טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRosenberg Orit
SubjectThe Physiological and Pharmacological Role of the
Translocator Protein 18kDa TSPO in Human
Osteoblast Like Cells
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Moshe Gavish
Full Thesis textFull thesis text - English Version


Abstract

Control of cell metabolism in connective tissue is crucial for human locomotor system maintenance. Therefore further understanding of the cellular signaling pathways, that are involved in metabolic stimulation of bone producing cells, i.e. osteoblasts, may provide an important insight for the future development of additional effective therapeutic tools for the maintenance of bone mass. The mitochondrial permeability transition pore (MPTP) regulates the cellular energetic homeostasis and therefore might be a target for pharmacological regulation of the human osteoblast. Accordingly the TSPO, which is one of the MPTP components, might be a target for this purpose. Therefore our intention in this study was to identify the potential role of TSPO in the physiology of human osteoblast-like cells. For this purpose the following specific objectives were planned: Identification of the TSPO in human osteoblast-like cells, investigation of the functional importance of TSPO in proliferation, energy consumption and production, in human osteoblasts, by using specific TSPO ligands, synthetic (PK11195 , Ro5-4864 and FGIN-1-27) and endogenous (protoporphyrin IX), determination of the role of TSPO in the regulation of the cell cycle of human osteoblast-like cells, determination of the role of TSPO ligands in the human osteoblast-like cell death induction, assessment of the possible interaction between TSPO and hexokinase 2 in osteoblast energy consumption (glucose incorporation) and energy production (ATP generation), assessment of interactions of TSPO ligands with other MPTP components (VDAC, ANT, hexokinase 2). The skatchard analysis showed that the [3H]PK 11195 binding to human osteoblast  TSPO  is saturable with a single population of binding sites (r = 0.92, 0.95). The equilibrium dissociation constants (Kd) were 9.15 and 9.34 nM and maximal binding capacity values (Bmax) were 7672,7691 fmol/mg protein.  This is a first report on the abundant existence of TSPO in human osteoblast-like cells. All TSPO ligands cause a shifting of the cell cycle to a lower energy demand state, while preventing cell death by different cellular pathways. The protoporphyrin IX acts stoichiometrically to prevent hexokinase 2 - VDAC 1 interactions, while PK 11195, which has a smaller molecular weight, interacts directly with VDAC 1 without interference with hexokinase 2 binding. Ro5-4864 and FGIN-1-27 act similarly to PK 11195 but also have several characteristics of protoporphyrin IX. We suggest that these synthetic TSPO ligands interact both directly with VDAC 1 and interrupt hexokinase 2 - VDAC 1 interactions. This ability reveals a protective fine tuning mechanism of these cells adaptation for a lower energy supply.