Periodic Reporting for period 3 - BONEPHAGY (Defining the role of the FGF – autophagy axis in bone physiology) Periodo di rendicontazione: 2020-01-01 al 2021-06-30 Sintesi del contesto e degli obiettivi generali del progetto The fibroblast growth factor (FGF) signaling pathways have been recognized as essential regulators of vertebrate skeletal development. The FGF family comprises of 18 secreted signaling proteins that bind to and activate four receptor tyrosine kinase molecules (FGFRs). The binding of FGF ligands to FGFRs induces receptor dimerization and activation, leading to the activation of multiple intracellular effectors. Mutations in FGF ligands and receptors result in at least 14 different types of human genetic disorders characterized by defective skeletal development and growth. A notable example is achondroplasia (ACH), the most common form of dwarfism (frequency about 1:15000), which is prevalently due to a G380R amino acid substitution in FGFR3. FGFR3 is a negative regulator of bone growth and these mutations activate the receptor resulting in a gain-of-function. Consistently, mutations in FGFR3 leading to loss of function cause skeletal overgrowth, camptodactyly, tall stature, and hearing loss (CATSHL) syndrome in humans. Genome-wide association (GWA) studies have identified variants in FGFR4 that are associated to human growth. In addition, FGF9 and FGF18 ligands regulate bone and cartilage homeostasis and are important during fracture bone repair and osteoarthritis. Many pathways are controlled by FGF signaling in chondrocytes and osteoblasts and their deregulation most likely contribute to the pathogenesis of FGF-related skeletal dysplasia. Thus, the identification of new intracellular effectors of FGF signaling in cartilage and bone is an important ongoing topic of research. Indeed, a deeper understanding of the processes regulated by FGF will not only shed light on new mechanisms regulating bone growth and homeostasis, but also provide new potential therapeutic avenues for the treatment of FGF-skeletal dwarfism. My laboratory demonstrated that FGF signaling is a major regulator of autophagy during post-natal bone growth (Cinque L, et al. Nature 2015). Autophagy is a catabolic process involved in the degradation and turnover of intracellular substrates. It relies on the biogenesis of autophagosomes (autophagic vesicles, AVs), double membrane vesicles that sequester cytosolic substrates and target them to the lysosomes (Lys) where they are degraded by resident hydrolases. The work proposed in the grant BONEPHAGY aims to: a) Define the molecular mechanisms by which FGF signaling control autophagy FGF-autophagy axis. b) To determine the physiological relevance of autophagy regulation by FGF signaling.c) Develop new therapeutic approaches for disorders due to mutations in the FGF signaling pathway. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti With the support of BONEphagy we have characterized the different intracellular responses elicited by FGF signaling in chondrocytes. We have combined different -omics approaches, such as RNA-sequencing, quantitative proteomic and phospho-proteomic analyses to characterize the intracellular signaling pathways leading from activation of the FGF receptors to the regulation of autophagy and lysosome biogenesis. These findings were subsequently validated in vivo using transgenic mice harboring gain- and loss- of function mutations in multiple components of the FGF pathway. A manuscript is currently under revision. In addition, we are performing a systematic identification of the autophagy substrates in chondrocytes and osteoblasts before and after stimulation with FGF. We have already demonstrated that a fractions of newly synthesized procollagen molecules inappropriately folded is cleared from the endoplasmic reticulum by a selective form of autophagy, known as ER-phagy. This work was recently published in EMBO Journal (Forrester et al. EMBO 2018). Furthermore, we are investigating whether the enhancement of autophagy and lysosomal catabolism is beneficial for the treatment of genetic skeletal disorders, such as achondroplasia and lysosomal storage disorders. We have recently demonstrated that the pharmacological enhancement of autophagy ameliorates the skeletal manifestation in 2 different mouse models of lysosomal storage disorders. This work was recently published in Journal of Clinical Investigation (Bartolomeo et al. 2017). Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) We have characterized a novel signaling pathway that is activated by FGFs in chondrocytes. These data might be important for the identification of potential therapeutic targets for treating genetic skeletal dysplasia and growth disorders. In the next 2.5 years we plan to evaluate whether this signaling cascade plays any physiological role during skeletal growth and whether it participates to the pathogenesis of FGF-related skeletal disorders. We have identified the molecular mechanisms mediating procollagen clearance from the ER via autophagy. In the next years, we plan to identify new autophagy substrates in chondrocytes and osteoblasts through mass-spectrometry analysis on purified lysosomes. We have provided proof of principle that modulation of autophagy might be beneficial for the treatment of skeletal abnormalities in lysosomal storage disorders. In the next years we plan to investigate whether manipulation of the signaling pathway we have identified in chondrocytes might be beneficial for the treatment of FGF-related skeletal disorders.
