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Simulated Microgravity Reduces Focal Adhesions and Alters Cytoskeleton and Nuclear Positioning Leading to Enhanced Apoptosis via Suppressing FAK/RhoA-Mediated mTORC1/NF-κB and ERK1/2 Pathways

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Simulated Microgravity Reduces Focal Adhesions and Alters Cytoskeleton and Nuclear Positioning Leading to Enhanced Apoptosis via Suppressing FAK/RhoA-Mediated mTORC1/NF-κB and ERK1/2 Pathways

Tuo Zhao et al. Int J Mol Sci.

Abstract

Simulated-microgravity (SMG) promotes cell-apoptosis. We demonstrated that SMG inhibited cell proliferation/metastasis via FAK/RhoA-regulated mTORC1 pathway. Since mTORC1, NF-κB, and ERK1/2 signaling are important in cell apoptosis, we examined whether SMG-enhanced apoptosis is regulated via these signals controlled by FAK/RhoA in BL6-10 melanoma cells under clinostat-modelled SMG-condition. We show that SMG promotes cell-apoptosis, alters cytoskeleton, reduces focal adhesions (FAs), and suppresses FAK/RhoA signaling. SMG down-regulates expression of mTORC1-related Raptor, pS6K, pEIF4E, pNF-κB, and pNF-κB-regulated Bcl2, and induces relocalization of pNF-κB from the nucleus to the cytoplasm. In addition, SMG also inhibits expression of nuclear envelope proteins (NEPs) lamin-A, emerin, sun1, and nesprin-3, which control nuclear positioning, and suppresses nuclear positioning-regulated pERK1/2 signaling. Moreover, rapamycin, the mTORC1 inhibitor, also enhances apoptosis in cells under 1 g condition via suppressing the mTORC1/NF-κB pathway. Furthermore, the FAK/RhoA activator, toxin cytotoxic necrotizing factor-1 (CNF1), reduces cell apoptosis, restores the cytoskeleton, FAs, NEPs, and nuclear positioning, and converts all of the above SMG-induced changes in molecular signaling in cells under SMG. Therefore, our data demonstrate that SMG reduces FAs and alters the cytoskeleton and nuclear positioning, leading to enhanced cell apoptosis via suppressing the FAK/RhoA-regulated mTORC1/NF-κB and ERK1/2 pathways. The FAK/RhoA regulatory network may, thus, become a new target for the development of novel therapeutics for humans under spaceflight conditions with stressed physiological challenges, and for other human diseases.

Keywords: ERK1/2; FAK; NK-κB; RohA; SMG; apoptosis; cytoskeleton; focal adhesion complex; mTORC1; nuclear positioning.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Simulated microgravity induces BL6-10 cell apoptosis. BL6-10 tumor cells cultured underground condition (1 g) and SMG (µg) for 24 h were stained with Annexin V-FITC and propidium iodide (PI), and then analyzed by flow cytometry. Data represent the mean ± SD of three independent experiments. * p< 0.05 versus different groups.
Figure 2
Figure 2
Simulated microgravity alters the cytoskeleton and inhibits the expression of F-actin and actin-binding protein. (A) BL6-10 cells were cultured in chamber slides for one day at 1 g or µg. The cells were also stained with FITC-phalloidin (green) and analyzed by light microscopy and fluorescence microscopy. Panels (a,b) using 10× magnification; panels (c,d) using 40× magnification; (B) Lysates prepared from BL6-10 cells cultured for three days at 1 g or µg were subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes and blotted with the indicated antibodies. Western blot band signals were quantified by chemiluminescence. Densitometric values were normalized to matching GAPDH controls. Data represent the mean ± SD. * p < 0.05 versus different groups. One representative experiment of three is shown.
Figure 3
Figure 3
Simulated microgravity reduces focal adhesions and inhibits FAK and RhoA signaling. (A) BL6-10 cells were cultured in medium of chamber slides for one day under ground conditions (1 g) or SMG (µg). The cells were stained with anti-paxillin (green) and anti-vinculin (red) antibodies followed by observation under a fluorescence microscope using 40× objectives (formation of cellular focal adhesions (a,c), arrows); (B) Western blotting analysis. Lysates were harvested from BL6-10 cells cultured for three days under 1 g or µg and subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes. Blots were stained with various antibodies and analyzed by chemiluminescence. Bands were qualified using Imaging Lab software (Bio-Rad). Densitometric values were normalized to the GAPDH control; (C) RhoA activity analysis. BL6-10 cells (three days) under 1 g and µg were subjected to RhoA activity assay by using a G-LISA RhoA Activation Assay Biochem kit. Data represent the mean ± SD of three independent experiments. * p < 0.05 versus different groups. One representative experiment of two is shown.
Figure 4
Figure 4
Simulated microgravity suppresses the mTORC/NF-κB pathway. (A,B) Lysates prepared from BL6-10 cells cultured for two days at 1 g or µg were subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes and blotted with indicated antibodies. Western blot band signals were quantified by chemiluminescence. Densitometric values were normalized to matching GAPDH controls. Data represent the me an ± SD of three independent experiments; (C) BL6-10 cells cultured for three days at 1 g or µg in a Lab-Tek1 II Chamber SlideTM System were fixed with paraformaldehyde, and subsequently incubated with anti-p-NF-κB (S337) (green) antibody and then incubated with FITC-labeled goat-anti-rabbit secondary antibody. Slides were covered using Prolong Gold Antifade Reagent with DAPI (blue) and observed by confocal microscopy. Panels (a,b) using 20× magnification; panels (c,d) using 50× magnification. * p < 0.05 versus different groups. One representative experiment of three is shown.
Figure 5
Figure 5
Rapamycin inhibits the mTORC1/NF-κB pathway resulting in the enhancement of apoptosis in cells under the 1 g condition. (A) Western blotting analysis. Lysates were harvested from BL6-10 cells cultured for three days under 1 g or 1 g +rapamycin and subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes. Blots were stained with various antibodies and analyzed by chemiluminescence. Bands were qualified using Imaging Lab software (Bio-Rad). Densitometric values were normalized to the GAPDH control. Data represent the mean ± SD of three independent experiments; (B) Confocal microscopy analyses. BL6-10 cells cultured for two days under 1 g or 1 g +rapamycin were observed by confocal microscopy. Panels (a,b) using 20× magnification; panels (c,d) using 50× magnification. One representative experiment of three is shown; (C) BL6-10 tumor cells cultured under ground conditions (1 g) and SMG (µg) for 24 h were stained with Annexin V-FITC and propidium iodide (PI), and then analyzed by flow cytometry. Data represent the mean ± SD of three independent experiments. * p < 0.05 versus different groups.
Figure 6
Figure 6
Simulated microgravity decreases nuclear positioning and down-regulates the ERK1/2 pathway. (A) BL6-10 cells cultured for two days at 1 g, µg, or µg + CNF1 in a Lab-Tek1 II Chamber SlideTM System were fixed with paraformaldehyde, and subsequently incubated with indicated antibodies and then incubated with FITC-labeled secondary antibodies, and observed by confocal microscopy (300× magnification). One representative experiment of three is shown; (B,C) Lysates prepared from BL6-10 cells cultured for two days at 1 g, µg, or µg + CNF1 were subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes and blotted with indicated antibodies. Western blot band signals were quantified by chemiluminescence. Densitometric values were normalized to matching GAPDH controls. Data represent the mean ± SD of three independent experiments. * p < 0.05 versus different groups.
Figure 7
Figure 7
CNF1 restores focal adhesions and NEPCs and activates FAK/RhoA, mTORC1/NF-κB, and ERK1/2 pathways leading to apoptosis reduction in cells under SMG. (A) BL6-10 cells were cultured in chamber slides for one day at 1 g, µg, and µg + CNF1, and cells were stained with anti-paxillin, and paxillin spots were counted for each cell. Approximately 20 cells were analyzed per experimental condition; (B) Lysates were harvested from BL6-10 cells cultured for two days under 1 g or µg and subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes. Blots were stained with various antibodies and analyzed by chemiluminescence. Bands were qualified using Imaging Lab software (Bio-Rad). Densitometric values were normalized to the GAPDH control; (C) RhoA activity analysis. BL6-10 cells were cultured for three days at 1 g, µg, and µg + CNF1 and were subjected to RhoA activity assay by using G-LISA RhoA Activation Assay Biochem kit. Data represent the mean ± SD of three independent experiments; (D) BL6-10 cells cultured for three days at 1 g or µg in a Lab-Tek1 II Chamber SlideTM System were fixed with paraformaldehyde, and subsequently incubated with anti-p-NF-κB (S337) (green) antibody and then incubated with FITC-labeled goat-anti-rabbit secondary antibody. Slides were covered using Prolong Gold Antifade Reagent with DAPI (blue) and observed by confocal microscopy. Panels (ac) using 20× magnification; panels (df) using 50× magnification; (E) BL6-10 tumor cells cultured under ground conditions (1 g) or SMG (µg) and SMG (µg) + CNF1 for one day were stained with Annexin V-FITC and propidium iodide (PI), and then analyzed by flow cytometry. * p < 0.05 versus different groups. One representative experiment of three is shown.
Figure 8
Figure 8
Schematic diagram presenting pathways where simulated microgravity reduces focal adhesions and alters the cytoskeleton and nuclear positioning, leading to enhanced cell apoptosis via suppressing FAK/RhoA-regulated mTORC1/NF-κB and ERK1/2 pathways pathways (solid up-arrows: enhance; solid down-arrows: reduce).

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