Download CD44 Mediated Endocytosis of Hyaluronan by Chondrocytes

CD44 Mediated Endocytosis of Hyaluronan by Chondrocytes
+Ariyoshi W, Takahashi N, Knudson CB, Knudson W
+East Carolina University, Greenville, NC
[email protected]
INTRODUCTION:
CD44 is a cell surface receptor for the extra cellular matrix macromolecule hyaluronan (HA). In addition, CD44 mediates the endocytosis of
HA leading to its subsequent degradation within lysosomes. We reported
that CD44 receptor mediated uptake of HA leading to its degradation
intercellularly is the principal pathway for HA turnover by chondrocytes
[1]. We hypothesized that CD44 receptor mediated endocytosis of HA
provides a mechanisms whereby chondrocytes to regulate local proteolytic
events in the turnover of aggrecan proteoglycan (PG). When PG is
processed within the extracelular matrix by MMP or aggrecanases, the
residual PG and link protein domains remain attatched to HA. We
addressed the question as to whether these fragments were co-internalized
with HA via CD44 mediated endocytosis by detection of the aggrecanasederived ITEGE neoepitope. Thus, the focus of this study is to determine
the mechanistic link between HA endocytosis and PG degradation.
MATERIALS AND METHODS:
Chondrocyte preparation: Chondrocytes were isolated from the
metacarpophalangeal joints of 18-24 month old adult bovine steers and
grown as high-density monolayers (2.0 x 106 cells/ 35mm dish) in a 1:1
mixture of DMEM/Ham’sF12 medium containing 10% FBS and incubated
at 37ºC in an atmosphere containing 5% CO2 [2].
Immunofluorescene microscopy: Chondrocytes were incubated with or
without IL-1 (10 ng/ml) for 0- 5 days. Some cells were preincubated with
100 U/ml Testiculat HA’ase and proteinase inhibitor (Sigma) for 1 h to
remove the matrix. At the end of the treatment the cells were fixed and
pemeabilized. Cells were then blocked, followed by incubation with an
ITEGE neoepitope antibody (kindly provided by Dr. Amanda J Fosang,
University of Melbourne, Australia) [3]. Bound antibody was detected
with cyamine-3-conjugated anti-rabbit secondary antibody. At the end of
the each procedure, the cells were washed and mounted onto glass slides
in media containing DAPI nuclear stain.
Western blotting of Aggrecanase derived neoepitope: Chondrocytes were
brought to 5 % serum condition and then incubated with 10 ng/ml IL-1 for
0-5 days. At time of harvest, the cultured chodrocytes were first treated
with testiclular hyaluronidase containing proteinase inhibitors (Sigma,
USA) in order to release and separate the membrane-bound extracellular
matrix fraction. Next, total protein was extracted from the cells in
monolayer using Cell Lysis Buffer (Cell Signaling Technology, USA) to
prepare the intracellular fraction. Sample aliquots of equibvalent protein
were loaded and separated by 10% SDS-PAGE gels. The ITEGE
neoepitope antibody [3] was used for analysis.
Inhibition of CD44-mediated HA internalization: Freshly isolated
chondrocyetes were allowed to recover for 2 days in high density
monolayer and then released using a Pronase/collagenase P solution. The
cell suspensions were then mixed with chondrocyte Amaxa nucleofector
solution containning 5 ug CD44 siRNA or control siRNA and processed
using a nucleofection technique (Amaxa Biosystems, USA). After 24
hours the medium was replaced with fresh medium and IL-1 stimullation
commenced 48 hours post transfection. Total proteins was extracted from
the cells and subjected to Western blotting analysis using specific antibody
for ITEGE or CD 44.
RESULTS:
To define the location of the ITEGE epitope, IL-1 treated cells were
examined by imunofluorescene microscopy. Staining with ITEGE was
detected within the pericellular matrix adjustant to the chondrocytes. After
5 days of IL-1 treatment, chondrocytes had clearly defined intracellular
immunofluorescent vesicles, probably secondary lysosomes, revealed after
removing the matrix with HA’ase, strongly suggesting that the ITEGE
epitope is endocytosed (Fig 1A).
To further define the cellular distribution of ITEGE epitope, the
membrane-bound extracellular matrix and the intracellular fraction were
isolated. The ITEGE epitope in each fraction was detected by western blot
analysis. Immunoreactivity to ITEGE was detected in both fractions from
all days of culture, commencing from day 0. The presence of ITEGE
epitope in unstimulated cultures suggests that MMPs or ADAMTSs may
be involved in the unstimulated, or “ base-line” release of aggrecan from
chondrocytes in culture. Treatment of chondrocytes with IL-1increased
the amounts of ITEGE in both fractions compared with control. In the
membrane bound extracelular matrix, ITEGE up-regulation reached a
maximum level at day 1 in response to IL-1, and appeared to be reduced
from day 3. In contrast, intracellular ITEGE epitope appeared to be
enhanced in days 3-5. This results also suggest that the chondrocytes
encytosed ITEGE epitope in response to IL-1 stimulation (Fig. 1B).
To define whether CD44 was required for endocytosis, the expression of
CD44 was inhibited via RNA interference. Chondrocytes were transfected
with an siRNA directed against CD44 or control siRNA. Following
transfection, the cells were cultured with or without IL-1 for 72 hours. As
shown in Fig 2, the protein level of CD44 was inhibited by the CD44
siRNA. Interestingly, accumulation of intracellular ITEGE bands was also
coordinately diminished in chondrocytes transfected with CD44 siRNA.
These results suggest that increased of intracellular ITEGE epitope
following IL-1 treatment is depemdent on CD44-mediated endocytosis.
DISCUSSION:
The major goal of this study was to determine the regulation of PG
degradation via the CD44-mediated internalization of HA decorated with
PG fragmentds. In many cell systems such as chondrocytes, local,
cell-mediated endocytosis of HA is the primary mechanism for turnover of
this extracellular matrix macromolecule.
During osteoarthritis
extracellular matrix turnover is substantially elevated, and the question
remains as to how these cells affect an increase in HA internalization and
aggrecan turnover. A report from Fosang et al. [4] demonstrated using
confocal microscopy, the detection of ITEGE eitope inside chondrocytes
of cartilage tissue slices that had been treated with IL-1.
Our
immunolocalization data (Fig.1) demonstrated IL-1 induced up-regulation
of ITEGE epitope both in the extracellular matrix and intracellularly.
Assuming that ADAMTS4/5 cleavage of aggrecan occurs extracellularly,
the intracellular accumulation of ITEGE suggests that internalization of the
ITEGE G1 domain of PG has occurred. If the endocytosis of ITEGE is
mediated by way of CD44 endocytosis of HA, alternation of CD44
expression would coordinately affect accumulation of intracellular ITEGE.
Using CD44 siRNA, CD44 protein expression was inhibited by ~50% (Fig
2). Under the same conditions, accumulation of intracellular ITEGE was
also inhibited by ~50%. These results suggest that HA internalization via
CD44 serves to complete the catabolism of aggrecan.
Figure 1. Localization of ITEGE G1 domains by Immunoflurorescene
microscopy (A) and Western blotting analysis (B)
REFERENCES: [1]Hua Q. et al. J Cell Sci 1993; 106:365-375 [2] Ohno S,
et al. J Biol Chem 2006; 271: 17952-60 [3] Fosang A J. et al. Biochem J
1995; 337-343 [4] Fosang A J. et al. J Biol Chem 2000; 275:33027-33037
Acknowledgements: Supported in part by NIH grants RO1-AR43384
(WK), RO1-AR39507 (CBK)
Poster No. 412 • 55th Annual Meeting of the Orthopaedic Research Society