Reduction of anabolic signals and alteration of osteoblast nuclear morphology in microgravity

15 Pages·2006·0.39 MB·English

Journal of Cellular Biochemistry 99:435–449 (2006)
Reduction of Anabolic Signals and Alteration of
Osteoblast Nuclear Morphology in Microgravity
Millie Hughes-Fulford,1,2,3* KarstenRodenacker,4and Uta Ju¨tting4
1Northern California Institutefor Research andEducation, San Francisco, California
2DepartmentofMedicine, University ofCalifornia, San Francisco,California
3DepartmentofVeteran Affairs, LaboratoryofCell Growth and Differentiation,San Francisco, California
4GSF—National Research Centerfor Environment and Health,Institute ofBiomathematicsand Biometry,
Ingolsta¨dter Landstraße1, D-85764 Neuherberg, Germany
Abstract Bone loss has been repeatedly documented in astronauts after flight, yet little is known about the
mechanism of bone loss in space flight. Osteoblasts were activated during space flight in microgravity (mg) with and
withouta1gravity(1g)fieldand24geneswereanalyzedforearlyinduction.Inductionofproliferatingcellnuclearantigen
(PCNA), transforming growth factor b (TGFb), cyclo-oxygenase-2 (cox-2), cpla2, osteocalcin (OC), c-myc, fibroblast
growthfactor-2(fgf-2),bcl2,bax,andfgf-2messageaswellasFGF-2proteinweresignificantlydepressedinmgwhen
comparedtoground(gr).Artificialonboardgravitynormalizedtheinductionofc-myc,cox-2,TGFb,bax,bcl2,andfgf-2
messageaswellasFGF-2proteinsynthesisinspaceflightsamples.Innormalgravity,FGF-2inducesbcl2expression;we
foundthatbcl2expressionwassignificantlyreducedinmicrogravityconditions.Sincenuclearshapeisknowntoelongate
intheabsenceofmitogenslikeFGF-2,weusedhigh-resolutionimage-basedmorphometrytocharacterizechangesin
osteoblast nuclear architecture under microgravity, 1 g flight, and ground conditions. Besides changes in cell shape
(roundish/elliptic),otherhigh-resolutionanalysesshowclearinfluencesofgravityontheinnernuclearstructure.These
changesoccurinthetexture,arrangement,andcontrastofnuclearparticlesandmathematicalmodelingdefinesthesingle
cell classification of the osteoblasts. Changes in nuclear structure were evident as early as 24 h after exposure to
microgravity. This documented alteration in nuclear architecture may be a direct result of decreased expression of
autocrine and cell cycle genes, suggesting an inhibition of anabolic response in mg. Life on this planet has evolved
inanormalgravityfieldandthesedatasuggestthatgravityplaysasignificantroleinregulationofosteoblasttranscription.
J.Cell.Biochem.99:435–449,2006. (cid:1)2006Wiley-Liss,Inc.
Keywords: microgravity;growthregulation;cytometry;morphometry;photometry;fgf-2;bcl2
Aprojectedbonelossof20–30%inastronauts and human studies, investigators found that
isoneofthemajorphysiologicalshowstoppers loss of weight bearing bone can be as high as
in the proposed 30-month manned mission to 3% per month andthis loss isprimarilydueto
Mars.Forthefirstthreedecadesofspaceflight, lack of new osteoblast growth in space flight
scientists have discovered systemic changes in [Wronski and Morey, 1983; Nicogossian et al.,
the body, which included loss of bone and in- 1989; Marie et al., 2000; Vico et al., 2000].
creasesinurinarycalcium.Fromtheseanimal Although flight opportunities have been lim-
ited, the volume of data about microgravity
effectsoncellproliferationanddifferentiationis
accumulating. It has been demonstrated that
cellgrowthanddifferentiationofosteoblastsis
Grant sponsor: NASA; Grant numbers: NAG-2-1086, inhibited or altered in a microgravity environ-
NAG-2-1286, NCC-2-136; Grant sponsor: Department of ment [Fitzgerald and Hughes-Fulford, 1996;
VeteransAffairsResearchServiceMeritReview.
Hughes-Fulford and Lewis, 1996; Carmeliet
*Correspondenceto:MillieHughes-Fulford,PhD,Director,
et al., 1998; Landis et al., 2000; Granet et al.,
Lab of Cell Growth, VA Medical Center Mail Code 151F,
2001] suggesting that the osteoporosis seen in
4150ClementStreet,SanFrancisco,CA94121.
E-mail:[email protected] spaceflight is occurring at the cellular level.
Received12January2006;Accepted15February2006 Alteredgeneexpression,reductionintranscrip-
DOI10.1002/jcb.20883 tion factors, and reductions in growth factor
(cid:1)2006Wiley-Liss,Inc.
436 Hughes-Fulford et al.
related proteins have also been observed in portion of the cells exhibited a spindle shape
othercellsinmicrogravity[deGrootetal.,1990; after 4 days in microgravity, with the most
CubanoandLewis,2000;Goodwinetal.,2000]. dramatic change observed being the elongation
In2001,Lewisetal.founda5.9-foldreductionin of nuclear shape [Fitzgerald and Hughes-Ful-
message for TGFa in space-flown lymphocytes ford,1996].
[Lewisetal.,2001]. Since it has been suggested that changes in
Inthisreport,wefoundsignificantreductionin nucleararchitectureandcontrolofgeneexpres-
expression ofseveral osteoblast genesincluding sionareinterrelated[Debnathetal.,1999],we
fibroblast growth factor-2 (fgf-2), cyclo-oxyge- analyzed message expression of other genes
nase-2(cox-2),c-myc,transforminggrowthfactor involvedincellgrowthandcorrelatedmorpho-
b (TGFb), proliferating cell nuclear antigen logical changes between osteoblasts grown in
(PCNA),osteocalcin(OC),andbaxinosteoblasts microgravity,1gorgr.Inthisstudywereport
grown in mg. Since fgf-2, TGFb, and cox-2 are significant microgravity-induced alterations
osteoblastautocrine/paracrinefactorsimportant in transcription factor message and protein
intheG1-Sphasetransition[Canalisetal.,1991; expression accompanied by dramatic changes
Hughes-Fulford et al., 1992; Stachowiak et al., in osteoblast cell morphology. The changes in
1994;Coffinetal.,1995;KeresztesandBoonstra, gene expression observed in osteoblasts in mg
1999],itispossiblethattheirdecreasedexpres- werenotobservedinosteoblastsmaintainedin
sionmightbelinkedwithinhibitionofactivation anin-flight1gfield,implyingagravityrequire-
ofbonegrowthinmicrogravity. ment for normal osteoblast growth. These
Since fgf-2 is a known osteoblast autocrine/ combinedchangesinosteoblastcellphysiology
paracrine factor important in the G1-S phase suggest thatthe decreasein bone formationin
transition[Canalisetal.,1991;Stachowiaketal., astronautsmaybeinpartduetomicrogravity-
1994;Coffinetal.,1995;KeresztesandBoonstra, inducedalterationsintheosteoblastatthegene
1999], we hypothesized that changes in fgf-2 expressionandsignaltransductionlevel.
expression may be linked to inhibition of bone
growth in mg. In this study, two groups of MATERIALSAND METHODS
osteoblasts onboard the Space Shuttle were
Preparation of Cells
exposedtomgfor19hbeforetheywerestimulated
withfetalcalfsera(FCS);onegroupwasgrownin The MC3T3-E1 osteoblast cell line clonally
microgravity, the other in an artificial 1 g derived from embryonic mouse calvaria
environment.Weanalyzedexpressionoffgf-2,a [Hiramatsu et al., 1983], was kindly provided
mitogen,andtranscriptionfactorthatpromotes to us by Dr. M. Kumegawa (Josai Dental
proliferation. University, Japan). The cell line was main-
As part of our study of fgf-2 induction, we tainedatlowpassagenumber.Cellsweregrown
examined concomitant changes in nuclear mor- in alpha minimal essential medium (aMEM)
phology of osteoblasts grown in microgravity. with 10% fetal calf serum (Hyclone Labs, Inc.,
Rijken[deGrootetal.,1991]firstnoticedchanges Logan, UT) supplemented with 2 mM L-gluta-
in cell shape in response to changed gravity in mine (Sigma, St. Louis, MO), 25 mM HEPES,
soundingrocketexperimentsin1991.Morpholo- and antibiotic–antimycotic solution (100 U
gicchangeswerealsoreportedinparabolicflight penicillin/ml,0.01 mgstreptomycin/ml,0.25mg
with osteoblasts exhibiting cytoplasmic retrac- amphotericinB/ml).Cellsweregrownina378C
tion and membrane ruffling in ROS/17/2.8 cells incubator with 5% CO . They were fed three
2
[Guignandon et al., 1995]. Increased PGE was times a week and passaged at confluence. For
2
alsofoundinmedia,accompaniedbysignificant spaceflight experimental samples, 120,000–
flight-induced changes that included a decrease 200,000 cells were plated onto non-coated,
in cell area and irregular shape in some cells sterile 11(cid:1)22 mm glass coverslips (Thomas
[Guignandonetal.,1995].Indomethacinreduced Scientific, Swedesboro, NJ), placed in 6-well
the irregular shape changes, but not the loss of plates, and grown in 10% serum-containing
cell area. Osteoblasts grown in microgravity on aMEM overnight. Cell-coated coverslips were
STS-56 demonstrated a reduction of growth in then transferred into 2% serum-containing
osteoblasts activated in-flight, suggesting that mediumforspaceflightinplungerboxunits.In
microgravitycausedadecreaseincellprolifera- accordancewithNASAflightrules,theplunger-
tion resulting in changes in cytoskeleton. A box units were held for 17 h in the Shuttle at
Reduction of Anabolic Signals 437
mid-deck temperature prior to launch. This, CentrumvoorConstructieMechatronica(CCM;
combined with low serum-containing medium, Neuenen, Netherlands). The hardware con-
placedtheosteoblastinaquiescentstatepriorto sisted of the CCM plungerbox and its Type I
Space Shuttle launch. In order to achieve our containerdevelopedforspaceflightcellculture.
goaltostudychangesinnuclearmorphologyin The Type I container provided a second level
microgravity, we launched cells in a quiescent of fluid containment. The plungerboxes were
condition. Osteoblasts were sera activated on- designed to provide a sterile environment for
orbit and mg or 1 g samples collected in-flight cell growth activation and fixation in a micro-
(Fig.1).Therewerefoursamples(n¼4)foreach gravity condition. The plungerbox is composed
time point in mg, 1 g flight, and gr samples. oftwoindependentculturechambersthateach
NASA’sLSLE(LifeSciencesLaboratoryEquip- holdtwo11mm(cid:1)22mmglasscoverslips.Each
ment) freezer, was used for sample storage on culture chamber was a separate sample. For
the Space Shuttle. Supplements, such as dex- each condition a sample size of n¼4 indepen-
amethasone b-glycerol phosphate and ascorbic dent cultures was used. Each separate culture
acid were not added to the media since these chamber has a series of reservoirs filled by
agentsareknowntodirectlyeffectgeneexpres- either10%serum-containingaMEMorfixative,
sionandcellmorphologyoftheosteoblast. whichcanbetransferredintoandoutofthecell
culture compartment by manually releasing a
Biorack Facilityand Osteo Hardware
spring-loadedplunger.
CellsweregrownintheBiorack,amultiuser
Experiment Time Line
facility,whichconsistsofincubatorswithvari-
ablegravitycentrifuges,acooler,afreezer,and Quiescent cells were stored in the Space
a sealed glovebox. Two identical Biorack Shuttlemid-decklockeruntil19hafterlaunch.
modules were used: one remained on earth (gr The astronauts then transferred the samples
samples) and the other was integrated into intoa378Cincubatorfor1hbeforestimulating
SpaceHab and flown on each of the Space thecellstogrowinthe1gcentrifugeormgstatic
Shuttle flights STS-76, STS-81, and STS-84. rackbychangingtheirmediafrom2%serumto
Biorackhasanimportantadvantageoverother 10% serum-containing medium (T¼0). mg and
microgravityfacilitiesinthatitprovidesasmall 1gsampleswerefixedduringflightat24hwith
radius(78mm),slowrotating(107.0(cid:2)0.5rpm) 3.7%formaldehydesolutionandwerestoredat
centrifuge for artificial 1 g in orbit. Because of þ58C for the remainder of the Space Shuttle
their proximity, both the mg and 1 g samples mission, while the GITC samples were frozen
experience identical launch vibrations, accel- untilreturn.Therewerefourseparateindepen-
erations,cosmicradiation,andotherunknown dentsamplesforeachgravityconditionateach
conditions of flight. In addition, an identical timepoint(Fig.1).
experiment was performed in the Biorack
module on earth gr samples with a 2 h delay BIOCHEMICAL MEASUREMENTS
fromin-flightproceduresinordertocontrolfor
FGF-2 Protein Analysis
any off-nominal operations during the flight.
The Osteo (the name ofthisflight experiment) Analysis of FGF-2 was completed with a
spaceflighthardwarewasdesignedaccordingto QuantitiesELISAImmunoassay(R&DSystems,
EuropeanSpaceAgency(ESA)specificationsfor Inc., Minneapolis, MN) according to manufac-
use in the Biorack facility and constructed by turer’s instructions. Cellular protein was col-
lected from the GITC subnatant as previously
described[FitzgeraldandHughes-Fulford,1999].
Analysis ofGene Expression
TableIshowstheoverviewofgeneexpression
measured in these experiments. RNA was ex-
tracted and purified by a modified acid guan-
idiumthiocyanate/phenol/chloroformextraction
method previously described [Fitzgerald and
Fig. 1. Time line of experiment from handover. 19 h pre-
Hughes-Fulford, 1996; Barry et al., 1999;
launch, activation in-flight, and termination of experiment
in-flight. FitzgeraldandHughes-Fulford,1999].
TABLE I. OsteoblastGeneExpressioninMicrogravity
GENE STS-76,81,and84composite
Timepoint #Ofcycles Present Significantchange
18s T¼0 18 Yes No
T¼3 18 Yes
T¼24 18 Yes
CPHI T¼0 22 Yes No
T¼3 24 Yes
T¼24 24 Yes
IL-1a T¼0 42 No —
T¼3 42 No
T¼24 42 No
cox-2 T¼0 34 Yes Yes
T¼3 30 Yes
T¼24 30 Yes
cox-1 T¼0 40 No —
T¼3 40 No
T¼24 40 No
cpla2 T¼0 32 Yes Yes
T¼3 32 Yes
T¼24 32 No
EP1 T¼0 37 Yes No
T¼3 37 Yes
T¼24 37 Yes
EP2 T¼0 40 No —
T¼3 40 No
T¼24 40 No
EP3 T¼0 — —
T¼3 47 No
T¼24 47 No
LDLr T¼0 38 — —
T¼3 38 No
T¼24 38 No
CyclinA T¼0 38 Yes No
T¼3 38 Yes
T¼24 38 Yes
CyclinE T¼0 38 — No
T¼3 38 Yes
T¼24 38 Yes
PCNA T¼0 42 Yes No
T¼3 42 Yes
T¼24 42 —
c-myc T¼0 38 Yes Yes
T¼3 38 Yes
T¼24 38 Yes
cjun T¼0 35 No —
T¼3 35 No
T¼24 35 No
TGFb1 T¼0 38 Faint Yes
T¼3 38 Yes
T¼24 38 No
FGF-2 T¼0 34 — Yes
T¼3 34 No
T¼24 34 Yes
Actin T¼0 30 Yes No
T¼3 30 Yes
T¼24 30 Yes
Fibronectin T¼0 34 Yes No
T¼3 34 Yes
T¼24 34 Yes
Collagen T¼0 33 No —
T¼3 33 No
T¼24 33 No
Osteocalcin T¼0 — Yes
T¼3 38 Yes
T¼24 38 Yes
b1integrin T¼0 47 No —
T¼3 47 No
T¼24 47 No
Bax T¼0 30 Yes Yes
T¼3 30 Yes
T¼24 30 Yes
bcl2 T¼0 34 Yes Yes
T¼3 38 Yes
T¼24 38 Yes
OsteoblastswereflownontheShuttleinaquiescentstate;afterinsertionintoorbit,thecultureswereactivatedwithFCS.Manyofthe
genesthathadsignificantlychangedexpressioninmicrogravityareearlypleiotopicgenesthatnormallyareincreasedbygrowthfactors
foundinFCS.Significantchangesatthecollectiontimepoints(Fig.1)areseeninFigures2and3(n¼4).
Reduction of Anabolic Signals 439
RT-PCR Analysis Cell Morphology
Osteoblasts were fixed on coverslips with
The semi-quantitative RT-PCR analysis and
3.7% formaldehyde in phosphate buffered sal-
primer sequences for many of the genes have
ineduringspaceflightandstoredat58Cfor1–
been described [Fitzgerald and Hughes-Ful-
2 weeks before staining. There was no visible
ford, 1996; Barry et al., 1999; Fitzgerald and
difference between control samples that were
Hughes-Fulford, 1999]. To correct for small
immediately stained and samples stored in
variations between experiments, each PCR
formaldehydeat58C.Fornuclearvisualization,
productwas compared toan internal standard
coverslipswerestainedwithHoechstdyenum-
ofcyclophilinor18Sproductsderivedfromthe
ber 33258 and rinsed in distilled water. The
sameRTreaction.Sincesamplesizewassmall
dried osteoblast coverslips were mounted onto
(200,000cells),RNAcontentwasheldconstant
slidesandphotographedwithaZeissAxioscope
andlinearRT-PCR wasaccomplished by vary-
microscope at 40(cid:1) and 100(cid:1) magnification.
ing the number of PCR cycles. Primers were
Slides were processed at the same time under
designed in this laboratory to span introns to
identical conditions. Photographic exposure
eliminate the possibility of detection of DNA.
was taken at identical exposure times and
PCRconditionswereestablishedsothatampli-
conditions(TablesVIandVII;Figs.4–7,9).
fication reaction was stopped in the linear
range. Primers not previously described are
IMAGING METHODS
18s F-TCA AGA ACG AAA GTC GGA GG; R-
GGA CAT CTA AGG GCA TCA CA; CPHI F- Image Acquisition
CGT CTC CTT TGAG CTG TTT GCA GAC R-
TableIIshowsnumberofimagesatthetotal
CATAATCATAAACTAACTCTGCAATCCA
number of cells evaluated. Images were digi-
GC;fgf-2F-AACTACAACTTCAAGCAGAAG
tized from the color film slides with an Epson
AGAGAR-TTAAGATCAGCTCTTAGCAGA
Expression 836XL scanner into Adobe Photo-
CAT; bcl2 F ACTTGTG GCCCA GATAGG-
shop(AdobeSystems,Inc.,MountainView,CA)
CACCCAG; R CGACTTC GCCGAGATG
(Table III and Fig. 4). Images were stored as
TCCAGC CAG; IL-1b F-GTC TCT GAA TCA
RGB (red, green, blue) TIF format image files
GAAATCCTTCTATCR-ATGTCAAATTTC
(Fig.5).FromtheseRGBimages(Fig.5a)only
ACT GCT TCA TCC; cpla2 F-GGA TTC TCT
theREDchannelimage(Fig.5R)wasusedsince
GGTGTGATGAAGG,R-CCCAATCTGCAA
thenuclearstructureisbestrepresentedbythis
ACATCAGCep1F-CTGGAGGGGCGTGTC
channel.
ATTTC,R-GCTGCGGAGGGTGGCTGTGG
ep2F-CTACGCCGCCTTCTCTTACATG,R- Image Preprocessing and Segmentation
GATGTCTTTCACCACGTTTGGC;ep3CAT
All image analysis was performed with IDL
CACCATGATGGTCACTGGC,R-GTCTTC
software(InteractiveDataLanguage,Research
ATGTGGCTGGGATACC;LDLrF-CAATGT
System,Inc.,Boulder,CO).Forsegmentation,a
CTCACCAAGCTCT,R-TCTGTCTCGAGG
globalluminancevaluewasautomaticallyesti-
GGT AGC T; cyclin A F AAA CAG CCT GCC
matedfromeveryimageLandusedforthresh-
TTCACCATTCAR-ATATTCTTCTCCCAC
oldingoftheimagecontentintobackgroundand
CTCAACCA;cyclinE,F-CAAGTGGCCTAT
rough cell nuclei mask M (Fig. 6). Cell cluster
GTCAACGAR-ACTGCTGGGTGGGGGTGT r
consisting of touching or occluding cells were
CA; PCNA F-CAC CAA ATC AAG AGA AAG
delineatedbymanualinteraction.Bordertouch-
TT, R-GAG AAT GGA GAG AAA AGA AA; c-
ingcellsweredeletedresultinginmaskM.The
mycF-CCAACAGGAACTATGACCTCG,R-
CCACATACAGTCCTGGATG;cjunF-GGA
TABLE II. NumberofImagesandCells
AACGACCTTCTATGACGATGCCCTCAA,
Evaluated
R-GAACCCCTCCTFCTCATCTGTCACGTT
CTT; collagen, F-GTC CCC CTG GCT CTG
Number Number
CTG GTT, R- TTT GGG TTG TTC GTC TGT
Abbreviation ofimages ofcells Description
TTC; b1 Integrin, TGT TCA GTG CAG CCT
gr 3 62 Normalgravity
TCA,R-CCTCATACTTCGGATTGACC;bax
1g(flight) 5 88 Gravityby
F-CAGCTCTGAGCAGATCATGAAGACA, centrifuge
mg(flight) 3 52 Staticinflight
R-GCCCATCTTCTTCCAGATGGTGAGC.
440 Hughes-Fulford et al.
TABLE III. Three-ClassJackknifedClassificationResultsWiththeFour
MostImportantFeaturesandTheirCorrespondingF-Values
(AlsoseeTableVI)
% gr 1g mg Feature F-value
gr 75 47 13 2 P2A 49.7
1g 58.0 22 51 15 HETERO 28.4
mg 88.5 1 5 46 GCV 22.7
Total 71.3 RHA 11.3
cuttinglinesfortheedgeoftouchingcellswere set only those features were used in the
setinthecenteroftheoverlappingoroccluding, selection steps that were considered indepen-
typically lens-shaped areas. Nuclei with large dent of possible global acquisition influences
overlap with others were rejected from further such as relative features and which were
analysis.Forfeatureextraction,theREDchan- univariatesignificant.Typically,featuresmea-
nel, image R (Fig. 5R) was transformed to the suring area or total fluorescent intensity were
fluorescence density image F (Fig. 5c) by the only used after normalization, for example,
formula: particle area divided by nuclear area. Due to
the small number of cells, only up to seven
255(cid:3)R
F ¼(cid:3)log (three-class) or six (two-class) features respec-
maxð255(cid:3)RÞ
tivelywerestepwiseselectedonthebasisofF-
Thisisanalogoustothewell-knowntransfor- statisticstoavoidoverfitting.Thevalueforthe
mationfromtransmittedlightTtoextinctionE. first chosen feature is univariate whereas the
Extinctionisequivalenttotheopticaldensityin following F-values are multivariate reflecting
quantitativemicroscopy. the impact of results after using this feature
togetherwiththealreadyselectedfeatures.The
T
E¼(cid:3)log classification matrices show the results of the
maxT hold-one-out (jackknifed) method because no
testsetwasavailable.Allstatisticalevaluations
Feature Extraction
were done at the 95% confidence level. For
From each cell nucleus, represented by its display of the three-class discrimination, the
mask M and the corresponding region in the canonical variables are used which are linear
fluorescentdensityimageF,numerousquanti- combinations of the selected features. Linear
tative features were calculated describing discriminant analysis results in the so called
shape, photometric, and textural properties. discriminant function defined by a ((n(cid:3)1)-
The latter are understood as properties of the classes(cid:1)m-feature) matrix D. The canonical
arrangement of fluorescent particles, which in variablesCANarethandefinedas
thiscaseisDNA,visualizedbyHoechst33528. 8 9
feature
Thesefeaturesaredescribedindetailelsewhere < 1=
CAN ¼D(cid:4) (cid:4)(cid:4)(cid:4)
[RodenackerandBengtsson,2003](Fig.7).For
:feature ;
n
classification, selected features are briefly
describedinTables6and7.Theyareillustrated ThisistheproductofthediscriminantmatrixD
by image sequences showing the changing and the selected features. This is a method to
featurevaluewithappropriatecellimages. projecttheobservationsforvisualization.
RESULTS
Statistics and Classification
Gene Expression and Protein Levels
All statistical evaluations were completed
inMicrogravity
using SAS (SAS Institute, Inc., Cary, NC) and
BMDP(StatisticalSoftware,Inc.,LosAngeles, Weanalyzedinductionof24osteoblastgenes
CA)programpackages.Allcellsfromspecimens using linear semi-quantitative RT-PCR. We
belongingtothesameexperimentalconditions found reduced gene expression in 10 of these
were pooled and stepwise linear discriminant genes; no change in 9 of the genes and copy
analysis was applied. From the whole feature number was too low to measure in the
Reduction of Anabolic Signals 441
Fig.2. Analysisofgeneexpressionat3hunder1gflight,mgflight,andground.N¼4,significancebetween
1gandmglevels:P<0.001,significanceingroundandmglevels:P<0.01(barsdenotes(cid:2)SD).
remaining genes examined. Osteoblasts were genous levels of cellular fgf-2 message and
activated during space flight in mg with and protein in these microgravity grown samples.
without a 1 g field and results compared to gr Inourstudies,wefoundthatthefgf-2message
samples. Induction of PCNA, TGFb, cox-2, induction was depressed 24 h after activation
cpla2, osteocalcin, c-myc, fgf-2, bcl2, and bax (Fig. 3). Moreover, when the cellular protein
were significantly depressed in mg when com- levels were measured in these cells, we found
pared to gr (Figs. 2 and 3). Artificial onboard that FGF-2 protein synthesis was also signifi-
gravitywasabletonormalizetheinductionofc- cantly reduced in mg flight, however, the
myc,cox-2,TGFb,fgf-2,bax,andbcl2aswellas presenceofartificialgravityin-flightincreased
FGF-2proteinsynthesisinspaceflightsamples. theabilityofthecellstosynthesizefgf-2(Fig.3)
Therewasnochangeinquiescentosteoblast 24hafterserumactivation.Finally,changesin
PGE levels (gr 415(cid:2)163 vs. mg 453(cid:2)189). bcl2 message(Fig.3)inductionparallel thatof
2
Eventhoughthecox-2messagewasreducedat fgf-2 message. The bcl2 message was signifi-
24 h, the PGE levels were high. However, in- cantly decreased in microgravity compared to
2
flight at 24 h, both the 1 g and the mg samples groundand1gsamples.
had a significant rise in PGE levels: 1 g
2 Three-Class Case
6,065(cid:2)870 and mg 6,959(cid:2)1132 versus gr
3,904(cid:2)1673(P<0.005). This results in the most discriminative fea-
Since it has been previously demonstrated turesP2A,HETERO,GCV,RHA,RHUA,NR1,
that a lack of mitogen causes elongation of and HLM3. A short description of the features
nuclei [Foote et al., 1996], we measured endo- selected is given in Tables 6 and 7. Cell nuclei
442 Hughes-Fulford et al.
Fig. 3. Analysisofgeneexpressionat24hunder1gflight,mgflight,andground.N¼4,significance
between1gandmglevels:P<0.001,significanceingroundandmglevels:P<0.01(barsdenotes(cid:2)SD).
showcleardifferencesinshapeandtexture.The ing and the less pronounced change between
firstselectedfeature(P2A)isshape,remaining background inside the nucleus and bright
features are all texture related. Variation of particle(GCV).
P2Aisbasedonthedeviationfromcircularityto TheclassificationresultislistedinTable3and
ellipticity. Further differences are reflected by illustrated in Figure 8 using the 1st and 2nd
several features sensitive to the different canonicalvariables(CAN1,CAN2)withtheclass
arrangement of bright fluorescent particles discriminationlines.Usingthree-classdiscrimi-
(HETERO)insidethedarkernucleussurround- nationanalysis,itisclearthatthegrandmgcell
Fig.4. Originalfluorescenceimage(gr)(left),mg(right).
Reduction of Anabolic Signals 443
Fig. 5. Effect of different filters on discrimination of features (a) original fluorescence image, (b) light
intensityimage,(c)fluorescentintensityFfromREDchannelwithfrequencydistribution,(R)REDwith
frequencydistribution,(G)GREEN,and(B)BLUEintensitychannel.(Colorfigurecanbeviewedintheonline
issue,whichisavailableatwww.interscience.wiley.com.)
populations are significantlydifferent from one From ground (gr) to 1 g (in-flight) (Table 4):
another.ThefirstcanonicalvariableCAN1alone The selected features HETERO, ELNENL,
permits a nearly complete discrimination at RHA, and NR4 represent pure textural
aboutCAN1¼0.8ofthemgcellsfromtheothers changes.
(seeFig.8).Asequenceofcellimagesisdisplayed From 1 g (in-flight) to mg (Table 5a): The
inFigure9usingthefirstcanonicalvariableas sequenceofselectedfeaturesstartswithP2A,a
order criteria. Note the change of particle shapefactorshowingtheinfluenceofgravityon
structurefromlefttoright. growth form, followed by texture features
HLM3andRHUA.
Two-Class Cases
From ground (gr) to mg (Table 5b): For
Theywereanalyzedtofindoutmoreaboutthe completenessthedirecttransitionfromground
changes from one experimental condition to (GR)tomgisalsooutlined.Chosenfeaturesare
anotherintermsofmostselectivefeatures. HETERO,RHUA,andRATIO.Thefirsttwoare
pure textural and the last is a shape feature
similartoP2A.
The two-class discrimination analysis pro-
videdacomparableresultwiththeheterogene-
ity of nuclear texture (HETERO) showing the
greatest difference between gr and mg (correct
class94.7%).Asimilardifference,withalower
correlationwasobservedfor1gandmg(correct
class 86.4%). Furthermore, the 1 g cells have
very similar morphology to the ground cells
(correctclass78.7%).Inotherwords,theground
and1gsamplesaremoresimilartoeachother
than they are to mg cells. The morphological
deviationofmgcellsmaybeduetotheinfluence
of gravity on the arrangement of nuclear
Fig. 6. Segmentation with mask M by threshold (red) and
r particlesasaresultofalteredgeneexpression.
manualcuttinglines(green).(Colorfigurecanbeviewedinthe
Allotherselectedfeatures(HLM3,RHUA,NR2,
online issue, which is available at www.interscience.wiley.
com.) NC9) except the last are features of particle
444 Hughes-Fulford et al.
Fig.7. Illustrationsoffeatureextractionforonenucleus.
arrangement. RATIO reflects an additional mg. Since changes in autocrine, growth, cytos-
shapeinfluence. keletal,andtranscriptionfactorsmaybeimpor-
tant in osteoblast physiology in spaceflight
DISCUSSION [Cubano and Lewis, 2000; Goodwin et al.,
2000], we analyzed 24 osteoblast genes under
Combatingspaceosteoporosisisakeyfactor
mg, 1 g, and gr conditions. Of the genes with
in planning long-range space missions of
significantlyalteredexpression,therewerefour
greaterthan6months.Inordertounderstand
cell cycle related, one osteoblast specific, two
underlyingcellularmechanismsofdisuseosteo-
immediate early, and two growth factor genes
porosis, osteoblasts were grown in mg and 1 g
reducedatoneorbothofthetimepoints.Many
on the Space Shuttle and were analyzed for
genes had no significant change in message
changes in gene expression and morphology
level,theseincluded18S,H4histone,fibronec-
after growth stimulation. We noted visual
tin, b-actin, cyclophilin, and prostaglandin E
differences in nuclear shape in cells growth in 2
receptor 1 (EP1). In contrast, there were nine
genes that had a significant reduction in
message induction in mg, these included cox-2,
cpla2, PCNA, bcl2, bax, c-myc, TGFb, OC, and
fgf-2 suggesting a change in anabolic response
intheabsenceofgravity.
Of particular interest was the reduction in
cox-2 message in the presence of high levels of
PGE . Cox-2 is a key enzyme in the mainte-
2
nanceofboneinresponsetostressandexercise
[Forwood, 1996; Forwood et al., 1998]. More-
over, cox-2 is a feed-forward enzyme, which is
induced by its own product, PGE [Pilbeam
2
etal.,1995].Inmg,thisfeed-forwardregulation
becomes uncoupled, with PGE levels signifi-
2
cantly increased in both the 1 g (P<0.05) and
the mg (P<0.001) osteoblasts when compared
to gr controls. The underlying mechanism for
this uncoupling is currently under study
using modeled mg and understanding the loss
offeed-forwardinductionofcox-2atthecellular
Fig.8. Three-classdiscriminationresultusingthe1stand2nd
levelmayhelpusunderstandthelossofbonein
canonicalvariableswithdiscriminationlinesbetweenrespective
classes. astronauts.