Effect Of Ferroportin Deficiency In Myeloid Lineage Cells On Iron Metabolism And Osteoclast Differentiation And Fuction

Objective To observe the effect of deletion of ferroportin?Fpn?in myeloid lineage cells on iron metabolism and osteoclast differentiation and function.Methods Ferroportin?also know as Scl40a1?is a sole iron exporter in mammalian cells.We obtained the Fpn-floxed mice,in which the exons 6 and 7 of murine Slc40a1 gene were flanked by two loxP sites,from the Jackson Laboratory and crossed them with Lysozyme M-Cre?LysM-Cre?mice to generate conditional knockout mouse strains with specific deletions of Fpn in myeloid lineage cells.The Fpn-flox/flox;Cre/+mice were designated as myeloid(Fpn^?LysM)Fpn knockout mice.The Fpn-+/+;Cre/+littermates were used as controls.Both long bones and vertebrae from 2-month old conditional knockout mice and controls were imaged in a Mirco CT.Meanwhile,The histomorphometric measurements of trabecular bone and osteoclast number and surface were done with a digitizer tablet interfaced to a Zeiss Axioscope with a drawing tube attachment.On the other hand,we also detected the serum level of N-terminal propeptide of type I procollagen?PINP?,a global marker of bone formation,to assess whether Fpn-deletion affects osteoblasts.In order to determine how Fpn-deletion affects cellular iron content in osteoclast precursors,we conducted Tf-Fe⁵99 up-taken assay in bone marrow macrophages?BMMs?derived from control and Fpn^?LysMLysM mice.And next,we set out to identify the effects of increased/decreased cellular iron on osteoclastogenesis in vitro.The control and Fpn-null BMMs were cultured with M-CSF and RANKL for 3 and 4 days before fixation.The cells were then stained for tartrate-resistant acid phosphatase?TRAcP?,an osteoclast differentiation marker.The total number of multi-nucleated,TRAcP⁺osteoclasts were counted.Besides,BMMs were cultured on cortical bovine bone slices like before.The m RNA expression of osteoclast marker genes,Acp5?encoding TRAcP?,Nfatc1?encoding key osteoclast transcription factor NFATc1?,and Ppargc1b?encoding PGC-1??,detected by real-time quantitative PCR,both in Fpn-null osteoclast lineage cells and control ones.And the protein levels of CTSK,NFATc1,and PGC-1?detected by Western Blot in the course of osteoclast differentiation.As we all know,cell proliferation,differentiation,and survival of osteoclast precursor cells are critical cellular processes regulating osteoclastogenesis in vivo and in vitro.To figure out which of these processes are influenced by increased cellular iron,we performed BrdU labeling followed by flow-cytometry analysis of control and Fpn-deletion macrophages,and used Cell-Death ELISA assay to analyze the survival of osteoclast precursor cells.We next tried to determine whether increased/decreased cellular iron induced by Fpn-deficiency modulates downstream signaling pathways of M-CSF and/or RANKL in macrophages by Western Blot.Results Deletion of Fpn caused a mild increase in cellular iron level in osteoclast lineage cells.However,loss of Fpn in precursor led to increased osteoclastogenesis and declined bone mass in female mice.In vitro mechanistic studies revealed that elevated intracellular iron promoted macrophage proliferation and enhanced expression of NFATc1 and PGC-1?,two transcription factors critical for osteoclast differentiation,but had no effects oncytoskeleton organization/actin-ring formation and bone resorption activity.In addition,The moderate iron excess induced by Fpn-deletion had no effects on osteoclast survival.Conclusion Deletion of ferroportin in myeloid lineage cells caused a mild increase in cellular iron accumulation,which promoted macrophage proliferation,.Fpn-deficient bone marrow macrophages exhibited accelerated osteoclast formation in vitro and declined bone mass in female mice.Briefly,our work indicate that intracellular iron level regulated by Fpn is critical for mitochondrial metabolism,osteoclastogenesis,and skeletal homeostasis in mice.