Classical and Novel TSPO Ligands for the Mitochondrial TSPO Can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling.

It is known that knockdown of the mitochondrial 18 kDa translocator protein (TSPO) as well as TSPO ligands modulate various functions, including functions related to cancer. To study the ability of TSPO to regulate gene expression regarding such functions, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 15 min, the classical TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. These changes also included gene products that are part of the canonical pathway serving to modulate general gene expression. These changes are in accord with real-time, reverse transcriptase (RT) PCR. At the time points of 15, 30, 45, and 60 min, as well as 3 and 24 h of PK 11195 exposure, the functions associated with the changes in gene expression in these glioblastoma cells covered well known TSPO functions. These functions included cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was corroborated microscopically for cell migration, cell accumulation, adhesion, and neuronal differentiation. Changes in gene expression at 24 h of PK 11195 exposure were related to downregulation of tumorigenesis and upregulation of programmed cell death. In the vehicle treated as well as PK 11195 exposed cell cultures, our triple labeling showed intense TSPO labeling in the mitochondria but no TSPO signal in the cell nuclei. Thus, mitochondrial TSPO appears to be part of the mitochondria-to-nucleus signaling pathway for modulation of nuclear gene expression. The novel TSPO ligand 2-Cl-MGV-1 appeared to be very specific regarding modulation of gene expression of immediate early genes and transcription factors.

Novel TSPO Ligand 2-Cl-MGV-1 Can Counteract Lipopolysaccharide Induced Inflammatory Response in Murine RAW264.7 Macrophage Cell Line and Lung Models.

We assessed the anti-inflammatory activity of the TSPO ligand 2-Cl-MGV-1. Lipopolysaccharide (LPS) was used to induce inflammatory response in a murine RAW264.7 macrophage model (LPS: 100 ng/mL) and a mouse model (C57BL/6) of lung inflammation (LPS: 5 mg/kg). In the macrophage model, the presence of 2-Cl-MGV-1 (25 µM) caused the LPS-induced elevation in nitrite levels to decrease by 70% (p < 0.0001) and interleukin (IL)-6 by 50% (p < 0.05). In the mouse model, 2-Cl-MGV-1, administered 30 min before, or co-administered with, an LPS injection, significantly inhibited the elevation in serum IL-5 levels (both by 65%; p < 0.001 and p < 0.01, respectively). 2-Cl-MGV-1 administration to mice 30 min before LPS injection and 1 h thereafter significantly inhibited the elevation in IL-1ß serum levels (both by 63%, p < 0.005). IL-6 elevation was inhibited by 73% (p < 0.005) when 2-Cl-MGV-1 was administered 30 min before LPS, by 60% (p < 0.05) when co-administered with LPS, and by 64% (p < 0.05) when administered 1 h after LPS. All cytokine assessments were conducted 6 h post LPS injection. Histological analyses showed decreased leukocyte adherence in the lung tissue of the ligand-treated mice. 2-Cl-MGV-1 administration 30 min prior to exposure to LPS inhibited inflammation-induced open field immobility. The beneficial effect of 2-Cl-MGV-1 suggests its potential as a therapeutic option for inflammatory diseases.

Inhibitory Effects of the Two Novel TSPO Ligands 2-Cl-MGV-1 and MGV-1 on LPS-induced Microglial Activation.

The 18 kDa translocator protein (TSPO) ligands 2-Cl-MGV-1 and MGV-1 can attenuate cell death of astrocyte-like cells (U118MG) and induce differentiation of neuronal progenitor cells (PC-12). Lipopolysaccharide (LPS) is a bacterial membrane endotoxin that activates cellular inflammatory pathways by releasing pro-inflammatory molecules, including cytokines and chemokines. The aim of the present study was to assess the immuno-modulatory effect of TSPO ligands in activated microglial cells. We demonstrated that the TSPO ligands 2-Cl-MGV-1 and MGV-1 can prevent LPS-induced activation of microglia (BV-2 cell line). Co-treatment of LPS (100 ng/mL) with these TSPO ligands (final concentration- 25 µM) reduces significantly the LPS-induced release of interleukin-6 (IL-6) from 16.9-fold to 2.5-fold, IL-ß from 8.3-fold to 1.6-fold, interferon-γ from 16.0-fold to 2.2-fold, and tumor necrosis factor-α from 16.4-fold to 1.8-fold. This anti-inflammatory activity seems to be achieved by inhibition of NF-κB p65 activation. Assessment of initiation of ROS generation and cell metabolism shows significant protective effects of these two novel TSPO ligands. The IL-10 and IL-13 levels were not affected by any of the TSPO ligands. Thus, it appears that the ligands suppress the LPS-induced activation of some inflammatory responses of microglia. Such immunomodulatory effects may be relevant to the pharmacotherapy of neuro-inflammatory diseases.