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 Pdf file on cDNA microarray reveals the alterations of cytoskeleton-related
genes in osteoblast under high magneto-gravitational
environment
Airong Qian, Shengmeng Di,
Xiang Gao, Wei Zhang, Zongcheng
Tian, Jingbao Li, Lifang Hu, Pengfei
Yang, Dachuan Yin, and Peng
Shang*
Key Laboratory for Space Bioscience and Biotechnology, Faculty of Life
Sciences, Institute of Special Environmental Biophysics, Northwestern Polytechnical University, Xi’an 710072, China
*Correspondence address. Tel: +86-29-88460391;
Fax: +86-29-88491671; E-mail: [email protected]
The diamagnetic levitation as a novel ground-based model
for simulating a reduced gravity environment has been widely applied in many
fields. In this study, a special designed superconducting magnet, which can
produce three apparent gravity levels (0, 1, and
Keywords superconducting magnet;
high magnetogravitational environment; simulated weightlessness; cytoskeleton;
microarray
Received: November 27, 2008 Accepted: March 18, 2009
Introduction
Bone loss caused by microgravity is one of the most common and serious
health problems that the astronauts face in space environment [1,2]. Studies on
spaceflights lasting more than 12 months have shown that due to microgravity,
astronauts on long missions may lose as much as 20% of their bone mass [3].
However, the mechanism of bone loss induced by microgravity is still not clear.
It has been hypothesized that gravity affects cells via three ways, including
the roles of cell organelle or molecules as being the cell gravity receptors,
adaptation response caused by physical or chemical reaction surrounding cells,
and the theory of bifurcations. Most researches are prone to the theory of
bifurcations, namely the direct vs. indirect effects at the cellular level
[4,5]. Mesland [6] has predicted that cells are a
nonlinear dynamical system and minute gravity will dramatically affect cell
behaviors when particles in cells are in the state of the gravity
sensitive-window. Thus, nonlinear state transitions (i.e. bifurcations) at the
molecular level might function to amplify relatively weak gravity signals at
the cellular level and the amplification methods may be through cytoskeleton
system and intercellular network system.
Cytoskeletons consist of three basic types of filaments and associated
protein molecules arranged into chains, maintain cells shape, help cells move,
and hold the nucleus in place. The cytoskeleton, as the load-bearing architecture
of the cell, plays a role in mechanotransduction [7].
Cytoskeletons have tensegrity to balance compression
with tension, and yield to forces without breaking [8]. National Aeronautics
and Space Administration (NASA) is interested in cytoskeletons because
cytoskeletons respond to gravity [9]. But what happens when gravity vanishes?
How do cells replicate and maintain their genomes, including the regulation of
their proliferative capacity and survival? Studies reported that changes in
cell shape and function were observed in response to changed gravity [10,11].
Our previous results also showed that simulated weightlessness by means of
diamagnetic levitation markedly affected the cytoskeleton alteration of osteoblast (A.R. Qian,
unpublished data).
Due to the cost expense and the limited opportunities for spaceflight
experiment, it is very necessary to develop ground-based models. Diamagnetic
levitation technology is a novel-simulated weightless technique and has
recently been applied in life science research. Although at present the studies
on the biological effects of high magneto-gravitational environment (HMGE)
produced by superconducting magnet with large gradient have just started, the
rapid-developing trends have been highlighted. Many studies have utilized this
technique, such as those on frogs, frog embryos, and cell cultures of plants. Valles et al. [12] have carried out the magnetic levitation-based Martian and Lunar
gravity simulator with the support from NASA. Brooks et al. [13] have reported that the leaves of
transgenic plants produce resonant-type stress response in strong magnetic
fields and in magnetic levitation (low gravity) environments (17 T, B , 25 T) but null
response in roots. It has been reported that the magnetic levitation inhibits microtubule
self-organization, which is consistent with the results reported in spacecraft
[14]. Hammer et al. [15] have reported that magnetic levitation of MC3T3 osteoblast
cells can be taken as a ground-based simulation of microgravity. These findings
indicated that magnetic levitation can be used as a new ground-based model for
simulating weightless environment. In this study, a superconducting magnet
(JMTA-16 T 50 MF) that can provide large gradient high magnetic field [B.(dB/ dz)
_ –1500 T2/m _ 1100 T2/m] was taken as a model to investigate its effects on gene expression
profile of osteoblast. In this inhomogeneous magnetic
field, a repulsive force will act on diamagnetic materials (cells) so that
different apparent gravities (0, 1, and
Materials and Methods
Cell cultures
The human osteoblast-like cell line MG-63 was
purchased from the Cell Collection Center of Shanghai (
Total RNA isolation and microarray preparation
Total RNA was isolated from MG-63 cells exposed to HMGE and controls for
24 h using Trizol method as recommended by the
manufacturer’s
protocol (Invitrogen,
Microarray data analysis
A powerful computer workstation loaded with a GeneChip
Operating Software (GCOS) was used to analyze the scanned image and to obtain
scaled quantitative information. Comparison analysis was used to analyze the
differences in gene expression profile in MG-63 cells exposed to 0, 1,
Quantitative real-time PCR analysis
After MG-63 cells were cultured in HGME for 24 h, total RNA was extracted
using the Trizol method (Invitrogen).
RNA was reverse transcribed and processed for PCRs
according to the protocol provided with the kits (TaKaRa,
SYBR Green real-time relative quantitative RT–PCR analysis was performed with SYBR Premix Ex TaqTM according to the manufacturer’s protocol (TaKaRa) on an MJ Research (Bio-Rad, In this study, the 2–DDCt method
of relative quantification was used to estimate relative changes in
cytoskeleton-associated gene expression in MG-63 cells exposed to HMGE. The DDCt calculation for the
relative quantification of the cytoskeleton-associated gene was used as
follows:
Eq.
where exp and cont are the experimental and control groups, respectively.
Each sample was performed in quadruplex wells, and
the Ct of each well was recorded at the end of the reaction. The mean and
standard deviation (SD) of the four Cts were
calculated. The changes in cytoskeleton gene expression, normalized to 18S
under HMGE conditions, were calculated according to the following equation:
amount of target _
2–DDCt [19].
Western blot analysis
After MG-63 cells were cultured in HMGE for 24 h, the cells were collected
and re-suspended in lysis buffer. The lysis solution was centrifuged at
Statistical analysis
Statistically significant differences were determined by Prism statistical
software (GraphPad Software Inc.,
Results
Effects of HMGE on gene expression profiles From the scatter graph
of expression genes in MG-63 cells exposed to HMGE (Fig. 1), the hybridization effect of the whole
microarray was very good and came up to the requirements of gene expression
analysis. Common genes significantly expressed (red) were in the majority and
there were also some indistinctively or uncertainly expressed genes (yellow).
Differentially expressed genes (blue) are clearly shown in Fig. 1. The eight green
diagonals indicated 2-, 3-, 10-, and 30-fold changes of expression,
respectively, between two samples. By performing microarray studies, we were
able to analyze the global changes in the gene expression profiles of MG-63
cells exposed to HMGE. SLR _1 or SLR
_ –1 was a significant dividing
value to analyze the expression profile, which indicates difference between two
groups more than two times. Among 54,613 gene probes examined with the
microarray, only 572 genes were down-regulated and 415 genes were up-regulated
to statistically significant levels .2 folds
in
Effects of HMGE on cytoskeleton-associated gene expression
The cytoskeleton, as the load-bearing architecture of the cell, is very
sensitive to altered gravitational forces and disruption of the cytoskeleton
will result in the alteration of cellular structure and function [1,2].
Microarray results showed that the expression of SPTBN1 (spectrin, b, non-erythrocytic 1) and supervillin genes under Thirteen cytoskeleton-associated genes sensitive to HMGE were selected
from microarray data and SYBR Green-based real-time PCR was used to verify the
effects of HMGE on cytoskeleton-associated gene expression at mRNA levels.
After being normalized by internal control genes, the relative gene expression
levels in experimental groups were obtained comparing with those of control
groups. And then, the differences in gene expression between
Effects of HMGE on cytoskeleton-associated protein
expression
In order to screen the cytoskeleton-related genes sensitive to gravity, paxillin, WASF2, WIPF1, and talin
1 were selected on the basis of the results of microarray and real-time PCR and
were further verified by western blot assay at protein level (Fig. 3). The western blot
results showed that WASF2 and paxillin expressions
were significantly decreased in
Discussion
The cytoskeleton-related genes in osteoblast
that are sensitive to HMGE have been identified by cDNA
microarray for the first time in this study. The novel and most significant
finding of this study is that exposure of osteoblasts
to HMGE (0, 1, and Microgravity/weightlessness-induced bone loss in humans and animals has
been reported to be mediated at least in part by decreased osteoblast
function [20]. In evaluating expressions of .40,000 human genes in MG-63 cells exposed to HMGE at 24 h and
corresponding ground controls, we found some differential genes on the basis of
typical cell functions, such as proliferation, cell adhesion, apoptosis, cell
cycle, cell communication, response to stress, and response to external
stimulus. These results suggest that the characteristics of MG-63 cells may be
altered by HMGE and MG-63 cells themselves may be sensitive to altered gravity
levels and magnetic field levels. Our previous work showed that HMGE affected osteoblast-like cell MG-63 morphology, adhesion,
proliferation, and secretion [16]. Alterations in the morphology, cytoskeleton,
and gene expression for growth factors and matrix proteins are observed in osteoblastic cells in vitro under microgravity conditions [20]. Pardo et al. [21] have reported that simulated microgravity using the random
positioning machine inhibits differentiation and alters gene expression
profiles of 2T3 preosteoblasts. It has been reported
that diamagnetic levitation changes growth, cell cycle, and gene expression of Saccharomyces cerevisiae [22,23].
It has also been reported that the cytoskeleton is the sensor of gravity
in cells and very sensitive to gravity alteration and that microtubule
self-organization is particularly dependent on gravity [24]. Mechanotransduction may be mediated at multiple locations
inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton [7]. The effects of
weightlessness on microtubule self-organization can be studied using
ground-based equipment and the findings closely resemble that of the
spaceflight experiment [25]. Alenghat and Ingber [26] have reported that all signals point to
cytoskeleton, matrix, and integrins. We therefore
wonder what will happen when gravity disappears or diminishes and what changes
in gene expression profile will occur when the cytoskeleton relaxes. cDNA microarray revealed that the expression of 13
cytoskeleton-related genes (adducin 3, coactosin-like 1, filamin A, SORBS3, CDC42BPB, tropomodulin 3, talin 1, SPTBN1, supervillin, WASF2, WIPF1, plectin 1, and paxillin) was significantly altered by HMGE. The expression of 13 cytoskeleton-related
genes was all up-regulated in diamagnetic levitation (Both of PCR and microarray analysis showed that WASF2 and WIPF1 gene expressions were up-regulated in PCR analysis of talin 1 and SORBS3 under high magnetic field Based on the data of the microarray and real-time PCR, we performed western
blot assay to examine the expression levels of four selected proteins (talin 1, paxillin, WASF2, and
WIPF1) with specific antibodies. Accordant with PCR and microarray analysis,
western blot analysis showed that WIPF1 expression were significantly increased
in
Acknowledgements
We would like to thank Prof. Jize Shi in
Funding
This work was supported by grants from the National Natural Science
Foundation of
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