How Microgravity Affects the Biology of Living SystemsView this Special Issue
Review Article | Open Access
Fiona Louis, Christophe Deroanne, Betty Nusgens, Laurence Vico, Alain Guignandon, "RhoGTPases as Key Players in Mammalian Cell Adaptation to Microgravity", BioMed Research International, vol. 2015, Article ID 747693, 17 pages, 2015. https://doi.org/10.1155/2015/747693
RhoGTPases as Key Players in Mammalian Cell Adaptation to Microgravity
A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions.
Microgravity has been demonstrated to have profound effects on both cellular and molecular levels, including changes in cell morphology [1, 2], alterations of proliferation, growth or differentiation [3, 4], modification of gene expression [5–7], and changes in signal transduction cascades [5, 8]. Single undifferentiated cells in vitro respond to altered conditions of gravity, but not all sensors and upstream regulators are known, which limits our understanding of cell sensitivity to microgravity-related conditions and even more to microgravity per se.
There are numerous observations strengthening the idea that cytoskeletal structures and cell surface receptors connected to them play an important role in the regulation of the differentiation potential of stem cells . As changes of shape and of the inner cytoskeletal architecture are common cell responses under conditions of real or simulated microgravity , the idea of cytoskeletal involvement in the cellular response to microgravity seems obvious. Moreover, stem cells or multipotent cells are recognized as being sensitive to mechanical stresses, which are known to influence cell commitment [10, 11]. The idea that not only terminally differentiated cells but also multipotent cells are sensitive to microgravity explains why even limited effects on cell commitment could have dramatic consequences. Small GTPases of the Rho family are known to control several aspects of cell dynamics (vesicular transport, traffic, cytoskeleton turnover) [12, 13] and appear to be the key players when trying to gain a better understanding of the effects of microgravity on differentiated and multipotent cells.
This review first attempts to highlight the fact that structures involved in mechanotransduction pathways are responsible for adaptation to microgravity: it will be explained that structural changes observed in cells exposed to real and simulated microgravity may result from specific RhoGTPase regulations. Then, the degree to which the effects of microgravity are important controllers of multipotent cell commitment will be discussed, highlighting the critical role of RhoGTPases in these regulations. The monitoring of RhoGTPase activities in conditions of microgravity is still a challenge as it is a dynamic process that controls other highly dynamic processes such as actin polymerization or focal adhesion turnover. In order to decipher cell adaptation in conditions of microgravity, the community is in need of a live imaging technology, like the one from Pache et al. , but that can be set up in flight! We are conscious of all the difficulties of using Förster resonance energy transfer- (FRET-) based biosensors dedicated to RhoA (Ras homolog gene family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1), two important actors of this GTPases family, under conditions of microgravity, and we are convinced that research groups that are successful with these types of sensors will provide very exciting results that will eliminate many confounding factors related to conditions of microgravity, such as launch vibrations. We predict that many specific GAP and GEF (resp., RhoGTPases inhibitors and stimulators) will turn out to be key players in cell adaptation to microgravity-related conditions in the future.
2. Mechanotransductors as Gravity Sensors
Discussions of whether an in vitro single cell or a cell population can sense changes in the gravitational field are very controversial. The currently most unknown research area involves the mechanism by which the physical event of g-force susception (by invagination, sedimentation, or buoyancy) becomes the biological process of g-force perception. Despite this, an enormous body of experimental data undoubtedly indicates that several types of cultured cells are sensitive to gravity [16, 17]. If, in fact, cells do not fall (collapse), it is because they are supported in some way. This support takes the form of a mechanical stress, set up by the intermolecular forces in response to the distortion produced by gravity. In conditions where gravity is limited (microgravity) (such as those found in an orbiting vehicle) there is thus no distortion produced, and consequently, there is no (limited) mechanical stress.
It seems that undifferentiated cells have structural elements that may play the role of “gravitational sensors” and “sense” the intensity of a mechanical tension and that several intracellular processes can depend on the value of the gravitational force. Theoretical considerations suggest that the forces involved are too small to trigger any response to the changed environment. Several research teams think that these effects are mostly caused by changes at the tissue and organ level  and that such environmental changes are stronger and more diverse  (e.g., lung, heart, and kidney become larger while spleen or pancreas get smaller in rats ). In conclusion, gravitational effects have been considered significant for cells with a diameter of no less than 10 μm . Thus, microgravity seems to alter mammalian cells as compared to bacterial cells which are normally too small.
Actors in the mechanotransduction chain represent key elements involved in microgravity adaptation. Nature provides clear examples of defined mechanoreceptors in eukaryotes such as the statoliths in plants and the otoliths of the inner ear in most species of vertebrates. Similar specialized cells of the sense organs detect pressure (touch) and vibrations and communicate these physical stimulations to the nerves of the afferent pathway up to the brain.
It thus seems that undifferentiated mammalian cells do indeed have structural elements that may play the role of a “gravitational sensor” and “sense” the intensity of a mechanical tension and that many intracellular processes (adhesion, proliferation, survival, contractility, migration, extracellular matrix (ECM) architecture, gene expression, etc.) can depend on the intensity of the gravitational force. The identification of cell structures capable of acting as gravisensors in in vitro cells still remains a problem. The general view of mechanosensing is that the overall cell is sensitive and is not a particular element.
In our opinion, the most significant element (primum movens) that may impact on cytoskeletal dynamics under microgravity is the displacement of the nucleus. The location of the nucleus is probably dictated by a tension equilibrium between the cyto- and nucleoskeletons and we can imagine that these tensions are constantly changing (in response to signals) and that the nucleus probably oscillates continuously . A microgravity environment may influence the oscillating behavior of the nucleus  and then trigger a series of mechanical adjustments that may modulate cell shape and structures, as well as functions by way of transcription activities.
In response to changes in nucleus location, cytoskeletal structures and integrins might be solicited for cell adaptation. The cytoskeleton is a network of three interconnected systems of filaments: the actin microfilaments, the microtubules, and the intermediate filaments. They condition the shape of the cells and the major mechanical functions such as adhesion, polarization, directional migration, as well as proliferation, survival, or apoptosis, gene expression, and architectural organization of their supporting scaffold .
Experiments in real and simulated conditions of microgravity have shown that cytoskeletal modulations can occur quickly after variations in gravity have taken place. Numerous articles have reported on changes within 30 min of the onset of a microgravity simulation, affecting from focal adhesions to signal transduction. Nevertheless cell response can be observed only after few seconds following gravitational changes, for example, in parabolic flight experiments. After only 22 seconds of microgravity, ML-1 thyroid cancer cells showed no sign of apoptosis or necrosis, but the F-actin and cytokeratin cytoskeleton was altered . Endothelial cells also demonstrated no signs of death (after 31 parabolas of 22 seconds) but had a cytoplasmic rearrangement and an alteration of cytoskeleton gene expressions . Concerning mesenchymal stem cells, morphologic characteristics of apoptosis cells (cell shrinkage, membrane blebbing, nuclear chromatin condensation, etc.) and decreased cell viability (rate of apoptosis up to 56.95%) were reported 12 h after parabolic flight experiment. The F-actin stress fibers and microtubules were disrupted and the expression of p53 (mRNA and protein levels) was upregulated . So, gravity-induced response of cells can occur very early, within seconds.
The reorganization of the cytoskeleton is believed to govern the modifications in size and shape of cells and nuclei as well as the patterning, number, and maturation of focal adhesions. The structures of the cytoskeleton, nuclei, and integrins may claim, to varying degrees, to fulfill the role of gravisensors .
The most likely candidates to assume the role of these structures are various elements of the cytoskeleton, the nucleus, intracellular organelles, and also certain cell surface receptors (integrins), which interact both with cytoskeletal structures and the extracellular matrix. These structures are able to sense constraints and deformations in the matrix which are caused either by a gravitational or mechanical field and convert this signal into intracellular messengers, which then give rise to a cellular response to the changes in gravity [21, 27]. It is also noteworthy that the cytoskeleton and integrins are not the primary sensors but react in response to their regulatory proteins (controllers of polymerization/destabilization agent).
Numerous cellular processes are controlled by gravity, for example, calcium signaling, mechanotransduction, ligand-receptors interactions, and cell-cell communications, which are all linked . During these mechanisms, cell density is important because force transmission is greatest at cell-cell and cell-substrate focal contacts where signaling molecules are concentrated or clustered (i.e., integrin clustering) . Indeed, transmission of forces from outside the cell through cell-matrix and cell-cell contacts appears to control the maturation or disassembly of these adhesions which rearrange the organization and contractile activity of the cytoskeleton. The cytoskeletal tensions formed at adhesions mediate mechanical signalling . Thus, vinculin phosphorylation determines whether cadherins transmit force and can produce biologically distinct functions .
In microgravity, gravity-induced breakage of cell-cell adhesions is reduced. So, cell-cell interaction was shown to be promoted in absence of gravity . Cell adhesion protein expression, specifically proteins found in tight junctions and adherens junctions, was upregulated resulting in enhanced cell-cell contact between cells (endothelial cells ). Also, increased levels of E-cadherin were observed in 3D tumor constructs cultured in simulated microgravity .
In osteoblasts, a downregulation of cell-cell adhesion proteins, such as catenin, is observed  and also a reduction in adhesion proteins such as vinculin and extracellular matrix proteins such as fibronectin . To explain this phenomenon, Levenberg et al. showed that there is an autoregulatory pathway that is activated by the presence of cell-cell or cell-substrate adhesion sites. So, when cell-cell adhesion is enhanced, cell-matrix adhesion is decreased . These adhesion processes are also dependent on Ca2+ signaling pathways, such as cell-cell adhesion via E-cadherin. This Ca2+ dependence is through activation of the protein kinase C (PKC) second messenger system, as well as activation of phospholipase C (PLC), which in turn activates a signaling cascade, resulting in the release of intracellular Ca2+ . This release of intracellular calcium, facilitating the binding of cadherins and β-catenin to the actin filaments comprising the cytoskeleton, resulted in increased strength of cell-cell contacts .
And several teams actually found a calcium release in vascular smooth muscle cells after 14 days of hindlimb unloading  and a downregulation of Calcium channel after 28 days . Also, a reduction in intracellular calcium concentration is observed after 2 days of simulated microgravity in chondrocytes  as well as in neurons . Moreover, in neutrophils, PKC pathway is inhibited under microgravity leading to a decrease in intracellular concentration of Ca2+ .
All the structural changes observed in cells subjected to microgravity-related conditions are dictated/controlled by dynamic molecular switches of the GTPase family (Figure 1). Small RhoGTPases mainly control the regulation of intracellular traffic and are responsible for cytoskeletal dynamics .
3. RhoGTPases: Mechanosensitive Molecular Switches
RhoGTPases, found in all eukaryotic cells, are key regulatory molecules which link surface receptors to the organization and turnover of the cytoskeleton, govern the formation of cell-matrix adhesions, and uphold the transcriptional control of gene expression, cell survival, and proliferation . They are members of the Ras superfamily of small GTP-binding proteins and are divided into three major classes: RhoA, Rac1, and Cdc42. GTPases are molecular switches that use a simple biochemical strategy to control complex cellular processes. They switch between two conformational states: a guanosine triphosphate- (GTP-) bound (“active”) state and another (“inactive”) state related to guanosine diphosphate (GDP). In their inactive forms, RhoGTPases are sequestrated in the cytoplasm, while upon signaling identified by integrins and growth factor receptors, they switch to their active forms and translocate to the cell membrane . There, they activate distinct and specific effector molecules which in turn regulate the organization of the cytoskeleton and cell-matrix adhesions, thus controlling cellular activities such as adhesion, and also affect cell proliferation and the expression of specific genes (Figure 2) . The cycle between the active and inactive forms is under the direct control of three groups of regulatory proteins. The guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to activate Rho proteins. The Rho proteins are then deactivated by GTPase-activating proteins (GAPs) which increase the intrinsic GTPase activity of the Rho protein, leading to the hydrolysis of GTP to GDP. The third group of proteins involved in the cycle of Rho signaling is guanine dissociation inhibitors (RhoGDI), which hide the isoprenyl groups of GTPases, an action that promotes the sequestration of inactive GTPases in the cytosol. The RhoGDIs also inhibit the release of GDP from the GTPase and contribute to the maintenance of GTPases in an inactive state. The Rho protein cycle is stimulated by agonists acting through G protein-coupled receptors (GPCRs), tyrosine kinase receptors, cytokine receptor activation, and mechanical stresses that mainly govern the activity of the GEFs . The best known actions of the RhoGTPases on mechanical parameters of the cytoskeleton can be underscored by the expression of constitutively active RhoA and Rac1 in cell lines. These modelsshow that RhoA activation leads to better cell spreading but lower mechanical properties, while Rac1 activation induces mechanotransduction . As we assume that exposure to gravitational stress is a mechanical stimulation, Rac1 might be rapidly induced in microgravity-related conditions. These results reveal the importance of RhoGTPases on mechanosensing, cell shape adaptation, or signal transduction. We will summarize below the different controls they can have on cellular mechanisms and metabolism.
4. RhoGTPases Control Cytoskeleton Dynamic
In microgravity, a qualitative and quantitative analysis of the structures of F-actin, β-tubulin, and vinculin has revealed a higher density of filamentous actin and a decreased organization in stress fibers. Exposing mesenchymal stem cells (MSCs) to low gravity affected the distribution of the different filaments and more specifically led to a significant reduction of the F-actin fibers [49, 50], extended filopodia, increased perinuclear distribution, and decreased density [15, 51]. Moreover, other research groups have found evidence of an accumulation of actin at the cell border [52, 53]. This loss of stress fibers is accompanied by an increase in monomeric G-actin content within the cells. The preceded alterations may be explained by a preferential reduction of RhoA activity.
Indeed, the activation of RhoA or Rac1 leads to the assembly of contractile actin:myosin filaments, protrusive actin-rich lamellipodia, and protrusive actin-rich filopodia, which in turn give rise to both the formation (actin polymerization) and the organization (filament bundling) of actin filaments. Thus, a number of studies (e.g., ) have shown that Rho kinase (ROCK) modulates the nonmuscle myosin II (NMM-II) activity by phosphorylation. Another protein, cofilin, regulates actin polymerization and filament elongation. Its phosphorylation leads to inactivation and occurs primarily through LIM kinases (LIMK), which are activated by Rac1-dependent kinases. Moreover, LIMK-dependent phosphorylation of cofilin can also be induced by RhoA acting through its target ROCK, which may be an important event in the stabilization of actin:myosin filaments . Microgravity leads to an alteration of the actin cytoskeleton and consequently to a decrease of integrin signaling that may be caused by the inhibition of RhoA activity. The absence of gravity increases the G-actin form, which reduces cofilin phosphorylation, and is consistent with a decrease in focal adhesions and thus stress fibers .
Finally, if a constitutively active RhoA is overexpressed, a recovery stress of the fibers is enabled, similar to what can be observed under normal gravity, and integrin signaling is restored as shown in MSCs .
Microtubules play critical roles in eukaryotic cells. They are key structural elements of the mitotic spindle apparatus during mitosis and interphase and serve as tracks upon which motor proteins transport vesicles and other components move throughout the cell . Several studies have mentioned perinuclear clustering in the microtubular network during microgravity [50, 59]. Also, the loss of the radial structure of microtubules has been observed after long stretches of time (4 h) in microgravity .
Microgravity has also been proposed to influence microtubules by affecting the self-organization of filaments. According to the theory on self-organization and in a series of in vitro studies with a change in gravity direction [61, 62] and microgravity , it was clearly shown that microtubule self-organization is sensitive to the direction and the magnitude of gravity, which may explain the results obtained under microgravity. Furthermore, the observed disorganization of microtubules may lead to a reduced rate of chromosome segregation during mitosis, while alterations of actin microfilaments and focal adhesions may also slow down cytokinesis and thus cell proliferation.
RhoGTPases regulate microtubule dynamics in different ways. Rac1 can phosphorylate at Ser16 of the microtubule plus-end-binding proteins (stathmins), which occurs in response to a number of extracellular stimuli . The effect of RhoA on microtubule dynamics is likely to be context-dependent. For instance, in migrating fibroblasts, RhoA promotes the formation of stabilized microtubules. Also, microtubules play a major role in defining cell shape and polarity through the specific interaction of their plus-ends with proteins at the cell cortex. This plus-end capture of microtubules has been attributed to a number of plus-end-binding proteins, whose activities are influenced by RhoGTPases . Altogether, results on microtubules observed in conditions of microgravity may be explained by an alteration of the RhoA and Rac1 activities.
Microgravity has also had an impact on intermediate filaments, which after 12 min in microgravity appeared as large bundles and aggregates in the vimentin network, that is, the most distributed of all intermediate filament proteins . ROCK phosphorylates intermediate filament proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the breakdown of local intermediate filaments and enables an efficient separation of intermediate filament networks . In fact, RhoA and Rac1 induce phosphorylation and reorganization of vimentin through kinases such as RhoA-associated protein kinase 2 (ROCK2), p21-activated kinase (PAK), Src kinase (family of nonreceptor tyrosine kinases), and tyrosine kinases .
Concerning lamins, which are nuclear intermediate filaments, Uva et al. showed DNA fragmentation in glial cells after 30 min of microgravity and explained the phenomenon by caspases causing lamina to collapse and chromatin to condense . Proteins linking nucleoskeleton and cytoskeleton complexes (LINC), thus connecting lamina to the cytoskeleton, have been found. When it comes to laminopathy models, in which this LINC complex is disrupted, they lead mostly to RhoA inhibition and lowered cytoplasmic elasticity, while actin and focal adhesion structures are mildly affected . Changes in nuclear structures, that we identified earlier as an important initiator of microgravity effects , might explain the RhoA activity inhibition and changes in cell tension evoked under microgravity.
Rac1 was shown to accumulate in the nuclear envelope in addition to being expressed in the nucleoplasm and seemed to have the same pattern as that reported for lamin B . This Rac1 accumulation was proven to promote cell division. In microgravity, the altered proliferation observed by Dai et al. or Damm et al. [70, 71] is controversial since Yuge et al.  rather found an increased proliferation in human mesenchymal stem cells. We thus suggest, based on our results obtained on rat osteosarcoma , that the lower proliferation might be explained by a reduced Rac1 activity in conditions of microgravity.
5. RhoGTPases as Regulators of Cell Adhesion and Matrix Remodeling
Integrins are transmembrane receptors that mediate the attachment between a cell and its surroundings, such as other cells or the ECM. In signal transduction, integrins convey information about the chemical composition and mechanical status of the environment into the cell. Therefore, in addition to transmitting mechanical forces, they are involved in cell signaling and the regulation of cell cycles, shapes, and motility .
Among the ligands of integrins can be mentioned fibronectin, vitronectin, collagen, and laminin. Then, adapter proteins such as talin and vinculin link the cytoskeleton to integrins, which attach the cell to the substrate, forming a focal adhesion. A variety of signaling proteins are associated with focal adhesions, including focal adhesion kinase (FAK), which is an important mediator of signaling at these centers. Forces are also transmitted to the substrate at these sites. In fibroblasts, local forces correlate with the area of focal adhesions and actomyosin contractility blocking results in a rapid disruption of focal adhesions .
In conditions of microgravity, a reduced focal adhesion-related area (frequently reported [35, 76]) can be explained by the lower tension applied to the cytoskeleton. This situation can be associated with an inactivation of RhoA, and as a result by decreased fibrillogenesis (fibronectin collagen) dramatically limiting integrin signaling. The proof of a reduced integrin signaling is that MSCs have been observed to display changes in the expression levels of collagen-specific integrins after 7 days of cultivation in a rotational bioreactor . In fact, activated expression of theα2-integrin has been seen during the course of MSC differentiation to osteogenesis . In addition, Loesberg et al. found a downregulation ofα1, β1, and β3 integrins after 48 h of simulated microgravity .
β1 integrin has been shown to be important for mediating the response of MSCs to mechanical stimulation . Upon application of fluid shear stress, an increase in alkaline phosphatase (ALP) activity and expression of osteogenic markers is observed, along with the activation of FAK and extracellular signal-regulated kinase 1/2 (ERK1/2). But when β1 integrins are blocked, FAK and ERK1/2 activation becomes inhibited . Phosphorylation of FAK has also been demonstrated to be important for osteogenic differentiation of human MSCs in response to tension . In microgravity-related conditions, the limitation of integrin signaling can be a plausible explanation for the reduced osteogenesis.
In addition, limitation of the integrin-mediated response can also reduce important negative regulatory pathways. Thus, growth of preadipocytes on a fibronectin matrix inhibits adipocyte differentiation and this effect is overcome when actin filaments are disrupted and promotes a rounding-up of cells . However, β1 in association withα5 binds to fibronectin, and Liu et al.  reported the presence of an expression switch fromα5 toα6 at the growth arrest stage of differentiation, which is consistent with an ECM change observed during adipogenesis. This switch is necessary in order to go from proliferation to differentiation of preadipocytes and can be explained by integrinsα6β1 that bind to laminin and can thus interfere with chromatin and gene regulation.
These two integrinsα5 andα6 are coordinately regulated by cyclic adenosine monophosphate (cAMP). Interestingly, cAMP has been shown to be activated in microgravity [83–85]. RhoA and cAMP have antagonistic roles in regulating cellular morphology . Thus, the excessive production of cAMP in microgravity may explain the limitation of RhoA activation during adipogenesis followed by the integrin switch ofα5 toα6 to promote adipogenesis. Also, it is well established that cAMP enhances the expression of both CCAAT-enhancer-binding proteins (C/EBP)α and β [87, 88] and initiates adipogenesis via the transcription factor CREB (cAMP response element binding protein) .
Concerning Rac1, cell adhesion to fibronectin (α5 integrin) but not to laminin (α6 integrin) is particularly efficient in activating Rac1 , leading to osteogenesis via β-catenin/Wnt pathways . In microgravity, fibrillogenesis is rapidly limited [92, 93], which explains the delay or absence of osteogenesis in multipotent cells. The extracellular domains of cadherins and β-catenin provide a link toα-catenin and the actin cytoskeleton . Upon tyrosine phosphorylation, β-catenin also plays a significant role in signaling when translocated to the nucleus to regulate cell proliferation .
Noritake et al.  have explained the increase in Rac1 during osteogenesis: until subconfluence, cell adhesions accumulate E-cadherins at the sites of cell-cell contacts which induce Rac1, and thus actin-meshwork formation and β-catenin, leading to osteogenesis. In fact, before E-cadherin-mediated cell-cell adhesion is established, GDP-Rac1 is sequestered in the cytosol by Rho GDI. When E-cadherins accumulate, GDP-Rac1 is converted to GTP-Rac1, through the action of a GEF, and is targeted to the plasma membrane releasing β-catenin linked to E-cadherin, which can go to the nucleus .
In addition, cell-to-cell physical contact via N-cadherin also plays a crucial role in regulating osteoblastic activity such as alkaline phosphatase activity and β-catenin signaling [98, 99]. Consequently, reduced cell-cell adhesion observed in microgravity, due to limited proliferation, may induce a decrease in Rac1 action and osteogenesis.
Moreover, it has been largely described that matrix rigidity affects osteogenesis. MSCs grown on collagen-I stiff gels (linking toα1 orα2-β1 integrins) have demonstrated activated osteogenesis, whereas softer collagen-I gels prime MSCs for a myogenic lineage . However, cytoskeleton and the dynamic mechanical balance that exists between cells and their ECM support appear as major players in several mechanotransduction pathways . Microgravity decreases the expression of collagen I [101–103], induces matrix metalloproteinases (MMP) production, and reduces the level of fibrillar collagen. Thus, it could be expected that altered conditions of gravity may change the mechanical properties of ECM (i.e., the stiffness). Several studies, for example, McBeath et al. or Shih et al. [104, 105], have shown that osteogenic differentiation becomes increased on stiffer matrices, as evident by type-I collagen, osteocalcin, Runx2 gene expressions, ROCK, FAK, and ERK1/2 induction and alizarin red S staining for mineralization. Consequently, FAK affects osteogenic differentiation through ERK1/2, whereas RhoA and ROCK regulate both FAK and ERK1/2 .
In microgravity, an initial modification of cytoskeletal dynamics might be at the origin of the following vicious circle: remodeling of a cytoskeleton is associated with a reduced internal tension (contractility) leading to the dispersion of FA. With such a reduction in FA, the cell tension cannot be restored and fibrillogenesis might be limited. Matrix deposition limitation and MMP activation (Rac1 dependent process [106, 107]) may further reduce the matrix stiffness, thus amplifying the dispersion of FA and reducing cell tension and fibrillogenesis. After a short exposure (from minutes to hours) to microgravity-related conditions (before fibrillogenesis, MMP production), the matrix stiffness is not modified. We can thus speculate that the ability of the cells to detect the stiff matrix they are normally seeded on has become rapidly impaired. Mechanical information is normally conveyed by ECM and cells by FA adaptation following tensegrity principles (equilibrium of internal and external tension) ; in microgravity it seems that the displacement of the nucleus (sensitive to G) conveys the mechanical stimulus and from a tensegrity perspective, the cell adapts to the reduced tension by lowering the ECM tension (interruption of fibrillogenesis and MMP production). The short-term adaptation of the cell to microgravity that we have described up to now seems to be characterized by a rapid reduction of RhoA and an increased Rac1 activity. Altogether, these studies revealed that the control of cytoskeleton remodeling by RhoGTPases impacts on cell adhesion signaling, limiting internal cellular tension as well as ECM fibrillogenesis, and triggers MMP production, thus limiting cell-matrix adhesion and survival.
6. RhoGTPases in Stem Cell Commitment
In simulated microgravity, cellular morphology is drastically changed after 7 days. The MSCs appear rounder and less firmly attached to their substrate than under conditions of normal gravity. Rather, they are very spread out and display a fibroblastic morphology .
Since the work by McBeath et al., we know that the shape of a cell affects its differentiation potential . Thus, MSCs that have been allowed to adhere over a larger area are able to differentiate towards the osteogenic lineage while cells adhering to a smaller area are restricted to the adipogenic lineage. These impacts on lineage commitment by mesenchymal stem cells seem to be regulated by shape-induced changes in the RhoGTPase activity and cytoskeletal tension . Yao et al.  showed that the cell shape itself is an inherent cue to regulate stem cell differentiation, both with and without external chemical induction factors. Thus, according to McBeath et al. , expressing dominant-negative RhoA causes MSCs to become adipocytes, while constitutively active RhoA induces osteoblastic or myocytic differentiation [110, 111].
Concerning Rac1, it has been shown to promote cell adhesion and spreading and thereby to prevent the cell shape change and the establishment of the cortical actin structure necessary for adipocyte formation . Adhering cells are characterized by an elaborate network of stress fibers and focal adhesions and are thus more prone to adopt a fibroblastic cell shape reflecting cytoskeleton tension [112, 113], which seems to be altered in conditions of microgravity.
The cell shape may also depend on the available substrate area and hence the cell density. However, if cellular growth is reduced in microgravity, the cell density will also be altered. Gao et al.  found that levels of RhoA activity did not vary substantially, but that the Rac1 activity was significantly higher in well-spread cells during early differentiation than in high-density cells.
They also demonstrated that Rac1 is necessary for osteogenesis and that constitutively active Rac1 inhibited adipogenesis, even if it is important for adipose commitment. Liu et al.  showed that an increase in preadipocyte density inhibited the RhoA activity and that a downregulation of the RhoA-ROCK pathway was required for both adipose lineage commitment and maturation [104, 111]. An increased cell density thus appeared to be critically important.
GTPases have also been shown to act in the cell cycle, mitosis, and cytokinesis. RhoGTPases influence the cyclin-dependent kinase (cdk) activity during the G1-Phase of the cell cycle. Thus, RhoGTPases control the organization of the microtubule and actin fibers during cell cycling. An inhibition of RhoA or Rac1 blocks the G1 progression in a variety of mammalian cell types [114, 115]. Also, Rac1 (but not RhoA) stimulates cyclin D1 transcription mediated by NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) [116, 117]. Thus, the necessity to downmodulate the Rac1 activity in adipogenesis is that Rac1 may prolong proliferation of preadipocytes, which is consistent with the reported effects of Rac1 on cyclin D1 [90, 118, 119]. In fact, Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division . Concerning the cell division itself, it has been shown that actin:myosin filaments, under the control of ROCK, are required at the cortex to allow positioning of the centrosomes . RhoA also plays a crucial role in the contractile ring function .
Microgravity affects the growth, proliferation, and differentiation of osteoblasts. Since the inhibition of RhoA, observed under microgravity, blocks G1 progression [114, 115], this may explain the altered proliferation and differentiation of osteoblastic cells and increased adipogenesis as summarized in Figure 3.
Furthermore, several cytoskeletal components, including Rac1 GTPase activating protein 1 (Rac-GAP1) and Tropomodulin 1, segregate asymmetrically during stem cell division, and overexpression of these proteins may enhance MSC commitment, as already proven with asymmetrical divisions of hematopoietic stem cells to progenitor cells .
7. RhoGTPases and Wnt/β-Catenin Signaling Crosstalk
Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. The canonical Wnt pathway leads to regulation of gene transcription, the noncanonical planar cell polarity pathway regulates the cytoskeleton via a RhoGTPase regulation that is responsible for the shape of the cell, and the noncanonical Wnt/calcium pathway regulates calcium inside the cell .
Mellor et al. found that Wnt signaling was inhibited in conditions of microgravity  and mouse osteoblasts subjected to simulated microgravity were found to have lower levels of several components of the Wnt/β-catenin signaling pathway. This may indicate, even indirectly, the activation of an adipogenic program under microgravity . Moreover, Wan et al.  recently demonstrated a changed RhoA and β-catenin signaling after 1 and 2.5 h, respectively, in clinorotated osteoblasts. They revealed that both the RhoA activity and the TCF/LEF (T-cell factor-1 and lymphoid enhancing factor-1) activity, a β-catenin recruiter, were downregulated by unloading. However, the inhibition of β-catenin signaling blocked the unloading-induced RhoA suppression, and dominant negative RhoA inhibited the TCF/LEF suppression, revealing a regulation loop between β-catenin, RhoA, and TCF/LEF. Furthermore, while β-catenin signaling seemed to be required for microgravity regulation of RhoA, this response was not mediated by the actin cytoskeleton or intracellular tension . The same was observed for Rac1/β-catenin signaling .
The Wnt canonical pathway involves the translocation of β-catenin to the nucleus, and β-catenin has been shown to promote osteogenic differentiation in early osteoblast progenitors in vivo . In contrast, other studies have suggested that canonical Wnt signaling may actually promote stem cell renewal and inhibit osteogenic differentiation of osteoprogenitor cells in vivo , as well as promoting stem cell renewal in human MSCs derived from bone marrow . Arnsdorf and colleagues  investigated the role of noncanonical Wnt signaling in mechanically induced osteogenic differentiation of MSCs. Exposure of MSCs to oscillatory fluid flow resulted in a translocation of β-catenin  and an upregulation of Wnt5a, which is capable of inducing both canonical and noncanonical pathways. Wnt5a is also necessary for the flow-induced activation of RhoA. However, the inhibition of Wnt5a did not affect the β-catenin translocation, which may instead be regulated by cadherin-catenin signaling. In addition, Santos et al.  showed that the activation of the RhoA/ROCK pathway by Wnt5a induced a downregulation of adipogenic markers. It was further reported that RhoA could also be activated by Wnt3a, one of the canonical Wnt family members , and that an inhibition of intracellular β-catenin decreased the RhoA activity .
Kim et al.  also found that Wnt signaling regulated the MSC differentiation into cardiomyocyte-like cells with a concomitant downregulation of RhoA and upregulation of Rac1. Concerning Rac1, it was shown to be a critical regulator in shear stress-driven β-catenin signaling in osteoblasts , and constitutively active Rac1 mutant caused a significant enhancement of the TCF/LEF activity.
These studies demonstrate that Wnt signaling is important for mechanically induced differentiation, through RhoA or Rac1 pathways. So, in conditions of microgravity, reduced RhoA, cell shape, and migratory behaviors can be explained by Wnt and β-catenin signaling. Finally, RhoGTPases are regulated by Wnt signaling, but in return, β-catenin location (translocation) is dependent on RhoGTPases. This intricate interplay between both regulatory elements makes them particularly important for the interpretation of microgravity effects.
8. RhoGTPases and Oxidative Stress
One of the first targets of Rac1 to be identified was p67phox, an essential structural component of the NADPH oxidase complex . Since then, Rac1 has been reported to promote reactive oxygen species (ROS) production in many cells and to mediate the activity of the Nox family [137, 138]. Consequently, Rac1 activation leads to the generation of ROS enabling adipogenesis commitment  and reducing osteoblastogenesis [140, 141]. Moreover, GTPases act on the antioxidant master gene Nrf2 (nuclear factor-like 2), which activates a protective adaptive response to oxidative stress through transcriptional activation of antioxidant defense genes .
RhoA is involved in Nrf2 phosphorylation, which is necessary for its activation . Nrf2 is a transcription factor for Hace1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1), and Hace1 binds and ubiquitylates Rac1 when the latter is associated with NADPH oxidase, thus blocking ROS generation by NOX [143, 144]. So, RhoA activation may limit ROS production and adipogenesis while Rac1 activation may support it. However, several research groups have reported that ROS causes RhoA activation [145, 146], while Nimnual et al. demonstrated that Rac1-mediated ROS production results in the downregulation of the RhoA activity . This is also required for Rac1-induced formation of membrane ruffles and integrin-mediated cell spreading. The GTPase regulation by oxidative cell status thus still remains unclear.
In line with these papers, several research groups, such as Versari et al., have found increased oxidative stress during space flight due to microgravity [148, 149] and cosmic radiations . As RhoA is decreased in microgravity, this could explain the increased production of reactive oxygen species. According to this paper, we can assume that Rac1 activities are increased in microgravity. An upregulated Rac1 activity fits well with enhanced ROS production and improved adipogenesis.
However, a higher Rac1 activity is also consistent with a higher ability for cell migration [151, 152]. Nevertheless, results of migration in space are controversial. Bone marrow cells from rats and human embryonic brain cells show a facilitated cell migration [153, 154], while bone marrow CD34+ cells have a lower migration potential in simulated microgravity . We can interpret the apparent discrepancies in migration results based on the time spent in microgravity: for short-term exposure (from minutes to hours), there are several reasons to believe that RhoA is decreased and Rac1 increased in line with their reciprocal inhibition , but for longer exposure (from hours to days), the Rac1-induced ROS production may increase RhoA activation [145, 146] and reduce the Rac1 activity limiting migration capabilities. The missing information in microgravity is related to the lack of measurements of specific RhoGTPase activities.
9. RhoGTPases Activities Monitoring in Microgravity
Meyers et al. showed a reduction in active RhoA (−88% (±2%)) and a decrease in phosphorylation of cofilin after 7 days in microgravity, in addition to the absence of stress fibers . If overexpression of active RhoA is carried out, this enables a recovery of stress fibers and restored integrin signals, similar to those observed in normal gravity in MSC . In simulated microgravity, a decrease in RhoA activity was also observed after 72 h [157, 158]. Unfortunately nothing is known about Rac1 activity. Zayzafoon et al. thus proposed a model in which the cytoskeleton is actually not the first sensor, but a secondary step affected by a gravity-sensitive sensor. In this model, it is the RhoA inactivation that is followed by cytoskeletal changes and transduction at FAs , which explains the alterations on MSC differentiations observed in microgravity. To our knowledge, our team is the first to perform RhoA and Rac1 monitoring during osteogenesis and adipogenesis in simulated microgravity using embryonic mesenchymal stem cells. C3H10T1/2 multipotent cells were cultured in modeled microgravity using NASA’s rotating wall vessels (RWV) or in control cultures under conditions of earth gravity for up to 8 days, seeded on collagen-coated microbeads (Cytodex 3, Sigma). The results presented in Figure 4 show significant decreases in both RhoA and Rac1 after long-term exposure to simulated microgravity. To our knowledge no comparison can be made with data obtained in real microgravity, unfortunately. Regardless of the limitation of the model when it comes to simulated microgravity-related conditions, these results clearly showed that downregulations of RhoA and Rac1 were compatible with enhanced adipogenesis and limited osteogenesis.
As preservation of active RhoGTPases in flight condition might be challenging, the recent validation of biosensors for imaging of active RhoA, Rac1, and Cdc42 represents an important step in understanding cell responses to microgravity. Despite the critical role of RhoGTPases that we describe in this review, there is a dramatic lack of data concerning the monitoring of their activities during exposure to microgravity particularly in real microgravity. These data are of crucial importance since cell adaptation is a dynamic process; we need to use available technologies such as fused fluorescent proteins and biosensors dedicated to following RhoGTPase activities in order to decipher cell adaptation in conditions of microgravity. On ground experiments, extensive biochemical and profiling studies on mechanotransduction pathways can be performed. In an automated spaceflight, the use of biosensors specific to molecules integrating many pathways such as RhoGTPases should be presented as a simplified and integrated view of cell mechanics. The community is in need of a live imaging data (already validated on ground ) that can be now used in flight conditions. We believe that groups that are successful in providing this type of integrated data will surprise our community whose thinking is limited by analysis of fixed images of cells and the monitoring of individual parameters.
RhoGTPases represent a unique hub for integration of biochemical and mechanical signals. As such, they are probably very rapidly involved in a cell’s adaptation to microgravity-related conditions. Published data describing this adaptation have reported on alterations of the cytoskeleton, adhesion, and fibrillogenesis as well as an enhancement of the ROS production and migration that can be explained by the specific regulation of RhoGTPases. To summarize the literature, we can speculate that after a short exposure of a cell to microgravity, the RhoA activity is depressed and the Rac1 activity increased. For long-term exposure, osteogenesis has been reported to be impaired and adipogenesis promoted. These changes in multipotent cell commitment fit nicely with prolonged depressed activities of both RhoA and Rac1 (Figure 5).
As we are convinced that focal adhesion and F-actin fibers are not the primary sensors of microgravity-related signals (but rather transducers or effectors of the response), many specific GAP and GEF (resp., RhoGTPase inhibitors and stimulators) will emerge as new players in the adaptation of cells to microgravity-related conditions. What are the mechanisms that explain the activation or inhibition of these GTPases regulators? As we try to establish that mechanosensors are involved in cell adaptation to microgravity we can predict that critical players identified in these extreme conditions will in return be recognized in the mechanobiology field.
|cAMP:||Cyclic adenosine monophosphate|
|CREB:||cAMP response element-binding protein|
|ERK1/2:||Extracellular signal-regulated kinase 1/2|
|FAK:||Focal adhesion kinase|
|FRET:||Förster resonance energy transfer|
|GDIs:||Guanine dissociation inhibitors|
|GEFs:||Guanine nucleotide exchange factors|
|GPCR:||G protein-coupled receptor|
|Hace1:||HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1|
|LINC:||Proteins linking nucleoskeleton and cytoskeleton complexes|
|MSC:||Mesenchymal stem cell|
|NF-κB:||Nuclear factor kappa-light-chain-enhancer of activated B cells|
|NMM-II:||Nonmuscle myosin II|
|Nrf2:||Nuclear factor (erythroid-derived 2-) like 2|
|Rac1:||Ras-related C3 botulinum toxin substrate 1|
|RhoA:||Ras homolog gene family, member A|
|ROCK2:||RhoA-associated protein kinase 2|
|ROS:||Reactive oxygen species|
|RWV:||Rotating wall vessels|
|SEM:||Standard error of the mean|
|Src family kinase:||Family of nonreceptor tyrosine kinases|
|TCF/LEF:||T-cell factor-1 (Tcf-1) and lymphoid enhancing factor-1 (Lef-1).|
Conflict of Interests
The authors declare that there is no conflict of interests regarding to the publication of this paper.
The study was partially funded by the European Space Agency (Microgravity Application Program, MAP “ERISTO”) (European Research in Space and Terrestrial Osteoporosis, Contract no. 14232/00/NL/SH) and the French Centre National d’Etudes Spatiales (CNES).
- P. J. Rijken, R. P. de Groot, W. Briegleb et al., “Epidermal growth factor-induced cell rounding is sensitive to simulated microgravity,” Aviation Space & Environmental Medicine, vol. 62, no. 1, pp. 32–36, 1991.
- M. Hughes-Fulford, “Function of the cytoskeleton in gravisensing during spaceflight,” Advances in Space Research, vol. 32, no. 8, pp. 1585–1593, 2003.
- A. Cogoli, “Fundamentals of space biology: research on cells, animals, and plants in space,” in Cell Biology, G. Clement, and K. Slenzka, Eds., pp. 121–170, Springer, New York, NY, USA, 2006.
- D. Grimm, P. Wise, M. Lebert, P. Richter, and S. Baatout, “How and why does the proteome respond to microgravity?” Expert Review of Proteomics, vol. 8, no. 1, pp. 13–27, 2011.
- R. P. de Groot, P. J. Rijken, J. Boonstra, A. J. Verkleij, S. W. de Laat, and W. Kruijer, “Epidermal growth factor-induced expression of c-fos is influenced by altered gravity conditions,” Aviation Space and Environmental Medicine, vol. 62, no. 1, pp. 37–40, 1991.
- T. G. Hammond, F. C. Lewis, T. J. Goodwin et al., “Gene expression in space,” Nature Medicine, vol. 5, no. 4, p. 359, 1999.
- Y. Liu and E. Wang, “Transcriptional analysis of normal human fibroblast responses to microgravity stress,” Genomics, Proteomics and Bioinformatics, vol. 6, no. 1, pp. 29–41, 2008.
- O. Ullrich, K. Huber, and K. Lang, “Signal transduction in cells of the immune system in microgravity,” Cell Communication and Signaling, vol. 6, article 9, 2008.
- P. S. Mathieu and E. G. Loboa, “Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape, mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways,” Tissue Engineering—Part B: Reviews, vol. 18, no. 6, pp. 436–444, 2012.
- J. C. Chen and C. R. Jacobs, “Mechanically induced osteogenic lineage commitment of stem cells,” Stem Cell Research and Therapy, vol. 4, article 107, no. 5, 2013.
- J. Eyckmans, G. L. Lin, and C. S. Chen, “Adhesive and mechanical regulation of mesenchymal stem cell differentiation in human bone marrow and periosteum-derived progenitor cells,” Biology Open, vol. 1, no. 11, pp. 1058–1068, 2012.
- A. B. Jaffe and A. Hall, “Rho GTPases: biochemistry and biology,” Annual Review of Cell and Developmental Biology, vol. 21, pp. 247–269, 2005.
- A. Hall, “G proteins and small GTpases: distant relatives keep in touch,” Science, vol. 280, no. 5372, pp. 2074–2075, 1998.
- M. Raftopoulou and A. Hall, “Cell migration: rho GTPases lead the way,” Developmental Biology, vol. 265, no. 1, pp. 23–32, 2004.
- C. Pache, J. Kühn, K. Westphal et al., “Digital holographic microscopy real-time monitoring of cytoarchitectural alterations during simulated microgravity,” Journal of Biomedical Optics, vol. 15, no. 2, Article ID 026021, 2010.
- L. B. Buravkova, “Problems of the gravitational physiology of a cell,” Human Physiology, vol. 36, no. 7, pp. 746–753, 2010.
- D. Ingber, “How cells (might) sense microgravity,” The FASEB Journal, vol. 13, pp. S3–S15, 1999.
- I. D. Pestov, “Fundamentals of gravitational biology,” Kosmicheskaia Biologiia i Meditsina, vol. 2, no. 1, 9 pages, 1997.
- M. Miyake, M. Yamasaki, A. Hazama, S. Nielsen, and T. Shimizu, “Effects of microgravity on organ development of the neonatal rat,” Uchu Seibutsu Kagaku, vol. 18, no. 3, pp. 126–127, 2004.
- E. C. Pollard, “Theoretical studies on living systems in the absence of mechanical stress,” Journal of Theoretical Biology, vol. 8, no. 1, pp. 113–123, 1965.
- D. E. Ingber, N. Wang, and D. Stamenović, “Tensegrity, cellular biophysics, and the mechanics of living systems,” Reports on Progress in Physics, vol. 77, no. 4, 2014.
- R. G. Bacabac, T. H. Smit, J. J. W. A. van Loon, B. Z. Doulabi, M. Helder, and J. Klein-Nulend, “Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm?” The FASEB Journal, vol. 20, no. 7, pp. 858–864, 2006.
- C. Ulbrich, J. Pietsch, J. Grosse et al., “Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton,” Cellular Physiology and Biochemistry, vol. 28, no. 2, pp. 185–198, 2011.
- J. Grosse, M. Wehland, J. Pietsch et al., “Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells,” The FASEB Journal, vol. 26, no. 2, pp. 639–655, 2012.
- R. Meng, H.-Y. Xu, S.-M. Di et al., “Human mesenchymal stem cells are sensitive to abnormal gravity and exhibit classic apoptotic features,” Acta Biochimica et Biophysica Sinica, vol. 43, no. 2, pp. 133–142, 2011.
- M. G. Tairbekov, “Molekulyarnye i kletochnye osnovy gravitatsionnoi chuvstvitel’nosti (Molecular and Cellular Fundamentals of Gravitational Sensitivity),” 2002.
- C. A. Lambert, C. M. Lapière, and B. V. Nusgens, “Biology of adherent cells in microgravity,” in Biology in Space and Life on Earth, R. N. Enno Brinckmann, Ed., pp. 123–155, Wiley-VCH, New York, NY, USA, 2007.
- T. B. Damm and M. Egli, “Calcium's role in mechanotransduction during muscle development,” Cellular Physiology and Biochemistry, vol. 33, no. 2, pp. 249–272, 2014.
- C. S. Chen, J. Tan, and J. Tien, “Mechanotransduction at cell-matrix and cell-cell contacts,” Annual Review of Biomedical Engineering, vol. 6, pp. 275–302, 2004.
- J. L. Bays, X. Peng, C. E. Tolbert et al., “Vinculin phosphorylation differentially regulates mechanotransduction at cell-cell and cell-matrix adhesions,” Journal of Cell Biology, vol. 205, no. 2, pp. 251–263, 2014.
- N. L. Cowger, E. Benes, P. L. Allen, and T. G. Hammond, “Expression of renal cell protein markers is dependent on initial mechanical culture conditions,” Journal of Applied Physiology, vol. 92, no. 2, pp. 691–700, 2002.
- G. L. Sanford, D. Ellerson, C. Melhado-Gardner, A. E. Sroufe, and S. Harris-Hooker, “Three-dimensional growth of endothelial cells in the microgravity-based rotating wall vessel bioreactor,” In Vitro Cellular & Developmental Biology: Animal, vol. 38, no. 9, pp. 493–504, 2002.
- M. Ingram, G. B. Techy, R. Saroufeem et al., “Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor,” In Vitro Cellular & Developmental Biology—Animal, vol. 33, no. 6, pp. 459–466, 1997.
- H. L. Nichols, N. Zhang, and X. Wen, “Proteomics and genomics of microgravity,” Physiological Genomics, vol. 26, no. 3, pp. 163–171, 2006.
- A. Guignandon, M. H. Lafage-Proust, Y. Usson et al., “Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions,” The FASEB journal, vol. 15, no. 11, pp. 2036–2038, 2001.
- S. Levenberg, B.-Z. Katz, K. M. Yamada, and B. Geiger, “Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands,” Journal of Cell Science, vol. 111, no. 3, pp. 347–357, 1998.
- J. A. Felix, V. V. Chaban, M. L. Woodruff, and E. R. Dirksen, “Mechanical stimulation initiates intercellular Ca2+ signaling in intact tracheal epithelium maintained under normal gravity and simulated microgravity,” American Journal of Respiratory Cell and Molecular Biology, vol. 18, no. 5, pp. 602–610, 1998.
- K. S. Ko, P. D. Arora, V. Bhide, A. Chen, and C. A. McCulloch, “Cell-cell adhesion in human fibroblasts requires calcium signaling,” Journal of Cell Science, vol. 114, part 6, pp. 1155–1167, 2001.
- P. N. Colleran, B. J. Behnke, M. K. Wilkerson, A. J. Donato, and M. D. Delp, “Simulated microgravity alters rat mesenteric artery vasoconstrictor dynamics through an intracellular Ca2+ release mechanism,” American Journal of Physiology: Regulatory Integrative and Comparative Physiology, vol. 294, no. 5, pp. R1577–R1585, 2008.
- M.-J. Xie, Y.-G. Ma, F. Gao et al., “Activation of BKCa channel is associated with increased apoptosis of cerebrovascular smooth muscle cells in simulated microgravity rats,” American Journal of Physiology: Cell Physiology, vol. 298, no. 6, pp. C1489–C1500, 2010.
- X. Li, S. Yang, S. Li, P. Jiang, and Z. Lin, “Effects of simulated microgravity on the alkaline phosphatase activity and intracellular calcium concentration of cultured chondrocytes,” Chinese Science Bulletin, vol. 44, no. 3, pp. 218–221, 1999.
- K. Meissner, J. R. Piqueira, and W. Hanke, “Fluorescent and dispersion experiments on biological membranes under micro-gravity,” Journal of Gravitational Physiology, vol. 11, no. 2, pp. P195–P196, 2004.
- A. Sundaresan, D. Risin, and N. R. Pellis, “Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity,” In Vitro Cellular & Developmental Biology—Animal, vol. 38, no. 2, pp. 118–122, 2002.
- A. Hall, “Rho GTpases and the actin cytoskeleton,” Science, vol. 279, no. 5350, pp. 509–514, 1998.
- S. Etienne-Manneville and A. Hall, “Rho GTPases in cell biology,” Nature, vol. 420, no. 6916, pp. 629–635, 2002.
- A. J. Ridley and A. Hall, “The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors,” Cell, vol. 70, no. 3, pp. 389–399, 1992.
- M. Chiariello, J. P. Vaqué, P. Crespo, and J. S. Gutkind, “Activation of Ras and Rho GTPases and MAP Kinases by G-protein-coupled receptors,” Methods in Molecular Biology, vol. 661, pp. 137–150, 2010.
- S. Servotte, Z. Zhang, C. A. Lambert et al., “Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases,” Protoplasma, vol. 229, no. 2–4, pp. 215–220, 2006.
- M. Hughes-Fulford and M. L. Lewis, “Effects of microgravity on osteoblast growth activation,” Experimental Cell Research, vol. 224, no. 1, pp. 103–109, 1996.
- M. A. Meloni, G. Galleri, G. Pani, A. Saba, P. Pippia, and M. Cogoli-Greuter, “Space flight affects motility and cytoskeletal structures in human monocyte cell line J-111,” Cytoskeleton, vol. 68, no. 2, pp. 125–137, 2011.
- S. I. M. Carlsson, M. T. S. Bertilaccio, E. Ballabio, and J. A. M. Maier, “Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization,” Biochimica et Biophysica Acta—Molecular Cell Research, vol. 1642, no. 3, pp. 173–179, 2003.
- L. B. Buravkova, P. M. Gershovich, J. G. Gershovich, and A. I. Grigor‘ev, “Mechanisms of gravitational sensitivity of osteogenic precursor cells,” Acta Naturae, vol. 2, no. 1, pp. 28–36, 2010.
- V. E. Meyers, M. Zayzafoon, S. R. Gonda, W. E. Gathings, and J. M. McDonald, “Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells,” Journal of Cellular Biochemistry, vol. 93, no. 4, pp. 697–707, 2004.
- F. Matsumura, “Regulation of myosin II during cytokinesis in higher eukaryotes,” Trends in Cell Biology, vol. 15, no. 7, pp. 371–377, 2005.
- K. Ohashi, K. Nagata, M. Maekawa, T. Ishizaki, S. Narumiya, and K. Mizuno, “Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop,” The Journal of Biological Chemistry, vol. 275, no. 5, pp. 3577–3582, 2000.
- V. E. Meyers, M. Zayzafoon, J. T. Douglas, and J. M. McDonald, “RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity,” Journal of Bone and Mineral Research, vol. 20, no. 10, pp. 1858–1866, 2005.
- M. Zayzafoon, V. E. Meyers, and J. M. McDonald, “Microgravity: the immune response and bone,” Immunological Reviews, vol. 208, no. 1, pp. 267–280, 2005.
- C. E. Walczak, “Microtubule dynamics and tubulin interacting proteins,” Current Opinion in Cell Biology, vol. 12, no. 1, pp. 52–56, 2000.
- F. Yang, Z. Dai, Y. Tan, and Y. Li, “Effects of altered gravity on the cytoskeleton of neonatal rat cardiocytes,” Microgravity Science and Technology, vol. 22, no. 1, pp. 45–52, 2010.
- M. L. Lewis, J. L. Reynolds, L. A. Cubano, J. P. Hatton, B. Desales Lawless, and E. H. Piepmeier, “Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat),” The FASEB Journal, vol. 12, no. 11, pp. 1007–1018, 1998.
- J. Tabony, N. Rigotti, N. Glade, and S. Cortès, “Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations,” Biophysical Chemistry, vol. 127, no. 3, pp. 172–180, 2007.
- C. Papaseit, N. Pochon, and J. Tabony, “Microtubule self-organization is gravity-dependent,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 15, pp. 8364–8368, 2000.
- H. Daub, K. Gevaert, J. Vandekerckhove, A. Sobel, and A. Hall, “Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16,” The Journal of Biological Chemistry, vol. 276, no. 3, pp. 1677–1680, 2001.
- L. Sciola, M. Cogoli-Greuter, A. Cogoli, A. Spano, and P. Pippia, “Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells,” Advances in Space Research, vol. 24, no. 6, pp. 801–805, 1999.
- H. Goto, H. Kosako, and M. Inagaki, “Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase,” Microscopy Research and Technique, vol. 49, no. 2, pp. 173–182, 2000.
- L. Chang and R. D. Goldman, “Intermediate filaments mediate cytoskeletal crosstalk,” Nature Reviews Molecular Cell Biology, vol. 5, no. 8, pp. 601–613, 2004.
- B. M. Uva, M. A. Masini, M. Sturla et al., “Microgravity-induced apoptosis in cultured glial cells,” European Journal of Histochemistry, vol. 46, no. 3, pp. 209–214, 2002.
- C. M. Hale, A. L. Shrestha, S. B. Khatau et al., “Dysfunctional connections between the nucleus and the actin and microtubule networks in laminopathic models,” Biophysical Journal, vol. 95, no. 11, pp. 5462–5475, 2008.
- D. Michaelson, W. Abidi, D. Guardavaccaro et al., “Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division,” Journal of Cell Biology, vol. 181, no. 3, pp. 485–496, 2008.
- Z. Q. Dai, R. Wang, S. K. Ling, Y. M. Wan, and Y. H. Li, “Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells,” Cell Proliferation, vol. 40, no. 5, pp. 671–684, 2007.
- T. B. Damm, A. Franco-Obregón, and M. Egli, “Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts,” Cell Cycle, vol. 12, no. 18, pp. 3001–3012, 2013.
- L. Yuge, T. Kajiume, H. Tahara et al., “Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation,” Stem Cells and Development, vol. 15, no. 6, pp. 921–929, 2006.
- A. Guignandon, C. Genty, L. Vico, M.-H. Lafage-Proust, S. Palle, and C. Alexandre, “Demonstration of feasibility of automated osteoblastic line culture in space flight,” Bone, vol. 20, no. 2, pp. 109–116, 1997.
- F. J. Alenghat and D. E. Ingber, “Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins,” Science's STKE: Signal Transduction Knowledge Environment, vol. 2002, no. 119, article PE6, 2002.
- N. Q. Balaban, U. S. Schwarz, D. Riveline et al., “Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates,” Nature Cell Biology, vol. 3, no. 5, pp. 466–472, 2001.
- N. Nabavi, A. Khandani, A. Camirand, and R. E. Harrison, “Effects of microgravity on osteoclast bone resorption and osteoblast cytoskeletal organization and adhesion,” Bone, vol. 49, no. 5, pp. 965–974, 2011.
- J. Gebken, B. Lüders, H. Notbohm et al., “Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2),” The Journal of Biochemistry, vol. 126, no. 4, pp. 676–682, 1999.
- W. A. Loesberg, X. F. Walboomers, J. J. W. A. Van Loon, and J. A. Jansen, “Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography,” Cell Motility and the Cytoskeleton, vol. 65, no. 2, pp. 116–129, 2008.
- L. Liu, C. Zong, B. Li et al., “The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system,” Journal of Tissue Engineering and Regenerative Medicine, vol. 8, no. 2, pp. 85–96, 2014.
- D. F. Ward Jr., W. A. Williams, N. E. Schapiro et al., “Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells,” Molecular and Cellular Biomechanics, vol. 4, no. 4, pp. 177–188, 2007.
- B. M. Spiegelman and C. A. Ginty, “Fibronectin modulation of cell shape and lipogenic gene expression in 3T3-adipocytes,” Cell, vol. 35, no. 3, part 2, pp. 657–666, 1983.
- J. Liu, S. M. DeYoung, M. Zhang, M. Zhang, A. Cheng, and A. R. Saltiel, “Changes in integrin expression during adipocyte differentiation,” Cell Metabolism, vol. 2, no. 3, pp. 165–177, 2005.
- P. Barbe, J. Galitzky, I. de Glisezinski et al., “Simulated microgravity increases β-adrenergic lipolysis in human adipose tissue,” The Journal of Clinical Endocrinology & Metabolism, vol. 83, no. 2, pp. 619–625, 1998.
- H. Maass, J. Transmontano, and F. Baisch, “Response of adrenergic receptors to 10 days head-down tilt bedrest,” Acta Physiologica Scandinavica, Supplement, vol. 144, no. 604, pp. 61–68, 1992.
- V. A. Convertino, J. L. Polet, K. A. Engelke, G. W. Hoffler, L. D. Lane, and C. G. Blomqvist, “Evidence for increased β-adrenoreceptor responsiveness induced by 14 days of simulated microgravity in humans,” American Journal of Physiology, vol. 273, no. 1, part 2, pp. R93–R99, 1997.
- J.-M. Dong, T. Leung, E. Manser, and L. Lim, “cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKα,” The Journal of Biological Chemistry, vol. 273, no. 35, pp. 22554–22562, 1998.
- Z. Cao, R. M. Umek, and S. L. McKnight, “Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells,” Genes & Development, vol. 5, no. 9, pp. 1538–1552, 1991.
- Q.-Q. Tang, M.-S. Jiang, and M. D. Lane, “Repressive effect of Sp1 on the C/EBPα gene promoter: role in adipocyte differentiation,” Molecular and Cellular Biology, vol. 19, no. 7, pp. 4855–4865, 1999.
- J. E. Reusch, L. A. Colton, and D. J. Klemm, “CREB activation induces adipogenesis in 3T3-L1 cells,” Molecular & Cellular Biology, vol. 20, no. 3, pp. 1008–1020, 2000.
- A. Mettouchi, S. Klein, W. Guo et al., “Integrin-specific activation of Rac controls progression through the G1 phase of the cell cycle,” Molecular Cell, vol. 8, no. 1, pp. 115–127, 2001.
- Q. Wan, E. Cho, H. Yokota, and S. Na, “Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts,” Biochemical and Biophysical Research Communications, vol. 433, no. 4, pp. 502–507, 2013.
- M. Hughes-Fulford and V. Gilbertson, “Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity,” FASEB Journal, vol. 13, no. 8, pp. S121–S127, 1999.
- A. Guignandon, C. Faure, T. Neutelings et al., “Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells,” The FASEB Journal, vol. 28, no. 9, pp. 4077–4087, 2014.
- F. H. Brembeck, M. Rosário, and W. Birchmeier, “Balancing cell adhesion and Wnt signaling, the key role of β-catenin,” Current Opinion in Genetics and Development, vol. 16, no. 1, pp. 51–59, 2006.
- F. M. van Roy and P. D. McCrea, “A role for kaiso-p120ctn complexes in cancer?” Nature Reviews Cancer, vol. 5, no. 12, pp. 956–964, 2005.
- J. Noritake, M. Fukata, K. Sato et al., “Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact,” Molecular Biology of the Cell, vol. 15, no. 3, pp. 1065–1076, 2004.
- M. Fukata and K. Kaibuchi, “Rho-family GTPases in cadherin-mediated cell-cell adhesion,” Nature Reviews Molecular Cell Biology, vol. 2, no. 12, pp. 887–897, 2001.
- C. H. M. Castro, C. S. Shin, J. P. Stains et al., “Targeted expression of a dominant-negative N-cadherin in vivo delays peak bone mass and increases adipogenesis,” Journal of Cell Science, vol. 117, no. 13, pp. 2853–2864, 2004.
- S. L. Ferrari, K. Traianedes, M. Thorne et al., “Role for N-cadherin in the development of the differentiated osteoblastic phenotype,” Journal of Bone and Mineral Research, vol. 15, no. 2, pp. 198–208, 2000.
- A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, “Matrix elasticity directs stem cell lineage specification,” Cell, vol. 126, no. 4, pp. 677–689, 2006.
- T. P. Stein and C. E. Wade, “Metabolic consequences of muscle disuse atrophy,” The Journal of Nutrition, vol. 135, no. 7, pp. 1824S–1828S, 2005.
- B. Nusgens, G. Chometon, A. Guignandon et al., “Role of the RhoGTPases in the cellular receptivity and reactivity to mechanical signals including microgravity,” Journal of Gravitational Physiology, vol. 12, no. 1, pp. 269–270, 2005.
- Z.-G. Zhang, C. A. Lambert, S. Servotte et al., “Effects of constitutively active GTPases on fibroblast behavior,” Cellular and Molecular Life Sciences, vol. 63, no. 1, pp. 82–91, 2006.
- R. McBeath, D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S. Chen, “Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment,” Developmental Cell, vol. 6, no. 4, pp. 483–495, 2004.
- Y.-R. V. Shih, K.-F. Tseng, H.-Y. Lai, C.-H. Lin, and O. K. Lee, “Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells,” Journal of Bone and Mineral Research, vol. 26, no. 4, pp. 730–738, 2011.
- D. L. Long, J. S. Willey, and R. F. Loeser, “Rac1 is required for matrix metalloproteinase 13 production by chondrocytes in response to fibronectin fragments,” Arthritis and Rheumatism, vol. 65, no. 6, pp. 1561–1568, 2013.
- D. C. Radisky, D. D. Levy, L. E. Littlepage et al., “Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability,” Nature, vol. 436, no. 7047, pp. 123–127, 2005.
- J. Settleman, “Tension precedes commitment—even for a stem cell,” Molecular Cell, vol. 14, no. 2, pp. 148–150, 2004.
- X. Yao, R. Peng, and J. Ding, “Effects of aspect ratios of stem cells on lineage commitments with and without induction media,” Biomaterials, vol. 34, no. 4, pp. 930–939, 2013.
- L. Gao, R. McBeath, and C. S. Chen, “Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin,” Stem Cells, vol. 28, no. 3, pp. 564–572, 2010.
- R. Sordella, W. Jiang, G.-C. Chen, M. Curto, and J. Settleman, “Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis,” Cell, vol. 113, no. 2, pp. 147–158, 2003.
- S. Huang, C. S. Chen, and D. E. Ingber, “Control of cyclin D1, p27Kip1, and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension,” Molecular Biology of the Cell, vol. 9, no. 11, pp. 3179–3193, 1998.
- S. Huang and D. E. Ingber, “The structural and mechanical complexity of cell-growth control,” Nature Cell Biology, vol. 1, no. 5, pp. E131–E138, 1999.
- M. F. Olson, A. Ashworth, and A. Hall, “An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1,” Science, vol. 269, no. 5228, pp. 1270–1272, 1995.
- M. Yamamoto, N. Marui, T. Sakai et al., “ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle,” Oncogene, vol. 8, no. 6, pp. 1449–1455, 1993.
- D. Joyce, B. Bouzahzah, M. Fu et al., “Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-κB-dependent pathway,” The Journal of Biological Chemistry, vol. 274, no. 36, pp. 25245–25249, 1999.
- J. K. Westwick, Q. T. Lambert, G. J. Clark et al., “Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways,” Molecular & Cellular Biology, vol. 17, no. 3, pp. 1324–1335, 1997.
- M. L. Coleman and C. J. Marshall, “A family outing: small GTPases cyclin' through G1,” Nature Cell Biology, vol. 3, no. 11, pp. E250–E251, 2001.
- A. J. Ridley, “Cyclin' round the cell with Rac,” Developmental Cell, vol. 1, no. 2, pp. 160–161, 2001.
- J. Rosenblatt, L. P. Cramer, B. Baum, and K. M. McGee, “Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly,” Cell, vol. 117, no. 3, pp. 361–372, 2004.
- M. Glotzer, “Animal cell cytokinesis,” Annual Review of Cell and Developmental Biology, vol. 17, pp. 351–386, 2001.
- S. B. Ting, E. Deneault, K. Hope et al., “Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2,” Blood, vol. 119, no. 11, pp. 2510–2522, 2012.
- R. Nusse and H. Varmus, “Three decades of Wnts: a personal perspective on how a scientific field developed,” The EMBO Journal, vol. 31, no. 12, pp. 2670–2684, 2012.
- L. Mellor, T. Bake, M. Hiremath, E. G. Loboa, and J. T. Oxford, “Simulated microgravity affects Wnt signaling in articular cartilage: possible implications for crosstalk between cartilage and subchondral bone,” in Proceedings of the 2014 NASA Human Research Program Investigators' Workshop, Galveston, Tex, USA, February 2014.
- M. Capulli, A. Rufo, A. Teti, and N. Rucci, “Global transcriptome analysis in mouse calvarial osteoblasts highlights sets of genes regulated by modeled microgravity and identifies A “mechanoresponsive osteoblast gene signature”,” Journal of Cellular Biochemistry, vol. 107, no. 2, pp. 240–252, 2009.
- Q. Wan, E. Cho, H. Yokota, and S. Na, “RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts,” Journal of Bone and Mineral Metabolism, vol. 31, no. 5, pp. 520–532, 2013.
- S. J. Rodda and A. P. McMahon, “Distinct roles for Hedgehog and caronical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors,” Development, vol. 133, no. 16, pp. 3231–3244, 2006.
- J.-B. Kim, P. Leucht, K. Lam et al., “Bone regeneration is regulated by Wnt signaling,” Journal of Bone and Mineral Research, vol. 22, no. 12, pp. 1913–1923, 2007.
- D. Baksh and R. S. Tuan, “Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem cells,” Journal of Cellular Physiology, vol. 212, no. 3, pp. 817–826, 2007.
- E. J. Arnsdorf, P. Tummala, and C. R. Jacobs, “Non-canonical Wnt signalling and N-cadherin related β-catenin signalling play a role in mechanically induced osteogenic cell fate,” PLoS ONE, vol. 4, no. 4, Article ID e5388, 2009.
- N. Case, M. Ma, B. Sen, Z. Xie, T. S. Gross, and J. Rubin, “β-Catenin levels influence rapid mechanical responses in osteoblasts,” The Journal of Biological Chemistry, vol. 283, no. 43, pp. 29196–29205, 2008.
- A. Santos, A. D. Bakker, J. M. A. De Blieck-Hogervorst, and J. Klein-Nulend, “WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase Rock,” Cytotherapy, vol. 12, no. 7, pp. 924–932, 2010.
- J. Rossol-Allison, L. N. Stemmle, K. I. Swenson-Fields et al., “Rho GTPase activity modulates Wnt3a/β-catenin signaling,” Cellular Signalling, vol. 21, no. 11, pp. 1559–1568, 2009.
- L. Peng, Y. Li, K. Shusterman, M. Kuehl, and C. W. Gibson, “Wnt-RhoA signaling is involved in dental enamel development,” European Journal of Oral Sciences, vol. 119, supplement S1, pp. 41–49, 2011.
- M.-H. Kim, M. Kino-oka, N. Maruyama, A. Saito, Y. Sawa, and M. Taya, “Cardiomyogenic induction of human mesenchymal stem cells by altered Rho family GTPase expression on dendrimer-immobilized surface with d-glucose display,” Biomaterials, vol. 31, no. 30, pp. 7666–7677, 2010.
- D. Diekmann, A. Abo, C. Johnston, A. W. Segal, and A. Hall, “Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity,” Science, vol. 265, no. 5171, pp. 531–533, 1994.
- J. D. Lambeth, “Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases,” Current Opinion in Hematology, vol. 9, no. 1, pp. 11–17, 2002.
- R. Takeya and H. Sumimoto, “Molecular mechanism for activation of superoxide-producing NADPH oxidases,” Molecules and Cells, vol. 16, no. 3, pp. 271–277, 2003.
- M. Almeida, E. Ambrogini, L. Han, S. C. Manolagas, and R. L. Jilka, “Increased lipid oxidation causes oxidative stress, increased peroxisome proliferator-activated receptor-γ expression, and diminished pro-osteogenic Wnt signaling in the skeleton,” The Journal of Biological Chemistry, vol. 284, no. 40, pp. 27438–27448, 2009.
- C.-L. Kao, L.-K. Tai, S.-H. Chiou et al., “Resveratrol promotes osteogenic differentiation and protects against dexamethasone damage in murine induced pluripotent stem cells,” Stem Cells and Development, vol. 19, no. 2, pp. 247–257, 2010.
- S. W. Lane, S. de Vita, K. A. Alexander et al., “Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development,” Blood, vol. 119, no. 3, pp. 736–744, 2012.
- M.-K. Kwak, K. Itoh, M. Yamamoto, T. R. Sutter, and T. W. Kensler, “Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione,” Molecular Medicine, vol. 7, no. 2, pp. 135–145, 2001.
- M. K. Cho, W. D. Kim, S. H. Ki et al., “Role of Gα12 and Gα13 as novel switches for the activity of Nrf2, a key antioxidative transcription factor,” Molecular & Cellular Biology, vol. 27, no. 17, pp. 6195–6208, 2007.
- M. Daugaard, R. Nitsch, B. Razaghi et al., “Hace1 controls ROS generation of vertebrate Rac1-dependent NADPH oxidase complexes,” Nature Communications, vol. 4, article 2180, 2013.
- A. Y. Chi, G. B. Waypa, P. T. Mungai, and P. T. Schumacker, “Prolonged hypoxia increases ros signaling and RhoA activation in pulmonary artery smooth muscle and endothelial cells,” Antioxidants and Redox Signaling, vol. 12, no. 5, pp. 603–610, 2010.
- D. Kondrikov, R. B. Caldwell, Z. Dong, and Y. Su, “Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis,” Free Radical Biology and Medicine, vol. 50, no. 11, pp. 1689–1698, 2011.
- A. S. Nimnual, L. J. Taylor, and D. Bar-Sagi, “Redox-dependent downregulation of Rho by Rac,” Nature Cell Biology, vol. 5, no. 3, pp. 236–241, 2003.
- S. Versari, G. Longinotti, L. Barenghi, J. A. M. Maier, and S. Bradamante, “The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment,” The FASEB Journal, vol. 27, no. 11, pp. 4466–4475, 2013.
- T. P. Stein, “Space flight and oxidative stress,” Nutrition, vol. 18, no. 10, pp. 867–871, 2002.
- I. Testard, M. Ricoul, F. Hoffschir et al., “Radiation-induced chromosome damage in astronauts' lymphocytes,” International Journal of Radiation Biology, vol. 70, no. 4, pp. 403–411, 1996.
- M. Fukata, M. Nakagawa, and K. Kaibuchi, “Roles of Rho-family GTPases in cell polarisation and directional migration,” Current Opinion in Cell Biology, vol. 15, no. 5, pp. 590–597, 2003.
- C. D. Lawson and K. Burridge, “The on-off relationship of Rho and Rac during integrin-mediated adhesion and cell migration,” Small GTPases, vol. 5, no. 1, Article ID e27958, 2014.
- T. Mitsuhara, M. Takeda, S. Yamaguchi et al., “Simulated microgravity facilitates cell migration and neuroprotection after bone marrow stromal cell transplantation in spinal cord injury,” Stem Cell Research and Therapy, vol. 4, no. 2, article 35, 2013.
- P. A. Plett, R. Abonour, S. M. Frankovitz, and C. M. Orschell, “Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells,” Experimental Hematology, vol. 32, no. 8, pp. 773–781, 2004.
- A. Espinosa-Jeffrey, P. M. Paez, V. T. Cheli, V. Spreuer, I. Wanner, and J. De Vellis, “Impact of simulated microgravity on oligodendrocyte development: implications for central nervous system repair,” PLoS ONE, vol. 8, no. 12, Article ID e76963, 2013.
- K. Burridge and K. Wennerberg, “Rho and Rac take center stage,” Cell, vol. 116, no. 2, pp. 167–179, 2004.
- A. Higashibata, M. Imamizo-Sato, M. Seki, T. Yamazaki, and N. Ishioka, “Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation—molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor,” BMC Biochemistry, vol. 7, article 19, 2006.
- X. Zhang, Y. Nan, H. Wang et al., “Model microgravity enhances endothelium differentiation of mesenchymal stem cells,” Naturwissenschaften, vol. 100, no. 2, pp. 125–133, 2013.
- K. Hamamura, G. Swarnkar, N. Tanjung et al., “RhoA-mediated signaling in mechanotransduction of osteoblasts,” Connective Tissue Research, vol. 53, no. 5, pp. 398–406, 2012.
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