Studies On The Mechanism Of STMN1 Transcriptional Regulation In Mouse Osteoblasts And Osteoclasts

Osteoporosis is a highly heritability complex disease, its genetic pathogenesis and therapeutic method haven’t been fully elucidated. Therefore, to investigate the osteoporosis gene regulation manipulation through mouse animal model, will benefit for discovering the pathogenesis and drug development. In Agricultural production field, high reproduction and production of cow, sow and hen, are vulnerable to bone loss and easy to suffer from osteoporosis, even lead to fatal damage. Husbandry researchers are inclined to adjust the Ca P concentration in animal food to diminish the loss to osteoporosis, however they can’t eliminate the harm fundamentally. It is little known about the genetics pathogenesis of osteoporosis in human, especially in livestock, so it is very important to investigate the genetic susceptible gene/locus to osteoporosis. In this dissertation, based on the basic principles of cytoskeleton microtubules (MTs) regulate various cell signaling pathways that are involved in bone cell function, we inhibited MT assembly by microtubule-targeting drugs stimulates osteoblasts differentiation and bone formation; with the purpose of discover the mechanism of MT in bone metabolism, we use STMN1 deficient mouse model, which is the endogenic inhibitor of MT, to discover STMN1 functions in bone development.1. We firstly characterized the bone mass phenotypes of STMN1 deficiency in postnatal mouse. Through dual energy X-ray absorptiometry (DEXA) technique,we found that BMD decreased 7% and 13% in lmonth and 3month old mouse separately, compared with wild type control littermates (p<0.05). Next, we analyzed trabecular and cortical bone microstructure by Micro-CT, results showed that Trabecular BMD、Cortical bone BMD、Tb.N、Tb.Th、BV/TV attenuated significantly compared with wild type control littermates (p<0.05).2. Bone histomorphometry analyses:By H&E staining (osteoblast special staining)、Trap staining(osteoclast special staining)、Von Kossa staining(bone mineralization staining)、Alizarin Red S staining(bone mineralization staining), we found that STMN1 deficiency resulted in osteoblasts decrease, simultaneously osteoclasts increase, led to bone mass decrease.3. Immuno fluorescence MT structure after STMN1 over-or-down regulation: overexpression STMN1 in mouse osteoblastic cells MC3T3-E1 and osteoclastic precurse cells Raw264.7, and also isolation primary calvarial osteoblastic cells from STMN1 deficient mouse, immuno fluorescence of anti-a-Tubulin-FITC showed that overexpression STMN1 increased the assembly of MT, and vice versa.4. To investigate the bone loss regulation mechanisms after STMN1 deficiency, we analyze the gene expression levels after over-or-down regulation STMN1, such as positive correlation with osteoblastic marker genes, BMP2、Runx2、Colla1、 Osteocalcin; and negative correlation with osteoclastic marker genes、RANKL、ATP6、Cathepsin K. Collectively, these results suggest that stathmin, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone.5. Promoter analysis of BMP2 gene was performed by bioinformatics predictions in combine with promoter serial deletion experiments. STMN1 deficiency increased MT assembly, inhibited Gli2 protealysis, and then enhanced Gli2 transactivation ability, which in ture actived target gene of BMP2 protein expression levels. All together, we herein presume that STMN1 regulations through "STMN1-Gli2-BMP2" network in bone.In conclusion, we herein investigated the new pathway manipulation of STMN1 in bone metabolism through knockout mouse model. STMN1 can regulate the MTs structure, then activate transcription factor, up-regulate downstream target genes. These findings will lead to further discover the pathogenesis of osteoporosis and milk fever of cows, as well as provide fundamental theory regarding the seclecion of resistance to osteoporosis drugs.