Abstract
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.
Highlights
Various studies have shown that the 18 kDa translocator protein (TSPO) is involved in numerous functions, including glioblastoma tumorigenicity in its various aspects, e.g., cell proliferation, cell viability, etc. [1,2,3,4,5]
The presentation of the Results is divided into 5 parts: (1) PK 11195 effects on gene expression in general; (2) Potential effects of such gene expression changes on function, uncovered by pathway analysis; (3) Microscopic correlates at cellular and intracellular levels in association with changes in gene expression due to PK 11195 exposure; (4) Actual observations of phenotypic effects of PK 11195 exposure that were predicted by pathway analysis; (5) Comparison with effects of a more advanced TSPO ligand (2-Cl-MGV-1) on gene expression
To answer the question whether nuclear gene expression can be modulated via the mitochondrial TSPO: (i) we first of all studied the effects of the classical TSPO ligand PK 11195 on gene expression in general, in a time dependent fashion. (ii) We applied pathway analysis to predict potential functional implications. (iii) We designed microscopic studies to study whether and how TSPO location could be associated with modulation of gene expression i.e., to determine whether location of TSPO could change between mitochondria and nucleus. (iv) Microscopy was applied to study whether functions were modulated as predicted by our pathway analysis. (v) comparisons with effects on gene expression by a more advanced TSPO ligand 2-Cl-MGV-1 were undertaken
Summary
Various studies have shown that the 18 kDa translocator protein (TSPO) is involved in numerous functions, including glioblastoma tumorigenicity in its various aspects, e.g., cell proliferation, cell viability, etc. [1,2,3,4,5]. By regulating ROS generation, ∆ψm transitions, ATP production, Ca2+ release, and NADH levels, TSPO may be a key mitochondrial protein providing mitochondria the capability to regulate gene expression in the cell nucleus. Apart from targeting the immediate question of regulation of nuclear gene expression by TSPO ligands, we address the question whether modulation of gene expression may be associated with well-known TSPO functions This was another reason to focus on PK 11195 at 25 μM, as by application of this concentration we expected to be better able to connect the present gene expression study with previous functional studies and their results that applied the same PK 11195 concentration. The study would show whether TSPO function associated with modulation of nuclear gene expression could correlate with known TSPO functions, and might possibly reveal hitherto unknown TSPO functions
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