Báo cáo khóa học: Large aggregating and small leucine-rich proteoglycans are degraded by different pathways and at different rates in tendon pot

Large aggregating and small leucine-rich proteoglycans are degraded
by different pathways and at different rates in tendon
Tom Samiric, Mirna Z. Ilic and Christopher J. Handley
School of Human Biosciences, La Trobe University, Melbourne, Victoria, Australia
This work investigated the kinetics of catabolism and the
catabolic fate of the newly synthesized
35
S-labelled proteo-
glycans present in explant cultures of tendon. Tissue from
the p roximal r egion o f bovine deep flexor tendon was incu-
bated with [
35
S]sulfate for 6 h and then placed in explant
cultures for periods of up to 15 days. The amount of radi-
olabel associated with proteoglycans and free [
35
S]sulfate lost
to the medium a nd retained in the matrix w as determined for
each d ay i n c ulture. I t w as shown that t he rate of catabolis m
of radiolabelled small proteoglycans (decorin and biglycan)
was s ignificantly slower (T
½
> 20 days) c ompared w ith the
radiolabelled large proteoglycans (aggrecan and versican)
that were rapidly lost from the tissue (T
½
 2 days). Both
the small and large newly synthesized proteoglycans were
lost from the matrix with either intact or proteolytically
modified core proteins. When explant cultures of tendon
were maintained either at 4 °C or in the presence of the

lysosomotrophic
2
agent ammonium chloride, inhibition of
the cellular catabolic pathway for small proteoglycans was
demonstrated indicating the involvement of cellular activity
and lysosomes in the catabolism of small proteoglycans. It
was e stimated from these studies that approximately 60% of
the radiolabelled s mall proteoglycans t hat were l ost from t he
tissue were degraded by the intracellular pathway present
in tendon cells. This work shows that the pathways of
catabolism for large aggregating and small leucine-rich
proteoglycans are different in tendon and this may reflect
the roles that these two populations of proteoglycans play
in the maintenance of the extracellular matrix of t endon.
Keywords: catabolism; proteoglycan; tendon.
The extracellular matrix of tendon is composed of parallel
bundles of collagen, which endows the tissue with tensile
strength and its ability t o transmit f orce generated by muscle
to bone. A lso present within the e xtracellular matrix o f
tendon are two groups of proteoglycans that can be
distinguished on the basis of their size. The small leucine-
rich proteoglycans m ake u p a pproximately 8 0% of the total
proteoglycans present in the tendon with decorin being the
predominant species and biglycan being present at lower
levels [1–3]. The remaining 20% of proteoglycans present i n
tendon are t he large a ggregating proteoglycans, versican
and aggrecan, which are present in similar levels [1–3].
Tendon cells are responsible for the synthesis and
degradation of extracellular proteoglycans. Studies investi-
gating the catabolism of the chemical pool of aggrecan and

versican by tendon in explant culture have revealed that this
process i nvolves t he proteolytic cleavage of the c ore p roteins
of these proteoglycans by aggrecanase activity as well as
other proteinases [1–3]. The catabolism of the chemical pool
of decorin and biglycan involves the loss of intact c ore
proteins from the tendon matrix as well as limited proteo-
lytic cleavage [1–3].
We have previously studied the kinetics of catabo lism of
newly synthesized proteoglycans in bovine collateral liga-
ment [4] and demonstrated that the catabolism of newly
synthesized
35
S-labelled large proteoglycans was rapid
(T
½
 2 days) and involved p roteolytic cleavage of
the core protein. O n the other h and,
35
S-labelled small
proteoglycans were lost from the tissue at a slower rate
(T
½
 20 days) and were either lost from the tissue with a n
intact or partially degraded core protein, or were internalized
by the c ells and c ompletely degraded w ithin the lys osomes of
the cells [4]. Indeed, i t has been shown t hat the cellular uptake
of small proteoglycans is mediated b y recep tor proteins
present in the plasma membranes of fibroblasts [5,6].
This study was undertaken to determine the metabolic
fate of newly synthesized

35
S-labelled proteoglycans by
tendon in order to elucidate the specific processes and
pathways that are involved in the catabolism of newly
synthesized proteoglycans present in a dense collagenous
connective tissue and to compare the resulting r adiolabelled
catabolic products with those reported by us for the
chemical pool present in the tissue [1].
Experimental procedures
Materials
Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s
nonessential amino ac ids, penicillin and streptomycin were
purchased from CSL (Melbourne, Victoria, Australia).
Sephadex G-25 (as prepacked PD-10 columns) was
from Pharmacia (Uppsala, Sweden). Aqueous solution of
Correspondence to C. J. Hand ley, School of Hum an Biosciences, L a
Trobe University, 3086, Vict oria, A ustralia. Fax: +61 39479 5784,
Tel.: +61 39479 5800, E-mail:
Abbreviation: GdnHCl, guanidine h ydrochloride.
Enzymes: chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4);
keratanase from Pseudomonas sp. (EC 3.2.1.103).
(Received 16 June 2004, revised 21 July 2004, accepted 27 July 2004)
Eur. J. Biochem. 271, 3612–3620 (2004) Ó FEBS 2004 doi:10.1046/j.1432-1033.2003.04307.x
[
35
S]sulfate (carrier free) was from DuPont New England
Nuclear (Boston, MA, USA). Keratanase (from Pseudo-
monas sp.; EC 3.2.1.103) was obtained f rom Sigma Chem ical
Co. (St. Louis, MO, USA) and chondroitinase ABC
(protease free; from Proteus vulgaris; EC 4.2.2.4) from ICN

Biochemicals (Costa Mesa, CA, USA). Adult bovine meta-
carpophalangeal joints were obtained from a local abattoir.
Tendon explant cultures
Deep flexor tendon proximal to the bifurcation was dissected
from a s ingle m etacarpophalangeal j oint of aone-to-two year
old steer. The tendon was then chopped into small pieces and
incubated in sulfate-free medium (1 g tissue per 10 mL
medium) containing 200 lCiÆmL
)1
[
35
S]sulfate at 37 °Cfor
6 h. The sulfate-free medium contained 0.13
M
sodium
chloride, 4.74 m
M
potassium chloride, 2.54 m
M
calcium
chloride, 1.9 m
M
magnesium chloride, 10 m
M
glucose,
1.0 m
ML
-glutamine, 1.19 m
M
potassium dihydrogen phos-

phate, 0.02 gÆmL
)1
Phenol Red and was buffered with
25 m
M
HEPES adjusted to pH 7 .4 with sodium hydroxide
[7]. Sulfate-free medium was used in order to increase the
incorporation of [
35
S]sulfate into proteoglycans as it has
previously been reported that the rate of incorporation of
[
35
S]sulfate i nto proteoglycans by tendon is considerably less
than other connective tissues such as articular cartilage, and
this necessitated the use of high specific radioactivity
[
35
S]sulfate [8]. It was also shown that the rate of incorpor-
ation of [
35
S]sulfate into proteoglycans was linear over the
6 h incubation period. After washing the tissue e xtensively in
DMEM to remove most of the unincorporated radioisotope,
duplicate samples containing 100 ± 20 mg of tissue were
distributed into individual sterile preweighed plastic vials
containing 4 mL of DMEM. DMEM contains sufficient
chemical levels of sulfate (0.8
M
magnesium sulfate) so that

the specific radioactivity o f radiolabelled sulfate present i n or
produced by the explant cultures will be considerably
reduced, thereby decreasing the level of re-use of [
35
S]sulfate
by the cells of the cultures.
The c ulture medium was collected and r eplaced daily with
4 mL of fresh DMEM. The collected medium was stored at
)20 °C in the presence of proteinase inhibitors [9]. At the
end of the culture p eriod, the tissue was extracted with 4
M
guanidine hydrochloride (GdnHCl) in the presence of
proteinase inhibitors at 4 °C for 72 h, followed by 0.5
M
NaOH at 21 °C for 24 h.
Determination of the percentage of
35
S-labelled
proteoglycans remaining in the matrix of tendon
explant cultures
To determine the percentage of
35
S-labelled proteoglycans
remaining i n t he matrix of tendon cultures on each day after
incubation with [
35
S]sulfate, 0.5 mL a liquots of the medium
fractions, G dnHCl and NaOH extracts were applied to
columns of Sephadex G-25 (PD-10 columns) equilibrated
andelutedwith4

M
GdnHCl, 0.1
M
Na
2
SO
4
,0.05
M
sodium acetate, 0.1% (v/v) Triton X-100, pH 6.1. The
35
S-labelled material that eluted in the excluded volume of
the column was attributable to
35
S-labelled macromolecules
in the medium that were originally derived from proteo-
glycans present in tendon matrix. The
35
S-labelled material
which e luted in the total volume was shown to r epresent free
[
35
S]sulfate. The rate of loss of
35
S-labelled proteoglycans
from the matrix of explant cultures was calculated from the
amount of
35
S-labelled macromolecules in the medium on
each day o f culture and t hat remained in the matrix at the

end of t he culture period. From these d ata t he percentage of
35
S-labelled proteoglycans remaining in the matrix was
plotted as a function of time in culture [10].
Separation of
35
S-labelled proteoglycans remaining in
the matrix of tendon explant cultures by size exclusion
chromatography
Aliquots (1 mL) of the G dnHCl extracts obtained f rom
tissue after predetermined times in culture were applied to a
column of Sepharose CL-4B (1.3 · 87.0 cm) equilibrated
andelutedwith4
M
GdnHCl, 5 0 m
M
sodium acetate buffer,
0.1% (v/v) Triton X-100 pH 5.8. Fractions of 1 mL were
collected at a flow rate of 6 mLÆh
)1
and assayed for
35
S-radioactivity. From these data the percentage of
35
S-labelled large and small proteoglycan species remaining
in the matrix at d ifferent times i n culture was determin ed [10].
Detection of
35
S-labelled proteoglycan core proteins
remaining in the matrix or released into the medium

of tendon explant cultures by fluorography
Tissue was dissected from a single metacarpophalangeal
joint and incubated with [
35
S]sulfate for 6 h as described
above. The tissue was maintained in D MEM alone for up t o
10 days. The culture medium was collected and replaced
daily. After predetermined times in culture, the tissue was
extractedwith4
M
GdnHCl as described above. Proteo-
glycans were isolated from tissue extracts and medium
samples by ion-exchange chromatography on Q-Sepharose
as described previously [1].
The s amples were then dialysed again st distilled H
2
O
containing proteinase inhibitors, lyophilized, and dissolved
in 1 mL of 0.1
M
Tris/0.1
M
sodium acetate pH 7.0
containing proteinase inhibitors [10]. The dried samples
were then digested with chondroitinase ABC (0.0375 U)
and keratanase (0.075 U) at 37 °C for 24 h in the presence
of proteinase inhibitors [11]. S amples were subjected t o
electrophoresis on a 4–15% gradient polyacrylamide/SDS
slab gel. The gel was then fixed in a solution of 30% (v/v)
methanol and 10% (v/v) acetic acid for 30 min, soaked in

Amplify for 30 min, dried and exposed to X-ray film at
)20 °C for approximately 40 days
3
[10].
Intracellular degradation of
35
S-labelled small
proteoglycans
In or der t o determine the rate o f i ntracellular degradation of
35
S-labelled small proteoglycans, bovine tendon from a
single metacarpophalangeal joint was incubated with
[
35
S]sulfate for 6 h as described above and then maintained
in DMEM alone for 5 days to allow time for loss of the
35
S-labelled large proteoglycans from the tissue cultures.
For the subsequent days (days 6–15) the tissue was
maintained in culture under the conditions described below.
The rate of intracellular proteoglycan catabolism by tendon
explants was determined from the amount of radiolabelled
Ó FEBS 2004 Catabolism of proteoglycans in tendon
1
(Eur. J. Biochem. 271) 3613
sulfate appearing in the medium on each day. For this,
aliquots of the culture medium were applied to Sephadex
G-25 (PD-10) columns and the amount of [
35
S]sulfate

determined fr om the amount of
35
S-radioactivity i n t he total
volume of the columns. The rate of release of
35
S-labelled
proteoglycans into the culture medium in these experiments
was determined by the
35
S-radioactivity that eluted in the
excluded volume on Sephadex G-25 columns. The percent-
age of
35
S-labelled proteoglycans remaining in the matrix
was determined as described above.
Treatment of data
Previous work has shown that there is variation between
animals in the absolute rates of metabolism of macro-
molecular components o f the extracelullar matrix o f
synovial connective tissues. Because the amount of tissue
was limiting, this only e nabled points to be r epeated in
duplicate. Therefore, individual experiments were repeated
at least three times using tissue from different animals.
Results
Determination of the loss of
35
S-labelled proteoglycans
from the extracellular matrix of tendon explant cultures
Explants of bovine tendon were in cubated with [
35

S]sulfate
for 6 h and then maintained in culture in DMEM for up to
10 days. Medium fractions w ere collected daily and the
tissue extracted at the end of the culture period with 4
M
GdnHCl followed b y 0 .5
M
NaOH as des cribed above.
Approximately 75% of
35
S-labelled proteoglycans were
extracted from the tendon matrix with GdnHCl (data not
shown). T he medium fractions and the GdnHCl and NaOH
extracts were analyzed by size exclusion chromatography on
Sephadex G-25 as described above. All the radiolabelled
material appearing in the total v olume of the columns was
showntobefree[
35
S]sulfate because it was all precipitated
by barium acetate (data not shown). Figure 1 shows the rate
at which
35
S-labelled proteoglycans and free [
35
S]sulfate
appeared in medium samples on e ach d ay in culture. During
the first two days of culture the appearance of free
[
35
S]sulfate in the culture medium was attributable to

unincorporated [
35
S]sulfate following incubation of the
tissue with [
35
S]sulfate on day 0. Over the subsequent days
(days 3–10), both the free [
35
S]sulfate and
35
S-labelled
proteoglycans appeared in the culture medium at a similar
rate. Any re-use of free [
35
S]sulfate during the first two days
of explant culture would be minimal as the specific
radioactivity of the radiolabelled s ulfate would b e markedly
reduced by the sulfate content of D MEM. Figure 2A shows
that there was a faster rate of loss of
35
S-labelled proteo-
glycans from the matrix in the first four days o f culture and
approximately 60% of
35
S-labelled proteoglycans remained
in the matrix after 10 days in culture.
Kinetics of loss of large and small
35
S-labelled
proteoglycans from the extracellular matrix of tendon

explant cultures
The a mount of
35
S-radiolabel ass ociated with large and
small proteoglycans remaining in the matrix of tendon
explants described in Fig. 2A was determined from tissue
extracts on days 0, 2, 4, 6, 8 and 10. These extracts were
subjected to gel filtration on a column of Sepharose
CL-4B eluted under dissociative conditions (Fig. 3). It is
evident that there are two
35
S-labelled proteoglycan p eaks,
a minor peak (K
av
 0.05) representing t he large p rote-
oglycans and a major peak (K
av
 0.5) representing the
small p roteoglycans. T he proportion of
35
S-radioactivity
associated with the large proteoglycans decreased from
16.6% on day 0 (the day of incubation with [
35
S]sulfate)
to 2.1% on day 10, whereas that f or the small proteo-
glycans showed an apparent increase from 83.4% on day
0 to 97.9% by day 10. This indicates that there is a
preferential loss of the newly synthesized
35

S-labelled large
proteoglycans from the extracellular matrix of tendon.
Although 40% of the
35
S-labelled proteoglycans was lost
from the extracellular matrix of tendon over the 10 day
culture period, the hydrodynamic size of each
35
S-labelled
proteoglycan species extracted from the matrix of the
tissue immediately after incubation of the tissue with
[
35
S]sulfate and at various time points in culture remained
constant. The presence of unincorporated [
35
S]sulfate early
in the culture period (days 0 and 2 ) is i ndicated in t he
elution profiles in the total volume of the column
4
.
The percentage of
35
S-labelled large and small proteo-
glycans remaining in the matrix at various times after
incubation with [
35
S]sulfate was determined by multiplying
the percentage of each proteoglycan species present in the
tissueextractsondays0,2,4,6,8and10ofthe

culture period (Fig. 3) by the percentage of
35
S-labelled
proteoglycans r emaining in the m atrix o n t he corresponding
day of culture (Fig. 2A). This was then expressed as the
percentage of the amount of each proteoglycan species
present i n the tissue on d ay 0 (Fig. 3; top) in order to
determine the kinetics of loss of the large and small
Fig. 1. Rate of appearance of
35
S-labelled proteoglycans and [
35
S]sul-
fate in to the c ulture medium of explant c ultures of tendon. The p roximal
region of bovine de ep flexor tendon was incubated with [
35
S]sulfate as
described in Experimental procedures, and cultured in DMEM alone
for10days.Therateofappearanceof
35
S-labelled proteoglycans (d)
and [
35
S]sulfate (s) into the culture medium from bovine tendon
explant cultures was determined by analysis of medium samples from
each day of culture period on columns of Sephadex G-25. The error
bars represent the range of duplicate samples.
3614 T. Samiric et al.(Eur. J. Biochem. 271) Ó FEBS 2004
35
S-labelled proteoglycans in tendon. Figure 2B shows that

the loss of
35
S-labelled small proteoglycans from the
extracellular matrix was much slower (T
½
> 20 days)
compared to the
35
S-labelled large proteoglycans (T
½

2 days). It is evident from Fig. 2B that over 85% of large
proteoglycans were lost from the tissue within the first six
days after incubation of the tissue with [
35
S]sulfate, whilst
only 20% of small proteoglycans were lost over this time
period.
Characterization of
35
S-labelled proteoglycans remaining
in the matrix and released into the medium of tendon
explant cultures by fluorography
To analyze the
35
S-labelled proteoglycan core proteins
isolated from either the tissue or released into the culture
medium, t endon was incubated with [
35
S]sulfate f or 6 h

prior to being maintained in culture in DMEM for up to
10 days. Radiolabelled proteoglycans present in the matrix
Fig. 2. Percentage of
35
S-labelled proteoglycans remaining in the
extracellular matrix of tendon explants cultures. (A) The proximal
region o f bovine deep flexor tendon was inc ubated with [
35
S]sulfate
and m aintained in D MEM for 10 days. The percentage of
35
S-labelled
proteoglycans remaining i n the matrix of tendon c ultures o n each d ay
after incubation with [
35
S]sulfate was determined as described in
Experimental procedures. The error b ar represents the r ange of
duplicate samples. (B) The percentage of
35
S-labelled large proteogly-
cans (d)and
35
S-labelled small proteoglycans (s)remaininginthe
tissue at each time after incubation of bovine tendon with [
35
S]sulfate
was dete rmined as described in Results. The error bars represent the
range of duplicate samples.
Fig. 3. Elution profiles on Sepharose CL-4B of the
35

S-labelled pro-
teoglycans remaining in the matrix of tendon cultures maintained in
DMEM. On the days indicated, tissue samples from the experiment
described in Fig. 2 were extracted with 4
M
GdnHCl and aliquots of
the
35
S-labelled proteoglycans we re app lied to a co lu mn of Se pharo se
CL-4B eluted with a buffer containing 4
M
GdnHCl. In each profile,
the amount of
35
S-labelled proteoglyc ans extracted fro m th e tissue o n
each day is expressed a s a pe rcen tage of the
35
S-labelled p roteoglycans
extracted on day 0. The values in parentheses refer to the relative
percentage of large and sm all p ro teoglyca n s pecie s p resent
7
on the day
of extraction.
Ó FEBS 2004 Catabolism of proteoglycans in tendon
1
(Eur. J. Biochem. 271) 3615
and culture medium were digested with chondroitinase
ABC and keratanase, which results in the removal of most
of the glycosaminoglycan chains but leaves
35

S-radio-
labelled glycosaminoglycan stubs associated with the core
protein. The partially deglycosylated core proteins were
then subjected to electrophoresis on a 4–15% gradient
polyacrylamide/SDS large gel followed by fluorography as
described in Experimental procedures. Figure 4 (lane i)
shows that three distinct high molecular mass bands above
300 kDa were present in tendon matrix immediately after
incubation with [
35
S]sulfate. Based on our previous work it
is likely that these bands represent intact core protein of
aggrecan and V
0
and/or V
1
splice-variants of versican [1].
With time in culture, a distinct band at  300 kDa
(indicated by asterisk) appeared, and remained in the
matrix over the culture period of 10 days (lanes ii and iii).
The precise identity of this band is not known but it is likely
to be a product of the proteolytic processing of the core
protein of aggrecan or versican. A series of weak bands
ranging b etween 80 to above 250 kDa were a lso present and
these are likely to represent degradation products of the
large proteoglycans that are retained in the m atrix. The
majority of rad iolabelled material present in the matrix of
fresh tendon was associated with the band ranging between
37 and 45 kDa. This band corresponds to the decorin core
protein; also present are small levels o f b iglycan core protein

[1]. A number of bands were also observed at 33 kDa and
below, which we have shown to be degradation products of
decorin [1].
A diffuse band at 50 kDa, which is likely to represent
intact fibromodulin or degradation products of large
proteoglycans, was also evident [10]. It was apparent that
the decorin core protein of 43 kDa (lane i) present in tissue
immediately after incubation with [
35
S]sulfate, decreased in
size with time in culture (lanes ii and iii) indicating
extracellular p rocessing of d ecorin core p rotein. It is possible
that newly synthesized decorin contains an intact amino-
terminal propeptide which is remove d with time in culture
by the action of proteinases present in the e xtracellular
matrix of the tissue [12,13]. Further proteolytic processing
of decorin core protein was shown by the presence of
additional distinct bands at 25 kDa and below (lanes ii and
iii). These observations indicate that degradation of core
proteins of newly synthesized small proteoglycans occurs
and that fragments are retained within the matrix.
Figure 4 (lanes iv and v) shows the proteoglycan core
proteins released i nto the medium of explant cultures after 3
and 6 days in culture, respectively. A number of distinct
bands of over 250 kDa and a series of bands ranging
between 75 and 160 kDa are present in t he medium and we
have previously shown that they represent catabolic prod-
ucts of aggrecan a nd versican [1]. It m ust b e pointed out that
the amount of
35

S-radioactivity associated with these high
molecular mass peptides is directly attributable to the high
density of sulfate groups associated with these large
proteoglycans.
Intracellular catabolism of
35
S-labelled small
proteoglycans by tendon explant cultures
Because it was shown that [
35
S]sulfate appeared in the
culture medium throughout the culture period (Fig. 1),
experiments were performed to determine if this was due to
intracellular d egradation of
35
S-labelled s mall proteogly-
cans. Bovine deep flexor tendon was maintained in culture
in DMEM for 5 days after incubation with [
35
S]sulfate to
allow for the loss of the majority of the radiolabelled large
proteoglycans (Fig. 2B). Cultures were then maintained in
DMEM at 37 °Cor4°C for a subsequent 10 days to
determine the effect of reduced cellular activity on the
appearance of free [
35
S]sulfate in the medium. In some
cultures, the temperature was switched at the mid-point of
the culture period to determine whether the effect of low
temperature on the generation of free [

35
S]sulfate was
reversible. Figure 5A shows that in cultures maintained at
37 °C, there was a continuous rate of formation of free
[
35
S]sulfate in the culture medium. The r ate of generation o f
[
35
S]sulfate in t he medium was reduced by over 90% in
cultures maintained at 4 °C, suggesting that metabolically
active cells were required for this process. This reduction
was also demonstrated when cultures were switched from
37 °Cto4°C on day 10 o f the culture period, whereas in
cultures that we re initially maintained at 4 °C, there was an
Fig. 4. Analysis of
35
S-labelled proteoglycan core proteins p resent in th e
matrix or medium of explant cultures of tendon. Newly synthesized
35
S-labelled p roteoglycans remaining in t he matrix or released into the
medium of tendon explant cultures after 10 days in culture were iso-
lated as described in Experimental procedures and d ige sted with
chondroitinase ABC and keratanase, prior to electrophoresis on a
4–15% polyacrylamide/SDS large gel. The gel was subjected to fluo-
rography as described in Experimental procedures. Lanes show pep-
tides present in (i) fresh tendon t issue, (ii) t issue after 6 days in culture,
(iii) tissue after 10 days in culture, (iv) days 1–3 pooled medium, and
(v) days 4–6 pooled medium. Approximate molecular mass of
observed pept ides are given.

3616 T. Samiric et al.(Eur. J. Biochem. 271) Ó FEBS 2004
apparent increase in the rate of [
35
S]sulfate appearance
when these cultures were switched to 37 °C, demonstrating
that this effect was reversible. In contrast to the rate of
formation of [
35
S]sulfate, Fig. 5B shows that there was an
increase by 40% of
35
S-labelled proteoglycans appearing in
the culture medium of tendon explants maintained at 4 °C.
The percentage of
35
S-labelled proteoglycans remaining in
the matrix of cu ltures maintained at 37 °C (calculated from
both the release of
35
S-labelled proteoglycans and the
appearance of [
35
S]sulfate with t ime in culture) w as
approximately 80% by the end of the culture period on
day 15 as shown in Fig. 5C. However, the loss of
35
S-labelled proteoglycans was reduced in cultures main-
tained at 4 °C, where about 95% of
35
S-labelled proteo-

glycans remained in the matrix by the end of the culture
period on day 15.
The work described above suggests that small proteo-
glycans a re tak en up by the cells and digested intracellularly.
To demonstrate that the lysosomal s ystem is involved i n the
appearance of free [
35
S]sulfate in the culture medium,
tendon cultures were maintained in DMEM contain ing
10 m
M
ammonium chloride following 5 days in culture in
DMEM alone. Ammonium chloride is a lysosomotropic
amine and acts by raisin g the intralysos omal pH which
inhibits the activity of lysosomal enzymes, and is known to
be an effective reversible inhibitor of lysosomal function at
low concentration [14]. Figure 6A shows that in cultures
maintained in DMEM containing 10 m
M
ammonium
chloride, the rate of [
35
S]sulfate appearing in the medium
was suppressed by approximately 78% compared with
control c ultures. This suppression was further demonstrated
when cultures were switched on day 10 from DMEM alone
to DMEM containing 10 m
M
ammonium chloride. When
cultures were switched on day 10 from DMEM containing

10 m
M
ammonium chloride to DMEM alone, the rate of
[
35
S]sulfate a ppearance was restored, demonstrating that
this effect was reversible. The rate of release of
35
S-labelled
proteoglycans into the culture medium was increased by
approximately 107% in cultures maintained in the presence
of ammonium chloride (Fig. 6B). The percentage of
35
S-labelled proteoglycans remaining in the matrix in
DMEM alone was approximately 80% by the end of the
cultureperiodonday15asshowninFig.6C.However,the
loss of
35
S-labelled proteoglycans was reduced in cultures
maintained in the presence of ammonium chloride, where
about 85% of
35
S-labelled proteoglycans remained in the
matrix by the end of the culture period on day 15.
Discussion
This study showed that the loss o f the large aggregating
proteoglycans (aggrecan and V
0
and/or V
1

splice-variants
of versican) that make up approximately 17% of the
35
S-labelled pool of newly synthesized proteoglycans was
rapid, with a half-life of about 2 days (Fig. 4). These
findings are consistent with studies using other joint
connective tissues such as articular cartilage [15] and
collateral ligament [10]. In the case of articular cartilage , it
has been shown that t he majority of newly s ynthesized
aggrecan remains closely associated with the chondrocytes
[16,17]. However, the majority of the chemical pool of
aggrecan resides in the interterritorial matrix and it is this
Fig. 5. Effect of r educ ed temperature on the rate of formation of
[
35
S]sulfate and release of
35
S-labelled proteoglyc ans from tend on
explant cultures. Explant cultures of deep flexor tendon were incubated
with [
35
S]sulfate as described in Experimental procedures and then
maintained in DMEM for 5 days prior to analysis. Tissue was sub-
sequently cultured for a further 10 days in DMEM at 37 °C(d),
DMEM at 4 °C(s), DM EM at 37 °C which was switched t o 4 °Con
day 10 ( ,), or D MEM at 4 °C which was switched t o 37 °C on day 10
(.). The culture medium was collected daily and analyzed for the
presence o f [
35
S]sulfate and

35
S-labelled proteoglycans. F rom this data,
(A) the rate of appearan ce of [
35
S]sulfate, (B) the rate of release of
35
S-labelled p roteoglycans, and (C) t he percentage of
35
S-labelled
proteoglycans remaining in the matrix were determined, as described in
Experimental p rocedur es. T he error bars represent the r ange of
duplicate samples over the remaining 10 days.
Ó FEBS 2004 Catabolism of proteoglycans in tendon
1
(Eur. J. Biochem. 271) 3617
population that is responsible for the biomechanical prop-
erties of cartilage. Work has shown that this population of
aggrecan turns over very slowly, with a half-life in excess of
3.5 years [18]. If this i s applied to the present s tudy, it is likely
that newly synthesized agg recan and v ersican may be closely
associated with tendon cells where the turnover is mediated
by proteolytic enzymes originating from tendon cells.
Indeed, we have shown that the catabolism of aggrecan in
tendon appears to be exclusively attributed to aggrecanase
proteinases whereas the catabolism of versican may involve
aggrecanase as well as othe r proteinases [1]. It is likely that
these enzymes are responsible for the rapid turnover of the
newly synthesized pool of large proteoglycans, as the
resulting radiolabelled co re protein fragments are of similar
size to those previously reported by our laboratory for the

chemical pools of large aggregating proteoglycans present
in tendon [1]. Furthermore, it has been reported that the
aggrecanase proteinases A DAMTS-4 and ADAMTS-5 are
expressed i n bovine tendon cells [2], bu t a t d ifferent stages of
development of the animal. We have observed the expres-
sion of both ADAMTS-4 and ADAMTS-5 in bovine
tendon cells from mature cattle (T. Samiric, M.Z. Ilic &
C.J. Handley, unpublished data)
5
.
In contrast to the rapid rate of loss of newly synthesized
large proteoglycans, the n ewly synthesized small proteo-
glycans were lost slowly from the matrix of tendon cultures
with a h alf-life of greater than 20 days, which is consistent
with fin dings f rom e arlier s tudies in explant cultures o f
tendon [19], articular cartilage [15] and ligament [4,10].
This slow loss of newly synthesized small proteoglycans
may be indicative of their association with other matrix
molecules, particularly Type I collagen fibres [20], and it is
possible that the turnover of this group of proteoglycans
may be coordinated with the turnover of other matrix
macromolecules. However, some of t he radiolabelled
decorin undergoes proteolytic cleavage and these products
are either retained within the matrix or lost to the culture
medium in a similar manner to that observed for the
chemical pool [1].
Approximately 60% of the
35
S-labelled decorin that was
lost from the matrix was taken up by t he tendon cells and

degraded within the lysosomal system. This was shown by
the gene ration o f f ree [
35
S]sulfate by t endon e xplant cultures
throughout the culture period. This finding is supported by
similar studies using ligament explant cultures [4]. The
cellular uptake and subsequent degradation of decorin has
been observed in a variety of cells of mesenchymal origin
[4,21]. It has been shown that the leucine-rich repeat region
of decorin binds to specific receptors present in the plasma
membrane and endo somes of s kin fibroblasts, osteosarcoma
cells and c hondrocytes [5,22]. Upon entering the c ell by
endocytosis, decorin is subsequently transported to the
lysosomes. Previous work has shown that at least two
intracellular pathways are involved in the c atabolism of
endogenously radiolabelled proteoglycans associated with
the c ell s urface in rat ovarian granulosa cells [23]. O ne
pathway leads to a rapid and complete intralysosomal
degradation resulting in th e release of [
35
S]sulfate. In the
second pathway, the rate of degradation is slower and
commences with extensive p roteolysis, generating glycos-
aminoglycan chains bound to peptides before final hydro-
lysis takes p lace [23].
Fig. 6. Effect of ammonium chlo ride on the r ate of f orma tion of
[
35
S]sulfate and release of
35

S-labelled proteoglycans from tendon
explant cultures. Explant cultures of deep fl exor tendon were incubated
with [
35
S]sulfate as described in Experimental procedures and then
maintained in DMEM for 5 days prior to analysis. Tissue was sub-
sequently cultured f or a further 10 days in DMEM alone (d), DMEM
containing 10 m
M
ammonium c hloride (s), DMEM alone which was
switched to DMEM containing 10 m
M
ammonium chloride on day 10
(,), or DMEM containing 10 m
M
ammonium chloride which was
switched to DMEM alone on day 10 (.). The culture medium was
collected daily and analyzed for the p rese nce of [
35
S]sulfate and
35
S-labelled proteoglycans. F rom t his d ata, (A) the rate of ap pearance
of [
35
S]sulfate, (B) t he rate of rele ase of
35
S-labelled pr oteoglycans, and
(C) the percentage of
35
S-labelled proteoglycans remaining in the

matrix were determined, as described in Experimental procedures. The
error b ars represent the range o f duplicate samples over the remaining
10 days.
3618 T. Samiric et al.(Eur. J. Biochem. 271) Ó FEBS 2004
This study indicates that the intracellular degradation of
decorin requires metabolically active cells including a
functional lysosomal system because this process was
inhibited at 4 °C a nd in the p resence o f a mmonium chloride
(Figs 5 and 6). In addition, when thes e treatments w ere
applied t o tendon explant c ultures there was an inhibition of
the intracellular degradation of decorin and a simultaneous
increase in the appearance of
35
S-labelledofdecorininthe
medium throughout the culture period (Figs 5 and 6), al beit
to different degrees. This enhanced loss of decorin by the
pathway that results in the loss of decorin from the
extracellular matrix has also been observed in ligament
explant cultures [4]. It has previously been reported that
decorin is only taken up by cells if it is not bound to other
extracellular matrix molecules [24]. However, little is known
about the nature of interactions of newly synthesized
decorin with other extracellular components and its distri-
bution within the matrix of fibrous connective tissues. It is
possible t hat a proportion of newly synthesized decorin
remains located close to the cell and may be loosely
associated with the cell membrane and/or extracellular
matrix. This pool of decorin is likely to be subjected to
intracellular degradation. The inhibition of the cellular
uptake of newly synthesized decorin appears to result in

more of this pool of decorin being lost from the tissue into
the culture medium. This may involve displacement of
newly synthesized decorin that is further away from the cell
and subsequent release to the medium, thus reducing
the accumulation of this proteoglycan within the extra-
cellular matrix of tendon. The low level of loss of radio-
labelled decorin suggests that a significant proportion of
the newly synthesized decorin is retained in the extra-
cellular m atrix i n strong interactions with other extracellular
matrix macromolecules where the core protein of this
proteoglycan can undergo p roteolytic processing (F ig. 4;
lanes ii and iii).
The work presented in this paper supports previous
observations which show that the catabolism of large and
small proteoglycans follow distinct separate pathways.
Furthermore, it is evident that in both tendon and ligament
[4] t he processes involved in the catabolism of proteoglycans
are similar and this is not unexpected considering the
similarity in the structure and organization of these two
dense connective tissues. In both tissues the intracellular
degradation pathway plays a significant role in the catabo-
lism of newly synthesized s mall proteoglycans. In the case of
tendon this pathway represents about 60% of the radio-
labelled pool of small proteoglycans and in t he case of
ligament represents 30% of this pool [4]. This raises the
question of whether this pathway is also involved in the
catabolism of the chemical pool of small proteoglycans that
are present in the extracellular matrix of these tissues.
Furthermore, the contribution of this intracellular pathway
of catabolism of small proteoglycans needs to be taken into

account in stu dies investigating the c atabolism o f small
proteoglycans in d ense connective tissues in pathological
conditions.
6
Acknowledgements
We wish to thank the Arthritis Foundation of Australia and the
Faculty of Health Sciences, La Trobe University for s upport.
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