ABSTRACT
BACKGROUND:SMAD family member 4(SMAD4)can promote bone remodeling in osteoporotic rats,but it is unclear whether SMAD4 interferes with the expression of iron metabolism related proteins in osteoporotic rats. OBJECTIVE:To explore the effect of SMAD4 overexpression on the expression of iron metabolism related proteins in osteoporotic rats. METHODS:Rats were randomized into sham group,ovariectomy group,transfection control group and SMAD4 overexpression group.Animal models of osteoporosis were established in the latter three groups by ovariectomy,and only adipose tissue was removed in the sham group.One week later,adenovirus was injected into the femoral bone marrow cavity.SMAD4 overexpression group and transfection control group were injected with adenovirus overexpressing SMAD4 gene and control empty virus,respectively.Index detection was performed at 1 month after injection.Micro-CT,hematoxylin-eosin staining and tartrate resistant acid phosphatase staining were used to detect bone formation and bone resorption in osteoporotic rats.ELISA was used to detect serum ferritin and hepcidin levels.Immunohistochemical staining was used to detect alkaline phosphatase,osteocalcin,receptor activator for nuclear factor-κB ligand and tartrate resistant acid phosphatase levels in femoral tissue.RT-qPCR was used to detect SMAD4,hepcidin,divalent metal transporter 1,transferrin receptor1 and ferroportin1 mRNA levels in femoral tissue.Western blot was used to detect SMAD4,alkaline phosphatase,osteocalcin,osteoprotegerin,receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps,hepcidin,divalent metal transporter 1,transferrin receptor 1,and ferroportin 1 protein levels. RESULTS AND CONCLUSION:In the sham group,bone trabeculae in femur tissue were intact,and almost no osteoclasts were found.In the ovariectomy and transfection control groups,the bone trabeculae were sparse and a large number of osteoclasts were present.In the SMAD4 overexpression group,the number of bone trabeculae was increased and the number of osteoclasts was decreased.Compared with the sham group,the ovariectomy group showed a significant reduction in the protein expression of SMAD4,alkaline phosphatase,osteocalcin,and osteoprotegerin in femoral tissue and hepcidin levels in serum and femoral tissue,while receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps protein levels,divalent metal transporter 1,transferrin receptor1,ferroportin1 mRNA and protein levels were significantly increased(P＜0.05).Compared with the transfection control group,the SMAD4 overexpression showed a significant increase in SMAD4,alkaline phosphatase,osteocalcin,and osteoprotegerin protein levels in femoral tissue and hepcidin levels in serum and femoral tissue,while the expressions of receptor activator for nuclear factor-κB ligand,tartrate resistant acid phosphatase,β-Crosslaps protein levels,divalent metal transporter 1,transferrin receptor1,and ferroportin 1 at mRNA and protein levels were significantly decreased(P＜0.05).To conclude,overexpression of SMAD4 promotes bone remodeling in osteoporotic rats by interfering with the expression of iron metabolism related proteins.
ABSTRACT
Objective:To identify the genetic variation in a mucopolysaccharidosis type Ⅱ(MPS Ⅱ)family, and conduct a functional study of iduronate-2-sulfatase(IDS): c.323A>C.Methods:A five-generation MPS Ⅱ family of 83 individuals including 4 patients from northern China was collected. Urine mucopolysaccharide and Alder-Reilly body were tested to assist the clinical diagnosis of MPS Ⅱ. IDS enzyme activity was detected on core family members. By the whole exome sequencing of a MPS Ⅱ patient in this family and bioinformatics analysis, the variant was screened and further identified by PCR-Sanger sequencing. Finally, to validate the function of the variant in vitro, the wild-type IDS overexpression plasmid(pCMV-hIDS-WT)and the IDS overexpression plasmid carrying the mutation site(pCMV-hIDS-c.323A>C)were transfected into COS-7 cells and the IDS activity was detected. Results:The proband(Ⅳ3)and Ⅳ4 were diagnosed as MPS Ⅱ by urine mucopolysaccharide, Alder-Reilly body, and IDS enzyme activity tests. Ⅳ3, Ⅳ4, Ⅲ19, and Ⅲ32 were determined to carry IDS: c.323A>C missense variant through the whole-exome sequencing, and diagnosed as MPS Ⅱ. Meanwhile, Ⅱ2, Ⅱ4, Ⅱ8, Ⅱ12, Ⅱ14, Ⅲ5, Ⅲ7, Ⅳ14 in the MPS Ⅱ family carried IDS: c.323A>C missense variant, and were excluded as MPS Ⅱ. The in vitro experiment in COS-7 cells showed that the missense mutation led to a significant decrease in IDS enzyme activity. Conclusion:The variant IDS: c.323A>C: p.Y108S significantly decreases the activity of IDS enzyme in vivo and in vitro, and it is identified as a pathogenic variant for MPS Ⅱ.