The Role And Mechanism Of LATS1 In Bone Remodeling

VI Osteoporosis is a bone metabolic disease associated with decreased bone mineral density,abnormal bone remodeling,and increased bone fragility fracture susceptibility.The number of osteoclasts in the bone marrow cavity of patients with osteoporosis increased significantly.Osteoclasts are myeloid cells derived from polymorphonuclear terminals,whose core function is to decompose the mineralized bone matrix.Therefore,Insight into osteoclast differentiation and its mechanism is of great significance for the prevention and treatment of osteoporosis.The conserved Hippo signaling pathway is considered to be the main determinant of cell fate,tissue homeostasis,and organ size.The NDR family kinase LATS1/2(large tumor suppressor 1/2)is the key kinase in the hippo signaling pathway.The activity of LATS1/2 directly determines the opening or closing of Hippo signaling and plays an important role in the maintenance of tissue homeostasis.In recent years,a large number of studies have shown that the Hippo signaling pathway is closely related to bone remodeling,but the role of LATS1 in bone mass maintenance remains unclear.We found that the expression of P-LATS1 in osteoclasts of patients with osteoporosis was significantly higher than that of the sham group.It suggested that LATS1 may be involved in the occurrence and development of osteoporosis.Furthermore,we revealed that LATS1 plays an important role in bone remodeling through in vitro cytological studies and animal experiments and preliminarily explored its regulatory mechanism.Firstly,we observed the Lats1-/-transgenic mice and found that compared with the control mice,the size and morphology of Lats1 knockout mice were significantly reduced,and the bone mineral density was abnormally increased.To find out the cause of abnormal bone remodeling in mice,we isolated the bone tissue of Lats1 knockout mice and stained the osteogenic and osteoclast differentiation marker genes.The results showed that the osteogenic and osteoclast differentiation marker genes of Lats1 knockout mice increased,but the mature osteoclast marker gene ctsk decreased significantly.The results demonstrated that the abnormal bone remodeling of Lats1 knockout mice was caused by the enhancement of osteogenic differentiation and the decrease of osteoclastic bone resorption.Osteoclasts are derived from monocyte precursor cells of the monocytemacrophage line.macrophage colony-stimulating factor(M-CSF)is required for proliferation and survival of the progenitors,and the receptor activator of NF-κB ligand(RANKL)is required for fusion and differentiation to osteoclasts.To demonstrate the regulatory effect of LATS1 on osteoclast differentiation,we have used the Cre-lox system and generate the osteoclast precursor cells-specific(Rank-Cre;Lats1f/f)and osteoclast cells-specific Lats1(Ctsk-Cre;Lats1f/f).It was found that Lats1 knockout could inhibit the differentiation of osteoclast precursor cells into osteoclasts but did not retrain the bone resorption of mature osteoclasts.This is contrary to the classical Hippo signaling family members MST2,RAFF2,YAP1 reverse regulation of osteoclast differentiation.All these results indicate that LATS1 plays an important role in osteoclast differentiation.To investigate the regulatory mechanism of LATS1 regulating osteoclast differentiation,we carried out a series of studies in raws cell lines.The results showed that RANKL could demerit LATS1 through the RANK/TRAF6/IKK/ NEMO pathways,and the activated LATS1 could affect the transcription of the NF-κB signal.In order to verify the effect of this signaling pathway on cell differentiation,we selected Rank-Cre;Lats1f/f for immunohistochemistry and in vitro differentiation experiments.The results showed that activation of P65 could rescue the inhibition of osteoclast differentiation caused by Lats1 knockout.These results suggest that LATS1 regulates osteoclast differentiation through the NF-κB signaling pathway.In order to clarify the mechanism of LATS1 activating NF-κB signaling pathway,we predicted the proteins A20,ELKS,and IKAP that may interact with LATS1 by using regphos software.Immunofluorescence and immunoprecipitation results showed that LATS1 and ELKS were bound to each other.Furthermore,we predicted that the site S37 of ELKS might be phosphorylated by LATS1.Western and immunohistochemical results indicated that LATS1 could directly phosphorylate the site S37 of ELKS.We analyzed its structure and found that the hydrogen bond between ELKS 37 site and its surrounding sites 38,27,40 was broken when the 37 site was inactivated.The results of immunoprecipitation also confirmed that the mutation of elks37 affected the binding of ELKS to LATS1 and IKK.These results indicate that LATS1 can affect the stability of ELKS and the NF-κB signaling pathway by acting on the 37 sites.In order to confirm the effect of LATS1 phosphorylation of ELKS ser 37 site on NF-κ B signaling,we constructed Elks1(S37A)knock-in mice(Elks+/S37A)and hybridized them to produce double copy point mutation ElksS37A/S37 A mice.Elks1(S37A)knock-in showed the same osteopetrosis phenotype as Rank-Cre;Lats1f/f.In conclusion,this project elucidates the detailed role and mechanism of LATS1 in bone formation,osteoclast differentiation and function through regulating the RANKL/NF-κB signaling pathway,which provides a new theoretical and practical basis for the maintenance of bone homeostasis and the occurrence of osteoporosis.