Hypergravity induces expression of cyclooxygenase-2 in the heart vessels

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced by various stimuli including mechanical stress and plays important roles in pathophysiological conditions. For example, gravitational stress has been shown to induce expression of COX-2 in bone tissues, which is essential for bone homeostasis. To investigate whether COX-2 is induced by gravitational loading in other tissues than bone, we exposed mice to hypergravity at 2G and 3G for 4 h. We demonstrate here that COX-2 is induced in the mouse heart vessels by hypergravity. Moreover, hypoxia-inducible factor (HIF)-1alpha and its downstream genes such as inducible nitric oxide synthase, vascular endothelial growth factor, and heme oxygenase-1 were induced in the heart simultaneously, while none of these genes were induced in the COX-2(-/-) mouse heart. Therefore, COX-2 induced in the heart helps protect the heart function against hypoxia under hypergravity condition through HIF-1alpha induction.     

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.     

Effects of hypergravity on expression of XTH genes in azuki bean epicotyls

Hypergravity produced by centrifugation caused inhibition of elongation growth and a decrease in the cell wall extensibility in azuki bean epicotyls ( Vigna angularis Ohwi et Ohashi). Also, hypergravity increased the molecular mass of xyloglucans, whereas it decreased xyloglucan-degrading activity in epicotyls. When the expression profiles of three xyloglucan endotransglucosylase/hydrolase ( XTH ) genes, VaXTHS4 , VaXTH1 and VaXTH2 , were analyzed under hypergravity conditions, the expression of VaXTHS4 , which shows only hydrolase activity, was downregulated in proportion to the logarithm of the magnitude of gravity (R = −0.94). However, the gene expression of VaXTH1 or VaXTH2 , which shows only transglucosylase activity, was not affected by gravitational conditions. When the seedlings that had been grown at 1  g were transferred to hypergravity conditions at 300  g , the downregulation of VaXTHS4 expression was detected within 1 h. By removal of hypergravity stimulus, VaXTHS4 expression was increased within 1 h. These results suggest that azuki bean epicotyls promptly regulate the expression level of only VaXTHS4 in response to gravity stimuli. The regulation of xyloglucan-hydrolyzing activity as a result of changes in VaXTHS4 expression may be involved in the regulation by gravity of molecular mass of xyloglucans, leading to modifications of cell wall mechanical properties and cell elongation. Lanthanum and gadolinium, potential blockers of mechanosensitive calcium ion permeable channels (mechanoreceptors), nullified the suppression of VaXTHS4 expression, suggesting that mechanoreceptors are responsible for inhibition by hypergravity of VaXTHS4 expression.     

Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered     

Increase in the level of arabinoxylan–hydroxycinnamate network in cell walls of wheat coleoptiles grown under continuous hypergravity conditions

Changes in the amount and composition of cell wall constituents in response to continuous hypergravity stimuli were studied in wheat ( Triticum aestivum L.) coleoptiles. The lengths of coleoptiles grown under hypergravity (300  g ) conditions for 2–4 days from germination stage were 60–70% of those of 1  g control. However, the net amounts of hemicellulosic polysaccharides and cellulose in hypergravity-treated coleoptiles increased progressively as much as those in the control coleoptiles. As a result, their contents per unit length of coleoptile largely increased under hypergravity conditions. In the hemicellulose fraction, the amounts of arabinose and xylose, the major components of the fraction, prominently increased in response to hypergravity. When hemicellulosic polysaccharides were separated into neutral and acidic polymers by an anion-exchange column, the amounts of the acidic fraction consisting of (glucurono)arabinoxylans were higher in hypergravity-treated coleoptiles than in control coleoptiles. The amounts of cell wall-bound ferulic acid and diferulic acid (DFA) increased dramatically in both 1  g control and hypergravity-treated coleoptiles. Particularly, the amounts of DFA in hypergravity-treated coleoptiles were significantly higher than those in control coleoptiles during the incubation period. These results suggest that continuous hypergravity increases the rigid network structures via arabinoxylan–hydroxycinnamate cross-links within cell wall architecture in wheat coleoptiles. These structures may have a load-bearing function and contribute to construct the stable cell wall against the gravitational force.     

Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2).

The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42+/-16% under hypergravity at 13 x g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COL1A2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERK 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.     

Proliferation and differentiation of Xenopus A6 cells under hypergravity as revealed by time-lapse imaging

Xenopus laevis A6 cells, which are cloned epithelial cells from the Xenopus kidney, differentiate into a dome structure when the cells reach confluence. We investigated the gravitational responses of A6 cellular motility during normal differentiation and differentiation under hypergravity conditions using centrifugation (1-100 x g). Progression to dome formation was analyzed by time-lapse micrography. Dome formation and increased expression of Na(+)/K(+)-adenosine triphosphatase were used as markers of differentiation. Interestingly, a high rate of cellular proliferation was observed at a low level of hypergravity (5 x g). Despite this, there was no difference in the time to dome formation between the control cells at primary cell density and those that differentiated under hyper- or hypogravity conditions. In conclusion, this experiment on amphibian cells revealed that the proliferation of A6 cells was strongly affected by gravity conditions, but the differentiation step appears to be controlled by an intra- or intercellular clock.     

Increase in expression level of alpha-tubulin gene in Arabidopsis seedlings under hypergravity conditions.

Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.     

Increased level of serum vascular endothelial growth factor by long-term exposure to hypergravity.

We have previously demonstrated that short-term exposure to hypergravity at 2G for 4 h induces expression of cyclooxygenase-2 (COX-2) in the mouse heart. Moreover, expression of vascular endothelial growth factor (VEGF) is also induced in the heart in a COX-2-dependent manner. Here, we demonstrate that long-term exposure of mice to 2G for 24 h resulted in a significant increase of serum VEGF level, although expression of COX-2 and VEGF in the heart decreased to the 1G-control level. Moreover, increase of serum VEGF was not suppressed by treatment with COX-2 inhibitor, indicating that VEGF was induced in a COX-2-independent manner. These results suggest that gravitational force contributes to maintenance of the serum VEGF level.     

Effects of 3-D clino-rotation on gene expression in human fibroblast cells

Continuous variation in the direction of the gravity vector leads to various cellular responses including modulation of gene expression. Complementary DNA (cDNA) array analyses are available to observe the variation of gene expression under different conditions. In this study, expression levels of 588 representative genes were compared using the Atlas human cDNA expression array in human fibroblast cells with and without 3-dimensional (3-D) clinostat. Five upregulated and 8 downregulated genes were detected. Among these genes, upregulation of XRCC1, and downregulation of ERB-B2 and p21(Cip1/Waf1) were confirmed by RT-PCR. These results suggested that the gene expression levels of XRCC1, ERB-B2 and p21(Cip1/Waf1) were modulated by vector-averaged microgravity induced by 3-D clinostat in human fibroblast cells. Our findings may be a basis for the biological study of 3-D culture systems.     

The physical basis of gravity stimulus nullification by clinostat rotation

The question of how rotation on a horizontal axis clinostat removes plants from the influence of the gravitational stimulus is answered. It is shown that appropriate horizontal axis clinostat rotation restricts the fall of intracellular particles to a quasi-circular path such that the position of the particle remains virtually stationary within cells. The displacement of the path of fall, due to centrifugal force, is then considered, and a method of determining the optimal rotation rate is developed from physical principles. This method selects the rotation rate which minimizes the volume of cytoplasm through which particles pass under the joint influence of centrifugal and gravitational forces. With the recognition that single axis clinostats are ineffective with large plants or for long experiments, a new type of clinostat is proposed on which intracellular conditions can be rendered virtually identical to those of plants in satellite free fall regardless of plant size or duration of experiment.     

Maternal reproductive experience enhances early postnatal outcome following gestation and birth of rats in hypergravity

A major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Recent spaceflight and centrifugation studies demonstrate that reproduction and ontogenesis in mammals are amenable to study under gravitational conditions that deviate considerably from those typically experienced on Earth (1 x g). In the present study, we tested the hypothesis that maternal reproductive experience determines neonatal outcome following gestation and birth under increased (hyper) gravity. Primigravid and bigravid female rats and their offspring were exposed to 1.5 x g centrifugation from Gestational Day 11 either through birth or through the first postnatal week. On the day of birth, litter sizes were identical across gravity and parity conditions, although significantly fewer live neonates were observed among hypergravity-reared litters born to primigravid dams than among those born to bigravid dams (82% and 94%, respectively; 1.0 x g controls, 99%). Within the hypergravity groups, neonatal mortality was comparable across parity conditions from Postnatal Day 1 through Day 7, at which time litter sizes stabilized. Maternal reproductive experience ameliorated neonatal losses during the first 24 h after birth but not on subsequent days, and neonatal mortality was associated with changes in maternal care patterns. These results indicate that repeated maternal reproductive experience affords protection against neonatal losses during exposure to increased gravity. Differential mortality of neonates born to primigravid versus bigravid dams denotes gravitational load as one environmental mechanism enabling the expression of parity-related variations in birth outcome.     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

Effects of gravity on early embryogenesis [correction of embryogeneis] in Caenorhabditis elegans.

The embryonic development of the nematode Caenorhabditis elegans was examined under different gravitational conditions. The embryos after fertilization normally hatched under hypergravity (200 G) or 3D-clinorotation, whereas the number of eggs laid from an adult hermaphrodite decreased and their hatching rate was reduced under the hypergravity condition. The first cleavage plane in the 1-cell embryo was slid to some extent by re-orientation of liquid culture vessel, but the pattern and timing of cleavages were not affected.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.