Hypoxia and Simulated Hypoxia Increase Nuclear Translocation of HIF-1 α and Decrease Proteoglycan Synthesis in
Articular Chondrocytes
*#Brucker P U; *Izzo N J; *Werkmeister L; #Vogt M T, #Fu F H, +*#Chu C R
+* Cartilage Restoration Laboratory, # Department of Orthopedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA
email address: [email protected]
Introduction:
Because of the avascularity of articular cartilage, chondrocytes likely
possess adaptations for survival in low oxygen tension. Chondrocytes in
the deeper layers of articular cartilage have been reported to exist in
oxygen tensions less than 1%.1 The transcription factor hypoxiainducible factor 1 α (HIF-1 α) induces a program of gene expression that
is critical for adapting tissues to a hypoxic environment.2 The aim of this
study was to test the hypotheses that HIF-1 α is differentially expressed
by bovine articular chondrocytes in different oxygen tensions and that
proteoglycan synthetic activity decreases at oxygen tensions and
conditions that also increase nuclear translocation of HIF-1 α.
Material and Methods:
Proteoglycan synthetic activity and cell viability: Osteochondral
explants of freshly slaughtered calves were harvested by standard coring
devices. Deeper chondral layers of the explants were exposed by
removal of 20% and 50% of the cartilage surface (intact cores as control
group). After cultivation in 21% O2, 2% O2, and hypoxia-simulated
conditions for 3 days proteoglycan synthetic activity was assessed by
measuring 35S-sulfate uptake and normalized by DNA content. Hypoxia
was simulated in 21% O2 by using cobalt chloride (300µM CoCl2),
which blocks the enzymatic degradation of HIF-1 α.3 Cell viability of
the osteochondral explants was assessed by fluorescent labeling with
propidium iodide and 5-chloromethylfluorescein diacetate.
Immunohistochemistry: Bovine articular chondrocytes were isolated
from the same specimens and cultured for 12 hours in 21% O2, 2% O2,
and cobalt chloride (21% O2). HIF-1 α was analyzed by indirect
immunofluorescence. In addition, counterstaining of the nuclei (DAPI)
and the Golgi complex (NBD C6-ceramide) was performed for
assessment of nuclear, perinuclear, or cytoplasmic localization of HIF1 α, respectively.
Western Blot: Nuclear extracts were prepared from bovine articular
chondrocytes after treatment with either 21% O2, 2% O2, or CoCl2 for 12
hours. The Western blot was performed by a polyacrilamide gel. For
both immunostaining and western blotting, Hep 3B cells were used as
positive and negative controls for HIF-1 α.4
Statistics: A General Linear Models Procedure (Tukey`s Test) was
performed for statistical analysis of the proteoglycan synthetic activity.
Results were considered significant at p<.05.
Results:
Proteoglycan synthetic activity and cell viability: After 3 days of
culture under the different oxygen tensions, no differences in cell
viability were noted (data not shown). Proteoglycan synthetic activity
was significantly (p=.0001) greater in 21% O2 than in 2% O2 for all
groups (Fig. 1). Exposure of the bottom 50% of the cartilage resulted in
a marked increase in total proteoglycan activity above control in
21% O2. The upregulation of total proteoglycan activity was not as great
in 2% O2 and in CoCl2. Simulation of hypoxia by CoCl2 led to similar
effects on proteoglycan synthesis as in hypoxic culture.
(arrowheads). Only cytoplasmic and perinuclear staining was seen in
21% O2 (arrows; Fig. 2). Nuclear counterstaining by DAPI and Golgi
staining by NBD C6-ceramide demonstrated an accumulation of HIF-1 α
within the Golgi complex (Fig. 3). Anti-HIF-1 α immunostaining in
Hep 3B cells showed equivalent staining pattern (data not shown).
Fig. 2:
Anti-HIF-1 α immunofluorescent staining of bovine articular
chondrocytes in monolayer cultures. In 2% O2 and in cobalt chloride, the
staining patterns of HIF-1 α overlap with the nuclei staining representing
nuclear translocation of HIF-1 α (pink color). In 21% O2, HIF-1 α is
stained primarily perinuclear. (Magnification: 400x)
Fig. 3:
Anti-HIF-1 α and Golgi complex immunofluorescent staining
of bovine articular chondrocytes in monolayer cultures. In 21% O2, the
perinuclear staining pattern of anti-HIF-1 α demonstrates co-localization
with the Golgi complex (yellow color) (Magnification: 600x).
Western Blot: In bovine articular chondrocytes, nuclear translocation of
HIF-1 α was strongly detected in 2% O2, but not in 21% O2 (Fig. 4).
Fig. 4:
Western Blot of HIF-1 α in Hep 3B nuclear extracts (A =
21% O2, B = 50µM CoCl2, C = 300µM CoCl2, D = 2% O2), and nuclear
extracts of bovine articular chondrocytes (BAC) (E = 21% O2, F =
2% O2).
Conclusion:
This study demonstrated upregulation of HIF-1 α in bovine articular
chondrocytes in conditions of low oxygen tension and simulated hypoxia
(CoCl2). We also showed accumulation of HIF-1 α in the Golgi complex
at 21% O2, with translocation of HIF-1 α to the nuclei at 2% O2 and in
the presence of CoCl2. Decreased proteoglycan synthesis was
demonstrated in both experimental conditions where HIF-1 α was
translocated to the nucleus. These results suggest that HIF-1 α may play
a role in modulating proteoglycan synthesis in bovine articular cartilage.
Acknowledgements: This study was supported by NIH AR046995
(CRC) and the Department of Orthopedic Surgery.
Fig. 1:
35
S-Proteoglycan synthesis in bovine cartilage explants.
Immunohistochemistry: HIF-1 α was strongly detectable in the
cytoplasm and the nuclei by immunofluorescence in 2% O2 and in CoCl2
References:
(1) Silver I, Phil Trans R Soc Lond B Biol Sci 1975. (2) Wang G L et al.,
J Biol Chem 1995. (3) Yuan Y et al., J Biol Chem 2003. (4) Wang G L et
al., Proc Natl Acad Sci 1994
51st Annual Meeting of the Orthopaedic Research Society
Paper No: 0003