Abnormal Iron Metabolism In Irp2 Deficient Mice Is Associated With Osteoporosis

Objective: To explore the effects of iron regulatory protein 2 on bone remodeling in aged mice,and the role of iron overloaded on the pathogenesis of Osteoporosis and its mechanism.Methods: 15-month-old healthy female Irp2 knockout(Irp2^-/- )mice were selected randomly as the experimental group?n=6,endogenous iron overload model?,and 15-month-old healthy female wild-type(Irp2^+/+)mice as the control group by genotype identification.The vertebrae?L6?were subjected to micro computed tomography?Micro-CT?scans and three-dimensional images were reconstructed.The bone tissue slices were treated with Prussian blue staining and HE staining to observe iron deposition and bone structure.The concentrations of iron,calcium,phosphorus in bone and iron in liver were determined by atomic absorption spectrometry.The expression of iron metabolism-related genes [Ferroportin-1?Fpn1?,Transferrin receptor-1?Tfr1?,Ferritin light chain?Ftl?,Hepcidin] and bone metabolism-related genes[Bone alkaline phosphatase?Balp?,Bone gla protein?Bglap?,Type ?collagen alpha 1 chain?Col??1?,Cathepsin K?Ctsk?,Nuclear factor of activated T-cells cytoplasmic 1?Nfatc1?,receptor activator of nuclear factor?B?Rank?,tartrate-resistant acid phosphonatase?Trap?] in bone tissue were detected by Realtime PCR.The expression of iron metabolism related proteins in bone tissue were detected by Western-blot.The levels of serum iron,calcium were evaluated by colorimetric method and microplate method,respectively.Enzyme-linked immunosorbent assays were performed to measure biochemical markers of bone turnover: bone formation markers indicate of osteoblast activity [Balp,Bglap and COL??1] and bone resorption markers indicate of osteoclast activity [Ctsk,Trap and C-terminal telopeptide of type ? collagen?CTX-1?].The level of serum 25 hydroxy vitamin D3[25?OH?D3] and 25-hydroxylase?CYP2R1?in liver were detected by ELISA.Results:1 Compared with wild-type control,the trabecular bone mass was markedly reduced?both of BMD and Tb.N?in Irp2^-/- mice.2 Compared with Irp2^+/+ mice,the bone iron deposition in Irp2^-/- group was significantly lower.Bone trabecular was more sparser and thinner,and obvious decreased bone density could be observed in Irp2^-/- mice by HE staining.3 Atomic absorption spectrometry showed that there was severe iron deposition in the liver of Irp2^-/- mice,whereas the levels of iron,calcium and phosphorus in the bone of Irp2^-/- mice were significantly decreased when compared with Irp2^+/+ mice.4 The m RNA expression of Hepcidin and Ftl was significantly increased in Irp2^-/- mice when compared with Irp2^+/+ mice.However,Fpn1 and Tf R1 gene expression were reduced in Irp2^-/- mice.5 The m RNA levels of Balp,Bglap,and Col ? ? 1,which reflect osteoblast activity,were all significantly reduced in Irp2^-/- mice.In contrast,the m RNA expression of osteoclast activity genes,Ctsk,Nfatc1,Trap and Rank,were all markedly upregulated in Irp2^-/- mice when compared with Irp2^+/+ mice.6 The protein levels of Ferritin-L,Ferritin-H,FPN1 and IRP1 in bone of Irp2^-/- mice were decreased as compared to Irp2^+/+ mice,whereas Tf R1 and Divalent Metal Transporter 1 without iron-responsive elements?DMT1-IRE?in bone of Irp2^-/- mice were markedly higher when compared with Irp2^+/+mice.7 Compared with the Irp2^+/+ group,serum level of iron was significantly decreased in Irp2^-/- group?64.71±5.21 ?mol/L vs 154.01±65.09 ?mol/L,P<0.01?.The levels of Balp?132.93±4.82 ?mol/L vs 166.66±18.83 ?mol/L?,Bglap?1.84±0.17 ?mol/L vs 2.86±0.19 ?mol/L?and COL ? ?1?20.21±0.91ng/ml vs 24.98±2.04 ng/ml?,which reflect osteoblasts activity,were significantly decreased in the Irp2^-/- group than their wild-type control?P<0.001?.The levels of Ctsk?72.42±1.82 ?mol/L vs 62.73±7.00 ?mol/L?,Trap?40.63±2.43 U/L vs 36.42±2.77 U/L?and CTX-1?2.74±0.33 ?mol/L vs2.19±0.42 ?mol/L?,which reflect osteoclasts activity,were significantly higher in the Irp2^-/- group as compared to their wild-type control?P<0.05?.Irp2^-/- mice had significantly lower vitamin D 25-hydroxylase activity?1.21±0.18 ng/mg vs 1.94±0.14 ng/mg,P<0.001?in liver and lower serum25?OH?D3?25.63±0.98 ?mol/L vs 28.40±1.66 ?mol/L,P<0.01?than Irp2^+/+mice.No obvious difference was found in serum calcium between the two groups.Conclusion:1.Irp2 gene knockout can lead to iron metabolic disorders and abnormal iron distribution in mice?iron overload in liver,iron content decreased in bone?.The expression of iron metabolism related gene and protein in the bone of Irp2^-/- mice have obvious and compensatory changes because of iron metabolic disorders.2 Both of Irp2^-/- mice and their control littermates developed osteoporosis.Irp2^-/- mice had severe osteoporosis.3 Iron metabolic disorder can aggravate the development of osteoporosis.Endogenous iron overload down-regulating of hepatic CYP2R1 activity,which leads to attenuation of serum 25?OH?D3.Taken together,Irp2 deficiency dysregulates iron metabolism in liver and bone,causing pathological effects on bone that ultimately leads to severe osteoporosis.