Functional Study Of MACF1 In Bone Development And Bone Formation In Mice

MACF1(Microtubule actin crosslinker factor1)is a huge structural protein.In mammalian cells,MACF1 is of great significance of stabilizing both the microtubule and the microfilament cytoskeleton dynamics to maintain cell morphology.MACF1 is also involved in the regulation of focal adhesion dynamics.In addition,as an important cellular structural protein in mammals,MACF1 also plays important roles in regulating cellular processes such as proliferation,migration,and differentiation.Previous studies in our group showed that MACF1 was involved in the regulation of osteogenic differentiation and bone formation.MACF1 was necessary for maintaining the morphology of MC3T3-E1 preosteoblasts and promoting cell proliferation in vitro,and regulated osteogenic differentiation through the β-catenin/TCF1/Runx2 signal axis in MC3T3-E1 preosteoblasts.Transfection of the MACF1 plasmid onto mouse skull was shown to increase bone mass and bone formation rate,and up-regulated expression levels of osteogenic differentiation-related marker genes.Although these results suggest that MACF1 is closely related to osteogenic differentiation and bone formation,these studies only focus MACF1’s preliminary function using experimental materials such as cell lines,no in vivo data are available to depict MACF1’s roles in regulating bone formation.As an important cytoskeletal crosslinker,what roles do MACF1 play during bone development and bone formation? In bone forming cells,how does MACF1 contribute to cytoskeleton integrity and osteogenic differentiation? Through what mechanism does MACF1 regulate osteogenic differentiation and bone formation? If there’s a potential correlation between MACF1 and osteoporosis? These issues need to be further studied.1.Research Objectives(1).To study the biological functions of MACF1 at different stages of bone development in mice;(2).To study the involvement of MACF1 in regulating cytoskeleton and cell function during osteogenic differentiation in MSCs;(3).To explore the molecular mechanism of MACF1 involved in the regulation of osteogenic differentiation.2.Materials and Methods(1).Bone-derived MSCs were collected from osteoporotic patients(60s vs 80s)and aged mice(6,12,15,18,21,22-month-old),and q PCR assay was used to detect the expression trend of MACF1 with age or degrees of osteoporosis;Further,Cre/Lox P technology was used to construct the mice model in which MACF1 was deleted specifically in MSCs;(2).In embryonic and neonatal MACF1 c KO mice,skeleton preparation,micro CT and paraffin sections were used to study the effect of MACF1 deletion on bone development;During adult and aged stage,DEXA,micro CT,three-point bending,plastic sections,serum ELISA,paraffin sections,calcein double-labeling and bone morphometric assay were used to study the effect of MACF1 deletion on bone mass,bone mechanical property,bone formation rate and bone remodeling;(3).Isolate MSCs from MACF1 c KO mice,and cell culture staining,immunofluorescent staining,q PCR,Western blot and electric cell-substrate impedance sensing were used to study the effect of MACF1 deletion on cytoskeleton integrity,proliferation,differentiation in MSCs;(4).In addition,Co-IP,i TRAQ-MS/MS and overexpression vectors with NLS signal were used to study the molecular mechanism of how MACF1 interacts with downstream transcription factor SMAD7 to regulate bone formation.3.Results and Conclusions(1).MACF1 positively regulates bone formation,loss of MACF1 significantly suppresses bone development and bone formation capability;During embryonic stage,loss of MACF1 retards suture fusion in the skull,and reduces mineralization capability in long bones;In adults,loss of MACF1 decreases bone mass and bone formation rate,reduces bone mechanical properties,suppresses osteoblastic functions and promotes osteoclastic functions on bone surfaces.In aged mice,loss of MACF1 decreases bone mass greatly,and promotes medullary adipogenesis.(2).MACF1 coordinates cytoskeleton morphology in MSCs,loss of MACF1 causes morphological abnormalities in microtubule cytoskeleton and focal adhesions;In MSCs isolated from the MACF1 c KO mice,loss of MACF1 perturbs the microtubule cytoskeleton,but leave the actin cytoskeleton intact;Further,loss of MACF1 changes the morphology of focal adhesions in MSCs,makes them bigger and slender.(3).MACF1 is essential for osteogenic differentiation,MACF1 interacts with and promotes SMAD7 nucleus translocation to regulate osteogenic differentiation;During osteogenic differentiation,loss of MACF1 impairs mineralization capability and inhibits expression level of osteogenic marker genes,show less mineralization capability.Proteomics assays show that MACF1 interacts with SMAD7.Cytoplasmic and nucleus SMAD7 negatively and positively controls osteogenic differentiation,respectively,and MACF1 positively regulates osteogenic differentiation and bone formation by promoting SMAD7 nucleus translocation.(4).Loss of MACF1 expedites aging phenotypes in bones,and is an important potential risk factor of senile osteoporosis.In bones from osteoporotic patients and MSCs from aged mice bone tissue,the expression level of MACF1 is negatively correlated with the severity of osteoporosis.Loss of MACF1 reduces bone mass and impairs bone microarchitecture,these inhibitory effects are even greater in aged mice.The results suggest that loss of MACF1 is significantly correlated with osteoporosis.Taken together,in this project,we studied the in vivo roles of MACF1 in regulating bone formation for the first time,and verified that MACF1 directly targets SMAD7 to positively regulate osteogenic differentiation,thus we revealed a potential relationship between MACF1 and osteoporosis.These results would not only improve the understanding of the functional diversity of MACF1,but also provide experimental basis for targeting MACF1 for diagnostic or therapeutic purposes of degenerative bone diseases such as osteoporosis.