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Functional & Integrative Genomics
ISSN 1438-793X
Volume 12
Number 2
Funct Integr Genomics (2012)
12:357-365
DOI 10.1007/s10142-012-0266-3
Preservation of bone mass and structure
in hibernating black bears (Ursus
americanus) through elevated expression of
anabolic genes
Vadim B.Fedorov, Anna
V.Goropashnaya, Øivind Tøien, Nathan
C.Stewart, Celia Chang, Haifang Wang,
Jun Yan, Louise C.Showe, Michael
K.Showe, et al.
1 23
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ORIGINAL PAPER
Preservation of bone mass and structure in hibernating black
bears (Ursus americanus) through elevated expression
of anabolic genes
Vadim B. Fedorov &Anna V. Goropashnaya &
Øivind Tøien &Nathan C. Stewart &Celia Chang &
Haifang Wang &Jun Yan &Louise C. Showe &
Michael K. Showe &Seth W. Donahue &Brian M. Barnes
Received: 21 October 2011 / Revised: 12 January 2012 /Accepted: 7 February 2012 /Published online: 18 February 2012
#Springer-Verlag 2012
Abstract Physical inactivity reduces mechanical load on
the skeleton, which leads to losses of bone mass and
strength in non-hibernating mammalian species. Although
bears are largely inactive during hibernation, they show no
loss in bone mass and strength. To obtain insight into
molecular mechanisms preventing disuse bone loss, we
conducted a large-scale screen of transcriptional changes
in trabecular bone comparing winter hibernating and sum-
mer non-hibernating black bears using a custom 12,800
probe cDNA microarray. A total of 241 genes were differ-
entially expressed (P< 0.01 and fold change >1.4) in the
ilium bone of bears between winter and summer. The Gene
Ontology and Gene Set Enrichment Analysis showed an
elevated proportion in hibernating bears of overex-
pressed genes in six functional sets of genes involved
in anabolic processes of tissue morphogenesis and de-
velopment including skeletal development, cartilage de-
velopment, and bone biosynthesis. Apoptosis genes
demonstrated a tendency for downregulation during hi-
bernation. No coordinated directional changes were
detected for genes involved in bone resorption, although
some genes responsible for osteoclast formation and
differentiation (Ostf1,Rab9a,andc-Fos) were signifi-
cantly underexpressed in bone of hibernating bears.
Elevated expression of multiple anabolic genes without
induction of bone resorption genes, and the down reg-
ulation of apoptosis-related genes, likely contribute to
the adaptive mechanism that preserves bone mass and
structure through prolonged periods of immobility dur-
ing hibernation.
Keywords Hibernation .Bone biosynthesis .Gene
expression .Apoptosis
Introduction
Mammalian hibernation is an adaptation involving metabol-
ic suppression to conserve energy during periods of low
food availability in highly seasonal or unpredictable envi-
ronments. Black bears hibernate for up to 6 months each
year, and during hibernation, bears remain largely immobile
and do not eat, drink, urinate, defecate, and they reduce
metabolic rate by 20–50%, yet maintain core body temper-
atures above 30°C (Nelson 1980; Tøien et al. 2011).
Electronic supplementary material The online version of this article
(doi:10.1007/s10142-012-0266-3) contains supplementary material,
which is available to authorized users.
V. B. Fedorov (*):A. V. Goropashnaya :Ø. Tøien :
N. C. Stewart :B. M. Barnes
Institute of Arctic Biology, University of Alaska Fairbanks,
Fairbanks, AK 99775, USA
e-mail: vfedorov@alaska.edu
C. Chang :L. C. Showe :M. K. Showe
Systems and Computational Biology Center, the Wistar Institute,
Philadelphia, PA 19104, USA
H. Wang :J. Yan
CAS-MPG Partner Institute for Computational Biology,
Shanghai Institutes of Biological Sciences,
320 Yue Yang Road,
Shanghai 200031, China
S. W. Donahue
Department of Biomedical Engineering,
Michigan Technological University,
309 Minerals and Materials Eng. Bldg., 1400 Townsend Drive,
Houghton, MI 49931, USA
Funct Integr Genomics (2012) 12:357–365
DOI 10.1007/s10142-012-0266-3
Author's personal copy
Physical inactivity decreases mechanical load on skel-
eton, which when prolonged leads to losses of muscle
and bone mass and strength in non-hibernating mamma-
lian species (Kaneps et al. 1997; Harlow et al. 2001).
Disuse-induced bone loss occurs by increases in osteo-
clastic bone resorption and/or decreases in osteoblastic
bone formation (Zerwekh et al. 1998). This unbalance
in resorption/formation leads to increased serum and
urinary calcium concentrations (Watanabe et al. 2004).
Although bears are largely inactive during hibernation,
they show no loss in bone mass and less loss in muscle
mass and strength than would be anticipated over such
a prolonged period of physical inactivity (Harlow et al.
2001; Floyd et al. 1990; McGee et al. 2008;McGee-
Lawrence et al. 2008,2009;Nelsonetal.1975). An
important adaptive consequence is that bears maintain
skeleton function and preserve mobility during and after
winter hibernation. This suggests that bears have unique
mechanisms to prevent disuse-induced bone loss and
reduce muscle atrophy during the inactivity of hiberna-
tion. Hibernating bears prevent bone loss by maintaining
balanced bone resorption/formation, and they maintain
normal serum calcium concentration despite anuria
during this long period of disuse (Floyd et al. 1990;
McGee et al. 2008; McGee-Lawrence et al. 2008,2009). The
molecular mechanisms that sustain balanced bone turnover
during hibernation are unknown, but there is some evidence
forendocrine or paracrine mechanisms (Donahue et al.
2006). For example, serum from hibernating bears
decreases apoptotic signaling in osteoblastic (bone form-
ing) cells compared to serum from non-hibernating
bears (Bradford et al. 2009). Furthermore, bear stem
cells may have the unusual ability to spontaneously
differentiate down an osteoblastic lineage and form
bone-like nodules (Fink et al. 2011). However, there
has not before been a genome-wide screening of tran-
scriptional changes in bone of hibernating bears to
identify functional groups of co-regulated genes and
provide insight into the molecular mechanisms that pre-
vent disuse-induced bone loss.
We previously developed genomic resources for the
black bear (Zhao et al. 2010) and detected transcriptional
changes at the genomic scale in liver, heart, and skeletal
muscle during hibernation (Fedorov et al. 2009; Fedorov et
al. 2011). In the context of preserving skeleton functionality,
an important finding was that protein biosynthesis genes
demonstrated elevated expression in hibernating bears. This
implies induction of translation and suggests activation of
energy expensive anabolic mechanisms that contribute to
the adaptive ability to reduce muscle atrophy over long
periods of fasting and immobility during hibernation
(Fedorov et al. 2009). In the present study, we use a custom
12,800 cDNA microarray to reveal transcriptional changes
in trabecular bone of hibernating bears compared to animals
sampled during summer. We conducted pathway analyses to
identify functional groups of co-regulated genes and
assessed the biological significance of the transcriptional
changes. We also assessed differences in the expression of
individual genes involved in bone formation, resorption, and
apoptosis. Transcriptional changes are considered in light of
previous findings such as balanced trabecular bone turn-
over (McGee-Lawrence et al. 2009) and reduced apoptosis
(Bradford et al. 2009) that prevent disuse osteoporosis during
hibernation.
Material and methods
Animals
We sampled the ilium bone from black bears including
animals reported on in a previous study (Fedorov et al.
2009). Bears (51–143 kg) were captured May–July (two
bears sampled in hibernation were captured in October)
from the field in Alaska and transferred to Fairbanks. In
order to decrease intragroup variation in gene expression,
only males >2 years old are compared in the experiments.
Summer active bears that were still feeding and active were
euthanized and sampled for tissues between late May and
early July (n05) and one bear was sampled on October 2
(Fig. 1). Food was withdrawn 24 h before these animals
were sacrificed. Bears in the hibernating condition were
euthanized for tissue sampling between March 1 and 11
(n05), about 1 month before expected emergence from
hibernation. These animals were without food since October
27. Animal protocols were approved by the University of
Alaska Fairbanks Institutional Animal Care and Use Com-
mittee (protocol nos. 02-39, 02-44, 05-55, and 05-56) and
USAMRMC Animal Care and Use Review Office (proposal
number 05178001).
Physiological monitoring and tissue harvesting
For monitoring of physiological conditions, bears were
instrumented as previously described (Fedorov et al.
2009). Briefly, core body temperature, ECG, and EMG
were monitored with radio telemetry. Beginning in late
November, bears were housed in individual undisturbed
outdoor enclosures that had dens with straw material for
nests. Oxygen consumption and respiratory quotient
weremonitoredwithopenflowrespirometrybydrawing
air from the closed dens. On the day of tissue harvest-
ing, bears were immobilized using Telazol (8–10 mg/kg)
and transported to a necropsy suite in a nearby building.
Oxygen consumption in the immobilized state was
checked on a subsample of animals, with an open flow
358 Funct Integr Genomics (2012) 12:357–365
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respirometry system during blood sampling, just prior to
euthanasia via a tracheal tube. Between the first distur-
bance of bears and the beginning of tissue sampling, 41–
65 min elapsed. Bears were euthanized by an intravenous
injection of pentobarbital with death assessed by termination
of heart beats as assessed with a stethoscope. Tissues collec-
tion followed immediately with samples frozen in liquid ni-
trogen within 12 min of death. One trabecular bone sample
80× 20 mm was cut from the ilium tuber coxae of each bear.
RNA preparation
Bone tissue was pulverized in a metal cylinder cooled in
liquid nitrogen with a piston and then transferred into Trizol
reagent with 0.1 volume of chloroform. The mix was centri-
fuged at 13,000×gfor 20 min at 4°C, and a clear aqueous
phase was added to 0.5 volume of 2-propanol and left for
10 min at room temperature. Then the mix was centrifuged
at 13,000×gfor 20 min at 4°C and the pellet was washed
with ethanol twice and resuspended in RNase-free water.
Additional RNA cleanup was performed with the Qiagen
RNeasy kit. All RNA samples were processed by DNase I
(Qiagen) treatment. RNA quality was evaluated with an
Agilent 2100 Bioanalyzer and concentration was measured
by using Nanodrop ND-1000.
Hybridization
RNA samples were hybridized with the two bear arrays
(BA01 and BA02) that contain 3,200 and 9,600 cDNA
probes representing unique annotated genes in the black
bear expressed sequence tags (ESTs) collection (Zhao et
al. 2010; Fedorov et al. 2011). Samples of total RNA were
linearly amplified with Illumina TotalPrep RNA Amplifica-
tion Kit (Ambion), and 1.6 μg of the amplified RNA was
labeled with 65 μCi of [33P]dCTP as previously described
(Kari et al. 2003). All RNA samples were amplified, la-
beled, and hybridized in the same batch. The hybridization
was carried out for 18 h at 42°C in 4 ml of MicroHyb buffer
(Invitrogen). Filters were rinsed at room temperature with
2× SSC/1% SDS to remove residual probe and MicroHyb
solution and then transferred to preheated wash solutions in
a temperature-controlled shaking water bath. Filters were
washed twice for 30 min in 1.5 l of 2× SSC/1% SDS at
Fig. 1 Gene set enrichment analysis results for the bone biosynthesis
(ossification) category. The ossification category is enriched by upre-
gulated genes in the ilium bone of hibernating black bears. An expres-
sion data set sorted by correlation with hibernating phenotype and the
corresponding heat map with red for upregulated and blue for down-
regulated genes during hibernation are shown on the left.Dates on the
top indicate time of tissue sampling from each bear. Plot of the running
sum for enrichment score (ES) in the data set (top) and location of
genes (hits) from the GO category in the list ranked according to
expression differences (middle) and the ranked list metric (bottom)
are shown on the right
Funct Integr Genomics (2012) 12:357–365 359
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50°C and then once for 30 min in 1.5 l of 0.5× SSC/1% SDS
at 55°C and once for 30 min in 1.5 l of 0.1× SSC/0.5% SDS
at 55°C. Filters were then exposed to phosphorimager
screens for 4 days and scanned at 50-μm resolution in a
Storm Phosphorimager. Image analysis was performed with
the ImaGene program (Biodiscovery).
Microarray data analysis
Hybridization signals were corrected for background, nor-
malized and one-way ANOVA test was used to select genes
that exhibited significant differences between hibernating and
summer active bears (Fedorov et al. 2011). A Pvalue <0.01
and |log
2
fold change|>0.5 were set as cutoffs for significant
differences in expressed genes, corresponding to the mean
false discovery rate (FDR) around 24%. The FDR was calcu-
lated using random permutation as described by Storey and
Tibshirani (2003). Lists ofall significant genes on the array and
differentially expressed genes with cutoffs of Pvalue <0.05
and |log
2
fold change|>0.5 were uploaded to Gene Ontology
(GO) miner (http://discover.nci.nih.gov/gominer/index.jsp).
The false discovery rate was assessed by resampling all the
significant genes on the array (Zeeberg et al. 2003,2005). In
addition to GO miner analysis, we verified enrichment in
significant GO categories of the biological processes by using
Gene Set Enrichment Analysis (http://www.broad.mit.edu/
gsea/index.jsp). Genes were ranked according to the correla-
tion between their expression values and the phenotype class
(hibernating and summer active phenotypes) distinction by
using the signal to noise ratio. An enrichment score (ES) that
reflects the degree to which genes involved in category are
overrepresented at the extremes (upregulated genes at the top
and downregulated genes at the bottom) of the entire ranked
list of genes was calculated. The ES wasnormalized to account
for the size of the category gene set presented in the experi-
ment, yielding a normalized enrichment score (NES). A cutoff
of 25% for false discovery rate of gene set enrichment was
used as this value was suggested appropriate for exploratory
studies (Subramanian et al. 2005). We also used Gene Set
Enrichment Analysis (GSEA) to test enrichment in selected
gene sets that were reported to be important for bone metabo-
lism. These gene sets were obtained from Molecular Signa-
tures Database (www.broadinstitute.org/gsea/msigdb/index.
jsp). All microarray data series were submitted to NCBI Gene
Expression Omnibus with accession number GSE35796.
Quantitative real-time PCR
We validated the microarray experiments by 220 quantita-
tive real-time PCR (RT PCR) tests using the same total RNA
samples. Twenty genes were tested. Hint1 was selected as a
reference gene for bone based on the stability of expression
values across all samples obtained from the microarray
experiments and then tested by RT PCR. All bear samples
showed similar expression values for Hint1 with low stan-
dard deviation in multiple RT PCR tests. Total RNA con-
centrations were measured with a NanoDrop ND-1000
spectrophotometer, and cDNA was synthesized from
0.5 μg of total RNA from each sample. The reverse tran-
scription was carried out with MiltiScribeTM reverse tran-
scriptase (Applied Biosystems) with oligo d(T)16 primer in
25-μl reactions at 25°C for 10 min, 48°C for 30 min, and at
95°C for 5 min. The synthesized cDNA was diluted four
times with RNase-free water, and 4 μl of diluted cDNA was
used in the 20-μl volume real-time PCR. Primers were
designed with the Primer3 software (http://frodo.wi.mit.
edu/primer3/) using bear EST sequences (Table S1). Real-
time PCR was performed in triplicates with Power SYBR
Green PCR Master Mix (Applied Biosystems) on an ABI-
7900 HT. Cycling parameters were 50°C for 2 min of
incubation, 95°C for 10 min of Taq activation, and 40 cycles
of 95°C for 15 s and 60°C for 1 min. Controls with no
template were set to exclude contamination, and controls
with no reverse transcriptase but all other components were
taken to exclude nonspecific amplification from genomic
DNA. Specificity of amplification was checked with the
melting curve analysis and agarose gel electrophoresis. Four
tenfold dilutions of a sample with mixed cDNA were used
for a standard curve for each primer set for calculating RT
PCR efficiency. We tested a difference in gene expression
between hibernating and summer active black bears with
P<0.10 as cutoff according to Pfaffl (2001). We calculated
the fold change in level of expression of a target gene
relative to a reference gene for each sample and then com-
pared the values for each group using Student’sttest as
described by Livak and Schmittgen (2001).
Results
Body temperature and metabolism
At the time of sampling between March 1 and 11, hibernating
bears had core body temperatures of 34.5±0.5°C (mean ± SD,
n05) and minimum rates of oxygen consumption of 0.078±
0.001 ml g
−1
h
−1
(n04), when measured over at least a 0.5-
hinterval2–9 h before euthanasia (Fedorov et al. 2009). In
three bears anesthetized before euthanasia, body temperature
had decreased to 33.6± 1.0°C and metabolic rate had increased
to 0.105± 0.012 ml g
−1
h
−1
. Ambient temperatures during the
study period in winter were −10°C to −35°C with short
periods with extremes of −43°C to 5°C. Temperatures within
bear dens were about 10°C above the outside temperature.
Bears lost 4.3±0.6% (n05) of their body mass per month
during the 4–5-month hibernation period. In two summer
active bears, fasted for 24 h and anesthetized before
360 Funct Integr Genomics (2012) 12:357–365
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euthanasia metabolic rates were 0.232 ml g
−1
h
−1
(range 0.252
to 0.213 ml g
−1
h
−1
) and body temperatures averaged 37.2°C.
Thus, immediately prior to tissue sampling, metabolic rate of
anesthetized hibernating bears was 45.4% of that of anesthe-
tized summer bears, with a 3.5°C lower body temperature.
Differentially expressed genes
Signals from 2,602 of 3,200 probes (81%) on bear array BA01
showed median intensities that were above background,
whereas 9,473 of 9,600 (99%) probes showed significant
signals on bear array BA02. To identify genes that were
differentially expressed in hibernating compared to summer
active bears, we used P<0.01 and |log
2
FC|>0.5, where FC is
fold change (the mean expression value in the hibernating
bears divided by the mean expression value in the summer
active bears) as the criteria for differentially expressed genes
as previously reported for other tissues (Fedorov et al. 2009,
2011). A total of 241 genes (2.6% of all genes with significant
signals) were differentially expressed in bone during hiberna-
tion (Table S2). All but two differentially expressed genes
demonstrated changes in expression that were less than four-
fold differences (|log
2
FC|<2). Of the significantly differen-
tially expressed genes, we identified 71 (29.5%) genes that
were overexpressed and 170 (70.5%) genes that were under-
expressed in bone during hibernation.
The estimated mean false discovery rate of 24% indicates
that the expected proportion of false positives in the list of
differentially expressed genes from array experiments is
relatively high. To obtain an experimental estimate of the
false discovery rate, we conducted quantitative real-time
PCR tests for 20 randomly selected genes that showed
differences in expression at P<0.05 in the array hybridiza-
tion. Eighteen out of 20 genes (90%) tested showed signif-
icant changes in the same direction as the array results
(Table 1, Fig. 2). The observed value of 10% for the
false discovery rate is reasonable for this exploratory study.
An important point of support for microarray results comes
from the highly significant positive correlation (r00.89, P<
0.0001) between fold changes for true positive in RT PCR and
microarray experiments. Expression fold changes do not de-
pend on significance level (Pvalue) of individual genes, and
thus, they are not sensitive to false discovery rate that results
from multiple testing.
Functional gene sets enriched by differentially expressed
genes
Genes with a significant hybridization signal on the arrays
were classified according to their GO categories of biolog-
ical processes. GO categories with less than five differen-
tially expressed genes detected were excluded from the
analysis (Zeeberg et al. 2003). Significant enrichment of
biological processes categories by differentially expressed
genes was validated by the results of GSEA (Table 2;
Fig. 1). GSEA ranks all genes with significant signals in
the experiment; thus, its results do not depend on the selec-
tion of genes above cutoffs for significance of expression
differences and false discovery (Subramanian et al. 2005).
During hibernation, the proportion of overexpressed genes
was significantly elevated for six gene sets involved in ana-
bolic processes of tissues morphogenesis and development
(Table 2). Among upregulated genesets, there was the skeletal
development category that includes genes involved in carti-
lage development and ossification (Table 3). Although the
ossification category demonstrated enrichment by upregulated
genes only at P00.084 level in the GO miner and included six
downregulatedgenes,mostlyinvolvedinbonemineralization,
the GSEA showed overall significant (P00.023) upregulation
of bone biosynthesis genes (Fig. 1). Phagocytosis was the only
biological processes category with significantly elevated pro-
portion of downregulated genes (Table 2).
Apart from validation of the GO miner result, we also
used GSEA to test enrichment in selected gene sets known
to be important for bone homeostasis. Similar to bone bio-
synthesis, bone morphogenetic protein signaling pathway
(GO:0030509, 12 genes on the array) showed elevated pro-
portion of upregulated genes (NES01.39; FDR00.13). In
contrast, the bone mineralization category (GO:0030282)
was represented by ten genes on the microarray and was
enriched by downregulated genes (NES0−1.43; FDR 0
0.062) during hibernation. No significant directional changes
(FDR00.576) were detected in expression of seven genes
involved in bone resorption (GO:0045453). However, two
genes that induce osteoclast formation and bone resorption,
small GTP-binding Rab9 protein (Rab9a) and osteoclast stim-
ulating factor-1 (Ostf1), were both downregulated (Table S2)
during hibernation. Receptor activation of NF-κbligand
(RANKL) signaling pathway (Biocarta: M2602, two genes
on the array) that plays an important role in resorption during
bone remodeling showed downregulation (NES 0−1.32;
FDR00.085) during hibernation. In the RANKL gene set, c-
Fos gene, an important mediator of osteoclast differentiation,
was the most downregulated (Table S2). The apoptosis gene
set including all apoptosis-related genes from Molecular Sig-
natures Database was represented by 202 genes on the micro-
array and demonstrated a clear tendency for downregulation
during hibernation with 38 genes being downregulated and
overrepresented at the bottom of the GSEA ranked list of
apoptosis genes (NES0−1.22; FDR00.11).
Discussion
We sampled hibernating bears in early March after at least
4 months of continuous hibernation and when they were 4–
Funct Integr Genomics (2012) 12:357–365 361
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6 weeks from emergence and their resumption of summer
active levels of metabolism, body temperature, and feeding
(Fedorov et al. 2009). All bears included as summer active
or hibernating animals showed physiology and behavior
typical for bears during summer and winter seasons (Tøien
et al. 2011).
Of the genes that were differentially expressed in bone
during hibernation, 70.5% were downregulated. This con-
trasts to the lower proportions of downregulated genes that
were previously reported for the liver (48% underexpressed
genes), skeletal muscle (46% underexpressed genes), and
heart (25% downregulated genes) in hibernating bears
(Fedorov et al. 2009,2011). The decrease in the transcrip-
tion levels for a number of genes may reflect a lower level of
homeostatic activity in bone compared to other organs as-
sociated with prolonged period of immobility and skeleton
unloading during hibernation.
Our study reveals coordinated induction in transcription
of genes involved in anabolic processes of tissues develop-
ment and formation including osteogenesis and cartilage
development. This finding implies elevation of some forms
of anabolic activity in bone during hibernation. In contrast,
no coordinated directional changes were detected for genes
involved in bone resorption, although some genes responsi-
ble for osteoclast formation and differentiation (Ostf1,
Rab9a,andc-Fos) were significantly underexpressed in
bone of hibernating bears. Pronounced underexpression of
the c-Fos gene, member of RANKL signaling pathway, is
Table 1 Gene expression differences obtained in microarray experiments and tested with real-time PCR in bear bone
Gene symbol Gene name RT PCR Microarray
Plog
2
FC Plog
2
FC
B4galt6 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 0.100 −0.573 0.024 −0.843
Cav1 Caveolin 1, caveolae protein 0.039 1.395 0.023 1.055
Cd47 Leukocyte surface antigen CD47 precursor 0.012 −0.823 0.001 −1.300
Cfl1 Cofilin-1 0.380 n/a 0.012 −1.429
Dcn Decorin 0.005 1.142 0.008 0.955
Ect2 Epithelial cell transforming sequence 2 oncogene 0.040 −1.392 0.047 −0.792
Fn1 Fibronectin 0.013 1.334 0.027 1.192
Gpc3 Glypican 3 0.015 1.847 0.012 1.922
Hoxa10 Homeobox A10 0.003 1.181 0.008 0.831
Lnpep Leucyl/cystinyl aminopeptidase 0.080 0.561 0.041 0.631
Mapk8 Mitogen-activated protein kinase 8 0.180 n/a 0.006 0.837
Mef2c Myocyte enhancer factor 2 C 0.011 1.196 0.013 1.055
Omg Oligodendrocyte myelin glycoprotein 0.043 −1.053 0.009 −0.746
Prrx1 Paired related homeobox 1 <0.001 1.515 <0.001 1.488
Sdc2 Syndecan 2 0.050 0.422 0.013 0.889
Srgn Serglycin <0.001 −1.761 0.002 −1.863
Timp2 Tissue inhibitor of metalloproteinase 2 0.005 0.992 0.021 1.138
Ube2d3 Ubiquitin-conjugating enzyme E2D 3 <0.001 −0.581 0.039 −0.613
Yap1 Yes-associated protein 1 0.002 1.180 0.011 0.598
Zeb1 Zinc finger E-box binding homeobox 1 0.003 0.585 <0.001 0.841
Inconsistent significance levels are in bold
0.00
1.00
2.00
3.00
4.00
5.00
6.00
B4GALT6
CAV1
CD47
DCN
ECT2
FN1
GPC3
HOXA10
LNPEP
MEF2C
OMG
PRRX1
SDC2
SRGN
TIMP2
UBE2D3
YAP1
ZEB1
RT-PCR
Microarray
Fig. 2 Differentially expressed genes in black bear bone tissue con-
firmed with real-time PCR. Solid and open bars show normalized
expression values obtained in real-time PCR and microarray experi-
ments, respectively; error bars represent SD. The values were normal-
ized to the mean in summer active bears
362 Funct Integr Genomics (2012) 12:357–365
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remarkable as mice lacking this gene have overly dense
bone and decreased bone resorption due to reduced osteo-
clast differentiation (Takayanagi et al. 2002). Transcription-
al changes detected in our study may have implications
for an adaptive mechanism preserving bone mass and
structure through prolonged periods of immobility dur-
ing hibernation.
Bears demonstrate a unique ability to prevent bone loss
through prolonged periods of inactivity and skeleton disuse
during 4–6 months of winter hibernation. In other mammalian
Table 2 Selected Gene Ontology categories of biological processes significantly enriched with differentially expressed genes in black bear bone
GO category Total genes on array Changed genes GO Miner GSEA
Enrichment FDR NES FDR
Organ morphogenesis (GO:0009887) 400 34↑2.400 <0.001 1.74 0.008
Skeletal development (GO:0001501) 119 14↑3.268 0.008 1.59 0.077
Central nervous system development (GO:0007417) 218 18↑2.305 0.030 1.35 0.189
Muscle tissue development (GO:0060537) 117 13↑3.087 0.015 1.25 0.178
Cartilage development (GO:0051216) 33 6↑5.051 0.038 1.57 0.074
Ossification (GO:0001503) 49 6↑3.402 0.084 1.54 0.023
Phagocytosis (GO:0006909) 48 12↓3.111 0.023 −1.43 0.094
Arrows indicate direction of gene regulation in hibernating animals
FDR false discovery rate, NES normalized enrichment score
Table 3 Genes in selected Gene Ontology categories of the biological processes significantly enriched with differentially expressed genes in black
bear bone
GO category Gene name Gene symbol Pvalue Fold change (log
2
FC)
Skeletal development (GO:0001501) Decorin Dcn 0.008 0.955
Fibronectin 1 Fn1 0.027 1.192
Homeobox A10 Hoxa10 0.008 0.831
Insulin-like growth factor 1 receptor Igf1r 0.005 0.515
KIAA1217 Kiaa1217 0.029 1.083
Kruppel-like factor 10 Klf10 0.042 0.746
Mitogen-activated protein kinase 8 Mapk8 0.006 0.837
Myocyte enhancer factor 2C Mef2c 0.013 1.055
Paired related homeobox 1 Prrx1 <0.001 1.488
Retinol binding protein 4, plasma Rbp4 0.022 0.898
Schwannomin interacting protein 1 Schip1 0.025 0.769
SRY (sex determining region Y)-box 9 Sox9 0.010 0.568
Yes-associated protein 1 Yap1 0.011 0.598
Zinc finger E-box binding homeobox 1 Zeb1 <0.001 0.841
Cartilage development (GO:0051216) Decorin Dcn 0.008 0.955
Myocyte enhancer factor 2 C Mef2c 0.013 1.055
Paired related homeobox 1 Prrx1 <0.001 1.488
SRY (sex determining region Y)-box 9 Sox9 0.010 0.568
Yes-associated protein 1 Yap1 0.011 0.598
Zinc finger E-box binding homeobox 1 Zeb1 <0.001 0.841
Ossification (GO:0001503) Fibronectin 1 Fn1 0.027 1.192
Insulin-like growth factor 1 receptor Igf1r 0.005 0.515
Kruppel-like factor 10 Klf10 0.042 0.746
Mitogen-activated protein kinase 8 Mapk8 0.006 0.837
Myocyte enhancer factor 2C Mef2c 0.013 1.055
SRY (sex determining region Y)-box 9 Sox9 0.010 0.568
Funct Integr Genomics (2012) 12:357–365 363
Author's personal copy
species, mechanical unloading over periods of 4–17 weeks
resulted in bone loss of 9–29% due to decreases in bone
formation (McGee-Lawrence et al. 2008). Balanced remodel-
ing preserves the architecture and strength of hibernating
bear bone (Floyd et al. 1990;McGee-Lawrenceetal.2008,
2009; Pardy et al. 2004). Hibernating grizzly bears demon-
strate decreases in the overall number of remodeling sites
in the cortical bone (McGee et al. 2008), but no differences
in the total number of remodeling sites in trabecular bone in
the ilium (McGee-Lawrence et al. 2009). Ilium biopsies
from hibernating black bears, albeit a small sample size,
suggested some increase in trabecular bone remodeling
(with balanced resorption and formation) during hibernation,
which is believed to maintain bone structure and calcium
homeostasis (Floyd et al. 1990). The molecular mechanisms
that maintain constant and balanced trabecular bone remodel-
ing during hibernation, despite the challenge of physical in-
activity, are unknown.
Bone loss during immobility in mammals typically results
from unbalanced remodeling due to decrease in bone forma-
tion alone or both decreased bone formation and increased
resorption (McGee et al. 2008). Prolonged periods of mechan-
ical unloading during hibernation potentially inhibit anabolic
processes and promote reduction of bone formation. Coordi-
nated induction in transcription of genes involved in anabolic
processes implies elevation of anabolic activity in trabecular
bone (ilium) that may counteract disuse-induced reduc-
tions in osteoanabolic activity, thus, preventing bone loss
during hibernation. There was a lack of directional changes
in expression of the group of genes involved in bone resorp-
tion. However, there was significant downregulation of
three genes (Ostf1,Rab9a,andc-Fos) stimulating osteoclast
differentiation. This decreased gene expression may coun-
teract the mechanisms responsible for the increased osteoclas-
togenesis and bone resorption normally seen in disuse
conditions. We also found a reduction in the transcription of
genes responsible for bone mineralization, which is consistent
with histological data showing decreased mineral apposition
rate in the ilium (McGee-Lawrence et al. 2009). Decreased
mineral apposition rate may be part of globally reduced
metabolism for the conservation of metabolic energy during
hibernation.
It has been suggested that reduction in disuse-induced
osteoblast apoptosis contributes to the maintenance of bone
formation during hibernation (Bradford et al. 2009). Coor-
dinated suppression in transcription of apoptosis-related
genes detected in our study supports decreased apoptotic
activity in the ilium bone during hibernation. Underexpres-
sion of the two key apoptotic genes (Casp3,Casp7) during
hibernation is consistent with reduction of caspase 3/7 ac-
tivity detected in MC3T3-E1 osteoblasts cultured in
hibernation serum as compared to osteoblasts treated with
sera from fall and spring active black bears (Bradford et al.
2009). Seasonal changes in serum factors may maintain
normal osteoblastic activity and bone formation during hi-
bernation by reducing osteoblast loss due to apoptosis.
Our finding of increased expression of bone anabolic
genes without an increase in transcription of bone resorption
genes is similar to the elevated expression of anabolic genes
involved in protein biosynthesis and unchanged transcrip-
tional level of protein catabolism genes reported previously
for skeletal muscles of hibernating bears (Fedorov et al.
2009). In addition to preventing disuse-induced bone loss,
bears have a unique ability to preserve muscle mass and
strength during hibernation (Harlow et al. 2001; Lundberg et
al. 1976). Coordinated increase in transcriptional level of
anabolic genes involved in protein biosynthesis implies
induction of translation that may help prevent muscle atro-
phy during hibernation (Fedorov et al. 2009). Induction of
anabolic processes without increased catabolism (inferred
here from transcriptional changes) suggests that similar
adaptive mechanisms contribute to the preservation of bone
and muscle mass during prolonged periods of physical in-
activity and starvation (i.e., hibernation). Elevation of ener-
gy expensive anabolic processes is generally unexpected
because of the lack of dietary intake during hibernation,
which is an adaptive strategy involving metabolic suppres-
sion to conserve energy during periods of low food avail-
ability. However, the energy cost of increased anabolism in
bone and muscle may be an important trade-off with the
adaptive mechanisms that allow bears to maintain full mus-
culoskeletal function and preserve mobility during and im-
mediately after hibernation, thus promoting survival.
In conclusion, this study represents the first research
effort to elucidate transcriptional changes for thousands
of genes in trabecular bone during hibernation in com-
parison to summer active black bears. Elevated expres-
sion of multiple anabolic genes without induction of bone
resorption genes, as well as the downregulation of apoptosis-
related genes, likely contribute to the adaptive mechanisms
that preserve bone mass and structure through prolonged
periods of immobility during hibernation. Future studies
on the hibernating transcriptome in homogenous popu-
lations of different bone cell types (e.g., osteoblasts and
osteoclasts) with gene probes more specific for bone will
identify co-regulated functional groups of genes and provide
new insight to the molecular basis of unusual bone ho-
meostasis in hibernating bears. Understanding the mo-
lecular mechanism that prevents bone loss in hibernating
bears may identify novel therapeutic targets to improve treat-
ments for osteoporosis.
Acknowledgments We thank the Alaska Department of Fish and
Game for supplying bears. This work was supported by the National
Science Foundation EPSCOR program and USAMRMC (05178001).
364 Funct Integr Genomics (2012) 12:357–365
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