The Effect Of Nell-1 On Cartilage Homeostasis And Endochondral Ossification At Postnatal Stage

As a secreted protein expressed in osteochondral tissues,Nell-1 was first isolated and characterized in craniosynostosis patients as specifically upregulated within prematurely fusing sutures.Many experimental focuses were put on revealing the roles of Nell-1 in the development of craniofacial skeletons,together with bone regeneration in calvarial defects.Previously,the only mouse model to study Nell-1's loss of function was N-ethyl-N-nitrosourea(ENU)-induced Nell-1deficient mice.However,this mutant mice show severe osteochondral deformities,which can lead to neonatal death.Therefore,in this study,we generated conditional Nell-1 knockout mouse model(Nell-1Col2?1KO)with Col2?1-Cre to observe changes in osteochondral tissues at newborn stage(postnatal day 0),growth stage(1-month-old),and adult stage(3-month-old).Col2?1-Cre transgenic mice,which served as a negative control,exhibited no significant differences with the WT control with regards skeletogenesis.No significant differences were observed between male and female mice in terms of the pathological effects induced by Nell-1 deficiency.At newborn stage,appearance of WT and Nell-1Col2?1KO pups showed no obvious difference.Alcian Blue-Alizarin Red skeletal staining and ex vivo micro-CT scanning showed no defect or loss of osteochondral tissues in Nell-1Col2?1KO mice.But subtle shortness of femur of Nell-1Col2?1KO mice was appreciable by length measurements.The difference between WT and Nell-1Col2?1KO mice became more obvious with time.Conditional Nell-1 chondrocyte-specific knockout resulted in postnatal dwarfism.Reduced body length,tail length,and body weight were all displayed in Nell-1Col2?1KO mice.Additionally,Nell-1Col2?1KO mice showed osteoporotic phenotypes as early as 1 month.Specifically,dramatically changed length,bone mineral density,bone volume/tissue volume,trabecular number,trabecular thickness,trabecular separation,cortical volume,cortical thickness and cortical mineral density were all observed in Nell-1Col2?1KO femurs and tibias,accompanied by abnormal osteoblasts and osteoclasts.By bone histomorphometric analyses,we found that both mineral apposition rate and bone formation rate per unit of bone surface of trabecular bones under femoral and tibial growth plates were decreased in 1-month-old and 3-month-old Nell-1Col2?1KO mice,indicating Nell-1's effect on trabecular bone formation based on endochondral ossification.Furthermore,4-week dynamic observation by in vivo micro-CT showed retarded and less mineralization at secondary ossification centers in mutants,which indicates Nell-1 is a functional key factor in modulating endochondral ossification.Next,we observed the changes of growth plate cartilage in Nell-1Col2?1KO mice.Histologically,reduced staining intensities of Safranin O,Col-2 and Col-10 together with less Brd U positive chondrocytes and thinner proliferation zone were observed in Nell-1Col2?1KO growth plate,indicating conditional Nell-1 knockout affects proliferation and differentiation of growth plate chondrocytes.Consistent with these in vivo phenotypes,Nell-1Col2?1KO chondrocytes also showed decreased proliferation(detected by MTT assay),lighter Alcian Blue staining intensity,reduced gene expression levels of Col-2 and Aggrecan.Notably,all the losses caused by Nell-1 inactivation were partially recovered by applying rh NELL-1 in vitro,demonstrating the crucial effect of Nell-1 on epiphyseal homeostasis,and also on proliferation,differentiation and maturation of chondrocytes.To clarify related mechanisms,we studied the interactions between Nell-1 and Ihh-PTHr P signaling pathway.By immunohistochemistry,we found limited distributions and weaker expressions of Ihh,Patched-1,PTHr P and its receptor in Nell-1Col2?1KO growth plates.These reduced expressions in vivo were consistent with the protein expression levels observed in primary WT and Nell-1Col2?1KO chondrocytes,while application of exogenous rh NELL-1 significantly rescued these losses Additionally,the gene expression levels of key molecules in Ihh-PTHr P pathway(Ihh,Patched-1,Smo,Gli-1 and PTHr P),together with Col-2 and Aggrecan,were all reduced in Nell-1Col2?1KO chondrocytes.Application of rh NELL-1 in vitro rescued the decline in gene expression,while cyclopamine,an inhibitor of Ihh signaling pathway,affected rescue by rh NELL-1.These data showed that Nell-1 promotes proliferation and differentiation of chondrocytes by elevating Ihh signaling.Since Gli-1 is a key transcription factor in Ihh signaling pathway,and its nuclear accumulation is critical for its functions,we then studied the effect of Nell-1 on Gli-1 by observing Gli-1's nuclear expression on protein level by ICC and verifying by Western blotting.Both of them showed more nuclear Gli-1 expression in WT,while adding Nell-1 clearly increased the protein level of Gli-1 in Nell-1Col2?1KO nucleus.We further tested the expression of Gli-1's downstream target genes Hip-1 and N-Myc.Both of them decreased with Nell-1's inactivation,and increased with application of rh NELL-1.When WT chondrocytes were treated with Gli-1's inhibitor,GANT61,adding rh NELL-1 can no longer increase Hip-1 and N-Myc's gene expression level.Collectively,chondrocyte-specific Nell-1 deficiency may result in postnatal dwarfism and premature osteoporosis.Nell-1 is a pivotal modulator of postnatal epiphyseal homeostasis and endochondral ossification by up-regulating expressions of key molecules in Ihh-PTHr P pathway and Gli-1's nuclear expression.