcium axis in proteinuria in adriamycin-induced nephropathy rats

2019 ◽  
Author(s):  
Sisi Wang ◽  
Han Xu ◽  
Na Guan ◽  
Qijiao Wei ◽  
Yinghong Tao ◽  
...  
Keyword(s):  
Anion Channel ◽  
Ruthenium Red ◽  
Voltage Dependent Anion Channel ◽  
Podocyte Injury ◽  
Inositol 1 ◽  
Calcium Uniporter ◽  
Voltage Dependent ◽  
Trisphosphate Receptor ◽  
Glucose Regulated Protein

Abstract Background Podocyte injury plays a key role in the development of proteinuria. We previously found that the intracellular inositol 1, 4, 5-trisphosphate receptor (IP3R)- glucose-regulated protein 75 (Grp75)- voltage dependent anion channel 1 (VDAC1)- mitochondrial calcium uniporter (MCU) calcium axis contributes to podocyte injury in cultured mouse podocytes. Objective This study investigated whether the IP3R-Grp75-VDAC1-MCU calcium axis is involved in the development and improvement of proteinuria in nephropathy rats.Methods The expression of members of the IP3R-Grp75-VDAC1-MCU calcium axis in the renal cortex of a previously established adriamycin (ADR)-induced nephropathy rat model and cultured mouse podocytes was investigated by western blot analysis and immunohistochemical staining. The effects of ruthenium red (RR), an MCU inhibitor, oninteractions in the IP3R-Grp75-VDAC1-MCU calcium axis were investigated by in vitro co-immunoprecipitation assays.Results The overexpression and inhibition of members of the glomerular IP3R-Grp75-VDAC1-MCU calcium axis were accompanied by the development and improvement of proteinuria, respectively, in nephropathy rats. RR inhibited the upregulation of members of the IP3R-Grp75-VDAC1-MCU calcium axis induced by ADR and their interactions. Conclusions The IP3R-Grp75-VDAC1-MCU calcium axis is involved in proteinuria in ADR-induced nephropathy and can be inhibited by RR.

scholarly journals Altered skeletal muscle microtubule-mitochondrial VDAC2 binding is related to bioenergetic impairments after paclitaxel but not vinblastine chemotherapies

2019 ◽  
Vol 316 (3) ◽  
pp. C449-C455 ◽  
Author(s):  
Sofhia V. Ramos ◽  
Meghan C. Hughes ◽  
Christopher G. R. Perry
Keyword(s):  
Skeletal Muscle ◽  
Direct Interaction ◽  
Permeability Transition Pore ◽  
Anion Channel ◽  
Permeability Transition ◽  
Mitochondrial Bioenergetics ◽  
Voltage Dependent Anion Channel ◽  
Retention Capacity ◽  
Microtubule Stabilization

Microtubule-targeting chemotherapies are linked to impaired cellular metabolism, which may contribute to skeletal muscle dysfunction. However, the mechanisms by which metabolic homeostasis is perturbed remains unknown. Tubulin, the fundamental unit of microtubules, has been implicated in the regulation of mitochondrial-cytosolic ADP/ATP exchange through its interaction with the outer membrane voltage-dependent anion channel (VDAC). Based on this model, we predicted that disrupting microtubule architecture with the stabilizer paclitaxel and destabilizer vinblastine would impair skeletal muscle mitochondrial bioenergetics. Here, we provide in vitro evidence of a direct interaction between both α-tubulin and βII-tubulin with VDAC2 in untreated single extensor digitorum longus (EDL) fibers. Paclitaxel increased both α- and βII-tubulin-VDAC2 interactions, whereas vinblastine had no effect. Utilizing a permeabilized muscle fiber bundle preparation that retains the cytoskeleton, paclitaxel treatment impaired the ability of ADP to attenuate H2O2 emission, resulting in greater H2O2 emission kinetics. Despite no effect on tubulin-VDAC2 binding, vinblastine still altered mitochondrial bioenergetics through a surprising increase in ADP-stimulated respiration while also impairing ADP suppression of H2O2 and increasing mitochondrial susceptibility to calcium-induced formation of the proapoptotic permeability transition pore. Collectively, these results demonstrate that altering microtubule architecture with chemotherapeutics disrupts mitochondrial bioenergetics in EDL skeletal muscle. Specifically, microtubule stabilization increases H2O2 emission by impairing ADP sensitivity in association with greater tubulin-VDAC binding. In contrast, decreasing microtubule abundance triggers a broad impairment of ADP’s governance of respiration and H2O2 emission as well as calcium retention capacity, albeit through an unknown mechanism.

Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl−secretion

2000 ◽  
Vol 278 (5) ◽  
pp. G753-G764 ◽  
Author(s):  
Shahid Umar ◽  
Jason Scott ◽  
Joseph H. Sellin ◽  
William P. Dubinsky ◽  
Andrew P. Morris
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Cellular Proliferation ◽  
Neck Region ◽  
Anion Channel ◽  
Cellular Level ◽  
Cystic Fibrosis Gene ◽  
Mrna And Protein Expression

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl−secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl−secretion (2.3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl−current.

scholarly journals A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia

2020 ◽  
Vol 57 (11) ◽  
pp. 4467-4487
Author(s):  
Meredith K. Loth ◽  
Sara R. Guariglia ◽  
Diane B. Re ◽  
Juan Perez ◽  
Vanessa Nunes de Paiva ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Redox Homeostasis ◽  
Anion Channel ◽  
Translocator Protein ◽  
Lower Amount ◽  
Proximity Ligation Assay ◽  
Ros Production ◽  
Voltage Dependent Anion Channel ◽  
Murine Microglia ◽  
Translocator Protein 18 Kda

Abstract In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Gerstmann-Straüssler-Scheinker PRNP P102L-129V mutation

2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Isidro Ferrer ◽  
Margarita Carmona ◽  
Rosa Blanco ◽  
Maria Recio ◽  
R. San Segundo
Keyword(s):  
Oxidative Stress ◽  
Spinal Cord ◽  
Stress Responses ◽  
Anion Channel ◽  
Dystrophic Neurites ◽  
Voltage Dependent Anion Channel ◽  
Western Blots ◽  
Abnormal Accumulation ◽  
Stress Damage

AbstractNeuropathological and biochemical studies in a case of Gerstmann-Straüssler-Scheinker disease bearing the PRNP P102L-129V mutation showed numerous multicentric PrPres in the cerebral cortex, striatum, thalamus and cerebellum, PrPres globular deposits in the anterior and posterior horns of the spinal cord, and multiple granular PrPres deposits in the grey and white matter of the encephalon and spinal cord. Western blots with antiPrPres antibodies revealed several weak bands ranging from 36 to 66 kDa, weak bands of 29 and 24 kDa, a strong band of about 20 kDa, a low band of molecular weight around 15 kDa and a weaker band of about 7 kDa. Spongiform degeneration was absent. Hyper-phosphorylated 3R and 4R tau occurred in dystrophic neurites surrounding PrPres plaques, neuropil threads and, to a lesser degree, in the form of neurofibrillary tangles. Gel electrophoresis of sarkosyl-insoluble fractions and western blotting with anti-phospho-tau antibodies showed a pattern similar to that seen in Alzheimer disease cases run in parallel. Dystrophic neurites in the vicinity of PrPres plaques were enriched in voltage dependent anion channel thus suggesting abnormal accumulation of mitochondria. These changes were associated with increased oxidative damage in neurons and astrocytes, Finally, increased expression of active stress kinases, that have the capacity to phosphorylate tau in vitro, p38 (p-38-P) and SAPK/ JNK (SAPK/JNK-P) was found in cell processes surrounding PrP plaques. Together, these observations provide evidences of mitochondrial abnormalities, and increased oxidative stress damage and oxidative stress responses in GSS bearing the PRNP P102L-129V mutation.

scholarly journals Redox-dependent gating of VDAC by mitoNEET

2019 ◽  
Vol 116 (40) ◽  
pp. 19924-19929 ◽  
Author(s):  
Colin H. Lipper ◽  
Jason T. Stofleth ◽  
Fang Bai ◽  
Yang-Sung Sohn ◽  
Susmita Roy ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Anion Channel ◽  
Fatty Acid Transport ◽  
Dependent Manner ◽  
Outer Mitochondrial Membrane ◽  
Voltage Dependent Anion Channel ◽  
Ros Homeostasis ◽  
Parkinson’S Diseases ◽  
Voltage Dependent

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson’s diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a “governator” sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC’s flow of metabolites.

scholarly journals Rewiring of Cancer Cell Metabolism by Mitochondrial VDAC1 Depletion Results in Time-Dependent Tumor Reprogramming: Glioblastoma as a Proof of Concept

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1330 ◽  
Author(s):  
Arif ◽  
Stern ◽  
Pittala ◽  
Chalifa-Caspi ◽  
Shoshan-Barmatz
Keyword(s):  
Cancer Cell ◽  
Cell Metabolism ◽  
Anion Channel ◽  
Translocator Protein ◽  
Global Changes ◽  
Voltage Dependent Anion Channel ◽  
Oncogenic Signaling ◽  
Neuronal Markers ◽  
Long Period

Reprograming of the metabolism of cancer cells is an event recognized as a hallmark of the disease. The mitochondrial gatekeeper, voltage-dependent anion channel 1 (VDAC1), mediates transport of metabolites and ions in and out of mitochondria, and is involved in mitochondria-mediated apoptosis. Here, we compared the effects of reducing hVDAC1 expression in a glioblastoma xenograft using human-specific si-RNA (si-hVDAC1) for a short (19 days) and a long term (40 days). Tumors underwent reprograming, reflected in rewired metabolism, eradication of cancer stem cells (CSCs) and differentiation. Short- and long-term treatments of the tumors with si-hVDAC1 similarly reduced the expression of metabolism-related enzymes, and translocator protein (TSPO) and CSCs markers. In contrast, differentiation into cells expressing astrocyte or neuronal markers was noted only after a long period during which the tumor cells were hVDAC1-depleted. This suggests that tumor cell differentiation is a prolonged process that precedes metabolic reprograming and the “disappearance” of CSCs. Tumor proteomics analysis revealing global changes in the expression levels of proteins associated with signaling, synthesis and degradation of proteins, DNA structure and replication and epigenetic changes, all of which were highly altered after a long period of si-hVDAC1 tumor treatment. The depletion of hVDAC1 greatly reduced the levels of the multifunctional translocator protein TSPO, which is overexpressed in both the mitochondria and the nucleus of the tumor. The results thus show that VDAC1 depletion-mediated cancer cell metabolic reprograming involves a chain of events occurring in a sequential manner leading to a reversal of the unique properties of the tumor, indicative of the interplay between metabolism and oncogenic signaling networks.

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