The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

Abstract

KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation.

Keywords: KCNQ channels; differentiation; glutamate; matrix mineralization.

Figure 1

Regulation of human mesenchymal stem cell (hMSC) osteogenic differentiation by tetraethylammonium (TEA). Matrix mineralization was evaluated via Alizarin Red S staining. (A) Differentiated hMSCs in osteogenic-induction medium (OM) produced calcium deposits and were dyed red (right), while the cells cultured in growth medium (GM) were not stained (left). The hMSCs were differentiated for 10 days; (B) The OD values for Alizarin Red S staining showed that hMSCs cultured in OM had a greater calcium content than those in GM; (C) Scheme for the protocol of TEA treatment during hMSC osteogenic differentiation. Cells were pre-incubated with 10 mM TEA for 1 day without any differentiation additives. OM containing TEA was used to induce hMSC osteogenic differentiation; (D) The mRNA expressions of osteogenic differentiation markers were analyzed with qRT-PCR at day 8 of osteogenic differentiation. The mRNA expression of ALP was significantly increased. The expressions of BSP and OSC tended to increase, although not statistically significantly. There was no significant change in Runx2 expression; (E) Alizarin Red S staining demonstrated that hMSC osteogenic differentiation was increased by TEA. The hMSCs treated with TEA produced greater calcium contents; (F) The OD values demonstrated that TEA increased hMSC osteogenic differentiation. The OD values were normalized with the Alamar Blue Assay to consider remained cell numbers (n = 3). The data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005 compared to controls. Scale bar represents 200 μm. ALP: alkaline phosphatase; BSP: bone sialoprotein; OSC: osteocalcin; OD: optical density.

Figure 1

Regulation of human mesenchymal stem cell (hMSC) osteogenic differentiation by tetraethylammonium (TEA). Matrix mineralization was evaluated via Alizarin Red S staining. (A) Differentiated hMSCs in osteogenic-induction medium (OM) produced calcium deposits and were dyed red (right), while the cells cultured in growth medium (GM) were not stained (left). The hMSCs were differentiated for 10 days; (B) The OD values for Alizarin Red S staining showed that hMSCs cultured in OM had a greater calcium content than those in GM; (C) Scheme for the protocol of TEA treatment during hMSC osteogenic differentiation. Cells were pre-incubated with 10 mM TEA for 1 day without any differentiation additives. OM containing TEA was used to induce hMSC osteogenic differentiation; (D) The mRNA expressions of osteogenic differentiation markers were analyzed with qRT-PCR at day 8 of osteogenic differentiation. The mRNA expression of ALP was significantly increased. The expressions of BSP and OSC tended to increase, although not statistically significantly. There was no significant change in Runx2 expression; (E) Alizarin Red S staining demonstrated that hMSC osteogenic differentiation was increased by TEA. The hMSCs treated with TEA produced greater calcium contents; (F) The OD values demonstrated that TEA increased hMSC osteogenic differentiation. The OD values were normalized with the Alamar Blue Assay to consider remained cell numbers (n = 3). The data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005 compared to controls. Scale bar represents 200 μm. ALP: alkaline phosphatase; BSP: bone sialoprotein; OSC: osteocalcin; OD: optical density.

Figure 2

RT-PCR analysis of the K_V7 channels in osteoblast-like cells. The PCR products using cDNA from the MG-63 (A) and Saos-2 cells (B) were electrophoresed in a 1.6% agarose gel (n = 5).

Figure 3

Changes in K_V7 channel expression during osteoblastic differentiation. The relative expression levels of K_V7.2, K_V7.3, and K_V7.5 during osteoblast differentiation were analyzed by qRT-PCR. (A) In MG-63 cells, mRNA expressions of K_V7.2 and K_V7.5 were not significantly changed (n = 3). However, K_V7.3 transcripts decreased for 4 days after osteoblast induction, whereas, at day 14, the K_V7.3 mRNA level was considerably increased (n = 4); (B) In Saos-2 cells, mRNA expression of K_V7.3 was reduced 1 h after osteoblast induction, but was significantly augmented at day 14 (n = 4); (C,D) The expression of K_V7.3 proteins was increased at day 4 of osteoblast induction (n = 3). Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005. d: day.

Figure 4

Functional characteristics of K_V7.3 channel in MG-63 cells during osteoblast differentiation. (A) Representative current responses to depolarizing steps observed at day 0 of undifferentiated MG-63 cells. XE991-sensitive currents were obtained by the subtraction of the currents in controls from in the presence of XE991 (50 μM); (B) Current-voltage relationships in controls and in the presence of XE991 at day 0 (n = 3); (C) Representative current responses to depolarizing steps observed at day 2 of osteoblastic differentiation; (D) Current-voltage relationships in controls and in the presence of XE991 at day 2 (n = 4); (E) XE991-sensitive currents at days 0–4 were normalized by mean current value at day 0. At day 1 of osteoblast differentiation, normalized XE991-sensitive currents had no significant change. At days 2 and 3, the K_V7.3 currents were increased, but not statistically significant. Data are presented as mean ± SEM. * p < 0.05.

Figure 4

Functional characteristics of K_V7.3 channel in MG-63 cells during osteoblast differentiation. (A) Representative current responses to depolarizing steps observed at day 0 of undifferentiated MG-63 cells. XE991-sensitive currents were obtained by the subtraction of the currents in controls from in the presence of XE991 (50 μM); (B) Current-voltage relationships in controls and in the presence of XE991 at day 0 (n = 3); (C) Representative current responses to depolarizing steps observed at day 2 of osteoblastic differentiation; (D) Current-voltage relationships in controls and in the presence of XE991 at day 2 (n = 4); (E) XE991-sensitive currents at days 0–4 were normalized by mean current value at day 0. At day 1 of osteoblast differentiation, normalized XE991-sensitive currents had no significant change. At days 2 and 3, the K_V7.3 currents were increased, but not statistically significant. Data are presented as mean ± SEM. * p < 0.05.

Figure 5

Effect of flupirtine, linopirdine, and XE991 on MG-63 cell viability. The MTT assay was performed on MG-63 cells. (A) MG-63 cells were incubated in growth medium (GM) with 30 μM of flupirtine, 30 μM of linopirdine, and 10 μM of XE991. At 72 h, linopirdine, XE991, and flupirtine caused notable decreases in cell viability (n = 16); (B) MG-63 cells were cultured in osteoblast-induction medium (OM) with 30 μM of flupirtine, 30 μM of linopirdine, and 10 μM of XE991. The cell viability was not significantly influenced by OM containing linopirdine, XE991, or flupirtine for 72 h of treatment (n = 16). The values are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Figure 6

Regulation of osteoblastic differentiation by K_V7 channel in MG-63 and Saos-2 cells. (A) Alizarin Red S staining data showed that 30 μM of linopirdine and 10 μM of XE991 augmented mineralization in the extracellular matrix in MG-63 cells (n = 10). There was no significant change in the mineralized matrix with 30 μM of flupirtine (n = 3); (B) The OD values demonstrated that linopirdine and XE991 increased the amount of calcium deposits; (C) Alizarin Red S staining illustrated that mineralization of Saos-2 cells was increased with 30 μM of linopirdine and 10 μM of XE991 (n = 8), while 30 μM of flupirtine reduced the amount of calcium deposits (n = 7); (D) The OD values are shown parallel to Alizarin Red S staining results. Data are presented as mean ± SEM. *** p < 0.005. Scale bar represents 100 μm. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; OD: optical density.

Figure 7

mRNA expression of osteoblastic differentiation markers in MG-63 cells. The relative mRNA expression levels of osteoblastic differentiation markers, ALP, OSC, Runx2, and osterix, were measured with qRT-PCR and normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. (A) While linopirdine (30 μM) and XE991 (10 μM) increased ALP mRNA expression at days 7 and 10 of osteoblastic induction, flupirtine (30 μM) decreased ALP mRNA expression at days 7 and 10 (n = 3–7); (B) The mRNA expression level of OSC was increased by linopirdine or XE991 at days 7 and 10, respectively (n = 3–7); (C) mRNA expression of Runx2 was decreased by flupirtine at day 4 and by linopirdine at day 10 (n = 3); (D) Osterix gene expression was increased by linopirdine and by XE991 at day 10 of osteoblastic induction (n = 3–7). The values are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005. ALP: alkaline phosphatase; OSC: osteocalcin; CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Figure 8

Regulation of synaptic vesicle-related protein, synapsin, by K_V7.3 channel in MG-63 cells. Western blot analysis showed that K_V7.3 blockade by linopirdine (30 μM) or XE991 (10 μM) increased synapsin expression during osteoblast differentiation. However, K_V7 activation by flupirtine (30 μM) had no significant effect on the protein expression of synapsin (n = 3). Vinculin is used as a loading control for Western blot analysis. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Figure 9

Alterations of ERK1/2 phosphorylation by the K_V7 opener or K_V7.3 blockers in MG-63 cells. Western blot analysis showed that, while linopirdine (30 μM) and XE991 (10 μM) increased the expression level of ERK1/2 phosphorylation at days 4 and 7 of osteoblast induction, flupirtine (30 μM) had no significant effect on the level of ERK1/2 phosphorylation. Treatment with linopirdine or XE991 showed no increase of ERK1/2 phosphorylation at day 14 of osteoblast differentiation (n = 3). Flupirtine augmented the expression of ERK1/2 phosphorylation at day 14 (n = 3). Vinculin is used as a loading control for Western blot analysis. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; ERK1/2: extracellular-signal-regulated kinase 1/2; p-ERK: ERK1/2 phosphorylation.

Figure 10

Effect of K_V7 channel on glutamate release during osteoblastic differentiation in MG-63 cells. (A) On day 2, flupirtine (30 μM) significantly decreased glutamate release after inducing osteoblastic differentiation (n = 6). However, linopirdine (30 μM) or XE991 (10 μM) caused significantly increased glutamate release (n = 5); (B) On day 4, flupirtine also caused decreased glutamate release (n = 5), whereas XE991 notably increased the extracellular glutamate (n = 7). Linopirdine augmented the amount of glutamate, but not statistically significantly (n = 8). The values are presented as mean ± SEM. * p < 0.05; ** p < 0.01; and *** p < 0.005. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.

Figure 11

Suppressive effect of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainite receptor antagonist, on osteoblastic differentiation promoted by K_V7.3 blockers. (A) Alizarin Red S staining showed that CNQX (50 μM) co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced amounts ofcalcium deposits similar to those of the controls (n = 3). (B) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression (n = 3). (C) At day 4, CNQX treatment with XE991 significantly reduced the ALP mRNA expression. At day 7, CNQX with flupirtine, linopirdine, or XE991 attenuated the ALP level. (D) CNQX treatment with flupirtine, linopirdine, or XE991 showed no significant changes in the OSC levels. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Figure 12

Suppressive effect of MK801, an NMDA (N-methyl-D-aspartate) receptor antagonist, on osteoblastic differentiation promoted by K_V7.3 blockers. (A) Alizarin Red S staining showed that MK801 (50 μM) treatment co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced amounts of calcium deposits similar to those of the controls (n = 3); (B) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression (n = 3); (C) At days 4 and 7, MK801 treatment with linopirdine or XE991 attenuated ALP mRNA expression; (D) At day 7, MK801 treatment with flupirtine or linopirdine significantly reduced OSC mRNA expression. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; MK: MK801; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Figure 13

Counter-effect of riluzole, a glutamate release inhibitor, on osteoblastic differentiation promoted by K_V7.3 blockers. (A) Alizarin Red S staining showed that riluzole (30 μM) co-applied with flupirtine (30 μM), linopirdine (30 μM), or XE991 (10 μM) produced mineralization levels similar to those of the controls (n = 3); (B) The OD values are shown parallel to Alizarin Red S staining results. The relative mRNA expression levels of osteoblastic differentiation markers, including ALP and OSC, were measured with qRT-PCR and normalized against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression (n = 3); (C) At day 4, riluzole treatment with linopirdine or XE991 reduced the mRNA expression of ALP. At day 7, there was no significant change in ALP levels, although riluzole treatment with flupirtine had a tendency to increase these levels; (D) At day 7, riluzole treatment with linopirdine or XE991 reduced the OSC mRNA expression. Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01. Scale bar represents 100 μm. CONT: non-treated controls; RLZ: riluzole; FLU: flupirtine; LINO: linopirdine; XE: XE991; ALP: alkaline phosphatase; OSC: osteocalcin; OD: optical density.

Figure 14

Induction of intracellular type 1 collagen by K_V7 channel during osteoblast differentiation in MG-63 cells. Western blot analysis demonstrated that 30 μM of linopirdine or 10 μM of XE991 considerably increased the expression of type 1 collagen on day 7 of osteoblastic induction, while 30 μM of flupirtine attenuated type 1 collagens. CONT: non-treated controls; FLU: flupirtine; LINO: linopirdine; XE: XE991.