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(The FASEB Journal. 1999;13:S121-S127.)
© 1999
MILLIE HUGHES-FULFORD1
Laboratory of Cell Growth and Differentiation, Veteran's Affairs Medical Center and University of California Medical Center, San Francisco, 94121
1Correspondence: Millie Hughes-Fulford, Ph.D., Director, Laboratory of Cell Growth (151F), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. E-mail: milliehf@spacedu.com
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ABSTRACT |
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The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN)
mRNA levels, protein synthesis, and extracellular FN matrix accumulation after
growth activation in spaceflight. These osteoblasts produce FN extracellular
matrix (ECM) known to regulate adhesion, differentiation, and function in
adherent cells. Changes in bone ECM and osteoblast cell shape occur in
spaceflight. To determine whether altered FN matrix is a factor in causing
these changes in spaceflight, quiescent osteoblasts were launched into
microgravity and were then sera activated with and without a 1-gravity field.
Synthesis of FN mRNA, protein, and matrix were measured after activation in
microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G)
when compared to ground (GR) osteoblasts flown in a centrifuge simulating
earth's gravity (1-G) field 2.5 h after activation. However, 27.5
h after activation there were no significant differences in mRNA synthesis. A
small but significant reduction of FN protein was found in the 0-G
samples 2.5 h after activation. Total FN protein 27.5 h after activation showed
no significant difference between any of the gravity conditions, however,
there was a fourfold increase in absolute amount of protein synthesized during
the incubation. Using immunofluorescence, we found no significant differences
in the amount or in the orientation of the FN matrix after 27.5 h in
microgravity. These results demonstrate that FN is made by sera-activated
osteoblasts even during exposure to microgravity. These data also suggest that
after a total period of 43 h of spaceflight FN transcription, translation, or
altered matrix assembly is not responsible for the altered cell shape or
altered matrix formation of osteoblasts.—Hughes-Fulford, M., Gilbertson,
V. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged
after exposure to microgravity.
Key Words: growth activation • spaceflight • mRNA expression
| INTRODUCTION |
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THE UNIQUE ENVIRONMENT of spaceflight places unusual stress on,
and causes many physiological changes in, organisms that evolved in a 1-G
environment (1-3) . Some of the basic physiological changes include muscle
atrophy and loss of calcium and mineralized bone. The bone loss that
accompanies spaceflight is one of the most serious health hazards associated
with, and impediments to, long-term manned missions (4 , 5 ). Biomedical
studies of manned spaceflight have consistently indicated a continuous and
progressive loss of calcium and weight-bearing skeletal bone. Evidence from
animal studies suggests that the bone loss occurring in spaceflight is due to a
decrease in bone formation (6) . The decrease in bone formation and osteoblast
growth in microgravity is likely due to both indirect effects such as systemic
or hormonal changes in the body and direct effects such as cellular response to
the lack of mechanical stress.
Proper cell attachment to the extracellular matrix (ECM)2is required for normal cell growth (7) . Previous studies have demonstrated that bone exhibits altered ECM assembly of collagen (8-9) , actin (10) , and altered cell morphology and nuclear shape of osteoblasts (9-12) in microgravity. The cell surface adhesion protein, fibronectin (FN), mediates the attachment of a variety of cell types to other ECM components such as type I collagen, heparin, and proteoglycans. Cell binding to FN is mediated by integrins, which recognize the arginine-glycine-aspartic acid (RGD) sequence on the FN molecule. The integrins on the cytoplasmic surface bind talin and a-actinin, forming a link between ECM components and the cytoskeleton. Binding of FN can also be mediated by RGD-independent interactions such as a4ß1 and proteoglycan receptors including CD44 and syndecans (13) . FN is known to be a factor in cell shape and growth regulation (14 , 15 ). It has been demonstrated that FN adhesion, resulting in the ability of epithelial cells to spread over a planar surface, can promote cell cycle progression from G0 to S phase (16) . Although the mechanisms are still under investigation, FN has also been shown to increase mechanochemical signal transduction intermediates in fibroblasts (17) . FN is present during all stages of matrix-mediated bone formation with synthesis rates being high during cell proliferation and attachment (18) . In addition, blocking FN attachments has been shown to inhibit osteoblast differentiation and in mature bone cultures resulted in apoptosis (19) .
For these reasons we were interested in determining what effect microgravity would have on the FN message, protein, and matrix organization. To define the role of FN in the altered cell shape and altered ECM matrix formation we performed space shuttle experiments in Biorack hardware in the following gravity environments: ground (GR), 0-G flight (0-G), and 1-G flight (1-G) conditions. Early passage MC3T3-E1 osteoblasts were used because they have been shown to exhibit osteoblast behavior during both proliferation and differentiation stages of bone mineralization (20 , 21 ). We have demonstrated previously that these osteoblasts change cell shape in microgravity and that osteoblast genes are induced by application of gravity and vibration forces (22-24) . In this study, we asked whether FN plays an important role in microgravity-induced changes in cell shape and matrix formation. Therefore, analysis of FN gene induction, protein synthesis, and matrix formation was done on samples collected under different gravity conditions during spaceflight. Our results demonstrate that extended exposure (>24 h) to microgravity does not cause changes in FN synthesis and matrix.
| MATERIALS AND METHODS |
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Cell culture
The MC3T3-E1 osteoblast cell line is clonally derived from embryonic mouse
calvaria (17 , 18 ) and was kindly provided to us by Dr. M. Kumegawa (Josai
Dental University, Japan). The cell line was maintained at low passage. Cells
were grown in alpha minimal essential medium (a-MEM) with 10% fetal calf serum
(Hyclone Labs Inc., Logan, UT) supplemented with 2 mM L-glutamine
(Sigma, St. Louis, MO), 25 mM HEPES, and antibiotic-antimycotic solution (100
U penicillin/ml, 0.01 mg streptomycin/ml, 0.25 mg Amphotericin B/ml). Cells
were grown in a 37°C incubator with 5% CO2. They were fed three
times a week and passed when the cells reached confluence. For flight
experimental samples, 120,000–200,000 cells were plated onto non-coated,
sterile 11 x 22-mm glass coverslips (Thomas
Scientific, Swedesboro, NJ) placed in six-well plates, and grown in 10% serum
containing a-MEM overnight. Cell-coated coverslips were transferred into the
plungerbox units in 2% serum-containing medium for flight. In cooperation with
NASA flight rules, the plungerbox units were held for 17 h in the shuttle
at mid-deck temperature before launch. This, combined with low serum-containing
media placed the osteoblast in a quiescent state before space shuttle launch.
Many previous culture experiments sent into space have been actively growing at
the time of launch. Moreover, lack of refrigeration has previously limited
on-board sample preservation and therefore many experiments were terminated
after landing on earth. Even for the few experiments that had the
opportunity for on-board collection, the data were compromised by
flight-imposed limitations including the following: lack of sufficient sample
numbers, addition of supplements directly affecting gene expression, lack of
fresh media changes, and lack of on-board 1-G controls. To achieve our
goal of studying FN metabolism in microgravity, we launched cells that were in
a quiescent condition and were not sera activated until on-orbit in the
microgravity environment. There were four samples for each time point in 0-
and 1-G flight samples and ground samples. Refrigerator and freezer
space was available for reliable sample storage after collection in
microgravity. In contrast to some previous spaceflight experiments, media
supplements such as dexamethasone, ß-glycerol phosphate, and ascorbic acid
were not added to our media because these agents are known to directly affect
gene expression and cell morphology of the osteoblast.
Biorack facility and osteo hardware
Biorack is a multi-user facility that consists of incubators with variable
gravity centrifuges, a cooler, a freezer, and a sealed glovebox. Two identical
Biorack modules were used: one remained on earth and the other was integrated
into SpaceHab and flown on the space shuttle. Biorack has an important
advantage over other microgravity facilities in that it provides a small-radius
(78 mm) slow-rotating (107.0 ± 0.5 rpm) centrifuge. The centrifugal force
results in a 1-G on-board control. Because of proximity, both the 0-G
and 1-G samples experience identical launch vibrations, accelerations,
cosmic radiation, and other unknown conditions of flight. The only difference
between the 0-G and 1-G flight groups is the gravity parameter.
In addition, an identical experiment was performed in the Biorack module on
earth with a 2-h delay from in-flight procedures.
The "Osteo" experimental flight hardware was designed according to European Space Agency specifications for use in the Biorack facility and constructed by Centrum voor Constructie Mechatronica (CCM, Neuenen, The Netherlands). The hardware consisted of the CCM plunger box and its Type I container developed for spaceflight cell culture. The Type I container provided a second level of fluid containment. The plunger boxes were designed to provide a sterile environment for cell growth activation and fixation in a microgravity condition. The plunger box is composed of two independent culture chambers that each hold two 11 x 22-mm glass coverslips. Each culture chamber was a separate sample. For each condition a sample size of n = 4 was used. Each culture chamber has series of compartments filled by either 10% serum-containing a-MEM or fixative, which can be exchanged into and out of the cell culture compartment by manually releasing a spring-loaded plunger.
Experimental time line
Quiescent cells were stored in the mid-deck locker until 18 h after launch. The
astronauts then transferred the samples into a 37°C incubator (either in the
1-g centrifuge or 0-g static rack) for 1 h before stimulating the
cells to grow by changing their media from 2 to 10% serum-containing medium (t
= 0). Zero-G and 1-G samples were fixed during flight with a modified
guanidinium thiocyanate solution (for RNA preservation) t = 0, 2.5 h,
and 27.5 h or 3.7% formaldehyde solution (for morphology) at 27.5 h. Samples
fixed with the guanidinium thiocyanate solution or the formaldehyde solution
were stored at -20°C or +5°C, respectively, for the remainder of the space
shuttle mission. There were four separate samples for each gravity condition
at each time point.
RNA isolation, reverse transcription (RT), and polymerase chain reaction (PCR)
RNA from cultured MC3T3-E1 osteoblast cells was extracted by a modified
guanidinium thiocyanate method, which was based on the protocol previously
described by Chomczynski and Sacchi (25) . Linear RT-PCR was performed as
previously described (9 , 19 , 20 , 23-26 ). Because sample size was so small
(200,000 cells) RNA content was held constant and linear RT-PCR was
accomplished by varying the number of PCR cycles. PCR conditions were
established so that the amplification reaction was stopped in the linear range
and reaction products could be accurately quantified and compared.
Oligonucleotide primers were designed to span at least one intron in order to
detect any contaminating genomic DNA. FN primer sequences were designed from
Genebank sequences by MHF: sense, 5'-CCC CAG TGA TGT TAG CAG ACC; anti-sense,
5'-GAT GGC AAA AGA AAG CAG AGG. Operon Technologies, Inc. (Alameda, CA)
synthesized the oligonucleotides. PCR bands were identified by agarose gel
electrophoresis and photographed with a direct screen instant camera DS-34
(Polaroid Corp., Cambridge, MA). For quantification, the bands were scanned at
400 dpi with a LaCie Silver Scanner III (LaCie, Portland, OR) and stored as
TIFF files. The area and density of each band were determined using Sigma
Gel (SPSS, Inc., Chicago, IL). The total number of pixels was corrected to the
micrograms of RNA used for each RT-PCR. Sample size for all conditions was n
= 4. Descriptive statistics and unpaired Student's t test analysis was
performed with Sigma Stat v. 2.0 (SPSS, Inc.).
Protein isolation
Protein remaining in the guanidinium thiocyanate solution after the RNA
extraction was precipitated out of each sample using a method similar to that
previously described (27) . Briefly, 2.5 ml of isopropanol was mixed with the
remaining organic phase from the RNA extraction, incubated at room temperature
for 15 min, and centrifuged at 12,000 g for 10 min at 4°C. The
resulting protein pellet was washed three times in 4 ml of 0.3 M guanidine
hydrochloride in 95% ethanol. For each wash samples were incubated in the wash
solution at room temperature for 20 min followed by centrifugation. Protein was
resuspended in 2% sodium dodecyl sulfate (SDS). Total protein was quantified
for each sample in triplicate by DC Protein Assay (Bio-Rad Laboratories,
Hercules, CA) according to the manufacturer's protocol and read on a Dynatech
MR5000 96-well plate reader. The standard curve and protein quantification
results were interpreted with BioLinx 2.0 software (Dynatech Laboratories,
Chantilly, VA).
Western blots
Five micrograms of total protein for each sample were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5% polyacrylamide).
Two molecular weight markers were also loaded onto the gel: a prestained
molecular weight marker (Bio-Rad) was used to track the migration of the
samples through the gel and a Cruz MarkerTM (Santa Cruz
Biotechnology, Santa Cruz, CA), which is compatible with the horseradish
peroxidase-conjugated secondary antibody and can be visualized by
chemiluminescence, and was recorded directly onto the film. Proteins were
transferred onto nitrocellulose filters with 100 volts for 1 h. No detectable
protein, as measured by staining, remained in the gel after transfer. Filters
were blocked with 5% blotto (20 mM Tris, 150 mM NaCl, pH 7.6, with 5% nonfat
milk and 0.02% Tween-20) for 2 h. Primary antibodies for FN were purchased
from DAKO (Carpinteria, CA). Molecular weight markers and secondary antibodies
were purchased from Santa Cruz Biotechnology. Proteins were visualized by
enhanced chemiluminescence (Amersham Life Sciences Inc., Arlington Heights, IL)
and recorded on Kodak XAR-5 film. To ensure that the Western blot signal was
within a linear range, the amount of protein loaded on the gel, primary and
secondary antibody concentrations, and film exposure length were all optimized.
Films were digitized with an Epson Expression 836XL scanner into Adobe
Photoshop (Adobe Systems Inc., Mountain View, CA). Density analysis was
performed with the Sigma Gel program (SPSS, Inc.). All densitometry readings
were normalized to nanograms of total protein loaded onto the gel. Sample size
for each condition was n = 4. Statistical analysis was performed in
Sigma Stat and graphed with Sigma Plot (SPSS, Inc.).
Cell morphology
Osteoblasts were fixed on coverslips with 3.7% formaldehyde in
phosphate-buffered saline (PBS) during flight and stored at 5°C for 1–2
weeks before staining. There was no visible difference between control samples
that were immediately stained and samples stored in formaldehyde at 5°C. For
FN visualization, coverslips were blocked with 10% goat serum in PBS,
incubated with anti-FN (rabbit) primary antibody (DAKO), and then incubated
with anti-rabbit secondary labeled with Texas Red. Dried osteoblast coverslips
were mounted onto slides and photographed with a Zeiss Axioscope microscope at x40 and x100 magnification.
Slides were processed at the same time under identical conditions. Photographs
were taken at identical exposure times and conditions. Photographs were taken
at pre-determined areas of the samples. Slides were digitized with an Epson
Expression 836XL scanner into Adobe Photoshop for computer analysis with NIH
image. Controls not shown were incubated with protein blocker, in the absence
of FN antibody, and then with the secondary antibody for the appropriate
incubation times.
| RESULTS |
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FN matrix
To compare the organization of the FN portion of the ECM under different gravity
conditions, osteoblast cells were launched on the space shuttle in a
semi-quiescent state and were sera activated 19 h after reaching orbit.
Twenty-seven and one-half hours after activation, the osteoblasts, which were
grown on untreated glass coverslips in microgravity (0-G) or in a 1-G
centrifuge on board the space shuttle (1-G) or ground controls (GR),
were fixed with formaldehyde for visualization of the FN matrix on conclusion
of the flight.
Samples were treated with a polyclonal antibody that selectively recognizes cellular FN to immunolocalize the FN matrix. The majority of the FN matrix had a diffuse distribution throughout the ECM. After approximately 1 day in microgravity there were no apparent changes in the FN matrix orientation between the 0-G and the 1-G or GR samples (Fig. 1 ). Quantification of total fluorescence from randomly selected slides revealed no significant differences among the different gravity conditions (Table 1 ). As an additional control, in samples without primary antibody, there was no FN matrix seen (image not shown).
|
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FN protein synthesis
The Western blot shown in Figure 2 shows representative samples of the
relative amount of FN per cell because proteins were extracted from samples
plated with an equal number of cells. Most of the immunoreactive material
migrated to the position of authentic FN, although some lower molecular mass
was also seen. In control blots performed without primary antibody present,
these bands were not seen (data not shown). At the 2.5 h timepoint there was
a small but significant reduction of FN protein in the 0-G sample when
compared to GR samples (Fig. 3 ).This difference was lost at the later
timepoint, where there was no significant difference in total FN protein
between the various gravity conditions. FN protein content is evident 27.5 h
after activation. FN protein is shown to have increased by approximately
fourfold during the 25 h between the two time points.
|
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FN mRNA
To determine the level of regulation at which FN synthesis is controlled, FN
mRNA synthesis was measured using RT-PCR (Fig. 4
).A specific band
corresponding to the expected FN PCR product was in high copy number relative
to most of the other genes analyzed. This increase was more pronounced at the
27.5 h time point where we reduced the total amount of RNA in the RT-PCR
reaction in order to remain in a linear amplification range. Final densitometry
quantification was corrected to micrograms of RNA so both the 2.5 h and the
27.5 h time points could be directly compared.
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As shown in Figure 5 ,2.5 h after initiation of osteoblast growth in microgravity, there was a small but significant change in the total FN mRNA synthesis between the 0-G and 1-G samples, with mRNA synthesis for FN in the 0-G samples lower than 1 G. There was a trend for the GR samples to be higher, but it was not significant due to outlying data points. In contrast, at the 27.5 h time point, there is no significant difference in FN mRNA synthesis between samples grown in microgravity and those grown in a 1-G environment. In addition, at this later time point (27.5 h) the FN message was over twofold higher than at the 2.5 h timepoint.
|
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DISCUSSION |
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The loss of bone in spaceflight is thought to be due to a lack of osteoblast
activity and possibly to a defect in morphological assembly of the ECM.
Previous work has demonstrated a cell shape change in osteoblasts exposed to
microgravity after 15 or more parabolic weightless events or as long as 4 days.
Change in cell shape is heterogeneous, with a portion of the cells having nuclear
elongation and a smaller area of contact with the surface (9 , 10 ).
Experiments on STS-56 demonstrated that osteoblast cells had a change in cell
shape after 4 days of weightlessness (9) and, more recently, experiments by Kacena et al.
(11) demonstrated a significant change in osteoblast cell shape in microgravity
as early as 3 h and as late as 3 days. None of these experiments had the
convenience of an on-board 1-G centrifuge. We have recently published
evidence (12) that nuclear morphology as well as the cell morphology was
altered in microgravity when compared to on on-board 1-G control in
these Biorack experiments. The changes in actin cytoskeleton and the nucleus
were heterologous in 0-G with a portion of the cells showing nuclear
elongation. In this study, in an attempt to explain the changed osteoblast
cell shape, we examined the ECM FN on the Biorack samples.
FN plays a crucial role in growth and differentiation of osteoblasts (28) and is crucial for normal growth and cell spreading (15) . We considered FN as a potential moiety for controlling the altered cell shape seen in microgravity. This study was undertaken to assess the role of FN in alterations of cell shape and cell matrix under microgravity conditions. By launching quiescent osteoblasts and activating and collecting them in microgravity, we were able to analyze gene activation and FN metabolism under microgravity conditions. In addition, for the first time, a total study of cell regulation of protein message, polypeptide synthesis, and immunolocalization of matrix was accomplished in spaceflight samples.
Because protein and RNA were extracted from the same samples, we were able to analyze both the transcriptional and translational control of FN. Although there was a slight and significant change in both messenger RNA and protein 2.5 h after sera activation, at later timepoints FN was not regulated differently in microgravity. We do not know why the earlier time points show significantly lower levels of both FN mRNA and protein synthesis but it is possible that a slow response to sera activation could be causing a lag in response since it is known that growth factor-induced signal transduction is sensitive to gravity (28) . The presence of de novo synthesis of FN by MC3T3-E1 osteoblast is supported by results that show an accumulation of both FN message (more than twofold) and protein (more than fourfold) during the 25-h incubation period between the early and late collection times. This finding is consistent with reports of others which show that FN protein, unlike other osteoblast matrix molecules, is synthesized throughout osteoblast growth and differentiation (29) . These data suggest that microgravity-induced cell morphology alterations are not caused by an aberration in the FN portion of the ECM. Extrapolation of our findings also suggests that changes in FN are not a causal factor in the loss of osteoblast growth in spaceflight. However, it is possible that FN binding and receptor-mediated signaling may be affected in microgravity and play a role in other altered responses.
The analysis of FN matrix from parallel samples fixed in flight for immunofluorescence localization studies confirmed the Western blot analysis showing that FN protein synthesis was not affected by microgravity. In addition, these data reveal that FN protein had been synthesized and exported out of the cells. The results also demonstrate that typical organization of the matrix was occurring. Finally, our data shows that 1 day after sera activation in 0-G, the samples have an overall regulation of FN that is indistinguishable from 1-G on-board controls or ground controls. Taken together, it is likely that changes in bone matrix formation, cell shape, and loss of bone growth previously observed in microgravity are not dependent on changes in FN regulation.
| ACKNOWLEDGMENTS |
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This work was supported by NASA Grant NAG-2-1086 and the Department of Veteran's
Affairs. We thank Kimberly Gasuad for preparation of the fibronectin
immunofluorescence and Wayne Sezto and Tephillah Chi for their technical
assistance at Hanger L. We thank the space shuttle flight crew from STS-84,
especially cosmonaut/astronaut Elena V. Kondakova for her excellent execution
of the experiment in flight. We appreciate the support of the Bionetics crew at
Kennedy Space Center, the NASA team, and the ESA Biorack team.
| FOOTNOTES |
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2 Abbreviations: FN, fibronectin; ECM, extracellular
matrix; RGD, arginine-glycine-aspartic acid; GR, ground; a-MEM, a-minimal
essential medium; RT-PCR, reverse transcriptase-polymerase chain reaction;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS,
phosphate-buffered saline.
Received for publication September 16, 1998. Revision received February 8, 1999.
| REFERENCES |
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