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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 changes in the Cav1.2 subunit protein on LTCC currents.(a) MC3T3-E1 cells were transfected with Cav1.2 siRNA and negative control (NC) siRNA (70 nM) for 12 h, followed by postincubation for 48 and 72 h additional hours. Western blot analysis indicates the magnitude and duration of Cav1.2 subunit suppression (n = 4, α = 0.05, *P = 0.016). Bars represent the mean ± s.d. with two-tailed Student's t-test against control samples. (b) I–V curves for the siRNA NC group. (c) I–V curves for the siRNA group. (d) and (e) Comparison of changes in the LTCC current densities in MC3T3-E1 cells of the siRNA NC group (n = 12 cells) and the siRNA group (n = 13 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.036, #P = 0.013). 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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f5: Effects of changes in the Cav1.2 subunit protein on LTCC currents.(a) MC3T3-E1 cells were transfected with Cav1.2 siRNA and negative control (NC) siRNA (70 nM) for 12 h, followed by postincubation for 48 and 72 h additional hours. Western blot analysis indicates the magnitude and duration of Cav1.2 subunit suppression (n = 4, α = 0.05, *P = 0.016). Bars represent the mean ± s.d. with two-tailed Student's t-test against control samples. (b) I–V curves for the siRNA NC group. (c) I–V curves for the siRNA group. (d) and (e) Comparison of changes in the LTCC current densities in MC3T3-E1 cells of the siRNA NC group (n = 12 cells) and the siRNA group (n = 13 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.036, #P = 0.013). 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: We examined LTCC currents by knocking down Cav1.2 expression to further clarify whether the alterations in Cav1.2 expression are involved in the reduction of LTCC currents in osteoblasts. Western blotting was used to evaluate gene knockdown efficiency following siRNA transfection. As shown in Figure 5a, siRNA treatment resulted in an approximately 60% suppression of the protein at 48 h post-transfection, with significant suppression lasting up to 72 h (P < 0.05). Therefore, the cells were subjected to patch clamp at 48 h post-transfection, which is the period at which Cav1.2 expression was maximally suppressed. LTCC current densities were significantly lower at all command potentials between cells receiving scrambled or Cav1.2 siRNA, regardless of whether the LTCCs were activated by Bay K8644 (Figure 5b and 5c). The difference in the mean peak current densities at +10 mV between the Cav1.2 knockdown (−1.58 ± 0.26 pA/pF) and the control cells (−2.76 ± 0.34 pA/pF) was significant (P < 0.05, Figure 5d). Moreover, in the presence of Bay K8644, the mean peak current densities in cells from knockdown and control cells were −2.72 ± 0.34 and −4.75 ± 0.44 pA/pF, respectively, and the difference between the two groups was significant (P < 0.05, Figure 5e).


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 changes in the Cav1.2 subunit protein on LTCC currents.(a) MC3T3-E1 cells were transfected with Cav1.2 siRNA and negative control (NC) siRNA (70 nM) for 12 h, followed by postincubation for 48 and 72 h additional hours. Western blot analysis indicates the magnitude and duration of Cav1.2 subunit suppression (n = 4, α = 0.05, *P = 0.016). Bars represent the mean ± s.d. with two-tailed Student's t-test against control samples. (b) I–V curves for the siRNA NC group. (c) I–V curves for the siRNA group. (d) and (e) Comparison of changes in the LTCC current densities in MC3T3-E1 cells of the siRNA NC group (n = 12 cells) and the siRNA group (n = 13 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.036, #P = 0.013). 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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Related In: Results  -  Collection

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f5: Effects of changes in the Cav1.2 subunit protein on LTCC currents.(a) MC3T3-E1 cells were transfected with Cav1.2 siRNA and negative control (NC) siRNA (70 nM) for 12 h, followed by postincubation for 48 and 72 h additional hours. Western blot analysis indicates the magnitude and duration of Cav1.2 subunit suppression (n = 4, α = 0.05, *P = 0.016). Bars represent the mean ± s.d. with two-tailed Student's t-test against control samples. (b) I–V curves for the siRNA NC group. (c) I–V curves for the siRNA group. (d) and (e) Comparison of changes in the LTCC current densities in MC3T3-E1 cells of the siRNA NC group (n = 12 cells) and the siRNA group (n = 13 cells), regardless of whether the LTCCs were activated by Bay K8644 (α = 0.05, *P = 0.036, #P = 0.013). 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: We examined LTCC currents by knocking down Cav1.2 expression to further clarify whether the alterations in Cav1.2 expression are involved in the reduction of LTCC currents in osteoblasts. Western blotting was used to evaluate gene knockdown efficiency following siRNA transfection. As shown in Figure 5a, siRNA treatment resulted in an approximately 60% suppression of the protein at 48 h post-transfection, with significant suppression lasting up to 72 h (P < 0.05). Therefore, the cells were subjected to patch clamp at 48 h post-transfection, which is the period at which Cav1.2 expression was maximally suppressed. LTCC current densities were significantly lower at all command potentials between cells receiving scrambled or Cav1.2 siRNA, regardless of whether the LTCCs were activated by Bay K8644 (Figure 5b and 5c). The difference in the mean peak current densities at +10 mV between the Cav1.2 knockdown (−1.58 ± 0.26 pA/pF) and the control cells (−2.76 ± 0.34 pA/pF) was significant (P < 0.05, Figure 5d). Moreover, in the presence of Bay K8644, the mean peak current densities in cells from knockdown and control cells were −2.72 ± 0.34 and −4.75 ± 0.44 pA/pF, respectively, and the difference between the two groups was significant (P < 0.05, Figure 5e).

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