Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

UNLABELLED: Chronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. SIGNIFICANCE: This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration.

Electron microscope tomography of native membranes.

Membrane proteins are often present in low amounts in cells. Their function can be modulated by interactions with other proteins. Moreover, these complexes can be transiently formed, thus making them difficult to be isolated and to be purified. One way to overcome these difficulties is to visualize these complexes in situ in the cells. For such purpose, electron microscopy coupled to tomography is a promising approach that has been developed over the last decades.Mitochondria are a good example of organelles where many membrane proteins form different functional complexes within the outer and the inner membranes. The latter is either close to the former or projects within the matrix to form cristae. Structure of these cristae involves different proteins and can vary from lamellar to tubular forms in normal mitochondria. In pathological conditions, other mitochondrial morphologies have been described, for instance, vesicular structures for inner boundary membrane have been observed.

Overexpression of translocator protein in inflammatory bowel disease: potential diagnostic and treatment value.

BACKGROUND: Inflammatory bowel diseases (IBDs), such as ulcerative colitis and Crohn's disease, are chronic inflammatory disorders that increase the risk for colorectal cancer. The mitochondrial translocator protein (TSPO) is a high-affinity drug- and cholesterol-binding protein expressed in the colon and its expression is increased in colon cancers. The aim of this study was to investigate TSPO expression in IBD biopsies and to establish an animal model of IBD to examine the role of TSPO. In addition, we evaluated the potential use of TSPO drug ligands in diagnosing and treating IBD. METHODS: TSPO expression in IBD biopsies was evaluated using immunohistochemistry. IBD was induced in a rat experimental model via treatment with dextran sodium sulfate (DSS). Colon morphology, TSPO expression, and proinflammatory cytokine production were evaluated in addition to the effect of TSPO drug ligands on disease pathology. RESULTS: TSPO protein levels were elevated in the enterocytes of IBD biopsies. TSPO expression was localized to the enterocyte mitochondria. DSS treatment induced a time-dependent phenotype mimicking IBD with tissue injury and subsequent tissue regeneration. Coadministration of DSS and the TSPO drug ligands PK 11195 or Ro5-4864 increased both the rate of colon ulceration and regeneration, whereas administration of the TSPO drug ligand flunitrazepam partially prevented this pathology. These data correlated with changes in proinflammatory cytokine plasma levels, as well as increased cytokine production and secretion from the colon. CONCLUSIONS: TSPO may serve as a marker of the IBD repair process, and TSPO drug ligands should be further evaluated for IBD treatment.

Distribution, pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine.

BACKGROUND INFORMATION: The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high-affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. RESULTS: TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam-binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5-4864 and protoporphyrin IX showed 5-13-fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage-dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium-dependent chloride secretion in the duodenum and calcium-dependent chloride absorption in the ileum, but did not affect jejunum ion transport. CONCLUSIONS: The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands.

BACKGROUND INFORMATION: TSPO (translocator protein), previously known as PBR (peripheral-type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High-affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. RESULTS: Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam-binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, (3)H-labelled PK 11195, as shown by B(max) and K(d) values of 10.0+/-0.5 pmol/mg and 4.0+/-1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and alpha-adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K(+), Na(+), Cl(-) and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. CONCLUSIONS: High-affinity ligand binding to mitochondrial TSPO modulates neurotransmitter-induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.

Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl- secretion in HT-29 human colon cancer cells.

BACKGROUND INFORMATION: TSPO (translocator protein), known previously as PBR (peripheral-type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over-expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized. RESULTS: We investigated the expression and function of TSPO in the human colon cancer cells HT-29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam-binding inhibitor, in the cytosol. Radioligand binding studies using the specific high-affinity drug ligand [(3)H]PK 11195 and membrane fraction demonstrated saturable binding, with K(d) and B(max) values of 13.5+/-1.5 nM and 10.1+/-1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose-dependent rise in intracellular [Ca(2+)], which was unaffected by extracellular Ca(2+), but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT-29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca(2+)-dependent transepithelial Cl(-) secretion. This secretion was inhibited: (i) after removal of extracellular Cl(-); (ii) by apical addition of the Cl(-) channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate]; and (iii) by basolateral addition of the Na(+)-K(+)-2Cl(-) co-transporter inhibitor bumetanide. Furthermore, the intracellular Ca(2+) chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195-induced Cl(-) secretion. CONCLUSIONS: These findings indicate that TSPO is located in mitochondrial membranes of HT-29 and reveal that its activation induces a rise in cytosolic Ca(2+), leading to the stimulation of Cl(-) secretion.

In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding.

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.