A Study On The Region Specific Effect Of Leptin In Endochondral Ossification Of The Vertebral Epiphyseal Plate Chondrocytes

Objective We intend to observe at the cellular level of leptin on the proliferation and differentiation of limb and vertebral epiphyseal plate in a dose-dependent manner.Despite distinct anatomy and development of tibial and vertebral skeletons,knowledge remains to be elusive regarding the regulatory and molecular mechanisms that account for these differences.This research intends to study the specific significance of STAT and ERK signaling pathway in leptin on proliferation,differentiation and apoptosis of vertebral epiphyseal plate effects at the cellular level,compared with that of appendicular skeleton,to observe the region specific effect of leptin.The signaling pathways in these two regions are also distinct and subject to differential regulation by leptin.Methods Take the limb and vertebral growth plate chondrocytes of one week old female wild type mice(C57BL/6)in primary culture.After stimulus with different dose of leptin(0,10 ng/m L,50 ng/m L,100 ng/m L,1000 ng/m L,48h),we observe the different effect of different dose of leptin on the proliferation,chondrogenic differentiation,apoptosis and cell cycle of vertebral growth plate and tibial growth plate.Meanwhile,we observed the expression of BMP7 and Wnt3,which are important signaling molecules,and speculated that the possible vector factor for the specific role of leptin.To detect the changes of STAT and ERK signaling related proteins and their phosphorylation levels in leptin induced cell proliferation and differentiation and to observe the expression of BMP7 and Wnt3 of appendicular and axial skeleton when STAT and ERK signaling pathway specific blocking and non blocking.Results Cell cycle analysis showed that leptin may have the opposite effect on the vertebral and limb epiphyseal plate,the former is to inhibit cell proliferation and promote cell proliferation of the latter.Leptin promotes the expression of limb epiphyseal plate cell proliferation related genes(collagen II,aggregan)and hypertrophy related genes(collagen X,ALP),on the contrary,leptin inhibits the expression of these genes in vertebral epiphyseal plate,in a time and concentration dependent manner.Therefore,we conclude that a certain concentration of leptin promotes limb epiphyseal chondrocytes differentiation,but it inhibites the differentiation of vertebral epiphyseal chondrocytes.The cell apoptosis results indicates that the level of apoptosis of limb epiphyseal chondrocytes was significantly inhibited after leptin treatment,on the contrary,the vertebral epiphyseal chondrocytes,leptin treatment can significantly increase the apoptosis(including early and late apoptosis),and this effect was in a dose-dependent manner,i.e.the level of apoptosis increased with the concentration of leptin increased.BMP7 level in tibial epiphyseal chondrocytes is much higher than that in vertebral epiphyseal chondrocytes,which is consistent with the fact that chondrocytes in appendicular regions are usually active in proliferation and differentiation.Upon treatment of ERK inhibitor in tibial epiphyseal chondrocytes,BMP7 level did not change significantly.Yet when treated with the STAT inhibitor,BMP7 level was significantly diminished.Therefore,it is likely that modulation of leptin on the BMP7 levels in tibial chondrocytes is mainly through the STAT signaling pathway.On the contrary,in vertebral chondrocytes,leptin modulates BMP7 level mainly by the ERK pathway because inhibition of the ERK pathway significantly reduced the BMP7 level.In contrast to BMP7,Wnt3 does not differ significantly in both tibial and vertebral primary epiphyseal chondrocytes,suggesting Wnt3 may be secondary to the different phenotypes in the bone developmental programs among tibial and vertebral epiphyseal chondrocytes.However,in a similar manner,leptin may regulate Wnt3 level in tibial chondrocytes mainly through the STAT pathway,while such regulation may be mainly mediated by the ERK pathway in vertebral chondrocytes.Conclusions According to the experimental results of cell proliferation,cell cycle and apoptosis,leptin can promotes limb epiphyseal chondrocytes proliferation,and inhibits the proliferation of vertebral epiphyseal chondrocytes.Simultaneously,we also found that the status of these two types of primary cultured cells are not the same.In the normal condition(no leptin treatment and serum deprivation),the cells viability,cell proliferation and apoptosis level of limb epiphyseal plate were higher than that of vertebral epiphyseal plate,suggesting that the limb epiphyseal chondrocytes have stronger self-renewing and proliferating capabilities compared with vertebral chondrocytes in leptin-and serum-free cultures.We have revealed the effects of leptin on the growth of epiphyseal plate chondrocytes are opposite in distinct anatomic regions of appendicular and axial skeletons.Based on the current data,we further speculate that modulation of appendicular and axial skeleton developmental programs may be attributed significantly,if not completely,by regulation of leptin levels in the body.In summary,these results clearly show that the molecular mechanisms of the response to leptin are very different in tibial vs.vertebral epiphyseal plate chondrocytes.Leptin affects BMP7 level mainly through the STAT pathway in tibial chondrocytes,while such effects may be mainly through the ERK pathway in vertebral chondrocytes.Taken together,our results on the regulations of BMP7 and Wnt3 by leptin add another layer of complexity on the regulation of chondrogenic proliferation and differentiation in appendicular and axial regions of skeleton.