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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: Moreover, simulated microgravity increased miR-103 expression.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.

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

Immunocytochemistry of Cav1.2 LTCC in MC3T3-E1 cells in response to 48 h under simulated microgravity conditions.(a), (b) and (c) Staining of MC3T3-E1 cells under normal gravity conditions with the nucleic acid dye ToPro3 (a), rabbit anti-Cav1.2 antibodies (b), (a) and (b) merged (c), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (d), (e) and (f) Simulated microgravity-treated MC3T3-E1 cells stained with the nucleic acid dye ToPro3 (d), rabbit anti-Cav1.2 antibodies (e), (d) and (e) merged (f), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (g) MC3T3-E1 cells incubated with competing peptides for anti-Cav1.2. Cultures incubated with the competing peptide displayed slight green staining and comparable levels of nuclear staining. (h) MC3T3-E1 cells incubated with Alexa Fluor 488-conjugated secondary antibody in the absence of primary antibody.
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f3: Immunocytochemistry of Cav1.2 LTCC in MC3T3-E1 cells in response to 48 h under simulated microgravity conditions.(a), (b) and (c) Staining of MC3T3-E1 cells under normal gravity conditions with the nucleic acid dye ToPro3 (a), rabbit anti-Cav1.2 antibodies (b), (a) and (b) merged (c), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (d), (e) and (f) Simulated microgravity-treated MC3T3-E1 cells stained with the nucleic acid dye ToPro3 (d), rabbit anti-Cav1.2 antibodies (e), (d) and (e) merged (f), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (g) MC3T3-E1 cells incubated with competing peptides for anti-Cav1.2. Cultures incubated with the competing peptide displayed slight green staining and comparable levels of nuclear staining. (h) MC3T3-E1 cells incubated with Alexa Fluor 488-conjugated secondary antibody in the absence of primary antibody.

Mentions: The alteration of LTCC current and activity involves several significant components. The L-type Cav1.2 subunit is known to play a central role in the regulation of both LTCC current and activity; however, the roles of Cav1.2 in mediating the function of LTCCs under real or simulated microgravity conditions remain unclear. Therefore, we investigated whether Cav1.2 expression was altered under simulated microgravity conditions. We performed immunostaining for the Cav1.2 subunit in MC3T3-E1 cells to study the expression and cellular localization of Cav1.2 in cells under simulated microgravity conditions. In Figure 3, immunostaining for the Cav1.2 subunit in MC3T3-E1 cells is shown before and after exposure to 48 h of simulated microgravity conditions (Figure 3). Control cells stained for Cav1.2 showed abundant plasma membrane and intracellular localization, particularly on the cell surface (Figure 3b and 3c). In contrast, the 48 h simulated microgravity conditions decreased immunostaining for Cav1.2 (Figure 3f and 3g). Intracellular staining persisted but was less intense than that observed in control cells, and the staining for Cav1.2 in the cell periphery markedly decreased (Figure 3f and 3g). Images were compared with cells that had been incubated with Fluor 488-conjugated secondary antibody in the absence of primary antibody to determine the specificity of staining (Figure 3d). The signal specificity for the antibody was determined by incubating MC3T3-E1 cells with competing peptide and anti-Cav1.2 antibody (Figure 3h). Western blot analyses were performed to further confirm the results of immunostaining for the Cav1.2 subunit in MC3T3-E1 cells regarding protein expression. Cav1.2 expression in the two groups is shown in Figure 4a. Cav1.2 expression significantly decreased by approximately 50% under simulated microgravity conditions compared with that of the horizontal rotation controls (P < 0.05, Figure 4a).


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)

Immunocytochemistry of Cav1.2 LTCC in MC3T3-E1 cells in response to 48 h under simulated microgravity conditions.(a), (b) and (c) Staining of MC3T3-E1 cells under normal gravity conditions with the nucleic acid dye ToPro3 (a), rabbit anti-Cav1.2 antibodies (b), (a) and (b) merged (c), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (d), (e) and (f) Simulated microgravity-treated MC3T3-E1 cells stained with the nucleic acid dye ToPro3 (d), rabbit anti-Cav1.2 antibodies (e), (d) and (e) merged (f), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (g) MC3T3-E1 cells incubated with competing peptides for anti-Cav1.2. Cultures incubated with the competing peptide displayed slight green staining and comparable levels of nuclear staining. (h) MC3T3-E1 cells incubated with Alexa Fluor 488-conjugated secondary antibody in the absence of primary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4308706&req=5

f3: Immunocytochemistry of Cav1.2 LTCC in MC3T3-E1 cells in response to 48 h under simulated microgravity conditions.(a), (b) and (c) Staining of MC3T3-E1 cells under normal gravity conditions with the nucleic acid dye ToPro3 (a), rabbit anti-Cav1.2 antibodies (b), (a) and (b) merged (c), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (d), (e) and (f) Simulated microgravity-treated MC3T3-E1 cells stained with the nucleic acid dye ToPro3 (d), rabbit anti-Cav1.2 antibodies (e), (d) and (e) merged (f), with Alexa Fluor 488-conjugated anti-rabbit IgG as the secondary antibody. (g) MC3T3-E1 cells incubated with competing peptides for anti-Cav1.2. Cultures incubated with the competing peptide displayed slight green staining and comparable levels of nuclear staining. (h) MC3T3-E1 cells incubated with Alexa Fluor 488-conjugated secondary antibody in the absence of primary antibody.
Mentions: The alteration of LTCC current and activity involves several significant components. The L-type Cav1.2 subunit is known to play a central role in the regulation of both LTCC current and activity; however, the roles of Cav1.2 in mediating the function of LTCCs under real or simulated microgravity conditions remain unclear. Therefore, we investigated whether Cav1.2 expression was altered under simulated microgravity conditions. We performed immunostaining for the Cav1.2 subunit in MC3T3-E1 cells to study the expression and cellular localization of Cav1.2 in cells under simulated microgravity conditions. In Figure 3, immunostaining for the Cav1.2 subunit in MC3T3-E1 cells is shown before and after exposure to 48 h of simulated microgravity conditions (Figure 3). Control cells stained for Cav1.2 showed abundant plasma membrane and intracellular localization, particularly on the cell surface (Figure 3b and 3c). In contrast, the 48 h simulated microgravity conditions decreased immunostaining for Cav1.2 (Figure 3f and 3g). Intracellular staining persisted but was less intense than that observed in control cells, and the staining for Cav1.2 in the cell periphery markedly decreased (Figure 3f and 3g). Images were compared with cells that had been incubated with Fluor 488-conjugated secondary antibody in the absence of primary antibody to determine the specificity of staining (Figure 3d). The signal specificity for the antibody was determined by incubating MC3T3-E1 cells with competing peptide and anti-Cav1.2 antibody (Figure 3h). Western blot analyses were performed to further confirm the results of immunostaining for the Cav1.2 subunit in MC3T3-E1 cells regarding protein expression. Cav1.2 expression in the two groups is shown in Figure 4a. Cav1.2 expression significantly decreased by approximately 50% under simulated microgravity conditions compared with that of the horizontal rotation controls (P < 0.05, Figure 4a).

Bottom Line: Moreover, simulated microgravity increased miR-103 expression.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.

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