Identificazione di nuove molecole coinvolte nell'omeostasi degli osteoblasti

Osteoblasts are bone forming cells that work in cooperation with osteoclasts, which resorb bone, in a continuous cycle of bone remodeling. Since bone remodeling is crucial for preserving skeletal integrity and functionality, events, such as mechanical unloading, perturb this equilibrium, leading to pathological conditions, like disuse osteoporosis. In a previous work conducted in our laboratory, Lipocalin 2 (LCN2) and Pre-proenkephalin 1 (PENK1) were identified as the most up- and down-regulated genes in osteoblasts subjected to mechanical unloading. Starting from this data, during my PhD I focused my research to evaluate the involvement of these molecules in osteoblasts homeostasis, in particular in response to mechanical unloading. In Chapter 2 we confirmed PENK1 downregulation due to mechanical unloading in both human and animal models of mechanical unloading. We also observed high expression of Penk1 in mouse in mouse femurs and calvariae cleaned from bone marrow, and this expression progressively increased during osteoblast differentiation. Surprisingly, Penk1 knock out (Penk1-/-) mice did not show bone phenotype compared to the WT littermates; however silenced Penk 1 in mature osteoblasts we observed an impairment of the Wnt pathway, while primary osteoblasts isolated from Penk1 -/- mouse calvariae showed an impairment of their metabolic and Alp activities, along with a lower nodule mineralization ability, compared to cells isolated from WT mice. Moreover, in a CFU-Fibroblastic assay, using bone marrow cells isolated from tibias, we observed a decrease of ability to form Alp-positive colonies in Penk1-/- versus WT mice, suggesting a cell autonomous positive effect of Penk1 in osteoblasts. In line with this, treatment of osteoblasts with Met-enkephalin, Penk1 encoded peptide, increased Osx and Col1a1 mRNAs, and enhanced nodule mineralization. In the second part of this work (Chapter3) we investigated whether genetic ablation of this gene can counteract the inhibitory effect of microgravity on osteoblast differentiation. Our results demonstrated that, at least in vitro, Lcn2 genetic ablation counteracts the reduction of Runx2 and Osx transcriptional expression induced by microgravity. Moreover, Lcn2 ablation also prevents the increase of RANKL/OPG ratio observed in the conditioned media of WT osteoblasts cultured under unloading conditions. However, in the mice subjected to mechanical unloading, the lack of Lcn2 is not sufficient to counteract the bone loss due to mechanical unloading and disuse, although it is enough to prevent the number and the surface area of osteoblasts from decreasing, as compared to WT littermates. In conclusion, although future studies will be required to fully understand the mechanism of bone detrimental adaptation after mechanical unloading, the work reported in this dissertation adds another piece of knowledge, demonstrating the role that Penk1 and Lcn2 play in this event, as well as the contribution of Penk1 in osteoblast homeostasis. It is also important to point out that these studies are exploitable and applicable to those pathological circumstances in which patients experience disuse induced-bone loss due to of paralysis or neuromuscular disorders, as well as in case of long-duration space flights.