Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Sep;28(9):4077-87.
doi: 10.1096/fj.14-249714. Epub 2014 Jun 5.

Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells

Affiliations

Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells

Alain Guignandon et al. FASEB J. 2014 Sep.

Abstract

Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects.

Keywords: Cdc42; MG-63; RhoA; VEGF; spaceflight.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Morphological description of MG-63 cells after exposure to microgravity. A, B) Left panels: 1 g conditions (ground-based control; solid arrow). Right panels: microgravity (μg) conditions (dotted arrow). Cells were processed for detection of FAs by using vinculin antibody (A) and of stress fibers by using rhodamine phalloidin (B) staining. SiScr corresponds to cells transfected with scrambled sequences of SiRNA (SiRhoA, Rac1, and Cdc42) of their respective specific siRNA. Images clearly show that with SiScr and SiRhoA, the cells presented an absence of large focal contacts when compared to the 1 g control cells. It is noteworthy that SiRac1 and SiCdc42 cells exposed to microgravity presented an adhesive profile similar to that of the controls. Stars indicate major cell detachment. Scale bars = 10 μm. C) A minimum of 30 cells extracted from a minimum of 10 fields were used for quantification of focal contact morphometry, as well as the number of stress fibers and their area. Open bars, ground-based control cells; solid bars, microgravity-exposed cells. Error bars represent sd (n = 30). *P < 0.01.
Figure 2.
Figure 2.
A) Images of fibronectin deposited by MG-63 cells after exposure to microgravity. Left panels: 1 g conditions (ground-based control; solid arrow). Right panels: microgravity (μg) conditions (dotted arrow). Cells were processed for detection of fibronectin by using a specific antibody. SiScr corresponds to cells transfected with scrambled sequences of SiRNA (SiRhoA, Rac1, and Cdc42) of their respective specific SiRNA. Scale bar = 10 μm. B) A minimum of 15 fields were used for the quantification of the fibronectin area. Open bars, ground-based control cells; solid bars: microgravity-exposed cells. Error bars represent sd (n = 30). *P < 0.01.
Figure 3.
Figure 3.
mRNA expression of VEGF variants under 1 g and microgravity (μg) conditions. Values are expressed as percentages of VEGF165 in SiScr cells (100%). Open bars, ground-based control cells; solid bars, microgravity-exposed cells. Error bars represent sd (n = 6). *P < 0.01.
Figure 4.
Figure 4.
A) Images of VEGF deposition by MG-63 cells after exposure to microgravity. Left panels: 1 g conditions (ground-based control; solid arrow). Right panels: microgravity (μg) conditions (dotted arrow). Cells were processed for detection of fibronectin by using specific antibody. SiScr corresponds to cells transfected with scrambled sequences of SiRNA (RhoA, Rac1, and Cdc42) of their respective specific siRNA. Scale bar = 10 μm. B) A minimum of 15 fields were used for quantification of the mVEGF area. Open bars, ground-based control cells; solid bars, microgravity-exposed cells. Error bars represent sd (n = 30). *P < 0.01.
Figure 5.
Figure 5.
Graphic summary. Under ground-based control (1 g) conditions, RhoA activity is permissive for stabilization of contacts and fibrillogenesis. In this condition, the produced VEGF can be immobilized in the matrix (Fbn), and it can be speculated that the RhoA activity is able to limit that of Rac1 when in cooperation with integrins. Since we have established that the silencing of Rac1 blocked the increased production of soluble VEGF induced in microgravity (μg) conditions, we can link the Rac1 activity with VEGF production. In 1 g controls, RhoA activity may prevail over that of Rac1, as cells cultivated on stiff substratum are known to possess high RhoA activity. In microgravity, we propose that the reduction of VEGF entrapment by limitation of Fbn deposition and increase in soluble isoform of VEGF limits the inhibitory effect of focal contacts on Rac1 activity. An imbalanced increase in Rac1 activity in microgravity may explain why Rac1 prevails over RhoA. In SiRac1 cells, we believe that the inability of silenced cells to increase Rac1 protects them from microgravity-induced effects.

Similar articles

Cited by

References

    1. Vico L., Collet P., Guignandon A., Lafage-Proust M. H., Thomas T., Rehaillia M., Alexandre C. (2000) Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts. Lancet 355, 1607–1611 - PubMed
    1. Carmeliet G., Bouillon R. (1999) The effect of microgravity on morphology and gene expression of osteoblasts in vitro. FASEB J. 13(Suppl.), S129–S134 - PubMed
    1. Burger E. H., Klein-Nulend J. (1998) Microgravity and bone cell mechanosensitivity. Bone 22, 127S–130S - PubMed
    1. Hughes-Fulford M. (2003) Function of the cytoskeleton in gravisensing during spaceflight. Adv. Space Res. 32, 1585–1593 - PubMed
    1. Nabavi N., Khandani A., Camirand A., Harrison R. E. (2011) Effects of microgravity on osteoclast bone resorption and osteoblast cytoskeletal organization and adhesion. Bone 49, 965–974 - PubMed

Publication types

MeSH terms