Knockdown of SLC41A1 magnesium transporter promotes mineralization and attenuates magnesium inhibition during osteogenesis of mesenchymal stromal cells

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Magnesium transporter; Mesenchymal stromal cells; Mineralization; Osteogenic differentiation; SLC41A1.

Fig. 1

High concentration of extracellular magnesium inhibited mineralization of mouse MSCs during osteogenesis. a Alizarin Red S staining images (left) and quantification (right) of mouse MSCs (mMSCs) 12 days after osteogenic induction with normal (0.8 mM) and high (5.8 mM) extracellular magnesium concentration. Biological replicate N = 3 and technical replicate n = 3 for every biological replicate. b Images of mMSCs during osteogenic differentiation with normal and high extracellular magnesium concentration. Scale bar: 100 μm. c Cell number of differentiating mMSCs under osteogenic induction medium containing 0.8 and 5.8 mM magnesium for 0, 3, 6, and 9 days. Cell number was determined by counting the DAPI-positive cells (N = 3, n = 7) and normalized by cell number for day 0. Data presented as mean ± SEM (* p < 0.05). D: day

Fig. 2

High concentration of extracellular magnesium modulated osteogenic differentiation of mMSCs. qPCR results of mMSCs under osteogenic differentiation for 0, 3, 6, and 9 days (D0, D3, D6, D9) with normal and high extracellular magnesium concentration. Relative gene expression indicates fold-change of gene expression in comparison with gene expression of undifferentiated MSCs on D0 (N = 3, n = 3), presented as mean ± SEM (* p < 0.05)

Fig. 3

Slc41a1 knockdown promoted mineralization and attenuated magnesium inhibition. a Slc41a1 expression of undifferentiated mMSCs after Slc41a1 gene knockdown (N = 3, n = 3). b, c Alizarin Red S staining (left) and the quantification (right) of wild-type mMSCs (ctrl) and mMSCs with Slc41a1 gene knockdown (KD) 6 and 12 days after osteogenic induction with normal (N: 0.8 mM) and high (H: 5.8 mM) extracellular magnesium concentration (N = 3, n = 3). d Images of mMSCs with Slc41a1 gene knockdown before and 3 and 6 days after osteogenic induction with normal magnesium concentration. Scale bar: 100 μm. e Gene expression of undifferentiated mMSCs with Slc41a1 gene knockdown and 3 and 6 days after osteogenic induction (D0, D3, D6) under 0.8 and 5.8 mM magnesium concentration. Data normalized by the gene expression of undifferentiated Slc41a1-knockdown MSCs (D0) and presented as mean ± SEM (N = 3, n = 3) (* p< 0.05)

Fig. 4

Slc41a1 knockdown elevated MGP expression. a, b Mgp gene expression of control (ctrl) mMSCs and Slc41a1-knockdown mMSCs (KD) with normal and high extracellular magnesium concentration before and 3, 6, and 9 days after the osteogenic induction (D0, D3, D6, D9). Data normalized by the gene expression of undifferentiated ctrl and KD MSCs respectively (D0) (N = 3, n = 3). c Mgp gene expression of control and Slc41a1 KD-mMSCs with normal extracellular magnesium concentration. Data normalized by Mgp gene expression of undifferentiated control MSCs (D0) (N = 3, n = 3). d Extracellular secreted MGP concentration obtained by ELISA assay (N = 3, n = 3). MGP matrix gla protein. * p< 0.05

Fig. 5

Slc41a1 knockdown attenuated the high magnesium-induced inhibition of Wnt signaling. Gene expression of Wnt5a, β-catenin, and Dkk1 in control (ctrl) and Slc41a1 knockdown (KD) mMSCs with normal and high extracellular magnesium concentration before and 3 days after osteogenic induction (D0 and D3). Two-way analysis of variance followed by Tukey’s post-hoc tests was performed for multiple comparisons. p < 0.05 is defined statistically significant; same letters denote no significant differences. Data normalized by the gene expression of undifferentiated wild-type MSCs (D0) and presented as mean ± SEM (N = 3, n = 3)

Fig. 6

SLC41A1 modulated the localization of phosphorylated β-catenin. Immunofluorescent staining of phosphorylated β-catenin (red), phosphorylated p38 mitogen-activated protein kinases (green), and DAPI (blue) on wild-type (w.t.) MSCs and MSCs treated with siRNA (si) targeting SLC41A1 magnesium transporter. Staining was performed before osteogenic induction (day 0), as well as 3 and 6 days after the induction. Scale bar: 50 μm