Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: Uncovering the impact of endolysosomal Two-pore channel 2 (TPC2) on the autophagy pathway during osteoblastogenesis and osteoclastogenesis

Rational: Endo-lysosomal two-pore channels 2 (TPC2) are voltage-gated ion channels with distinct ion selectivity as are regulated by both NAADP and PI (3,5)P2 that control the release of Ca2+ and Na+, respectively. As the lysosomal function is fundamental for regulation of different aspects of the autophagy flux, TPC2 channels are also associated with control of autophagy. Interestingly, autophagy has an important role in regulation of bone cells differentiation and function. General objectives: This study aimed to give thorough insight to the interaction between TPC2 and the autophagy pathway in the control of osteoblast and osteoclast differentiation and function. Experimental design and methods: To achieve the purposes of this project, mesenchymal stem cells (MSCs) and human osteosarcoma cells (SAOS-2) were used to assess osteoblastogenesis and bone mineralization, respectively, while the peripheral blood mononuclear cells (PBMCs) have been used to study osteoclastogenesis. The cells have been treated with different pharmacological TPC2 inhibitors including naringenin, tetrandrine, MT-8 and SG-094 during their differentiation process. At the end, formation of osteoblasts and in vitro bone mineralization were evaluated by alkaline phosphatase, alizarin red S and Von Kossa staining methods, while the formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) and phalloidin staining methods. Western blot analysis was done to investigate the expression of osteoblast and osteoclast specific markers and autophagy-related molecules. Results: The inhibition of TPC2 activity stimulated osteoblast differentiation from MSCs as confirmed by alkaline phosphatase staining and western blot analysis for RUNX2 expression. Moreover, inhibition of TPC2 activity increased the bone mineralization by osteoblast-like (SAOS-2) cells. Interestingly, TPC2 inhibition in osteoblast and SAOS-2 cells resulted in reduced expression of p62 and LC3II/I while the expression of mammalian target of rapamycin complex 1 (mTORC1) as the master regulator of autophagy that negatively controls this pathway was increased; this, together with increased RUNX2 indicates promoted osteoblast differentiation. Furthermore, TPC2 inhibition showed a bi-phasic effects on osteoclastogenesis: formation of giant and multinuclear osteoclasts with evident F-actin ring was induced by TPC2 inhibitors at lower concentrations, while decreased osteoclast formation was observed in response to naringenin and MT-8 at high concentrations. Likewise, TPC2 inhibition downregulated the expression of LC3II/I and p62 and increased expression of mTORC1. Interestingly, inhibition of mTORC1 activity by rapamycin as an autophagy enhancer reversed the osteoclast differentiation induced by TPC2 inhibitors. Conclusion: Inhibition of TPC2 channel activity can increase osteoblast differentiation and bone mineralization and inhibits the completion of the autophagic flux. Enhanced expression of RUNX2/mTORC1 could be the reason for increased osteoblastogenesis due to TPC2 inhibition. Furthermore, low doses of TPC2 inhibitors stimulated osteoclast formation and blocked the autophagy flux termination by targeting mTORC1 and rapamycin opposed their stimulatory effects.