Dataset Information

Combined Effects of Proton Radiation and Simulated Microgravity on the Cell Viability and ALP Activity of Murine Osteoblast Cells.

ABSTRACT: Proton radiation (PR) and microgravity (μG) are two key factors that impact living things in space. This study aimed to explore the combined effects of PR and simulated μG (SμG) on bone function. Mouse embryo osteoblast precursor cells (MC3T3-E1) were irradiated with proton beams and immediately treated with SμG for 2 days using a three-dimensional clinostat. All samples were subjected to cell viability, alkaline phosphatase (ALP) activity and transcriptome assays. The results showed that cell viability decreased with increasing doses of PR. The peak ALP activity after PR or SμG alone was lower than that obtained with the non-treatment control. No difference in cell viability or ALP activity was found between 1 Gy PR combined with SμG (PR-SμG) and PR alone. However, 4 Gy PR-SμG resulted in decreased cell viability and ALP activity compared with those obtained with PR alone. Furthermore, Gene Ontology analysis revealed the same trend. These results revealed that PR-SμG may lead to reductions in the proliferation and differentiation capacities of cells in a dose-dependent manner. Our data provide new insights into bone-related hazards caused by multiple factors, such as PR and μG, in the space environment.

SUBMITTER: Ma L

PROVIDER: S-EPMC8669367 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Combined Effects of Proton Radiation and Simulated Microgravity on the Cell Viability and ALP Activity of Murine Osteoblast Cells.

Ma Liqiu L Kong Fuquan F Gong Yihao Y Wang Qiaojuan Q Liu Jiancheng J Sui Li L

Frontiers in public health 20211130

Proton radiation (PR) and microgravity (μG) are two key factors that impact living things in space. This study aimed to explore the combined effects of PR and simulated μG (SμG) on bone function. Mouse embryo osteoblast precursor cells (MC3T3-E1) were irradiated with proton beams and immediately treated with SμG for 2 days using a three-dimensional clinostat. All samples were subjected to cell viability, alkaline phosphatase (ALP) activity and transcriptome assays. Th ...[more]

PMID: 34917576

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Project description:Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2's induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity's inhibition on BMP2's osteogenic induction.

Project description:Simulated microgravity (SMG) poses substantial challenges to astronaut health, particularly impacting osteoblast function and leading to disuse osteoporosis. This study investigates the adverse effects of SMG on osteoblasts, focusing on changes in mitochondrial dynamics and their consequent effects on cellular energy metabolism and mechanotransduction pathways. We discovered that SMG markedly reduced the expression of osteoblast differentiation markers and promoted mitochondrial fission, as indicated by an increase in punctate mitochondria, a decrease in mitochondrial length, and a reduction in cristae density. These mitochondrial alterations are linked to elevated reactive oxygen species levels, a decrease in ΔΨm, and a metabolic shift from oxidative phosphorylation to glycolysis, resulting in decreased adenosine triphosphate production, which are all indicative of mitochondrial dysfunction. Our results showed that treatment with mitochondrial division inhibitor-1 (mdivi-1), a mitochondrial fission inhibitor, effectively inhibited these SMG-induced effects, thereby maintaining mitochondrial structure and function and promoting osteoblast differentiation. Furthermore, SMG disrupted critical mechanotransduction processes, by affecting paxillin expression, the RhoA-ROCK-Myosin II pathway, and actin dynamics, which subsequently altered nuclear morphology and disrupted Yes-associated protein signaling. Notably, treatment with mdivi-1 prevented these disruptions in mechanotransduction pathways. Moreover, our study showed that SMG-induced chromatin remodeling and histone methylation, which are epigenetic barriers to osteogenic differentiation, can be reversed by targeting mitochondrial fission, further highlighting the significance of mitochondrial dynamics in osteoblast function in an SMG environment. Therefore, targeting mitochondrial fission emerges as a promising therapeutic strategy to alleviate osteoblast dysfunction under SMG conditions, providing novel approaches to maintain bone health during prolonged space missions and safeguard the astronaut well-being.

Dataset Information

Combined Effects of Proton Radiation and Simulated Microgravity on the Cell Viability and ALP Activity of Murine Osteoblast Cells.

Publications

Combined Effects of Proton Radiation and Simulated Microgravity on the Cell Viability and ALP Activity of Murine Osteoblast Cells.

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