TRANSLOCATOR PROTEIN 18 DKA (TSPO) ENDOGENOUS LIGAND REGULATES THE METABOLISM OF HUMAN OSTEOBLAST

Abstract

Introduction: The mitochondrial Translocator Protein 18 kDa (TSPO, previously named as the peripheral benzodiazepine receptor - PBR) is involved in cellular respiration, steroidogenesis and apoptosis. In our recent study we reported on the role of the synthetic pharmacological ligands to the TSPO in enhancing human osteoblast catabolism. There is also a previous evidence of the existence of an endogenous ligands to the TSPO, but their role in the human osteoblast physiology hasn’t been verified yet. Porphyrine IX has been found having affinity to the TSPO. Therefore we hypothesize that human osteoblast metabolism might be mediated by the porphyrine IX and the mode of its action is similar the synthetic ligand to the TSPO.

Methods: Cell cycle of the cultured human derived osteoblast- like cells, following exposure to Porphyrine IX, endogenous ligand to TSPO, and N,N-di-n-hexyl 2-(4- fluorophenyl)indole-3-acetamide (FGIN-1–27), synthetic ligand to the TSPO, was determined by flow cytometry (FACS). These ligands’ affect on cell number, metabolic activity, i.e. cellular fluorodeoxyglucose ([¹⁸F]-FDG) incorporation and alkaline phosphatase activity, and cell death rate, i.e. LDH activity in the culture media, were assayed. The semi-quantitative response of TSPO to exposure to these ligands was estimated by Western blotting. Six samples of cultured cells for each condition were used. The t test was implemented for the statistical analyses. P values below.05 considered as statistically significant

Results: Cell count significantly decreased following exposure to FGIN-1–27 or porphyrine IX. Cellular [¹⁸F]-FDG incorporation and alkaline phosphatase activity were suppressed by both ligands. Cell cycle analysis showed a significant decrease in the fraction of cells in the G1 and G2/M phases when exposed to each ligand with a higher proportion of necrotic and apoptotic cells.

Western blotting showed a decrease in TSPO abundance following treatment by both ligands. LDH activity in culture media significantly increased following exposure to FGIN-1–27 or porphyrine IX.

Discussion: We show that FGIN-1–27 and porphyrine IX have a similar cell death inducing affect on human osteoblast-like cell in vitro. This affect is parallel to the inhibition of the cellular metabolism. Since both ligands similarly reduce the availability of TSPO we postulate that their mode of action is similar by affecting this mitochondrial structure with sub sequential induction of cell death, i.e. apoptosis and necrosis. Therefore we suggest that human osteoblast metabolism and cell cycle are mediated through TSPO and that porphyrine IX might be an active endogenous ligand to the TSPO having a regulatory affect on the human bone cell cycle.