The Function And Mechanism Of Deubiquitinase OTUB1 On Alveolar Ectasia And Bone Formation

Deubiquitinase OTUB1 plays an important role in immune inflammation,DNA damage repair,and tumorigenesis.Multiple mouse models reveal OTUB1 is essential in tissue homeostasis.Otub1 deletion in T cell promotes its responses to a self-antigen,while B-cell-conditional deletion Otub1 results in cell hyperplasia and autoimmunity induction.Astrocytes-cell-specific deletion of Otub1 are more likely to develop experimental autoimmune encephalomyelitis.Moreover,mouse lacking Otub1 in dendritic cell are easier die from chronic encephalitis in Toxoplasma gondii infection.All of this mouse model indicates the importance of OTUB1 in tissue homeostasis.However,the physiological roles of OTUB1 in mouse embryonic developmental are still poorly understood.The aim of this study is to analyze the potential biological role of OTUB1 in development by establish Otub1 knockout mouse model and to identify new physiological substrates of OTUB1.We successfully generated Otub1 systemic and osteoblast-specific knockout mouse models through Cre-Loxp technology.We found that Otub1 knockout mouse died immediately after birth and showed a cyanotic appearance.At the same time,Pulmonary buoyancy experiments suggest that Otub1 knockout mouse have no spontaneous breathing behavior after birth.Phenotypic analysis found that the destruction of synthesis and assembly of elastic and lipofibroblast differentiation leads to alveolar cavity without support,resulting Otub1 deletion mouse postnatal death.In addition,we found Otub1 knockout mouse developed a marked dwarfism,Alcian Blue and Alizarin Red staining reveal that cranial,sternal,and limb sclerite development delayed in Otub1 deletion mouse.Histological and cytological experiments further revealed that Otub1 deletion did not affect the proliferation and hypertrophy of chondrocytes,nor the mineralization process,but rather inhibited bone development by attenuating the differentiation of osteoblasts.Consistently,deletion of Otub1 in osteoblast cells manifest defects in bone formation,bone fragility and bone mass,and in vitro osteogenesis induction assays confirmed that Otub1 deletion would destruct differentiation of osteoblasts.We also detected a low level of Otub1 in primary-osteoporosis samples,and overexpression of OTUB1 significantly improved phenotypic features of ovariectomy mice,such as low bone mass,increased bone fragility and bone microarchitecture destruction.Tyrosine kinase receptor FGFR2 is a classical regulator of bone and lung development,and phenotypic-driven mechanism studies have found that FGFR2 is the physiological substrate of OTUB1.Ubiquitination levels of FGFR2 is increased and protein stability is decreased in the lung and osteoblasts of OTUB1 deficient mice.In-depth studies revealed that OTUB1 binds FGFR2 through its N terminal ubiquitin binding domain and antagonistic NEDD4 family HECT ubiquitin ligase Smurf1 regulate the ubiquitination of FGFR2,thereby effectively inhibit FGFR2 from lysosomal degradation by inhibiting the K48-linked polyubiquitination of FGFR2 independent its catalytic activity and maintain the protein stability of FGFR2,thereby maintaining homeostatic of lung and bone.Based on the above research,we delivered Fgfr2 to the joint cavity of Otub1 osteoblast-specific knockout mice via the AAV9 gene delivery system and found that the osteogenesis defects of Otub1 osteoblast specific knockout mouse is alleviated.In addition,we overexpressed Fgfr2 in lung fibroblasts,which also rescued the lipogenic differentiation ability of Otub1 knockout mice.All of these further confirmed the bone mass loss and diminished lipogenic differentiation of fibroblasts after Otub1 deletion are caused by the inactivation of FGFR2.In summary,this study used a knockout mouse model reveal that OTUB1 regulates the secretion and assembly of elastin and osteoblast differentiation through tyrosine kinase receptor FGFR2,which in turn affects lung development and bone formation.OTUB1 prevent FGFR2 from lysosomal degradation by removing K48-linked polyubiquitination of FGFR2 independent its catalytic activity.This study also confirmed that the ubiquitin ligase enzyme Smurf1 is a new E3 of FGFR2,OTUB1 antagonistic Smurf1 regulate the ubiquitination of FGFR2 and maintain the protein stability of FGFR2,and OTUB1 cooperate with Smurf1 maintain the ubiquitination and protein stability of FGFR2.