Microgravity influences circadian clock oscillation in human keratinocytes

Abstract

Microgravity and sudden changes of gravitational forces exert numerous effects on tissues, organs and apparatus. Responses to these forces variably applied to cells indicate the existence of mechanotransduction pathways able to modulate transcription. Oscillation of circadian clocks similarly influences many cellular and metabolic processes. Here we hypothesized that signals derived from changes of gravitational forces applied to epidermal cells might influence their physiology in harmony with the oscillation of the molecular clock. In this study, we describe amplified oscillations of Bmal1 circadian clock gene in human keratinocytes exposed to short simulated microgravity and to rapid variation of gravitational forces. We found that exposure to microgravity enhances the amplitude of the Bmal1 feedback loop sustained by an apparently lower variability of Rev-erbα transcription, while recovery from microgravity is characterized by increased amplitude of Bmal1 expression and elongation of the oscillatory periods of Bmal1 and Rev-erbα. These data highlight the existence of integrated signaling network connecting mechanosensitive pathways to circadian gene regulation.

Keywords: Bmal1; Circadian clock; FAK, focal adhesion kinases; GPCR, G-protein-coupled receptors; Keratinocytes; Mechanotransduction; Microgravity; RPM, Random Positioning Machine; Rev-erbα; hg, hypergravity; mAbs, monoclonal antibodies.

Fig. 1

Simulated microgravity modifies Bmal1 and Rev-erbα circadian oscillations in human keratinocytes. (A) Experimental scheme: untreated HaCaT cells or treated for 1 h with1 μM Dex for gene clock synchronization were exposed to gravitational forces of 1g or μg. Total RNA samples were collected every 6 h for consecutive 48 h and subjected to qRT-PCR analysis for Bmal1 and Rev-erbα mRNA expression. (B) Bmal1 mRNA transcripts show no oscillation in unsynchronized HaCaT cells exposed to either μg or 1g. Both Dex-synchronized cells cultures at 1g (Dex + 1g) or in μg (Dex + μg) show Bmal1 circadian oscillations, with higher Bmal1 transcription in cells exposed to μg (p < 0.05, one-way ANOVA). Rev-erbα mRNA show no oscillation in unsynchronized HaCaT cells exposed to either μg or 1g. Both Dex-synchronized cells cultures at 1g (Dex + 1g) or in μg (Dex + μg) show minimal increase of Rev-erbα circadian oscillations, with higher transcripts in cells exposed to μg. GAPDH mRNA expression is not subjected to circadian oscillations. All the experiments were done in triplicates and were evaluated for statistical significance (*p < 0.05) using the one-way ANOVA followed by Bonferroni post hoc test. Results are expressed as mean fold increase ± standard deviation (SD). (C–F) Unsynchronized and Dex-synchronized cells exposed to microgravity for 24 h were stained for immunofluorescence and nuclei are visualized by DAPI staining. Bar: 10 μm. Immunostaining with anti-cytokeratins antibody shows unmodified organization of cytosolic filaments in all the experimental conditions (C). Cells stained with anti-β1 integrin mAb display the expected signals localized on the cell surface and appear unmodified by the treatments (D). The proliferation rate, assessed in immunofluorescence with an anti-Ki67 polyclonal antibody, reveals positive cycling cells comparable in all the experimental conditions (E). The identification of apoptotic cells in immunofluorescence using the M30 mAb directed against the cleaved form of cytokeratin 18 shows similar numbers of positive cells for all the treatments (F).

Fig. 2

Bmal1 and Rev-erbα circadian oscillations during the recovery process from simulated microgravity. (A) Experimental scheme: HaCaT cells were exposed to μg or 1g for 60 h, then treated with 1 μM Dex for 1 h and recovered in 1g for consecutive 48 h. Total RNA samples were collected at 6 h, 12 h, 24 h, 30 h, 36 h, 42 h and 48 h during the recovery phase in 1g. Untreated cells exposed to 1g were used as control. (B) Comparative analysis of Bmal1 and Rev-erbα mRNA expression in Dex-synchronized HaCaT cells cultured either in 1g (1g + Dex) or in microgravity (μg + Dex). The kinetics of Bmal1 mRNA transcripts indicates that cells exposed to μg display atypical oscillation recovery as compared to cells recovered from 1g, different for intensity, period and time of peaking (*p < 0.05, one-way ANOVA), with a 5.5-fold increase of Bmal1 mRNA. Results are expressed as mean values ± standard deviation (SD). The Rev-erbα kinetics in synchronized cells either cultured at 1g or in the recovery phase is anti-phasic to Bmal1, with a period of 30 h and with amplitudes and peaks of equal intensity. GAPDH expression is used as an internal control. All the experiments were done in triplicates and evaluated for statistically significance using the one-way ANOVA test. Results are expressed as mean fold increase ± standard deviation (SD). (C–F) Cells were stained for immunofluorescence after 60 h of μg or 1g. Immunofluorescence was performed using specific antibodies and nuclei visualized by DAPI staining. Bar: 10 μm. Immunofluorescence with anti-cytokeratins mAb shows unchanged patterns of cytokeratins organization in HaCaT cells differently treated (C). Cells stained with anti-β1 integrins mAb do not show changes of β1-integrins dynamics in all the experimental conditions (D). Proliferative cells assessed in immunofluorescence using anti-Ki67 polyclonal antibody. There were no significant changes in the percentage of Ki67 positive cells among the various treatments (E). Number of apoptotic events evaluated by an anti-M30 staining shows no differences for all the conditions tested (F).

Fig. 3

Genes clock synchronization is not depending on the pharmacologic treatment. (A) Experimental scheme: HaCaT cells exposed to either μg or 1 g for 60 h were treated for 1 h with either Dex 1 μM or Forskolin (Forsk) 10 μM for synchronization and evaluated for Bmal1 expression after 12 h. (B) Comparative analysis of Bmal1 mRNA expression in Dex-synchronized HaCaT cells cultured either in 1 g (1g + Dex) or in microgravity (μg + Dex) reveals a 3.5-fold increase of Bmal1 mRNA in cells subjected to gravitational changes. (C) Bmal1 expression (3.5-fold increase) could be detected in μg + Forsk-treated cells as compared to 1g + Forsk-treated cells. Results are expressed as mean values ± standard deviation (SD). *p < 0.05, one-way ANOVA followed by Bonferroni post hoc test.