title: Knockdown of SLC41A1 magnesium transporter promotes mineralization and attenuates magnesium inhibition during osteogenesis of mesenchymal stromal cells.
abstract: Magnesium is essential for numerous physiological functions. Magnesium exists mostly in bone and the amount is dynamically regulated by skeletal remodeling. Accelerating bone mass loss occurs when magnesium intake is insufficient; whereas high magnesium could lead to mineralization defects. However, the underlying magnesium regulatory mechanisms remain elusive. In the present study, we investigated the effects of high extracellular magnesium concentration on osteogenic differentiation of mesenchymal stromal/stem cells (MSCs) and the role of magnesium transporter SLC41A1 in the mineralization process.
  
  
  
  Murine MSCs derived from the bone marrow of BALB/c mouse or commercially purchased human MSCs were treated with osteogenic induction medium containing 5.8 mM magnesium chloride and the osteogenic differentiation efficiency was compared with that of MSCs in normal differentiation medium containing 0.8 mM magnesium chloride by cell morphology, gene expression profile of osteogenic markers, and Alizarin Red staining. Slc41a1 gene knockdown in MSCs was performed by siRNA transfection using Lipofectamine RNAiMAX, and the differentiation efficiency of siRNA-treated MSCs was also assessed.
  
  
  
  High concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Early osteogenic marker genes including osterix, alkaline phosphatase, and type I collagen were significantly downregulated in MSCs under high concentration of magnesium, whereas late marker genes such as osteopontin, osteocalcin, and bone morphogenetic protein 2 were upregulated with statistical significance compared with those in normal differentiation medium containing 0.8 mM magnesium. siRNA treatment targeting SLC41A1 magnesium transporter, a member of the solute carrier family with a predominant Mg2+efflux system, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. High concentration of magnesium significantly upregulated Dkk1 gene expression and the upregulation was attenuated after the Slc41a1 gene was knocked down. Immunofluorescent staining showed that Slc41a1 gene knockdown promoted the translocation of phosphorylated β-catenin into nuclei. In addition, secreted MGP protein was elevated after Slc41a1 was knocked down.
  
  
  
  High concentration of extracellular magnesium modulates gene expression of MSCs during osteogenic differentiation and inhibits the mineralization process. Additionally, we identified magnesium transporter SLC41A1 that regulates the interaction of magnesium and MSCs during osteogenic differentiation. Wnt signaling is suggested to be involved in SLC41A1-mediated regulation. Tissue-specific SLC41A1 could be a potential treatment for bone mass loss; in addition, caution should be taken regarding the role of magnesium in osteoporosis and the design of magnesium alloys for implantation.
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan.|Division of Nephrology, Department of Medicine, Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, 33004, Taiwan.
authors.forename: Yu-Tzu
authors.initials: YT
authors.lastname: Tsao
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan.
authors.forename: Ya-Yi
authors.initials: YY
authors.lastname: Shih
authors.affiliation: Stem Cell Research Center, National Yang-Ming University, Rm. 825, Chih-Teh Building, No.322, Sec.2, Shih-Pai Rd, Taipei, 11221, Taiwan.
authors.forename: Yu-An
authors.initials: YA
authors.lastname: Liu
authors.affiliation: Stem Cell Research Center, National Yang-Ming University, Rm. 825, Chih-Teh Building, No.322, Sec.2, Shih-Pai Rd, Taipei, 11221, Taiwan. lyspub1@gmail.com.
authors.forename: Yi-Shiuan
authors.initials: YS
authors.lastname: Liu
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan. DAV47@tpech.gov.tw.|Stem Cell Research Center, National Yang-Ming University, Rm. 825, Chih-Teh Building, No.322, Sec.2, Shih-Pai Rd, Taipei, 11221, Taiwan. DAV47@tpech.gov.tw.|Taipei City Hospital, 145 Zhengzhou Road, Taipei, 10341, Taiwan. DAV47@tpech.gov.tw.|Department of Medical Research, Taipei Veterans General Hospital, Taipei, 11217, Taiwan. DAV47@tpech.gov.tw.
authors.forename: Oscar K
authors.initials: OK
authors.lastname: Lee
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects* / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D060449:Wnt Signaling Pathway; D000097568:Matrix Gla Protein
chemical_list: D000469:Alkaline Phosphatase; D055396:Bone Morphogenetic Protein 2; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008274:Magnesium; D015675:Osteocalcin; D053495:Osteopontin; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors; D000097568:Matrix Gla Protein; C525718:BMP2 protein, human; C501126:DKK1 protein, human; C476304:SLC41A1 protein, human; C506895:SPP1 protein, human; C478163:SP7 protein, human
keywords: Magnesium transporter; Mesenchymal stromal cells; Mineralization; Osteogenic differentiation; SLC41A1