Figures
Figure 1
TRPM7 levels and its role in SaOS-2 cells cultured in different concentrations of Mg2+ . SaOS-2 cells were cultured in 0.1, 1.0, and 5.0 mM Mg2+ . A ) After 96 h western blot was performed on protein lysates and antibodies against TRPM7 were utilized. Anti-β-actin antibodies were used as control of equal loading. A representative blot is shown. B ) SaOS-2 were silenced for TRPM7 with specific siRNAs. Scrambled, non-silencing sequences (NS) were used as controls. Western blot was performed as in (A ). The histogram shows the densitometry (TRPM7 vs β-actin) of three separate experiments ± standard deviation (*p ≤ 0.05). C ) SaOS-2 silenced or not for TRPM7 were counted after 96 h. The bars are the mean ± standard deviation of four separate experiments. p was calculated vs NS (**p ≤ 0.01). D ) NO release was measured with the Griess method. Data are expressed as the mean ± standard deviation of three separate assays (**p ≤ 0.01, ***p ≤ 0.001).
Figure 1
Figure 2
The effect of iNOS inhibitor on the release of NO and on the proliferation of Mg2+ -deficient SaOS-2 cells. SaOS-2 silenced or not for TRPM7 , were cultured in 0.1 mM Mg2+ for 96 h, in the presence or not of L-NIL. A ) The cells were counted as described. The bars are the mean ± standard deviation of four separate experiments (*p ≤ 0.05, **p ≤ 0.01). B ) Nitric oxide release was measured with the Griess method. Data are expressed as the mean ± standard deviation of three separate assays (**p ≤ 0.01).
Figure 2
Figure 3
MagT1 levels and its role in SaOS-2 cells cultured in different concentrations of Mg2+ . SaOS-2 cells were cultured in 0.1, 1.0, and 5.0 mM Mg2+ . A ) After 96 h western blot was performed with antibodies against MagT1. Anti-β-actin antibodies were used as control of equal loading. B ) SaOS-2 cells were silenced for MagT1 with specific siRNAs. Scrambled, non-silencing sequences (NS) were used as controls. Western blot was performed on protein lysates using antibodies against MagT1. β-actin was used as control of loading. The histogram shows the quantitative evaluation of MagT1 vs β-actin by densitometry on three separate experiments ± standard deviation (*p ≤ 0.05). C ) SaOS-2 cells were counted. The bars are the mean ± standard deviation of four separate experiments (*p ≤ 0.05). D ) Nitric oxide release was measured with the Griess method. Data are expressed as the mean ± standard deviation of three separate assays (**p ≤ 0.01, ***p ≤ 0.001).
Figure 3
Figure 4
SaOS-2 cultured in different extracellular Mg2+ concentrations were silenced for MagT1 with specific siRNAs or NS and treated with L-NIL (A ) or L-NAME (B ). After 96 h the cells were counted. The bars are the mean ± standard deviation of four separate experiments (*p ≤ 0.05, **p ≤ 0.01).
Figure 4
Authors
Dipartimento di Scienze Biomediche e Cliniche L. Sacco, Università di Milano, Milano
I-20157, Italy
* Correspondence: Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano, Via GB Grassi, 74 Milano 20157, Italy
A correct magnesium (Mg2+ ) intake is essential for bone health. In particular, Mg2+ deficiency inhibits the proliferation of osteoblast-like SaOS-2 cells by increasing nitric oxide (NO) production through the upregulation of inducible NO synthase. At the moment, little is known about the expression and the role of TRPM7, a channel/enzyme involved in Mg2+ uptake, and MagT1, a Mg2+ selective transporter, in SaOS-2 cells. Here, we demonstrate that TRPM7 is not modulated by different extracellular concentrations of Mg2+ and its silencing exacerbates growth inhibition exerted by low Mg2+ through the activation of inducible NO synthase and consequent accumulation of NO. Moreover, MagT1 is upregulated in SaOS-2 cultured in high Mg2+ and its silencing inhibits the growth of SaOS-2 cultured in media containing physiological or high Mg2+ , without any modulation of NO production. We propose that TRPM7 and MagT1 are both involved in regulating SaOS-2 proliferation through different mechanisms.