Pappa2 Gene Affects Mouse Hip Development Through IGF Signaling Pathway

Objectives:Developmental dysplasia of the hip(DDH)is one of the major causes of children disability and early osteoarthritis.In China,report an incidence of 1.5 to 20 cases of DDH per 1000 births,and the identified risk factors include hip delivery,primipara,oligohydramnios,high birth weight,straight leg swaddling,female and family history,etc.As a complex polygenic disease,Genetic factors and environmental factors are commonly accepted,but the pathogenesis of DDH is multifactorial and poorly understood.Among which genetic factors play an important role.Our previous study indicated that an SNP associated with the family was localized in the Pregnancy-associated plasma protein-A2(PAPP-A2)gene which suggest an association of PAPP-A2 with sporadic DDH susceptibility in a Chinese Han population.The PAPPA2 gene is located in the q24 region of chromosome 1,including 23 exons and 22 introns.As a metalloproteinase,PAPP-A2 can specifically hydrolyze insulin-like growth factor binding protein-5(IGFBP-5).IGFBP-5 is one of the six family members of insulin-like growth factor binding protein,and it is also one of the most abundant in skeleton.It can bind to insulin-like growth factor-1(IGF1)to form the complex of IGF-I-IGFBP-5,resulting in the reduction of free IGF1,thus reducing the bioavailability of IGF1.As an important factor related to growth and development,IGF regulates cartilage development and fibroblasts collagen synthesis.Acetabular cartilage dysplasia and soft tissue relaxation around hip joint are two main pathological factors of DDH.Therefore,the purpose of this study is to explore how PAPPA2 gene affects the development of cartilage and fibrous tissue through IGF signaling pathway,and then lead to DDH susceptibility.We first carried out in vitro cell experiments to observe the changes of IGF1 and collagen synthesis and cartilage proliferation in mouse fibroblasts and primary chondrocytes by up-regulating or down-regulating the expression of PAPP-A2 protein.The expression of Pappa2 gene was interfered by local transfection and intraamniotic injection of PAPP-A2 sgRNA lentivirus,and the differences of expression of IGF signaling pathway related proteinand development of hip joint between the two groups were compared.Methods:This research is divided into two parts.1.Effect of abnormal expression of PAPPA2 gene on collagen synthesis of mice fibroblast cell and proliferation of mice primary chondrocyteFibroblast were cultured in a culture plates until the cells converged.They were intervened by:(1)IGFBP-5 protein,(2)PAPP-A2 protein,(3)blank control,(4)PAPP-A2 sgRNA lentivirus,and(5)lentivirus in control group.PAPP-A2 protein and IGFBP-5 protein were interfered according to concentration gradient.Lentivirus transfection pretreatment was also carried out.Hydroxyproline hydrolysis was used to determine collagen synthesis in fibroblasts.Western blot and Real time RT-PCR were used to detect the expression of COL1A1,IGF1 protein and mRNA.Primary chondrocytes were cultured in a culture plates until the cells converged.They were intervened by:(1)IGFBP-5 protein,(2)PAPP-A2 protein,(3)blank control,(4)PAPP-A2 sgRNA lentivirus,and(5)lentivirus in control group.Western blot and Real time RT-PCR were used to detect the expression of ACAN,collagen type II COL2 and IGF1 in chondrocytes.2.The effect of low expression of Pappa2 gene on the proliferation of acetabulum and femoral head cartilage also collagen synthesis in soft tissue around acetabulum in miceICR female mice were directly injected with PAPP-A2 sgRNA lentivirus and control group lentivirus separately around the right and left hip at 4 days after birth(B4),and The mice were sacrificed at 14 days after birth(B14).The green fluorescent protein(GFP)fluorescence in the hips was observed using a fluorescence stereomicroscope to confirm successful transfection,then immunohistochemistry,western blot and real time RT-PCR were used to detect the expression of IGF1,COL1A1,COL2,ACAN,IGFBP-5 and PAPP-A2.PAPP-A2 sgRNA lentivirus was microinjected into the uterus of mice at 13 days of gestation(P13)via amniotic cavity.Pregnant mice were sacrificed one day before birth(P19)to obtain fetal mice interfered by lentivirus.The expressions of IGF,COL1A1,COL2,ACAN and PAPP-A2 were detected by immunofluorescence.ICR female mice were directly injected with PAPP-A2 sgRNA lentivirus and control group lentivirus separately around the right and left hip at 4 days after birth(B4),and The mice were sacrificed at 14 days after birth(B14).The morphological changes of the acetabulum were observed and the transverse diameter of the acetabulum was measured.The green fluorescent protein(GFP)fluorescence in the hips was observed using a fluorescence stereomicroscope to confirm successful transfection,paraffin sections were made and the safranine was dyeing with safranine and fast green.The ossification of chondrocyte layers(the resting cell layer,proliferating cell layer and mast cell layer)was observed.Results:1.In fibroblasts,the expression of collagen was up-regulated after when add PAPP-A2 protein.When IGFBP-5 protein was added,the expression of collagen was lower than that of control.2.The content of hydroxyproline increased and the IGF1 expression was up-regulated in fibroblasts interfered by PAPP-A2 protein,while the expression of IGF1 and COL1A1 was down-regulated in fibroblasts interfered by IGFBP5 protein.3.The content of hydroxyproline in fibroblasts transfected with PAPP-A2 sgRNA lentivirus was lower than that in control group,the expression of IGF1 and PAPP-A2 was down-regulated,and the expression of IGFBP5 was up-regulated.4.The expression of ACAN was up-regulated in primary chondrocyte interfered by PAPP-A2 protein,while the expression of ACAN and COL2 was down-regulated in primary chondrocyte interfered by IGFBP5 protein.5.The expression of PAPP-A2,ACAN and IGF1 in primary chondrocytes transfected with PAPP-A2 knockdown lentivirus was down-regulated,and the expression of IGFBP5 was up-regulated.6.The expression of PAPP-A2,ACAN and COL2 in acetabulum and femoral head cartilage was down-regulated and IGFBP5 was up-regulated at day B14 in mice with PAPP-A2 sgRNA lentivirus injected into hip joint.IGF1 expression in soft tissue around acetabulum was down-regulated.7.The expressions of PAPP-A2,IGF1,ACAN and COL2 in acetabular femoralhead cartilage and PAPP-A2,IGF1 and COL1A1 in periacetabular soft tissue were down-regulated at P19 day in mice with PAPP-A2 knockdown lentivirus amniotic cavity micro-injection.8.In mice withPAPP-A2 sgRNA lentivirus injected into the hip joint,the transverse diameter of the acetabulum increased,the acetabular fossa became shallow and the mast cell layer narrowed.Conclusion:1.In fibroblasts,PAPP-A2 protein interference can significantly increase the expression of collagen in fibroblasts,and there is a dose-dependent relationship.IGFBP-5 protein interference can the decrease the expression of collagen.2.The expression of Pappa2 in fibroblasts and primary chondrocytes was negatively correlated with the expression of IGFBP-5.The high expression of IGFBP-5 could reduce the expression of IGF1.3.Pappa2 can indirectly affect the expression of IGFBP-5 in the soft tissue around the acetabulum of mice.4.In the acetabular and femoral head cartilage of mice,the low expression of Pappa2 can indirectly affect the proliferation and differentiation of cartilage by affecting the expression of IGFBP-5.5.Low expression of Pappa2 can affect acetabular morphology in mice.