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Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts.

Sun Z, Cao X, Zhang Z, Hu Z, Zhang L, Wang H, Zhou H, Li D, Zhang S, Xie M - Sci Rep (2015)

Bottom Line: In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells.Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions.These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Aerospace Medicine, Ministry of Education, The Fourth Military Medical University, 710032, Xi'an, Shaanxi, China.

ABSTRACT
L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity.

No MeSH data available.


Related in: MedlinePlus

Effects of miR-103 knockdown on LTCC currents in MC3T3-E1 cells under simulated microgravity conditions.(a) I–V curves for the Con + miR-103 inhibitor NC group. (b) I–V curves for the Con + miR-103 inhibitor group. (c) I–V curves for the MG + miR-103 inhibitor NC group. (d) I–V curves for the MG + miR-103 inhibitor group. (e) and (f) Comparison of changes in the LTCC current densities in cells of the miR-103 inhibitor NC + MG group (red, n = 12 cells) and the miR-103 inhibitor + MG group (green, n = 14 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.032, #P = 0.006). The values are the mean ± s.d., and statistically significant differences were determined using a one-way ANOVA with a Bonferroni post hoc test.
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f8: Effects of miR-103 knockdown on LTCC currents in MC3T3-E1 cells under simulated microgravity conditions.(a) I–V curves for the Con + miR-103 inhibitor NC group. (b) I–V curves for the Con + miR-103 inhibitor group. (c) I–V curves for the MG + miR-103 inhibitor NC group. (d) I–V curves for the MG + miR-103 inhibitor group. (e) and (f) Comparison of changes in the LTCC current densities in cells of the miR-103 inhibitor NC + MG group (red, n = 12 cells) and the miR-103 inhibitor + MG group (green, n = 14 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.032, #P = 0.006). The values are the mean ± s.d., and statistically significant differences were determined using a one-way ANOVA with a Bonferroni post hoc test.

Mentions: Next, the influence of miR-103 on LTCC currents was investigated to further assess the role of miR-103 on the expression of Cav1.2. Under normal gravity conditions, the inward currents did not differ between the negative control group (Figure 8a) and the miR-103 inhibitor group (Figure 8b). However, the inward currents were larger at all command potentials in the miR-103 inhibitor group (Figure 8d) compared with the negative control group (Figure 8c) under simulated microgravity conditions in the absence or presence of Bay K8644. The LTCC current densities in the miR-103 inhibitor-transfected cells were significantly larger compared with those of the negative control group under simulated microgravity conditions (P < 0.05, Figure 8e and 8f). The difference in the mean peak current densities at +10 mV between the miR-103 inhibitor group (−2.86 ± 0.33 pA/pF) and the negative control group (−2.02 ± 0.38 pA/pF) was significant (P < 0.05, Figure 8e). The application of 10 μM Bay K8644 caused the maximum inward current density to increase by 1.6-fold with no change in the maximal activation voltage. In the presence of Bay K8644, the mean peak current densities in osteoblasts from the two groups were −4.34 ± 0.43 and −2.93 ± 0.32 pA/pF, and the difference between two groups was significant (P < 0.05, Figure 8f). Similar to the finding for Cav1.2 expression, miR-103 inhibitor transfection could not restore the LTCC currents back to the control levels (P < 0.05, Figure 8e and 8f). Additionally, miR-103 inhibitor had no effects on the LTCC currents in cells under normal gravity conditions (P > 0.05, Figure 8e and 8f).


Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts.

Sun Z, Cao X, Zhang Z, Hu Z, Zhang L, Wang H, Zhou H, Li D, Zhang S, Xie M - Sci Rep (2015)

Effects of miR-103 knockdown on LTCC currents in MC3T3-E1 cells under simulated microgravity conditions.(a) I–V curves for the Con + miR-103 inhibitor NC group. (b) I–V curves for the Con + miR-103 inhibitor group. (c) I–V curves for the MG + miR-103 inhibitor NC group. (d) I–V curves for the MG + miR-103 inhibitor group. (e) and (f) Comparison of changes in the LTCC current densities in cells of the miR-103 inhibitor NC + MG group (red, n = 12 cells) and the miR-103 inhibitor + MG group (green, n = 14 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.032, #P = 0.006). The values are the mean ± s.d., and statistically significant differences were determined using a one-way ANOVA with a Bonferroni post hoc test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f8: Effects of miR-103 knockdown on LTCC currents in MC3T3-E1 cells under simulated microgravity conditions.(a) I–V curves for the Con + miR-103 inhibitor NC group. (b) I–V curves for the Con + miR-103 inhibitor group. (c) I–V curves for the MG + miR-103 inhibitor NC group. (d) I–V curves for the MG + miR-103 inhibitor group. (e) and (f) Comparison of changes in the LTCC current densities in cells of the miR-103 inhibitor NC + MG group (red, n = 12 cells) and the miR-103 inhibitor + MG group (green, n = 14 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.032, #P = 0.006). The values are the mean ± s.d., and statistically significant differences were determined using a one-way ANOVA with a Bonferroni post hoc test.
Mentions: Next, the influence of miR-103 on LTCC currents was investigated to further assess the role of miR-103 on the expression of Cav1.2. Under normal gravity conditions, the inward currents did not differ between the negative control group (Figure 8a) and the miR-103 inhibitor group (Figure 8b). However, the inward currents were larger at all command potentials in the miR-103 inhibitor group (Figure 8d) compared with the negative control group (Figure 8c) under simulated microgravity conditions in the absence or presence of Bay K8644. The LTCC current densities in the miR-103 inhibitor-transfected cells were significantly larger compared with those of the negative control group under simulated microgravity conditions (P < 0.05, Figure 8e and 8f). The difference in the mean peak current densities at +10 mV between the miR-103 inhibitor group (−2.86 ± 0.33 pA/pF) and the negative control group (−2.02 ± 0.38 pA/pF) was significant (P < 0.05, Figure 8e). The application of 10 μM Bay K8644 caused the maximum inward current density to increase by 1.6-fold with no change in the maximal activation voltage. In the presence of Bay K8644, the mean peak current densities in osteoblasts from the two groups were −4.34 ± 0.43 and −2.93 ± 0.32 pA/pF, and the difference between two groups was significant (P < 0.05, Figure 8f). Similar to the finding for Cav1.2 expression, miR-103 inhibitor transfection could not restore the LTCC currents back to the control levels (P < 0.05, Figure 8e and 8f). Additionally, miR-103 inhibitor had no effects on the LTCC currents in cells under normal gravity conditions (P > 0.05, Figure 8e and 8f).

Bottom Line: In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells.Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions.These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Aerospace Medicine, Ministry of Education, The Fourth Military Medical University, 710032, Xi'an, Shaanxi, China.

ABSTRACT
L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity.

No MeSH data available.


Related in: MedlinePlus