Simulated Microgravity Inhibits MC3T3-E1 Preosteoblasts Proliferation By The Activation Of Mi R-103/Cav1.2 Signaling Pathway

Background The maintenance of bone mass and the development of skeletal architecture are dependent on mechanical stimulation. Numerous studies have shown that mechanical loading promotes bone formation in the skeleton, whereas the removal of this stimulus during immobilization or in microgravity results in reduced bone mass. Microgravity, which is the condition of weightlessness that is experienced by astronauts during spaceflight, causes severe physiological alterations in the human body. One of the most prominent physiological alterations is bone loss, which leads to an increased fracture risk. Long-term exposure to a microgravity environment leads to enhanced bone resorption and reduced bone formation over the period of weightlessness. An approximately 2% decrease in bone mineral density after only one month, which is equal to the loss experienced by apostmenopausal woman over one year, occurs in severe forms of microgravity-induced bone loss. Experimental studies have shown that real or simulated microgravity can induce skeletal changes that are characterized by cancellous osteopenia in weight-bearing bones, decreased cortical and cancellous bone formation, altered mineralization patterns, disorganized collagen and non-collagenous proteins, and decreased bone matrix gene expression. Decreased osteoblast function has been thought to play a pivotal role in the process of microgravity-induced bone loss. Both in vivo and in vitro studies have provided evidence of decreased matrix formation and maturation when osteoblasts are subjected to simulated microgravity. The mechanism by which microgravity, which is a form of mechanical unloading, has detrimental effects on osteoblast functions remains unclear and merits further research. Calcium is an important osteoblast regulator, and calcium channels, particularly LTCCs play fundamental roles in cellular responses to mechanical stimuli in osteoblastic lineage bone cells. Several lines of evidence have found that bone density increases and that bone resorption decreases when these calcium channels are activated in osteoblasts. The administration of the LTCC antagonist verapamil or nifedipine can substantially suppress mechanical loading-induced increases in bone formation in rats, suggesting that LTCCs mediate mechanically induced bone adaptation in vivo. The levels of the extracellular matrix proteins osteopontin and osteocalcin increased in periosteal-derived osteoblasts by applying strain alone or strain in the presence of the LTCC agonist Bay K8644 within 24 h post-load. This mechanically induced increase in osteopontin and osteocalcin was inhibited by nifedipine. Therefore, LTCCs play important roles in regulating osteoblasts proliferation and are sensitive to mechanical stimuli. Recent studies have shown that many factors participate in LTCCs regulation. Among them, mi RNAs, which is a small non-coding RNA molecule, has become the subject of many studies and functions in the silencing and post-transcriptional regulation of gene expression. mi RNAs function via base-pairing with complementary sequences within m RNA molecules. Some mi RNAs are reported to participate in regulating Cav1.2 expression in several types of cells, whereas their functions in osteoblasts have not beenconfirmed.Aims We performed these experiments to confirm the effects of simulated microgravity on osteoblasts proliferation, to determine whether simulated microgravity can affect osteoblasts LTCCs and to investigate the functions and mechanisms of mi R-103/Cav1.2 signaling pathway in regulating osteoblasts proliferation.Methods 1. Ed U labeling, PCNA expression detection and Cell Counting Kit-8 assay were performed to assess the effects of simulated microgravity on osteoblasts proliferation. 2. We conducted calcium imaging and patch clamp to explore the effects of simulated microgravity on LTCCs in osteoblasts. By using Western blotting, the expression of Cav1.2 was determined. In addition, the si RNA of Cav1.2 was constructed and western blotting was used to evaluate gene knockdown efficiency following si RNA transfection. Then, the cells were subjected to patch clamp at post-transfection. Moreover, we administrated the LTCC antagonist nifedipine or Cav1.2 si RNA before Cell Counting Kit-8 assay was performed to investigate osteoblasts proliferation under simulated microgravity condition. 3. The effects of simulated microgravity on Cav1.2 m RNA expression was observed by Northern blotting and q PCR. Additionally, q PCR was used to screen differential expression mi RNAs which directly targeted Cav1.2 m RNA. Furthermore, mimic and inhibitor of mi R-103 was constructed and q PCR analyzed mi R-103 levels in MC3T3-E1 cells after treated with mi R-103 mimic, mi R-103 inhibitor or their negative controls. Then, a mi R-103 inhibitor was transfected into MC3T3-E1 cells, and western blot analyses and patch clamp were performed to test for Cav1.2 expression and LTCCs currents density respectively. Liposome was used to transfect mi R-103 mimic or mi R-103 inhibitor into MC3T3-E1 cells, and we examined osteoblasts proliferation by Ed U labeling, PCNA expression detection and Cell Counting Kit-8 assay. Finally, mi R-103 mimic or inhibitor were co-transfected with Cav1.2 si RNA or its negative control by liposome, and Cell Counting Kit-8 assay wasconducted to examine osteoblasts proliferation.Results 1. Simulated microgravity suppresses the percentage of Ed U positive osteoblasts, down-regulates of PCNA expression and inhibits osteoblasts viability. 2. Simulated microgravity attenuates the Bay K8644-induced increase in the intracellular calcium concentration and reduces LTCC currents in osteoblasts. Cav1.2 protein level is down-regulated under simulated microgravity condition. And Cav1.2 knockdown reduces LTCC currents density. Moreover, Cav1.2 knockdown or LTCCs blocking inhibits osteoblasts proliferation under simulated microgravity condition. 3. Simulated microgravity up-regulates Cav1.2 m RNA and mi R-103 expression. A mi R-103 inhibitor partially counteracts the decrease in Cav1.2 protein level and LTCC currents density induced by simulated microgravity. Mi R-103 inhibits osteoblasts proliferation in response to simulated microgravity, and the inhibitory effects of mi R-103 oligos on osteoblasts proliferation under simulated microgravity condition are completely blocked when co-transfected mi R-103 mimic or inhibitor with Cav1.2 si RNA.Conclusions 1. Simulated microgravity inhibits osteoblasts proliferation. Simulated microgravity suppresses the percentage of Ed U positive osteoblasts, down-regulates of PCNA expression and inhibits osteoblasts viability. All these data reveal that simulated microgravity inhibits osteoblasts proliferation and lay the foundations for exploring the mechanisms. 2. Simulated microgravity substantially inhibits LTCCs in MC3T3-E1 osteoblast-like cells by suppressing Cav1.2 expression. And it may be a novel mechanism for the inhibitory effects of simulated microgravity on osteoblasts proliferation. 3. Simulated microgravity inhibits osteoblasts proliferation by the activation of mi R-103/Cav1.2 signaling pathway. This work may provide a novel mechanism of microgravity-induced adverse effects on osteoblasts proliferation, offering a new avenue to further investigate microgravity-induced bone loss.