1 Division of Orthopedic Rheumatology, Department of Orthopedic Surgery,
University of Erlangen-Nuremberg, 91054 Erlangen, Germany
2 Molecular Biology Section, Division of Biology, University of California San
Diego, La Jolla, CA 92093, USA
3 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA
Author for correspondence (e-mail:
rsjohnson{at}ucsd.edu)
Accepted 23 January 2003
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Summary |
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Key words: Chondrocytes, Hypoxia, HIF-1, Extracellular matrix
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Introduction |
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The morphogenesis of long bones begins with the condensation of embryonic
mesenchymal cells (Grigoriadis et al.,
1988). After initial condensation, the mesenchymal cells
differentiate into chondrocytes. At the embryonic growth plate, these
chondrocytes proliferate and synthesize type-II collagen, aggrecan, small
proteoglycans and glycoproteins
(Scott-Savage and Hall, 1979
).
During this differentiation process the chondrocytic protein expression shows
a strict temporal and spatial distribution pattern
(Kirsch et al., 1997
). When
the differentiation process continues, the chondrocytes become hypertrophic
and express type-X collagen (Buckwalter et
al., 1987b
; Kirsch and von der
Mark, 1991
). Finally, growth-plate chondrocytes mineralize and
undergo terminal differentiation followed by apoptotic cell death
(Buckwalter et al., 1987a
;
Gerstenfeld and Shapiro, 1996
;
Hatori et al., 1995
;
Kirsch et al., 2000
). We and
other groups have provided evidence that central areas of the embryonic growth
plate have diminished oxygen levels
(Rajpurohit et al., 1996
;
Schipani et al., 2001
). We
have recently shown that HIF-1 is involved in the regulation of growth arrest
and survival of embryonic growth plate chondrocytes in vivo
(Schipani et al., 2001
). The
loss of HIF-1
in growth-plate chondrocytes induced apoptotic cell death
(Schipani et al., 2001
).
Apoptotic chondrocytes, normally restricted to the zone of terminal
differentiation, were apparent specifically in central areas of the growth
plate lacking oxygen. Our experimental mouse model suggested that HIF-1 might
act as a survival factor for chondrocytes by regulating the expression of
glycolytic enzymes and cell-cycle regulators.
To test our hypothesis that HIF-1-mediated alterations in energy
generation of chondrocytes might lead to an insufficient expression of type-II
collagen and aggrecan, we cultured murine growth-plate chondrocytes and
deleted their HIF-1
gene. In addition, we determined the role of
HIF-1
in energy production and cell growth under normoxic and hypoxic
conditions in vitro. We found that HIF-1 has an essential and unexpected role
in extracellular matrix synthesis. This, coupled to its role in chondrocyte
survival, makes it a factor of central importance in cartilage
morphogenesis.
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Materials and Methods |
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Determination of deletion frequency by quantitative PCR
All murine newborns used displayed homozygous flanking of the
HIF-1 locus by loxP sites as described in detail
previously (Ryan et al., 1998
;
Sauer, 1998
). At day 1
post-plating, adherent chondrocytes were infected with adenovirus containing
either ß-galactosidase or Cre recombinase (generously supplied by Frank
J. Giordano, Yale University, New Haven, CT) to create wild-type chondrocytes
(+/+) or HIF-1
-deleted cells (-/-), respectively. Cells were incubated
with adenovirus containing medium for 24 hours, fresh medium was added and
cells were allowed to recover for at least 24 hours before starting the
experiments. Genomic DNA was prepared by digestion in 10 mM Tris-HCl pH 7.5,
100 mM NaCl, 10 mM EDTA, 0.5% SDS with 0.4 µg µl-1 proteinase
K (Roche) overnight at 65°C. DNA was extracted with
phenol-chloroform-isoamylaclohol (25:24:1, pH 8.2) and precipitated with three
volumes of ethanol and half a volume of 6 M ammonium acetate. DNA was
resuspended in 10 mM Tris, 1 mM EDTA, pH 8.0. Primer express software (Applied
Biosystems) was used to design forward, reverse and fluorescein-dye-tagged
oligonucleotides (Operon) for use in real-time PCR
(Table 1). Loss of the
conditional HIF-1
alleles was measured using 0.9 µM each
forward and reverse primers, 0.25 µM fluorescein-dye-tagged oligonucleotide
and TaqMan Universal Master Mix (Roche). Reaction conditions were: 95°C
for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1
minute. The degree of excision was calculated by comparison of intact
HIF-1
DNA relative to an unexcised gene, in this case
c-Jun. Deletion efficiency was determined at 60, 80, 100 and 200
viral particles per cell [multiplicity of infection (MOI)]. By this approach,
a MOI of 100 was found to result in deletion frequencies of 74.1% and
therefore all further experiments were conducted using MOI 100. Cultures
infected with adenovirus containing ß-galactosidase (MOI 100) served as
wild-type controls.
|
Lactic acid measurement
Supernatants from different chondrocyte cultures were collected after 22
hours under normoxic or hypoxic (0.5% oxygen) conditions. Lactic acid was
determined by a colorimetric assay (Sigma) at 540 nm according to the
manufacturer's instructions. Lactic acid levels were normalized to total
protein content using the Bradford assay (Biorad).
ATP measurement
To measure ATP, chondrocytes were cultured 22 hours either with (20%) or
without (0.5%) oxygen. The ATP Bioluminescence Assay Kit CLS II (Roche) was
used. The assay is based on the light-emitting oxidation of luciferin by
luciferase in the presence of extremely low levels of ATP. After collecting
the chondrocytes by scraping, cells were centrifuged for 10 minutes at 500
g in the cold. Chondrocyte pellets were resolved in boiling
100 mM Tris buffer containing 4 mM EDTA. Boiling was continued for another 2
minutes in order to inactivate NTPases. Cell remnants were removed by a
further centrifugation step at 1000 g. Supernatants were
separated and placed on ice. Determination of free ATP concentrations was as
outlined in the manufacturer's protocol. Light emission was measured at 562 nm
using a luminometer (Berthold). ATP levels were normalized to protein content
using the Bradford assay (Biorad).
RNA isolation and RT-PCR
Chondrocytes were lysed by addition of RNA-Bee (Tel-Test, Friendswood, TX)
directly to the six-well plates. Cell lysate was scraped and carefully
pipetted into a tube. After supplementation with bromochloropropane, tubes
were centrifuged at 14,000 g for 10 minutes at 4°C. After
carefully pipetting the aqueous phase, RNA was precipitated with isopropanol.
After a 13,000 g spin, the RNA pellet was washed in 70%
ethanol. RNA yield was spectrophotometrically determined. A digestion step
with DNase I (Gibco BRL) was introduced to avoid interference of genomic DNA
with the PCR reactions. For reverse transcription, the Superscript
First-Strand Synthesis System for reverse-transcription polymerase chain
reaction (RT-PCR) (Gibco BRL) with random hexamer primers was used according
to the manufacturer's instructions.
Real-time PCR
For PCR analyses, cDNAs from triplicate wells of three independent
experiments (22 hours hypoxia or normoxia) were diluted to a final
concentration of 10 ng µl-1. For PCR reactions TaqMan Universal
Mastermix (Applied Biosytems) was used. 50 ng cDNA was used as template to
determine the relative amounts of mRNA by real-time PCR (ABI Prism 7700
sequence detection system) using specific primers and probes; HIF and
Jun probes contained Minor-Groove-Binding elements (MGBs) (ABI
Systems) for further thermal stabilization. The reaction was conducted for as
follows: 95°C for 4 minutes, and 40 cycles of 15 seconds at 95°C and 1
minute at 60°C. 18S rRNA was amplified as an internal control. Cycle
threshold (Ct) values were measured and calculated by the Sequence detector
software. Relative amounts of mRNA were normalized to 18S rRNA (Applied
Biosystems) and calculated with the software program Microsoft Excel. Relative
mRNA contents were calculated as
x=2-Ct, where
Ct=
E
C and
E=Ctsample
Ct18S and
C=Ctcontrol
Ct18S.
Quantification of soluble VEGF isoforms in conditioned medium
Soluble vascular endothelial growth factor (VEGF) isoforms were determined
by using the DuoSet ELISA development KIT for mouse VEGF (R&D Systems).
Cell culture supernatants (wild-type and HIF-1-null cells) from
triplicates of three different experiments were harvested after exposure to
either 22 hours of normoxia or hypoxia, centrifuged at 2000 g
and stored at -20°C. Further VEGF ELISA was conducted according to the
manufacturer's instructions. VEGF concentrations were normalized to protein
content (Biorad).
Type-II collagen ELISA
For type-II collagen quantification the native type-II collagen detection
kit was used (Chondrex). Wild-type and HIF-1-null chondrocytes were
exposed to 44 hours of hypoxia or normoxia. After removing the media,
chondrocyte layer was washed with PBS twice. 0.5 ml 0.05 M acetic acid was
added directly to the cell layer. Cells were harvested by scraping. Cells were
transferred to a microcentrifuge tube and 50 µl 1% pepsin solution (in 0.05
M acetic acid) was added. Suspension was digested on a rotator overnight at
4°C. 50 µl of TSB (1 M Tris, 2 M NaCl, 50 mM CaCl2, pH 7.8)
was added and the pH was adjusted to 8.0. Following, suspension was incubated
with 50 µl of 0.1% pancreatic elastase (Sigma) in TSB for 30 minutes at
37°C. Suspension was centrifuged at 10,000 g for 5
minutes. Supernatant was diluted 1:10 with sample dilution buffer solution.
The further type-II collagen ELISA was conducted as recommended in the
manufacturer's instructions. Type-II collagen concentrations were normalized
to protein content (Biorad).
Quantification of alcian-blue stainable proteoglycans
Chondrocytes were cultured either with (20%) or without (0.5%) oxygen over
44 hours. Quantification of proteoglycans was conducted as described by
Kitaoka et al. (Kitaoka et al.,
2001). In brief, cells were fixed with 10% neutral buffered
formalin for 10 minutes at room temperature. After washing the cells twice
with PBS, cells were incubated with 3% acetic acid for 10 minutes.
Proteoglycans were stained with 1% alcian blue in 3% acetic acid (pH 2.5) for
30 minutes at room temperature. After washing the cell layer twice, alcian
blue was extracted with 500 µl dimethyl sulfoxide. Absorbance was measured
at 650 nm.
Statistical analysis
Data are given as mean ± standard deviation. For inference, the
statistical analysis software program StatView was used. Statistical
differences were identified using the unpaired Student's t test.
*P0.05, **P
0.01.
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Results |
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|
HIF-1 is required for energy generation by epiphyseal
chondrocytes
To determine whether chondrocytes lacking HIF-1 display deficient
energy generation, we measured free ATP levels in cells exposed to different
oxygen levels. We have recently shown that primary wild-type fibroblasts
generate increased ATP levels under hypoxia than under normoxia. Additionally,
HIF-1
-null fibroblasts showed a reduction of
50% of free ATP
(Seagroves et al., 2001
).
Interestingly, primary chondrocytes displayed a slight increase in free ATP in
hypoxia compared with cells cultured at 20% oxygen
(Fig. 2A). In primary
chondrocytes lacking HIF-1
and exposed to 22 hours of hypoxia, a
significant reduction of free ATP was seen.
|
We then measured lactic acid, the end product of anaerobic glycolysis. A
previous study of cartilage explants suggested the existence of a negative
Pasteur effect under hypoxic and anoxic conditions
(Lee and Urban, 1997).
However, our experiments did not confirm these results. Indeed, by contrast,
we found a significant increase in lactic acid under hypoxic conditions,
paralleling rising levels of free ATP and suggesting an enhanced glucose use
by anaerobic glycolysis (Fig.
2B). In primary chondrocytes with cre/loxP-mediated
deletion of HIF-1
, we detected a significant reduction of lactic acid
under hypoxia compared with wild-type cells. This suggests that the increased
glycolytic rate seen in wild-type cells is mediated by HIF-1
. In
addition, HIF-1
-null chondrocytes exposed to 20% oxygen revealed a
moderate decrease in lactic acid concentration, indicating that glycolysis is
also contributing to energy generation in epiphyseal chondrocytes under
aerobic conditions.
HIF-1 increases expression of Glut-1, PGK-1 and VEGF
To help to determine whether increased levels of ATP in wild-type
chondrocytes under 0.5% oxygen are generated mainly by anaerobic glycolysis,
real-time PCR analyses of phosphoglycerate kinase-1 (PGK-1) and glucose
transporter 1 (Glut-1) transcripts were conducted. As demonstrated in
Fig. 3A, we detected a
significant increase in PGK-1 mRNA levels under hypoxia compared to normoxia.
HIF-1-null chondrocytes showed a complete loss of the induced increase
in PGK-1 message. Mutant chondrocytes also had significantly reduced PGK mRNA
under normoxic conditions. Similar results were obtained for Glut-1
transcripts (Fig. 3B),
supporting the hypothesis that HIF-1
is responsible for sustaining
glucose use and glucose uptake in low oxygen tension conditions. It has been
previously shown that VEGF is a target gene of HIF-1
-mediated hypoxic
response (Semenza, 1998
;
Semenza, 2000
). In addition,
VEGF is known to be expressed by chondrocytes during normal development and
pathological conditions such as osteoarthritis
(Gerber et al., 1999
;
Pfander et al., 2001
).
Previous studies have clearly established the importance of VEGF for
metaphyseal angiogenesis, thus contributing to the maintenance of oxygen
levels in growth plate and surrounding tissues
(Carlevaro et al., 2000
;
Gerber et al., 1999
). We
detected a strong induction of VEGF mRNA levels in normal chondrocytes exposed
to 0.5% oxygen (Fig. 3C). The
increase in VEGF transcript levels was lost in HIF-1
-mutant cells.
Furthermore, even chondrocytes with HIF-1
deletion cultured under
normoxic conditions showed reduced VEGF mRNA. Similar results were obtained
examining protein by determination of soluble VEGF concentrations in
conditioned medium using a VEGF-ELISA (Fig.
4).
|
|
Extracellular matrix synthesis is controlled by HIF-1 under
hypoxia
Finally, to test whether HIF-1-mediated alterations in energy
production affect the expression of the main cartilage matrix proteins, we
determined mRNA levels of type-II collagen and aggrecan. The gene expression
profile of aggrecan was closely related to chondrocytic energy generation
(Fig. 5A). In HIF-1
-null
chondrocytes, aggrecan mRNA levels were 51% of wild type under hypoxia,
whereas only an 18% reduction was detected under ambient conditions. To test
further whether decreased transcript levels of aggrecan affected overall
proteoglycan synthesis, we measured the amounts of extractable proteoglycans
produced by cultured chondrocytes. Significantly more extractable
proteoglycans were detected in HIF-1
-null than in wild-type cultures
after 44 hours exposure to 0.5% oxygen
(Fig. 5B). Type-II collagen
mRNA levels were also diminished in mutant cells under hypoxia, to
76% of
wild-type levels (Fig. 6A).
Moreover, in accordance with a previous study on articular chondrocytes
(Hansen et al., 2001
), we
measured strongly increased concentrations of type-II collagen in wild-type
cultures under hypoxia compared with normoxia
(Fig. 6B). To determine whether
the reduced expression of extracellular matrix components in
HIF-1
-mutant cells under hypoxic conditions was due to a failure in
activating glycolysis, we treated chondrocytes with 2-deoxyglucose (2-DG), a
well characterized chemical inhibitor of glycolysis. Incubation with 2-DG
resulted in a highly significant reduction of type-II collagen expression
under both normoxic and hypoxic conditions
(Fig. 7), further supporting
the central importance of glycolytic energy production for matrix
synthesis.
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Discussion |
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In a previous study, we found that deletion of HIF-1 in the
cartilage led to spatially unrestricted cell division and ultimately necrosis
in the embryonic growth plate (Schipani et
al., 2001
). To analyse the role of HIF-1
in cartilage
biology in an in vitro setting, we isolated chondrocytes from murine neonatal
tissue. This allowed a more controlled evaluation of the role of HIF-1
in the growth and survival of chondrocytes under conditions of differential
oxygenation.
In contrast to our previous study, in which we found uncontrolled cell
growth of HIF-1-null chondrocytes, mutant cells in vitro displayed a
retarded exponential growth phase. This discrepancy might be explained by the
fact that chondrocytes in vitro are showing the typical cell growth phases,
whereas growth plate chondrocytes in vivo undergo strictly temporal and
spatial events such as proliferation, differentiation, cell cycle arrest and
apoptosis. However, no major changes in chondrocytic cell shape were detected
in mutant chondrocyte cultures.
We found that the generation of free ATP in wild-type chondrocytes is
slightly increased under hypoxic conditions, suggesting a highly effective
adaptation of chondrocytes to hypoxic microenvironments. Support for the
hypothesis that chondrocytes are able to exist at low oxygen tensions is
provided by Stockwell et al., who showed in a previous study that the per-cell
oxygen consumption of chondrocytes is less than 10% of hepatocyte oxygen
consumption (Stockwell,
1983).
We found that free ATP levels were significantly reduced in hypoxic
chondrocytes lacking HIF-1, to 51% of that of wild-type cells. Even
under normoxic conditions, null chondrocytes showed
33% reduced ATP
levels compared with their wild-type counterparts. In accordance with these
findings, articular chondrocytes have been found to have 70-80% reduced ATP
levels under normoxia, using antimycin as an inhibitor of mitochondrial
oxidative phosphorylation (Johnson et al.,
2000
). These results provide evidence that HIF-1
might act
as an essential element controlling a minor part of aerobic and a major part
of anaerobic energy production of epiphyseal chondrocytes. To characterize the
metabolic activity of primary chondrocytes further, levels of lactate (the end
product of anaerobic glycolysis) were determined. As seen in our previous
studies of HIG-1
-null fibroblasts, we here again demonstrated decreased
lactic acid levels in mutant cultures compared with wild-type cultures under
hypoxic conditions (Seagroves et al.,
2001
). We further show that hypoxia strongly induces the mRNA
expression of PGK-1 and Glut-1 (by 4- and 6.7 times, respectively), and show
that this induction is completely dependent on functional HIF-1
. These
results clearly indicate that HIF-1
is required for energy production
in epiphyseal chondrocytes. In addition, a significant reduction of PGK-1 and
Glut-1 mRNA levels was detected under normoxic conditions, suggesting an
important role for HIF-1
in controlling basal glucose metabolism.
In cartilage, VEGF is mainly expressed by hypertrophic growth-plate
chondrocytes and articular chondrocytes
(Horner et al., 1999;
Pfander et al., 2001
). In
addition to its well-characterized angiogenic properties, VEGF is known to
play a crucial role in long-bone development
(Gerber et al., 1999
). The
factor acts as a paracrine and autocrine mediator, centrally involved in
metaphyseal angiogenesis and new bone formation
(Carlevaro et al., 2000
). In
our previous study, we have clearly shown that VEGF expression is markedly but
not completely depressed in growth-plate chondrocytes lacking HIF-1
in
vivo (Schipani et al., 2001
).
Interestingly, although wild-type chondrocytes in vitro demonstrate increased
VEGF synthesis during hypoxia, this increase is completely lost in
HIF-1
-null cells, suggesting that VEGF regulation in vivo is more
complex and influenced by additional mechanisms (e.g. cell-cell or cell-matrix
interactions).
Finally, in order to determine whether diminished energy production during
hypoxia affects gene expression of matrix molecules, we analysed mRNA and
protein levels of aggrecan and type-II collagen. In the absence of
HIF-1, aggrecan expression is strongly decreased at the mRNA and
protein levels during diminished oxygen delivery. In murine embryos lacking
HIF-1
, strongly reduced type-II collagen signals were detected
specifically in central hypoxic areas of the growth plate, where energy
limitations are thought to be most evident
(Schipani et al., 2001
).
Protein and mRNA expression levels of type-II collagen were strongly reduced
in mutant cells exposed to hypoxia; this demonstrates that hypoxia-induced
matrix gene expression requires the presence of HIF-1
.
In summary, we have demonstrated the importance of HIF-1 to sustain
cell growth, to maintain energy generation and to allow matrix molecule
expression in the hypoxic microenvironment. This provides evidence for a novel
and exciting role for the transcription factor, in production and maintenance
of extracellular matrix in the critically important central region of the
growth plate of cartilage. Further study of this phenomenon will allow a
better understanding of how cartilage formation occurs in the context of the
challenges of hypoxia.
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Acknowledgments |
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Footnotes |
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References |
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