Abstract：
BACKGROUND: Osteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts\' effects on osteoporosis.
METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl\'s iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining.
RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice.
CONCLUSIONS: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.
METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl\'s iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining.
RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice.
CONCLUSIONS: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.
摘要：
背景：骨质疏松（OP）可能是由破骨细胞功能过度活跃引起的。由于骨吸收是一种独特的破骨细胞功能，因此抗骨质疏松症在组织修复和再生中具有相当大的治疗作用。在这项研究中,我们主要探讨破骨细胞对骨质疏松作用的潜在机制。
方法:使用RAW264.7细胞,并通过M-CSF和RANKL给药诱导破骨细胞和铁积累。我们研究了铁调素和二价金属转运蛋白1（DMT1）对卵巢切除术（OVX）诱导的骨质疏松症中铁积累和破骨细胞形成的影响。OVX诱导小鼠骨质疏松，并用铁调素(10、20、40、80毫克/千克，分别）和通过尾静脉注射过表达DMT1。铁调素,免疫组化染色检测SPI1和DMT1,蛋白质印迹和RT-PCR。生物信息学分析，荧光素酶测定,和染色质免疫沉淀(ChIP)证实铁调素是直接的SPI1转录靶标。通过激光扫描共聚焦显微镜检测铁的积累，Perl的铁染色和铁含量测定。使用抗酒石酸酸性磷酸酶(TRAP)染色评估破骨细胞的形成。
结果：我们发现RAW264.7细胞在暴露于M-CSF和RANKL时分化成破骨细胞，增加了破骨细胞生成相关基因的蛋白质水平，包括c-Fos,MMP9和Acp5。当施用M-CSF和RANKL时,我们还观察到较高浓度的铁积累。然而,Hepcidin抑制来自RAW264.7的原代破骨细胞的破骨细胞分化细胞并降低细胞内铁浓度。Spi-1原癌基因（SPI1）转录抑制铁调素的表达，DMT1增加，促进小鼠破骨细胞的分化和铁积累。SPI1的过表达显著降低了HAMP启动子的荧光素酶活性，增加了HAMP启动子的富集。此外，我们的结果表明，Hepcidin抑制小鼠破骨细胞和OVX小鼠的破骨细胞分化和铁积累。
结论：因此，研究表明，SPI1可以通过DMT1信号激活抑制OVX小鼠铁的积累和破骨细胞的形成。
方法:使用RAW264.7细胞,并通过M-CSF和RANKL给药诱导破骨细胞和铁积累。我们研究了铁调素和二价金属转运蛋白1（DMT1）对卵巢切除术（OVX）诱导的骨质疏松症中铁积累和破骨细胞形成的影响。OVX诱导小鼠骨质疏松，并用铁调素(10、20、40、80毫克/千克，分别）和通过尾静脉注射过表达DMT1。铁调素,免疫组化染色检测SPI1和DMT1,蛋白质印迹和RT-PCR。生物信息学分析，荧光素酶测定,和染色质免疫沉淀(ChIP)证实铁调素是直接的SPI1转录靶标。通过激光扫描共聚焦显微镜检测铁的积累，Perl的铁染色和铁含量测定。使用抗酒石酸酸性磷酸酶(TRAP)染色评估破骨细胞的形成。
结果：我们发现RAW264.7细胞在暴露于M-CSF和RANKL时分化成破骨细胞，增加了破骨细胞生成相关基因的蛋白质水平，包括c-Fos,MMP9和Acp5。当施用M-CSF和RANKL时,我们还观察到较高浓度的铁积累。然而,Hepcidin抑制来自RAW264.7的原代破骨细胞的破骨细胞分化细胞并降低细胞内铁浓度。Spi-1原癌基因（SPI1）转录抑制铁调素的表达，DMT1增加，促进小鼠破骨细胞的分化和铁积累。SPI1的过表达显著降低了HAMP启动子的荧光素酶活性，增加了HAMP启动子的富集。此外，我们的结果表明，Hepcidin抑制小鼠破骨细胞和OVX小鼠的破骨细胞分化和铁积累。
结论：因此，研究表明，SPI1可以通过DMT1信号激活抑制OVX小鼠铁的积累和破骨细胞的形成。
