The Role And Mechanism Of Sp7-expressing Osteoblasts-derived Indian Hedgehog In Regulating Osteoblast Differentiation And Bone Formation

BackgroundThe mammalian skeleton originates from three different embryonic cellular populations and is accomplished through two distinct ossification processes:intramembranous ossification and endochondral ossification.Endochondral ossification,which represents the primary mechanism of bone formation in vertebrates,is the main growth mode of trunk and limb bones.The transition from cartilage to bone tissue is tightly integrated with chondrocytes,osteoblasts,and angiogenesis during endochondral ossification,and impaired cartilage development and bone formation can lead to bone dysplasia,dwarfism,and other conditions.This vital developmental process is tightly regulated,temporally and spatially,by many factors,including transcription factors,hormones,and cytokines.Indian hedgehog is a cytokine that governs complicated endochondral ossification and is mostly expressed in pre-hypertrophic chondrocytes,and in osteoblast lineage cells.Mutations involving IHH can impair Ihh signal transduction activity and have been identified as pivotal pathogenic factors for brachydactyly type A1and acrocapitofemoral dysplasia.Current genetic studies suggest that chondrocyte-derived Ihh maintains normal development of the growth plate and osteogenesis by regulating chondrocyte proliferation,differentiation,and specification of osteoprogenitor cells.In addition,the BMP/SMAD signaling pathway is a key regulator of osteogenic differentiation and bone formation.The ligand Bmp2/4（a member of the bone morphogenetic protein family）is a secreted protein that plays an important role in osteogenesis and is mainly expressed in the perichondrium and periosteal osteoblasts,and induces phosphorylation of the downstream Smad1/5/8 complex by binding to the receptor,which in turn activates Runx2 transcription.Runx2 is an important transcription factor that regulates osteoblast differentiation and osteogenesis-related gene expression.Recent studies have shown that the BMP2/SMAD signaling pathway acts on the downstream of Ihh,and down-regulation of the Ihh gene resulted in a significant decrease in the expression of Bmp2.In summary,the functions of Ihh derived from osteoblast lineage cells in osteogenesis have not been surveyed,and the molecular mechanisms by which Ihh directly affects osteoblast precursor differentiation and mineralization remain to be elucidated.Therefore,elucidating the functions of osteoblastic Ihh in vivo,as well as further understanding the mechanisms by which Ihh affects the behavior of Sp7-expressing osteoblasts,will contribute to the development of new therapies for osteodysplasia and dwarfism deformity.Objective:（1）Dynamic observation of Cre protein recombinase,endogenous Sp7,and Ihh gene and protein expression in early developing tibial tissue by direct detection of green fluorescent protein,immunohistochemistry and in situ hybridization using Sp7-i Cre transgenic mice and Rosa26^ZSGreen mice to verify the co-localized expression of Sp7,Cre and Ihh in tibial perichondrium/periosteal osteoblasts during bone development;（2）Sp7-i Cre;Ihh^fl/flknockout mice were bred and genotyped and in vivo observation of the effect of Ihh on bone formation using this mouse model;（3）In vitro cellular assays were used to study the regulation of Ihh on osteoblast proliferation and differentiation,and to investigate the molecular mechanism of osteoblast Ihh gene regulation of cell differentiation and mineralization.In conclusion,using in vivo conditional knockout of Ihh gene in transgenic mice and Rosa26 tracer mice,combined with in vivo histological and in vitro cellular studies,we elucidated the role of osteoblast-derived Indian hedgehog factor in regulating cell differentiation and bone formation at the holistic,cellular and molecular levels,and preliminarily explored its mechanism of action.Methods:（1）A strain of Sp7-i Cre transgenic mice was constructed;Ihh-specific knockout mice(Sp7-i Cre;Ihh^fl/fl;Rosa26^-ZSGreen)were bred,and Ihh^fl/fl mice from the same litter were taken as a littermate control group.The methods were as follows:a.Extraction of genomic DNA from mouse tail tissues and identification of genotypes of two groups of mice by PCR amplification and agarose gel electrophoresis;b.Tracing dynamic expression of Sp7 promoter-mediated Cre recombinase during early development using Cre reporter mice;observing dynamic expression of endogenous Sp7 and Ihh genes and proteins using immunohistochemistry and in situ hybridization;c.Extraction of RNA from E18.5 embryonic mouse tibia,calvaria and primary osteoblasts,liver,kidney,lung,and heart tissues,RT-q PCR to detect the expression of Ihh gene in different tissues and to confirm the effective knockdown of Ihh specifically in bone tissue.The gross phenotypes of newborn mice with knockout Ihh gene and Ihh^fl/fl mice were observed,and the newborn mice’s length and body weight were measured.The bone tissue phenotype of mice was examined by X-ray examination and whole-skeleton staining with histological staining.（2）In vivo experiments to study the role of Ihh on bone formation in osteoblasts during early mouse development using Ihh-specific knockout Sp7-i Cre;Ihh^fl/fl mice.This was done as follows:a.The knockout Ihh group and control group mice in embryonic stages 13.5（E13.5）,14.5（E14.5）,16.5（e16.5）,and 18.5（E18.5）were collected.The dynamic phenotypes of bone growth and matrix mineralization in mice during early development were studied by whole-skeleton staining;b.The histomorphological characteristics of the tibia were further analyzed by staining with Safranin O and fast green,and the characteristics of tibial tissue mineralization and bone formation in early bone development were observed by von Kossa（calcium salt staining method）and Masson trichrome staining,respectively.c.Using tibial tissue sections from mice at each stage,we observed the expression of the runx-related transcription factors（Runx2）,type I collagen（Col1α1）,osteoprotegerin（Opg）and osteocalcin（Ocn）by immunohistochemistry and,also observed the expression of angiogenesis-related markers Vegfa and CD31 in two groups of mice with E18.5;d.An immunohistochemistry of collagen type II and collagen type X was performed using tibial tissue sections from mice at each stage,and the expression of PCNA,an indicator of proliferation,was also detected,and apoptosis was observed by an in situ apoptosis detection kit（TUNEL）;The expression of PCNA was detected,and apoptosis was observed by an in-situ apoptosis detection kit（TUNEL）.e.Total RNA was extracted from the tibial tissues of control and knockout Ihh mice on day E18.5 and the expression of osteogenic-related genes in tibial tissues was detected.（3）In vitro experiments were conducted to investigate the proliferation,differentiation,and mineralization of primary osteoblasts after knockdown of Ihh,to further investigate the role of Ihh in regulating osteoblast function,and to verify whether Ihh plays a role in regulating osteoblast differentiation through the downstream BMP2/SMAD/RUNX2 pathway.The methods were as follows:a.The expression of Bmp2 in E14.5,E16.5,and E18.5 mice after knockdown of Ihh was observed at the protein level by Anti-BMP2 staining;total RNA was extracted from E18.5 tibial tissues,and the expression of Bmp2 and downstream SMAD1 and SMAD5 in tibial tissues after knockdown of Ihh was observed at the gene level by real-time quantitative PCR;Primary osteoblasts were extracted and RT-PCR was performed to detect the expression of Bmp2in the primary cells of the knockout Ihh group and the control group.b.The proliferation of the two groups of cells was measured by CCK-8 assay and crystalline violet staining.After 7 and 14 days of osteogenic induction,the two groups of cells were stained with ALP,von Kossa（calcium salt staining）and ARS staining to observe cell differentiation and mineralization,respectively;Total RNA and protein were collected from the two sets of cells,and RT-q PCR and Western blot were used to determine m RNA and protein expression levels of osteogenesis-related genes,respectively.c.Primary osteoblasts from two groups of mice were extracted,and the knockout group cells and normal group cells were given osteogenic differentiation induction medium+rh BMP2 and osteogenic differentiation induction medium for 7 and 14 days,respectively,and ALP staining,von Kossa（calcium salt staining）and ARS staining were performed to observe cell differentiation and mineralization;total RNA and protein were extracted from the two groups of cells,and the expression of osteogenic-related genes at the gene and protein levels were detected by RT-q PCR and Western blot,respectively.c.During induction of differentiation,knockout group cells and normal group cells were given osteogenic induced differentiation medium+rb BMP2 and induced differentiation medium for 14days,respectively.ALP staining showed reduced alkaline phosphatase activity in knockout group osteoblasts,and the results of von Kossa and ARS staining indicated inhibition of cell differentiation and mineralization ability.Results:（1）Successful breeding and genotypic identification of Sp7-i Cre⁺;Rosa26^-ZSGreenand Sp7-i Cre⁺;Ihh^fl/fl;Rosa26^-ZSGreenmice.a.Fluorescence images of frozen sections of tibia showed the presence of GFP-positive cells in the perichondrium and bone collar at E14.5,and by E18.5,most of these cells were present in the periosteum and bone trabeculae;Immunohistochemical staining images of Sp7 showed co-localized expression of Sp7 and GFP in osteogenic lineage cells;immunohistochemistry and in situ hybridization of Ihh showed that Ihh was expressed in early development not only in pre-mast chondrocytes but also in sp7-positive cells in the periosteum and trabeculae.b.Amplification of genomic DNA from mouse tail or toe tissue by conventional PCR demonstrated that Sp7-i Cre;Ihh^fl/fl mice carry the Cre gene in vivo,indicating the successful construction of Sp7-i Cre;Ihh^fl/fl mice;in addition,Sp7-i Cre;Ihh^fl/fl mice were born in Mendelian proportions but died shortly after birth（due to rib dysplasia and softening of the thoracic structures leading to respiratory failure）.Neonatal mice exhibited severe bone dysplasia characterized by short limbs and dwarf deformity,but normal division of the interphalangeal joints;further quantitative analysis showed reduced body weight and significantly shorter body length（P<0.05）;X-ray results further showed a significant reduction in bone mineral density and bone volume in knockout Ihh mice;Whole skeleton staining further showed that in addition to the dysplasia of the long bones,other bones,such as ribs,clavicle,vertebrae,sternum,and mandible,were also smaller in size,while the low mineralization and wider sagittal suture of the cranial bones also indicated that intramembranous osteogenesis was also impaired.c.RT-q PCR analysis of RNA extracted from the tibiae of both groups of mice confirmed a significant deletion of the Ihh gene in the tibiae of the knockout group（～90%）;the level of Ihh in the skull was also significantly reduced by～50%.In addition,analysis of Ihh m RNA levels in the liver,lung,heart and kidney showed no statistically significant differences（P>0.05）,and the expression of Ihh was specifically reduced in bone tissue.d.H&E staining showed that the knockout mice had shortened and disorganized growth plates,shorter resting,proliferative and hypertrophic zones compared to control mice,as well as significantly reduced ossification and periosteal width.Von Kossa and Masson staining showed that the bone marrow cavity of the mutant mice was shortened and mineralization was restricted to the center of the tibia,but trabecular and cortical bone formation did not occur.（2）Deletion of Ihh in Sp7⁺osteoblasts delayed bone growth and matrix mineralization,resulting in severe bone dysplasia and abnormal growth plate morphology,but did not affect normal joint segmentation.a.Gross phenotypes and whole skeleton staining of two groups of mice at different time points showed that knockout of osteoblastic Ihh retarded bone growth and matrix mineralization,while joint segmentation was normal.The results of staining of tibial tissues at different time points with Safranin O&fast green,von Kossa and Masson showed that cortical and trabecular bone formation was impaired in knockout mice and affected normal growth plate formation;Immunohistochemical results demonstrated that the expression of Runx2 and Sp7,key transcription factors necessary for bone formation,and the bone matrix proteins type I collagen,osteoprotegerin,and osteocalcin were weak and restricted to the terminally differentiated chondrocytes in the central region;RT-q PCR results of tibial tissues from E18.5 mice further indicated reduced expression of osteogenesis-related genes;c.Immunohistochemical and PR-q PCR results of angiogenesis-related markers Vegfa and CD31 showed significantly reduced expression of Vegfa and CD31 in presumed primary ossification centers in tibiae of E18.5 mutant mice;in addition,immunohistochemical and RT-q PCR results showed that the expression of chondrocyte markers Col2α1 and Col10α1 was also reduced,and anti-PCNA staining and TUNEL staining results further indicated that deletion of Ihh resulted in decreased chondrocyte proliferation and increased apoptosis.（3）In vitro cellular assays showed that deletion of Ihh inhibited proliferation,differentiation and mineralization of primary osteoblasts,and that osteoblast-derived Ihh may exert osteogenic effects through the BMP2/SMAD/RUNX2 signaling pathway.a.As newborn mutants died shortly after birth,primary osteoblasts were isolated from the calvaria of E18.5 Ihh^fl/fl mice and Sp7-i Cre;Ihh^fl/fl mice and then cultured for proliferation and differentiation.A sufficient number of cells could be extracted from the cranial parietal bone tissue of both groups of E18.5 mice for in vitro cell experiments,and the cell morphology was basically normal.The results of the CCK-8 assay and crystalline violet staining showed that the primary osteoblasts of mutant mice grew relatively slowly and their proliferation ability was inhibited.The level of Ihh m RNA in primary osteoblasts of mutant mice cultured in growth medium for 7 and 14 days decreased by 53%and 63%,respectively.After 7 and 14 days of culture in the induced differentiation medium,we also observed that inactivation of Ihh also inhibited osteoblast mineralization;further RT-PCR results showed a significant decrease in the m RNA levels of osteogenesis-related genes.b.Immunohistochemical results of BMP2showed not only reduced perichondral osteoblasts and decreased Bmp2 expression in E14.5 mutant mice compared to Ihh^fl/fl mice in the same litter,but the expression of Bmp2 in periosteum and bone trabecular region was weak at E18.5.Quantitative RT-PCR analysis at the genetic level demonstrated significantly reduced expression of Bmp2 and Runx2 in the tibiae of mutant mice.The results of RT-PCR and Western blot assay showed that deletion of Ihh reduced the expression of osteogenic-related genes at the gene and protein levels,and also attenuated the BMP2/SMAD/RUNX2 pathway,Moreover,Moreover,the results of rh BMP2 rescue experiments showed that primary osteoblasts lacking Ihh recovered or partially recovered osteogenic and mineralizing functions after rh BMP2 treatment.Conclusion:In this study,we successfully constructed a novel Ihh knockout mouse model(Sp7-i Cre;Ihh^fl/fl),which for the first time specifically knocked out the Ihh gene in Sp7-expressing cells in vivo,revealing the important role of the Ihh gene in Sp7+osteoblasts in regulating cell differentiation and bone formation.The specific knockdown of the Ihh gene in osteoblastic lineage cells resulted in severe bone dysplasia characterized by short limbs and dwarf deformities,especially impaired cortical and trabecular bone formation and abnormal growth plate formation,but did not affect normal interphalangeal joint segmentation.Further in vitro experiments showed that Ihh derived from osteoblastic lineage cells may regulate osteoblast differentiation,mineralization,and bone formation through the BMP2/SMAD/RUNX2 signaling pathway.