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GENE EXPRESSION ANALYSIS OF CHONDROCYTE MECHANICAL RESPONSE BY LARGE SCALE DNA
MICROARRAY
+Kanbe, K; +Inoue, K; *Xiang, C; **Wei, L; **Chen, Q
+Tokyo Women’s Medical University/Daini Hospital, Tokyo, Japan
*NIH/NIMH, **Brown Medical School/Rhode Island Hospital
Introduction: Mechanical stress-induced matrix deformation plays a
fundamental role in regulating cellular activities. Abnormal mechanical
loading is a hallmark for osteoarthritis development. Previously, we have
shown that mechanical stress plays an important role in regulating
chondrocyte proliferation and differentiation. We also identified several
mechanoresponsive genes in cartilage, such as Indian hedgehog, type X
collagen, and matrilin-1, which happen to be up regulated in OA
cartilage. In this study, we test the hypothesis that mechanoresponsive
genes in cartilage play an important role for transducing mechanical
signals to chondrocytes. To test this hypothesis, we identified
mechanoresponsive genes in cartilage by large scale DNA microarray
analysis. Conventional studies of gene expression and regulation have
been restricted to analysis of individual genes. In contrast, DNA
microarray technology provides a format for simultaneous measurement
of the expression level of thousands of genes in a single hybridization
assay. With DNA microarray, DNA probes representing thousands of
cDNA clones are arrayed onto glass slides and hybridized with
fluorescence-labeled cDNA derived from chondrocyte mRNA. Our study
was designed to determine the change of gene expression profile in
mouse chondrocytes subjected to mechanical stress induced deformation.
We determined the alteration of gene expression in response to
mechanical stress with cDNA microarray containing 12,000 sequenceverified mouse genes, about a third of total mouse genome.
Methods: 3D chondrocytes culture system: Primary chondrocytes were
isolated from rib cages of new born mice by digestion with collagenase D
(3 mg/ml) for 6 hours. Chondrocytes were collected and seeded onto
culture plates in DMEM contained 10% FCS and 1% of penicillinstreptomycin. After overnight incubation, chondrocytes were suspended
by trypsin digestion. One-hundred micro liters of cell suspension
containing 1 million chondrocytes were applied to 2x2x0.25-cm Gelform
sponges presoaked with Hanks’ balanced salt solution (HBSS). After
overnight incubation, the sponges were subjected to cyclic deformation
with a Bio-Stretch device to induce 5% elongation for 15 min/h at 1 Hz
for periods of 2 and 4 days for microarray.
Chondrocyte growth analysis: Collagen sponges containing chondrocytes
were digested with 0.03% collagenase, followed by collection of
chondrocytes by centrifugation. Chondrocytes were counted with a
hemacytometer. The viability of cells was confirmed by trypan blue
exclusion assay.
Isolation of mRNA: Total RNA from chondrocytes cultured in sponges
was extracted with RNeasy mini kit. Quantification of mRNA including
type X collagen and Indian hedgehog (Ihh) was performed by real-time
quantitative reverse transcriptase (RT)-PCR with a Perkin-Elmer ABI
Prism 7700 sequence detection system.
DNA Microarray: Five micro grams of total RNA were reverse
transcribed in the presence of Aminoallyl dUTP. cDNA from stretched
chondrocytes was coupled with Cy5, while cDNA from non-stretched
chondrocytes was coupled with Cy3 as reference. After hybridization,
labeled slides were scanned with GenePix 400A. Microarray data were
analyzed by IPLab software.
Results: Identified mechanoresponsive genes consist of candidate
components of mechanotransduction pathways including extracellular
matrix protein, ion channels, cytoskeleton, and nuclear transcriptional
factors. About 1 percent of the genes alter their expression significantly in
response to mechanical stress. The amplitude of the change of gene
expression by mechanical stress is modest, with most changes below 3fold. Biomechanically up-regulated genes after 2 days of mechanical
loading include those encoding extracellular matrix such as perlecan, HA
binding protein, osteopontin and follistatin, those encoding cytoskeletal
proteins such as beta spectrin2 and vimentin, those encoding cytoplasmic
proteins such as elongation factor 2 and cholesterol 24-hydroxylase, and
those encoding nuclear proteins including SOX-1 (Table 1). Downregulated genes in response to mechanical stress include those encoding
membrane proteins ATPase, cytoplasmic protein retinol binding protein 1
and nuclear protein CLK2 (protein kinase). After 4 days of cyclic
deformation, up-regulated chondrocyte genes including those encoding
membrane proteins such as Pak3 binding protein, those encoding
cytoplasmic protein such as ubiquitin specific protease 25, and those
encoding nuclear proteins such as transcription factor CA150. Down
regulated genes include collagen alpha 2 (VI) chain precursor in the
matrix, NMDA1 in the membrane, and CLOCK in the nucleus.
Discussion: Our microarray data not only confirmed mechanosensitive
genes identified previously, such as osteopontin and glutamate receptor
NMDA1, but also suggested unexpected genes, such as those in retinoic acid
signaling and circadian clock regulation. Since this is one of the first
analyses of chondrocyte mechanical response using large scale DNA
microarray, validation of the microarray data at three different levels was
carried out. First, known mechanoresponsive genes such as matrilin-1 and
Indian hedgehog were confirmed to be up-regulated by mechanical stress in
mRNA samples for microarray by real-time RT-PCR. Thus, these RNA
samples can be used for microarray analysis. Second, mechanoresponsive
genes identified by microarray, such as osteopontin, is verified by real-time
RT-PCR from the same RNA samples. Therefore, microarray analysis is
accurate for quantifying mRNA changes. Third, mechanoresponsive genes
identified previously, such as osteopontin and NMDA1, are confirmed by
our microarray analysis. Therefore, our microarray analysis is reliable and
can be used to identify new mechanoresponsive genes. Interestingly, we
identified a group of extracellular matrix proteins in cartilage to be
mechanoresponsive molecules. They include perlecan, osteopontin, HA
binding protein and type VI collagen. Among them, osteopontin and type VI
collagen are known to be upregulated in OA cartilage. Perlecan is also
implicated to play a mechanical role during cartilage growth and
differentiation as its mutation causes severe chondrodysplasia with
dyssegmental
ossification.
In
conclusion,
identification
of
mechanoresponsive genes in cartilage may help to better understand the
mechanism underlying abnormal activation of genes in OA and other
pathological cartilage that experiences abnormal mechanical loading.
Location
Description
matrix
perlecan
1.947
matrix
HA binding protein
1.898
matrix
osteopontin
sexreted
follistain
1.799
cytoskeleton
B-spectrin 2
1.855
cytoskeleton
vimentin
1.847
cytoplasm
proteasome ru93
1.774
cytoplasm
(3158)ribosomal protein
1.764
cytoplasm
thiolase peroxisomal
1.764
cytoplasm
elomgation factor 2
1.731
cytoplasm
ribosomal protein L3
1.723
cytoplasm
ribosomal protein L18
1.715
cytoplasm
Laminin receptor
1.712
cytoplasm
aldehyde reductase
1.708
ER membrane
cholesterol 24-hydroxylase
1.891
nuclear
tax interaction protein
1.811
nuclear
SOX-1
1.721
nuclear
YY1 interaction protein
1.717
50th Annual Meeting of the Orthopaedic Research Society
Poster No: 0818
Ratio
1.85