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* INSERM U311, Etablissement de Transfusion Sanguine, 67065 Strasbourg;
INSERM U151, CHU Rangueil, 31054 Toulouse;
Wilson Hall, University of Alabama in Huntsville, Alabama 35899, USA; and
§ Laboratoire D'Immunologie, CHU Rangueil, 31054 Toulouse Cedex, France
1Correspondence: INSERM U311, Etablissement de Transfusion Sanguine, 67065 Strasbourg Cedex, France. E-mail: jason.hatton{at}etss.u-strasbg.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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varied in inverse proportion to the
g level for some experimental treatments. In addition to
these novel findings, the results confirm earlier studies which showed
that PKC is sensitive to changes in gravitational acceleration. The
mechanisms of cellular gravisensitivity are poorly understood but the
demonstrated sensitivity of PKC to this stimulus provides us with a
useful means of measuring the effect of altered gravity levels on early
cell activation events.—Hatton, J. P., Gaubert, F., Lewis,
M. L., Darsel, Y., Ohlmann, P., Cazenave, J.-P., Schmitt, D. The
kinetics of translocation and cellular quantity of protein kinase C in
human leukocytes are modified during spaceflight.
Key Words: microgravity • translocation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
2
). In immune cells microgravity has been reported to
inhibit mitogen-induced proliferation (3)
, interleukin
secretion (4)
, and the cytotoxic effects of cytokines
(5)
. Events early in intracellular signal transduction
after mitogen-induced cell activation appear sensitive to microgravity.
The expression of c-fos and c-jun mRNA in the
human epidermal carcinoma line A431 in response to agonists that
stimulate protein kinase C
(PKC)2 during 6 min of
microgravity was significantly reduced compared to samples at 1
g, whereas stimulation of gene expression by agonists that
activate Ca2+- and cAMP-dependent signaling was insensitive
to changes in gravitational acceleration (6)
. Other
experiments suggest that PKC-mediated signal transduction is sensitive
to changes in the gravitational acceleration and that resulting changes
in cell function may be due to modifications occurring at or before PKC
involvement during cellular activation and differentiation (reviewed in
ref. 7
). However, the mechanisms by which the cell can perceive a
change in gravity remain enigmatic. An initial step toward elucidating
these mechanisms is the identification of parameters of cell function,
which vary in proportion to g level and determining which
are the earliest gravisensitive events in cell activation.
PKC is a family of serine/threonine kinases that are believed to play a
key role in the regulation of cellular proliferation and
differentiation. PKC is present in all eukaryotes (8)
and
is the target of phorbol esters that are potent activators of
proliferation and differentiation (9)
. The classical
cPKCs, which include PKC-
, -ßI, -ßII,
and -
require diacylglycerol, phosphatidylserine, and
Ca2+ for activation, whereas the novel nPKCs , 
, 
,
and 
are calcium independent (10)
. The requirement for
diacylglycerol can be replaced by the synthetic phorbol ester analogs
(9)
. The atypical isoforms 
and 
are insensitive to
both diacylglycerol and Ca2+. PKC isoforms generally
translocate from one cellular compartment to another after activation
(11)
. In U937 cells PKC-ßI,
-ßII, and - isoforms show unique patterns of
intracellular distribution in unstimulated U937 cells, as well as
distinct patterns of redistribution after stimulation with phorbol
esters (12)
. Although individual PKC isoforms show some
substrate specificity in in vitro assays (13)
,
it is believed that the particular intracellular distribution of
individual isoforms ensures a narrow substrate specificity and hence
the possibility of distinct signaling functions for each isoform
(11)
. The targeting of individual PKC isoforms before and
after activation is believed to be regulated by isoform-specific
anchoring proteins (14)
. Clearly, translocation plays a
key role in signal transduction by individual PKC isoforms.
We previously reported that the total quantity and subcellular
localization of PKC in two human leukemic cell lines appears to vary in
proportion to g level after phorbol ester stimulation
(15)
. In both Jurkat (a T lymphoblast line) and U937 (a
monocytic line) the total quantity of PKC was elevated in microgravity
compared to 1 g, whereas the proportion of PKC in the
nucleus/cytoskeleton was highest in microgravity but decreased with
increasing g level. Conversely, the quantity of PKC in the
cytosol increased in proportion to g level, most probably
due to a redistribution of PKC from the nucleus/cytoskeleton. However,
in these experiments it was not possible to determine how the kinetics
of PKC translocation after phorbol ester stimulation nor the quantity
of individual PKC isoforms was affected by a change in g
level. Therefore, we investigated the translocation kinetic of PKC in
U937 cells from the cytosol to the particulate fraction (membrane,
insoluble cytoskeleton, and nucleus) after phorbol ester stimulation
under microgravity, 1-g, and 1.4-g conditions
during a space shuttle flight. In addition, the total amount of phorbol
ester binding PKC, as well as selected isoforms, was measured in U937
cells and purified human peripheral blood T cells after exposure to the
different g levels. U937 and T cells were used for this
study because the abundance and behavior of PKC isoforms in these cell
types are well characterized and both cell types have been reported to
be sensitive to exposure to microgravity (4
,
12
, 15-17
). An experiment aboard the space
shuttle is subject to many unique problems compared to experimentation
in a terrestrial laboratory. Therefore, we developed a specialized cell
culture system for these experiments and evaluated the response of U937
and T cells to culture in this device under conditions simulating those
anticipated for the space shuttle experiment. The results of these
optimization experiments are described in detail elsewhere
(18)
. To further understand the effect of environmental
conditions on PKC distribution and quantity we assessed PKC isoforms in
U937 cells under different culture conditions simulating those
encountered in the space shuttle experiment during spaceflight.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and PKC-ßII were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Tween-20 was obtained from Bio-Rad
France (Ivry-sur-Seine, France). GF/B glass microfiber filters were
obtained from Whatman (Maidstone, UK). The Bichrotinic Acid protein
assay kit and Supersignal-enhanced chemiluminescent substrate were
obtained from Pierce (Rockford, IL). Nitrocellulose membranes
(0.45-µm pore size) were obtained from Schleicher and Schuler
(Dassel, Germany). Anti-CD14, -CD19, and IgG2 antibody-coated magnetic
beads were obtained from Dynal France (Compiegne, France). Anti-CD56
antibody was obtained from Immunotech (Marseille, France). Genosys
blocking buffer was obtained from Genosys (Pampisford, UK).
Peroxidase-coupled goat anti-mouse and goat anti-rabbit polyclonal
antibodies were obtained from Jackson (West Grove, PA).
Phosphate-buffered saline (PBS) was obtained from Biomérieux
(Marcy l'etoile, France).
Cells
The human leukemic myelomonocytic cell line U937 and peripheral
human blood T cells were used in this study. U937 cells were obtained
from the American Cell Type Collection and checked for mycoplasma
contamination by a polymerase chain reaction assay. This cell line was
maintained in batch cell culture at 37°C/5% CO2
incubator at the Life Science Support Facility, Kennedy Space Center
before the space shuttle mission. Cells were maintained in RPMI-1640
medium containing 10% fetal bovine serum, 2 mM glutamine, 1% (v/v)
penicillin-streptomycin-neomycin antibiotic mix, with medium changes
every 2 days. At each medium change cell concentration was adjusted to
5 x 105 cells/ml.
Human T cells were purified from the peripheral blood of healthy donors through the use of the following protocol. Whole peripheral blood was subject to a 20-min centrifugation at 400 g on a Ficoll gradient to separate leukocytes from erythrocytes. After separation the leukocyte fraction was washed twice in RPMI-1640 medium with supplements before being incubated with Dynal magnetic beads coated with anti-CD14, CD-19, and CD-56 antibodies for 2 x 30 min at 4°C to eliminate monocytes, B cells, and natural killer cells. This treatment yielded better than 98% pure human T cells, as determined by cytofluorimetry. After purification the T cells were resuspended in medium at the concentration required for the experiment.
Space shuttle flight
The experiments under microgravity were performed aboard the
space shuttle Atlantis during the STS-76 mission launched
from the Kennedy Space Center on March 22nd, 1996. The shuttle carried
a pressurized laboratory module that contained the experimental
facilities used for our experiments. The flight lasted for 9 days,
which included 5 days docked to the Russian Mir station. The
microgravity level obtained aboard was generally better than
10-3 g.
Experimental devices used for space shuttle experiment
A specially designed cell culture/activation cassette known as
the General Cell Activation Kit-2 (GCAK-2) was used to perform the
experiment within the technical and safety constraints of space shuttle
flight. This cassette is described in detail elsewhere
(18)
. Briefly, each cassette consisted of a machined
polycarbonate block enclosing six independent culture activation units.
Each culture activation unit consisted of a main 500-µl cell culture
chamber that can be connected and isolated from two 150-µl daughter
chambers by rotating stainless steel vanes. The daughter chambers
contained activators and fixative/inhibitor solutions. Cell culture
suspensions and other liquids were introduced into the chambers through
filling ports. A movable piston in each chamber accommodated volume
changes. Injection of activator/fixative into the main culture chamber
was effected by opening the vane between chambers and pushing on the
piston. The European Space Agency Biorack facility was used to perform
experimental operations with the GCAK-2 cassettes during space shuttle
flight (19)
. This consisted of a 37°C incubator, without
CO2, with racks placed to expose samples to microgravity
and a centrifuge to expose samples to 1 g. Experimental
manipulations were performed in-flight by a crew member in a separate
glovebox. After completion of experimental manipulations, in-flight
samples were stored in either a -20°C freezer or a +5°C
refrigerator as detailed below in the experimental protocol.
PKC translocation and PKC isoform quantity during space shuttle
flight
Pre-flight, U937 cells were transferred to RPMI-1640 medium
supplemented to permit culture in the absence of CO2
[RPMI-1640 at pH 7.4, 25 mM HEPES, 12 mM sodium bicarbonate, 1 mM
sodium pyruvate, 2 mM L-glutamine, 10% (v/v)
heat-inactivated fetal bovine serum, 1% (v/v)
penicillin-streptomycin-neomycin antibiotic mix]. Cells were loaded
into GCAK-2 cassettes shortly before the cassettes were installed in
the middle deck of the space shuttle cabin 18 h before launch. The
cassettes remained in this area at ambient temperature (22.8–24.4°C)
for 77 h before being transferred to the 37°C (± 0.5°C)
incubator in Biorack. The long storage time at ambient temperature was
due to operational constraints of the space shuttle mission. For each
experimental condition one cassette was placed in the static racks of
the incubator, exposed to microgravity, while an identical cassette was
placed on a centrifuge within the incubator, which exposed samples to
an acceleration equivalent to terrestrial gravity (1 g). The
cassettes were incubated for 16 h at 37°C until the start of
experimental manipulations. After this incubation period, cassettes
were briefly transferred to the Biorack glovebox where a crew member
injected 100 ng/ml final concentration [3H]PDBu (2.5
µCi per sample) into the cell culture suspension, before immediately
replacing the cassettes in the incubator. Cassettes were incubated for
either 10 or 60 min at 37°C, then transferred back to the glovebox
where a digitonin-based inhibitor mix was added to the cell culture
suspension (0.5 mg/ml digitonin, 50 mM MOPS, pH 7.2, 150 mM NaCl, 5 mM
NaF, 5 mM EDTA, 10 mM EGTA, 2 mM PMSF, 1 mM
Na3MoO4, 10 µM ß-methylaspartic acid, 10
µg/ml soybean trypsin inhibitor). This both permeabilized the cell to
release cytosolic PKC and preserved the translocation state of PKC
(20)
. In addition, an unactivated control was performed
where [3H]PDBu was added to the cell culture, followed
immediately by the inhibitor mix. After addition of the inhibitor mix
the cassettes were transferred to -20°C until post-flight analysis
in our laboratory. In addition, an identical experiment was performed
on the ground, delayed 2 h with respect to the flight experiment,
with samples being exposed to 1 g in the Biorack incubator
static racks and 1.4 g on the centrifuge.
PKC isoform quantification during space shuttle flight
U937 cells (1.5 x 106 cells/ml) and purified
human peripheral T cells (2 x 106 cells /ml), were
loaded into GCAK-1 cassettes and subjected to the same conditions as
those in the PKC translocation experiment, described above, up to
addition of activator substances. U937 cells were stimulated with 100
ng/ml PDBu, whereas T cells were stimulated with a 0.4 µM A23187
ionophore/100 ng/ml PDBu mix. The cell cultures were incubated with the
activators for 10 or 60 min before addition of a cell lysing buffer (50
mM HEPES, pH 7.4, 100 mM NaF, 10 mM Na4PO2, 2
mM orthovanadate, 0.5 mM EGTA, 0.5 mM EDTA, 2 mM PMSF, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1% NP-40). In addition, an
unactivated control was performed by adding medium, followed by
inhibitors to the cell cultures. Due to logistical reasons it was not
possible to freeze samples in flight. Instead the cassettes were
transferred to 5°C storage immediately after addition of inhibitors
and stored for 7 days at this temperature until landing, after which
samples were frozen at -20°C until post-flight analysis. An
identical experiment was performed on the ground as described for the
PKC translocation experiment above.
Effect of culture environment on cell viability and PKC levels in
U937 cells
In ground-based studies the relationship between the age of the
cell culture, culture conditions, and PKC isoform quantities were
investigated in U937 cells. U937 cells were prepared at 2 x
106 cells/ml in RPMI-1640 with supplements [RPMI-1640 at
pH 7.4, 25 mM HEPES, 12 mM sodium bicarbonate, 1 mM sodium pyruvate, 2
mM glutamine, 10% (v/v) fetal bovine serum, 1% (v/v)
penicillin-streptomycin-neomycin antibiotic mix]. Cell suspension (2.5
ml) was loaded into either 25-cm2 culture flasks, or 2.5-ml
syringes. The culture flasks were incubated at 37°C in a 5%
CO2 atmosphere, whereas syringes were incubated either at
37 or 25°C. Samples were incubated for 0, 24, 48, 72, 96, and
120 h before the cell suspensions were recovered. Cell viability
after the incubation period was determined by trypan blue exclusion, as
detailed below. Samples were then processed as described below to
determine PKC isoform content.
[3H]PDBu assay of PKC translocation and total
cellular PKC quantity
Samples from the space shuttle experiment, which had been
labeled with [3H]PDBu and permeabilized with
digitonin/inhibitor mix in-flight, were processed as follows. The
samples were thawed, vortexed to break up cell aggregates, and
centrifuged at 100,000 g for 1 h at 4°C. The
supernatant was transferred to a separate tube, whereas the pellet
(containing nuclei, membrane, and insoluble cytoskeleton) was
resuspended in sample buffer A (50 mM MOPS, pH 7.2, 150 mM NaCl, 5 mM
NaF, 5 mM EDTA, 10 mM EGTA, 2 mM PMSF, 1 mM
Na3MoO4, 10 µM ß-methylaspartic acid, 10
µg/ml soybean trypsin inhibitor). Both fractions were filtered
through a GF/B filter washed three times with sample buffer A.
3H-labeling on each filter was determined by liquid
scintillation counting (Packard 1900 TR liquid scintillation analyzer,
Packard Instruments, Meridian, CT).
The total amount of PKC in U937 cell samples from the culture environment experiment was determined by the following method: 1.5 ml of cell suspension recovered from cell culture flasks or syringes was centrifuged at 400 g for 5 min. The cell pellet was then resuspended in 400 µl of ice-cold Buffer A and 100 µl of 125 ng/ml [3H]PDBu (0.4 µCi per sample) was added to each sample. Samples were incubated on ice for 30 min. Samples were filtered on a GF/B filter washed three times with sample buffer A. 3H labeling on each filter was determined by liquid scintillation counting as described above.
The nonspecific binding in the [3H]PDBu assay was assessed by incubating 1 x 106 U937 cells with either 100 ng/ml [3H]PDBu (2.5 µCi per sample) alone or 100 µg/ml unlabeled PDBu and 100 ng/ml [3H]PDBu (2.5 µCi per sample). Nonspecific binding was found to be less than 10% of total labeling under these conditions.
DNA concentration assay
A 20-µl aliquot of homogenized cell suspension was split into
two 10-µl aliquots for DNA determination by a DAPI-based technique
(21)
. Briefly, each aliquot was mixed with 1 ml sample
buffer containing 300 ng/ml DAPI, 10 mM Tris, 10 mM EDTA, and 100 mM
NaCl. DNA concentration was determined by fluorescence at 455 nm,
measured in a Jobin Yvon spectrofluorimeter JY3D.
Western blot assays of PKC isoforms
Samples in cell lysing buffer from the space shuttle experiment
were thawed post flight and sonicated for 3 x 5 s on ice to
break up any particulate matter. SDS was then added to the sample at a
final concentration of 1% w/v, followed by 30-min vortexing at 4°C.
Reducing buffer C (10% SDS, 25% ß-mercaptoethanol, 25% glycerol)
was then added and the samples boiled at 100°C for 5 min before
loading onto polyacrylamide gels. Two-milliliter aliquots of U937 cell
culture samples from the culture environment experiment were mixed with
10 ml of ice-cold PBS and centrifuged at 400 g for 5 min.
The pellet was resuspended in ice-cold PBS, centrifuged again, and the
pellet then resuspended in ice-cold RIPA buffer (Tris, 50 mM; pH 6.8;
leupeptin, 5 µg/ml; aprotonin, 5 µg/ml; aqueous PMSF, 10 µM).
Samples were then sonicated 3 x 5 s on ice, 1% w/v SDS
added, and then vortexed for 30 min at 4°C. Samples were then mixed
1:1 with Laemli buffer and boiled for 5 min (Tris · HCl, 50 mM;
pH 6.8; glycerol, 20%; NEM, 4 mM; SDS, 2%; and Pyronine-Y colorant).
Equal quantities of protein from each sample were loaded on a SDS-PAGE
minigel, migrated at 80 V, and then transferred onto nitrocellulose
membranes (Miniprotein electrophoresis kit, Bio-Rad). Membranes were
blocked for 1 h in 10% Genosys blocking buffer, 0.05% Tween-20
in Tris-buffered saline (TBS; 16 mM Tris · HCl, 3 mM Tris, 150
mM NaCl, pH 7.5) followed by 2 x 5 min in 0.5% Tween-20/TBS
before incubation with the primary antibody. Rabbit polyclonal
antibodies directed against PKC-ßII and PKC- were used
at between 0.1 and 0.25 µg/ml concentrations, depending on the
quantity of protein in the sample. A mouse monoclonal antibody reactive
with actin was used at 0.25–0.1 µg/ml concentrations. Membranes were
incubated with primary antibodies for 1.5 h in 4% blocking
buffer, 0.05% Tween-20 in TBS. After three washes in 0.5%
Tween-20/TBS, membranes were incubated for 1.5 h either with
peroxidase-coupled goat anti-rabbit or goat anti-mouse antibodies,
depending on the species of the primary antibody, in the same buffer
used for the primary antibodies. The membranes were then washed 4 x 5 min in 0.5% Tween-20 before ECL detection by Supersignal
according to the manufacturer's instructions. All manipulations were
performed at room temperature (20–25°C). The intensity of the bands
recorded on film were quantified by a computer image analysis (Biocom
Image Analysis System, Les Ulis, France).
Bichrotinic acid assay of protein concentrations
A commercial kit of the Bichrotinic acid (BCA) assay was used to
determine protein concentrations in samples. Briefly, samples were
diluted to between 1/10 and 1/50 initial concentration in water before
being mixed with the BCA reagents in a multi-well plate. Samples were
then incubated at 37°C for 30 min before measurement of sample
optical density at 540 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). There were no significant differences between the microgravity
and 1 g centrifuge samples nor between 1 g ground
and 1.4 g. The total amount of PKC binding phorbol ester
([3H]PDBu) per cell was elevated in both flight
conditions compared to the 1-g ground sample, whereas the
amount of PKC in the ground 1.4-g sample was significantly
lower than the 1-g ground sample (Fig. 2
). Again, there was no significant difference between microgravity
and 1-g on-board centrifuge samples.
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A significant translocation of PKC from the cytosol to the particulate
fraction (which includes the nucleus, membrane, and insoluble
cytoskeleton) following application of the radiolabeled phorbol ester
([3H]PDBu), which both activated and selectively labeled
PKC, was observed at all g levels (Fig. 3
). The proportion of total cellular PKC in the particulate sample
in unstimulated cells was similar for all conditions. A sharp increase
in the amount of PKC in the particulate fraction was observed 10 min
after phorbol ester stimulation at all g levels, however,
this increase was significantly higher in the flight samples than in
ground samples. Sixty minutes after stimulation the amount of PKC in
the particulate fraction remained elevated in both ground conditions (1
g ground and 1.4 g), whereas the amount of PKC in
this fraction in the ground samples returned to a similar level to that
in unstimulated cells. No significant differences in PKC translocation
were observed between microgravity and 1 g flight nor
between 1-g ground and 1.4-g samples.
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The amount of PKC- and PKC-ßII in U937 cells was
determined by Western blot. No significant differences in the
quantities of these isoforms were observed between any of the
g levels at the level of sensitivity of the experiment
(Fig. 4
). Likewise, in T cells that had been stored at ambient temperature
before loading into the Biorack 37°C incubator, no differences were
observed in PKC- quantities between g levels (Fig. 5A
). However, the quantity of PKC- in T cells that had been
stored at 5°C before Biorack load, and showed a strong inverse
correlation with g level (analysis of variance test
P < 0.01), with the highest quantity of PKC in the
microgravity sample and the lowest quantity in the 1.4-g
sample (Fig. 5B)
. No significant differences were observed in
PKC-ßII quantity in T cell levels between any of the
g levels, in either 5°C or ambient stored samples (data
not shown).
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Cell culture environment experiment
U937 cells were stored in cell culture flasks and syringes at
37°C, as well as syringes at 25°C, simulating some of the
conditions to which cells were exposed in the space shuttle experiment
(culture in a closed container, storage at ambient temperature). In all
conditions, cell viability, measured by trypan blue exclusion,
decreased with time (Fig. 6A
). However, the viability of cultures stored at 37°C in syringes
decreased rapidly after the 3rd day of culture, whereas the decrease in
viability in cultures stored in syringes maintained at 25°C was
minimal. In both 37°C syringe and flask cultures over 90% of the
D-glucose in the medium was used by the 3rd day of culture,
whereas over half of the initial concentration remained in 25°C
cultures at the 4th day of culture. These data are in agreement with
experiments performed in the cell culture cassettes used for the space
shuttle experiment reported separately (18)
, indicating
that syringe culture is a good analog of the conditions within the
cassette system.
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Changes in the quantity of both PKC- and PKC-ßII were
observed, the kinetics of which depended on storage conditions. For
cultures stored in syringes or culture flasks at 37°C a marked
increase in PKC- was observed that peaked on the 2nd day of culture,
followed by a rapid decline to undetectable levels by the 5th day of
culture (Fig. 7A
). For 25°C syringe cultures, PKC- gradually decreased with
time but was still detectable after 5 days of culture. The quantity of
PKC-ßII increased rapidly in 37°C flask cultures
reaching a peak more than twice the initial level after 3 days of
culture, before returning to baseline levels 5 days after the start of
the experiment (Fig. 7B)
. A small decrease in PKC-ßII
with time was observed in 37°C syringe cultures, whereas there was no
significant change at 25°C. No significant change in
[3H]PDBu binding was observed (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In this experiment
we used a [3H]PDBu to both activate and label with high
affinity PKC (22)
. Previously, we reported that the
binding of [3H]PDBu to PKC was not modified in
microgravity, whereas the intracellular distribution of PKC and
quantity of cellular PKC in U937 cells varied in proportion to
g level (15)
. In the experiments reported here
we attempted to determine whether the observed gravidependent PKC
distribution was a result of a redistribution of PKC before stimulation
with phorbol ester, or an alteration in translocation after application
of the phorbol ester.
A significant translocation of PKC from cytosol to the particulate
fraction (which includes membranes, insoluble cytoskeleton, and
nucleus) occurred, after application of [3H]PDBu, at all
g levels. Translocation from the site of activation to the
site of action is believed to be a critical step in signal transduction
by PKC and so any modification of this process may result in perturbed
PKC-dependent gene expression (11)
. Therefore, this result
is important because it demonstrates that PKC translocation does occur
in microgravity. In all conditions the quantity of PKC in the
particulate fraction before phorbol ester stimulation was similar,
indicating that the intracellular distribution of PKC in unstimulated
cells was insensitive to g level. However, there are
significant differences in the translocation kinetic of PKC between the
two flight conditions (microgravity and 1-g centrifuge) and
two ground conditions (1 g static and 1.4 g). As
shown in Figure 3
the translocation of PKC to the particulate fraction
in the flight samples is biphasic, with a strong translocation at 10
min, but returning to baseline levels by 60 min. In contrast, the
translocation in the ground samples is initially slower, but remains
sustained at 60 min. Due to the technical constraints of the space
shuttle experiment it was not possible to examine the extent of PKC
translocation at intermediate time points, so we cannot determine from
these data the exact time of maximum translocation. Inhibition of
PKC- translocation in cardiac myocytes results in suppression of
norepinephrine-induced inhibition of contraction (23)
,
whereas in variants of U937 cells with defective microtubule
organization, association of PKC-ßII with microtubules
was inhibited, resulting in suppression of integrin secretion
(24)
Therefore, the alterations in PKC translocation
observed in our experiments could result in modification of
PKC-mediated signal transduction and cell function.
There was no significant difference between microgravity and
1-g on-board centrifuge conditions, nor between
1-g ground and 1.4-g samples in the PKC
translocation kinetic and cell growth rate. Due to operational and
technical constraints of the space shuttle mission all flight samples
were stored at ambient temperature (22.8–24.4°C) in microgravity for
more than 3 days before transfer to the 37°C incubator for 16 h,
with the 1-g samples only exposed to 1 g when
loaded into the centrifuge in the 37°C incubator. Hence, the
1-g flight samples were in fact exposed to microgravity for
an extended period before exposure to 1 g. Likewise, the
1.4-g samples were exposed to 1 g on the ground
before transfer to 1.4-g centrifuge in the 37°C incubator.
Therefore, a plausible explanation for the observed data is that the
cells were sensitive to the prevailing g level at ambient
temperature and changes that occurred during this time were not readily
reversed by subsequent exposure to higher g levels on the
centrifuge. This conclusion is supported by data from previous
experiments which showed that the distribution of PKC in U937 cells
exposed to microgravity for 7 h followed by 1 g for
8 h was intermediate between that of cells exposed to microgravity
continuously and those exposed to 1 g without interruption
(15)
However, because we did not have a continuous
in-flight 1-g control, we cannot exclude the possibility
that other spaceflight factors, such as launch acceleration and
vibration or cosmic radiation (i.e., HZE particles) contribute to the
observed difference between flight and ground samples.
The total amount of [3H]PDBu binding PKC per cell was
significantly elevated in flight compared to 1-g ground
samples, whereas the quantity of PKC in the 1.4-g samples
was significantly lower than 1-g ground. There was no
significant difference in PKC levels between the two flight conditions,
but the quantity of PKC in the 1.4-g samples is
significantly lower than in the 1-g ground samples. This is
an interesting finding because it suggests that the quantity of PKC
accumulated during microgravity exposure in the 1-g flight
samples remained unchanged when the cells were transferred to 1
g. However, the quantity of PKC in 1-g cultivated
cells apparently decreased on transfer to 1.4 g, indicating
that changes in total PKC levels in microgravity may not be readily
reversible on subsequent exposure to 1 g, whereas a transfer
from 1 g to hypergravity induces a change in PKC levels. A
proportional increase in total cellular PKC with decreasing
g level is consistent with our previous observations
(15)
, but the apparent irreversibility of the effects of
microgravity are unexpected. These data therefore suggest that exposure
to microgravity results in some unique alterations in cell behavior not
encountered at higher g levels.
No significant differences in the quantity of PKC- and
-ßII in U937 cells were detectable between any of the
g levels at the level of sensitivity of the experiment. This
result is nevertheless compatible with the [3H]PDBu assay
data because PDBu labels all isoforms of PKC, except PKC and so the
amount of PKC detected by this technique is the sum of several
different isoforms. In addition, the sensitivity of Western blotting to
detect changes in the PKC levels in the small quantity of protein
available for the analysis was limited compared to the
[3H]PDBu binding assay. However, there was a tendency for
an elevation in quantity of both isoforms in the 1-g flight
samples, suggesting that this treatment may have influenced the
quantity of these isoforms. The quantity of PKC- in T cells stored
at 5°C before loading into the Biorack 37°C incubator, showed a
strong inverse correlation with g level (Fig. 5B)
, whereas
no significant differences between g levels was noted for
samples stored at ambient temperature before Biorack load (although
there was a tendency toward elevated quantities of this isoform in the
microgravity sample). At 24°C the metabolism of the T cells is still
active, but at 5°C metabolism is greatly reduced, so it is likely
that the cold-stored T cells were relatively insensitive to the
g level during storage before Biorack load. This result
appears to support our conclusion that the quantity of PKC varies in
proportion to g level and that exposure of the cells to
microgravity during ambient storage before Biorack load causes changes
in the cell that are not readily reversed on subsequent exposure to
other g levels at 37°C. However, these effects seem
specific to PKC-, since no significant difference in quantities of
PKC-ßII could be detected between any g levels
for either ambient or cold stored T cells.
The growth of U937 cells in flight, assessed from DNA concentration,
was significantly lower than in ground conditions. Reduced growth of
mammalian cells in microgravity has been observed by several other
groups (25-27)
. Regardless of the potential mechanisms of
inhibition of cell growth in microgravity, different rates of growth
between flight and ground cultures would result in altered rates of
nutrient depletion, oxygen utilization, and changes in medium pH.
Metabolic and oxidative stresses are known to activate PKC isoforms in
diverse cell types (28-31)
, so the possibility exists
that some of the changes observed in the space shuttle experiment are
due to these secondary stress effects. Therefore, we tested this
hypothesis in an experiment simulating the effect of different cell
culture conditions, analogous to those in the space shuttle
experiments, on the quantity of individual PKC isoforms and
[3H]PDBu binding in U937 cells. Sterile hypodermic 2-ml
syringes were used to simulate culture in the GCAK-2 cassettes.
Comparison of cell viability data between these two culture systems
confirmed that syringes are good analogs of the cassette culture
environment (18)
. A rapid decrease in cell viability after
the 3rd day of culture was noted in both 37°C syringe and flask
cultures, with the rate of decrease being highest in 37°C syringes.
Because in both cases medium D-glucose was almost
completely depleted by the 4th day of culture, this result suggests
that nutrient depletion and hypoxia were major contributors to reduced
viability in the 37°C syringe cultures. No significant changes in
[3H]PDBu binding PKC with age of cell culture was
observed under any of the experimental conditions, suggesting that the
alterations in total [3H]PDBu-labeled PKC observed in the
space shuttle experiment are not simply caused by different levels of
medium and oxygen utilization between flight and ground conditions.
However, significant changes were observed in the quantities of
individual PKC isoforms in U937 cells with age of culture and culture
conditions (Fig. 7)
PKC- shows a biphasic change in quantity with
age of culture in both 37°C syringe and flask cultures, increasing in
quantity up to the 2nd day of culture before declining to undetectable
levels by the 5th day of culture. In contrast, a marked change in
PKC-ßII levels were observed only in 37°C flask
cultures with a doubling in quantity by the 3rd day of culture before a
decrease to baseline levels by the 5th day of culture. Therefore, given
the changes in PKC isoforms with culture age and condition, we cannot
exclude the possibility that culture environmental stress contributed
to changes in PKC localization and quantity observed in U937 cells in
the space shuttle experiment. However, in T cells gravidependent
changes in PKC levels were also observed that are unlikely to be due to
these secondary effects because these cells were in a quiescent,
non-proliferating state.
The results of our experiment aboard the space shuttle show that marked
changes in PKC localization and quantity do occur in U937 and T cells
during spaceflight. However, the mechanisms underlying these effects
remain unclear. A detailed discussion of potential mechanisms for
gravisensing have been reviewed (7)
. A change in gravity
level could be perceived either directly by individual cells or
indirectly through changes in the extracellular environment. In
macroscopic fluid volumes, such as a culture flask, sedimentation and
buoyant (Bernard) convection are important physical forces that are
driven by gravity. Absence of these phenomena in microgravity could
have important consequences for cells in culture. The metabolic heat
generated by a single cell is on the order of 1 nW (D. Jones, personal
communication), which is several orders of magnitude greater than
thermal noise, so collectively this heat may help to drive, under
1-g conditions, convection currents in cell cultures that
could improve mixing and transport of nutrients to the cells compared
with microgravity. Likewise, under 1-g conditions,
nonadherent cells of the type used in our experiments sediment to the
floor of the culture container, which increases the possibility of cell
to cell contact and mechanical interaction with the culture vessel,
compared with cells in suspension, as would be the case for cells under
microgravity. Although the cell types used in our experiments
proliferate in an anchorage-independent manner, complete inhibition of
attachment to the cell culture vessel has been reported to inhibit
proliferation (32)
. In contrast, it is much less obvious
how change in gravity could be directly perceived by a single cell. The
weight of a single cell is many orders of magnitude smaller than other
physical forces acting on the cell, such as surface tension, internal
forces in the cytoskeleton, electrostatic fields, or thermal noise
(e.g., brownian motion) in fluids, so it seems unlikely that a change
in gravity could be perceived against the much larger background noise
of other forces (33)
. However, there are alternative
mechanisms by which gravity may be perceived directly through
intracellular structures. In vitro, the polymerization of
microtubule has been observed to behave as a nonlinear system
(34)
. Nonlinear systems are thermodynamic systems that are
not stable, far from equilibrium, which attempt to reach a stable state
by dissipating energy to form patterns. As the system starts to move
toward a stable state it passes through a point known as the
bifurcation point. At this point the system is very sensitive to small
environmental forces, too small to affect the equilibrium state, which
can strongly influence the type of patterns formed. Similar patterns of
microtubule organization have been observed in vivo, in
plant cells, peripheral nerve cells, and invertebrate embryos,
suggesting that this biophysical process may act in vivo (35
and Tabony, personal communication). It is interesting to note that the
patterns formed by microtubules in vitro are sensitive to
direction of gravity at the bifurcation point, potentially offering a
mechanism by which gravity could be perceived intracellularly. There is
a close association between microtubules and certain PKC isoforms,
notably PKC- and -ßII (12
,
24
), so alterations in microtubule organization could
result in changes in PKC isoform distribution. Lewis et al. observed
disorganized microtubules in Jurkat cells during the early part of a
spaceflight, along with inhibited cell proliferation and enhanced
expression of apoptosis markers (27)
. In T cells the
polarization of microtubule organization toward antibody-stimulated T
cell receptors was lost in microgravity (36)
. However,
whether these changes are due to external factors or direct effects of
microgravity on the cytoskeleton are unclear.
In summary, we have shown that a key component of intracellular signal transduction, PKC, varies in intracellular distribution and quantity according to g level. Although this result could provide a partial explanation for alterations in cell function observed under microgravity, we are still far from identifying the causal mechanisms of gravisensitivity. Many possibilities exist, but a first step toward elucidating the mechanisms is to determine the timescales of perception of altered gravitational stimulation, so that the number of different possible mechanisms can be narrowed down based on theoretical calculations of the minimum time required to perceive a change. In addition, great care is required to identify and eliminate any potential artefacts in the experimental techniques employed. Thus, the demonstrated gravisensitivity of PKC provides us with a useful marker of the effect of altered g level on early cellular activation events for kinetic experiments of this kind.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 24, 1998.
Revision received October 27, 1998.
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REFERENCES |
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