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

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.

Regulator of G Protein Signaling Protein 12 (Rgs12) Controls Mouse Osteoblast Differentiation via Calcium Channel/Oscillation and Gαi-ERK Signaling

“…LTCCs, via Ca 2+ influx, play a key role in regulating intracellular Ca 2+ homeostasis of OBs . Thus, we used Bay K8644, an agonist of LTCCs, to determine whether LTCCs partly contribute to the restrain of Ca 2+ influx after Rgs12 deletion .…”

“…Change of intracellular Ca 2+ concentration was accessed and recorded for 45 min as mentioned in the first paragraph of section Ca 2+ imaging. Once the cells reached a stable baseline level of cytosolic Ca 2+ , the Ca 2+ ‐free ISO was exchanged for a Ca 2+ ‐free ISO containing 1µM TG (Sigma‐Aldrich) to trigger Ca 2+ release from the ER, a normal ISO containing 1.3mM CaCl 2 to induce extracellular Ca 2+ influx, or a normal ISO containing 10µM Bay K8644 (Sigma) to induce L‐type Ca 2+ channel amplified influx . Changes of intracellular Ca 2+ intensity were normalized by the initial Ca 2+ level of cell itself.…”

miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

“…8,44 The skeletal deconditioning, such as reduced bone mass, altered mineralization patterns and decreased expression of bone matrix genes, has been described in astronauts or animal models under microgravity conditions. 8 38,40 Decreased osteoblast proliferation is thought to play a central role in microgravity-induced bone loss. 8 38,40 Decreased osteoblast proliferation is thought to play a central role in microgravity-induced bone loss.…”

“…36,38,39 To confirm the microgravity achieved by this clinostat, we tested the effects of the simulated microgravity on biological characteristics of primary mouse osteoblasts in this experimental condition ( Figure S1). The clinostat (Astronaut Research and Training Center, China) is an effective tool to simulate microgravity.…”

“…The fluorescence was captured and observedusing an inverted microscope linked to a laser scanning confocal microscope (FluoView1000; Olympus) 18,38. After fixation, cells were washed once with PBS and resuspended in PBS/0.1% Triton X-100 and incubated with 50 U DNase-free RNaseA (Calbiochem, Germany) (30 minutes, room temperature).…”