Detection of35S-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 meta
Trang 1Large 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 synthesized35S-labelled
proteo-glycans present in explant cultures of tendon Tissue from
the proximal region of bovine deep flexor tendon was
incu-bated with [35S]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 [35S]sulfate lost
to the medium and retained in the matrix was determined for
each day in culture It was shown that the rate of catabolism
of radiolabelled small proteoglycans (decorin and biglycan)
was significantly slower (T½> 20 days) compared with the
radiolabelled large proteoglycans (aggrecan and versican)
that were rapidly lost from the tissue (T½ 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 4C or in the presence of the lysosomotrophic
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 estimated from these studies that approximately 60% of the radiolabelled small proteoglycans that were lost from the 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 tendon 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 to transmit force generated by muscle
to bone Also present within the extracellular matrix of
tendon are two groups of proteoglycans that can be
distinguished on the basis of their size The small
leucine-rich proteoglycans make up approximately 80% 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 in
tendon are the large aggregating 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 involves the proteolytic cleavage of the core proteins
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 core
proteins from the tendon matrix as well as limited
proteo-lytic cleavage [1–3]
We have previously studied the kinetics of catabolism of newly synthesized proteoglycans in bovine collateral liga-ment [4] and demonstrated that the catabolism of newly synthesized 35S-labelled large proteoglycans was rapid (T½ 2 days) and involved proteolytic cleavage of the core protein On the other hand, 35S-labelled small proteoglycans were lost from the tissue at a slower rate (T½ 20 days) and were either lost from the tissue with an intact or partially degraded core protein, or were internalized
by the cells and completely degraded within the lysosomes of the cells [4] Indeed, it has been shown that the cellular uptake
of small proteoglycans is mediated by receptor proteins present in the plasma membranes of fibroblasts [5,6] This study was undertaken to determine the metabolic fate of newly synthesized 35S-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 radiolabelled 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 acids, 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 Handley, School of Human Biosciences, La
Trobe University, 3086, Victoria, Australia Fax: +61 39479 5784,
Tel.: +61 39479 5800, E-mail: C.Handley@latrobe.edu.au
Abbreviation: GdnHCl, guanidine hydrochloride.
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)
Trang 2[35S]sulfate (carrier free) was from DuPont New England
Nuclear (Boston, MA, USA) Keratanase (from
Pseudo-monassp.; EC 3.2.1.103) was obtained from Sigma Chemical
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 single metacarpophalangeal joint of a one-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[35S]sulfate at 37C for
6 h The sulfate-free medium contained 0.13M sodium
chloride, 4.74 mM potassium chloride, 2.54 mM calcium
chloride, 1.9 mM magnesium chloride, 10 mM glucose,
1.0 mM L-glutamine, 1.19 mMpotassium dihydrogen
phos-phate, 0.02 gÆmL)1 Phenol Red and was buffered with
25 mMHEPES adjusted to pH 7.4 with sodium hydroxide
[7] Sulfate-free medium was used in order to increase the
incorporation of [35S]sulfate into proteoglycans as it has
previously been reported that the rate of incorporation of
[35S]sulfate into proteoglycans by tendon is considerably less
than other connective tissues such as articular cartilage, and
this necessitated the use of high specific radioactivity
[35S]sulfate [8] It was also shown that the rate of
incorpor-ation of [35S]sulfate into proteoglycans was linear over the
6 h incubation period After washing the tissue extensively 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 (0.8Mmagnesium sulfate) so that
the specific radioactivity of radiolabelled sulfate present in or
produced by the explant cultures will be considerably
reduced, thereby decreasing the level of re-use of [35S]sulfate
by the cells of the cultures
The culture medium was collected and replaced 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 period, the tissue was extracted with 4M
guanidine hydrochloride (GdnHCl) in the presence of
proteinase inhibitors at 4C for 72 h, followed by 0.5M
NaOH at 21C for 24 h
Determination of the percentage of35S-labelled
proteoglycans remaining in the matrix of tendon
explant cultures
To determine the percentage of35S-labelled proteoglycans
remaining in the matrix of tendon cultures on each day after
incubation with [35S]sulfate, 0.5 mL aliquots of the medium
fractions, GdnHCl and NaOH extracts were applied to
columns of Sephadex G-25 (PD-10 columns) equilibrated
and eluted with 4M GdnHCl, 0.1M Na2SO4, 0.05M
sodium acetate, 0.1% (v/v) Triton X-100, pH 6.1 The
35S-labelled material that eluted in the excluded volume of
the column was attributable to35S-labelled macromolecules
in the medium that were originally derived from
proteo-glycans present in tendon matrix The35S-labelled material
which eluted in the total volume was shown to represent free [35S]sulfate The rate of loss of35S-labelled proteoglycans from the matrix of explant cultures was calculated from the amount of35S-labelled macromolecules in the medium on each day of culture and that remained in the matrix at the end of the culture period From these data the percentage of
35S-labelled proteoglycans remaining in the matrix was plotted as a function of time in culture [10]
Separation of35S-labelled proteoglycans remaining in the matrix of tendon explant cultures by size exclusion chromatography
Aliquots (1 mL) of the GdnHCl extracts obtained from tissue after predetermined times in culture were applied to a column of Sepharose CL-4B (1.3· 87.0 cm) equilibrated and eluted with 4MGdnHCl, 50 mMsodium 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
35S-radioactivity From these data the percentage of
35S-labelled large and small proteoglycan species remaining
in the matrix at different times in culture was determined [10]
Detection of35S-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 [35S]sulfate for 6 h as described above The tissue was maintained in DMEM alone for up to
10 days The culture medium was collected and replaced daily After predetermined times in culture, the tissue was extracted with 4M 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 samples were then dialysed against distilled H2O containing proteinase inhibitors, lyophilized, and dissolved
in 1 mL of 0.1M Tris/0.1M 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 37C for 24 h in the presence
of proteinase inhibitors [11] Samples were subjected to 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
Intracellular degradation of35S-labelled small proteoglycans
In order to determine the rate of intracellular degradation of
35S-labelled small proteoglycans, bovine tendon from a single metacarpophalangeal joint was incubated with [35S]sulfate for 6 h as described above and then maintained
in DMEM alone for 5 days to allow time for loss of the
35S-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
Trang 3sulfate 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 [35S]sulfate
determined from the amount of35S-radioactivity in the total
volume of the columns The rate of release of35S-labelled
proteoglycans into the culture medium in these experiments
was determined by the35S-radioactivity that eluted in the
excluded volume on Sephadex G-25 columns The
percent-age of35S-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 of the extracelullar matrix of
synovial connective tissues Because the amount of tissue
was limiting, this only enabled points to be repeated in
duplicate Therefore, individual experiments were repeated
at least three times using tissue from different animals
Results
Determination of the loss of35S-labelled proteoglycans
from the extracellular matrix of tendon explant cultures
Explants of bovine tendon were incubated with [35S]sulfate
for 6 h and then maintained in culture in DMEM for up to
10 days Medium fractions were collected daily and the
tissue extracted at the end of the culture period with 4M
GdnHCl followed by 0.5M NaOH as described above
Approximately 75% of 35S-labelled proteoglycans were
extracted from the tendon matrix with GdnHCl (data not
shown) The 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 volume of the columns was
shown to be free [35S]sulfate because it was all precipitated
by barium acetate (data not shown) Figure 1 shows the rate
at which 35S-labelled proteoglycans and free [35S]sulfate
appeared in medium samples on each day in culture During
the first two days of culture the appearance of free
[35S]sulfate in the culture medium was attributable to
unincorporated [35S]sulfate following incubation of the
tissue with [35S]sulfate on day 0 Over the subsequent days
(days 3–10), both the free [35S]sulfate and 35S-labelled
proteoglycans appeared in the culture medium at a similar
rate Any re-use of free [35S]sulfate during the first two days
of explant culture would be minimal as the specific
radioactivity of the radiolabelled sulfate would be markedly
reduced by the sulfate content of DMEM Figure 2A shows
that there was a faster rate of loss of35S-labelled
proteo-glycans from the matrix in the first four days of culture and
approximately 60% of35S-labelled proteoglycans remained
in the matrix after 10 days in culture
Kinetics of loss of large and small35S-labelled
proteoglycans from the extracellular matrix of tendon
explant cultures
The amount of 35S-radiolabel associated 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 two35S-labelled proteoglycan peaks,
a minor peak (Kav 0.05) representing the large prote-oglycans and a major peak (Kav 0.5) representing the small proteoglycans The proportion of 35S-radioactivity associated with the large proteoglycans decreased from 16.6% on day 0 (the day of incubation with [35S]sulfate)
to 2.1% on day 10, whereas that for 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 synthesized35S-labelled large proteoglycans from the extracellular matrix of tendon Although 40% of the35S-labelled proteoglycans was lost from the extracellular matrix of tendon over the 10 day culture period, the hydrodynamic size of each35S-labelled proteoglycan species extracted from the matrix of the tissue immediately after incubation of the tissue with [35S]sulfate and at various time points in culture remained constant The presence of unincorporated [35S]sulfate early
in the culture period (days 0 and 2) is indicated in the elution profiles in the total volume of the column
The percentage of 35S-labelled large and small proteo-glycans remaining in the matrix at various times after incubation with [35S]sulfate was determined by multiplying the percentage of each proteoglycan species present in the tissue extracts on days 0, 2, 4, 6, 8 and 10 of the culture period (Fig 3) by the percentage of 35S-labelled proteoglycans remaining in the matrix on the corresponding day of culture (Fig 2A) This was then expressed as the percentage of the amount of each proteoglycan species present in the tissue on day 0 (Fig 3; top) in order to determine the kinetics of loss of the large and small
Fig 1 Rate of appearance of35S-labelled proteoglycans and [35 S]sul-fate into the culture medium of explant cultures of tendon The proximal region of bovine deep flexor tendon was incubated with [ 35 S]sulfate as described in Experimental procedures, and cultured in DMEM alone for 10 days The rate of appearance of 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.
Trang 435S-labelled proteoglycans in tendon Figure 2B shows that
the loss of 35S-labelled small proteoglycans from the
extracellular matrix was much slower (T½> 20 days)
compared to the 35S-labelled large proteoglycans (T½
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 [35S]sulfate, whilst
only 20% of small proteoglycans were lost over this time
period
Characterization of35S-labelled proteoglycans remaining
in the matrix and released into the medium of tendon
explant cultures by fluorography
To analyze the 35S-labelled proteoglycan core proteins
isolated from either the tissue or released into the culture
medium, tendon was incubated with [35S]sulfate for 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 35S-labelled proteoglycans remaining in the
extracellular matrix of tendon explants cultures (A) The proximal
region of bovine deep flexor tendon was incubated with [35S]sulfate
and maintained in DMEM for 10 days The percentage of35S-labelled
proteoglycans remaining in the matrix of tendon cultures on each day
after incubation with [ 35 S]sulfate was determined as described in
Experimental procedures The error bar represents the range of
duplicate samples (B) The percentage of 35 S-labelled large
proteogly-cans (d) and 35 S-labelled small proteoglycans (s) remaining in the
tissue at each time after incubation of bovine tendon with [35S]sulfate
was determined as described in Results The error bars represent the
range of duplicate samples.
Fig 3 Elution profiles on Sepharose CL-4B of the 35S-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 were applied to a column of Sepharose CL-4B eluted with a buffer containing 4 M GdnHCl In each profile, the amount of 35 S-labelled proteoglycans extracted from the tissue on each day is expressed as a percentage of the 35 S-labelled proteoglycans extracted on day 0 The values in parentheses refer to the relative percentage of large and small proteoglycan species present
of extraction.
Trang 5and 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 [35S]sulfate Based on our previous work it
is likely that these bands represent intact core protein of
aggrecan and V0 and/or V1 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 between 80 to above 250 kDa were also present and
these are likely to represent degradation products of the
large proteoglycans that are retained in the matrix The majority of radiolabelled 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 of biglycan 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 [35S]sulfate, decreased in size with time in culture (lanes ii and iii) indicating extracellular processing of decorin core protein It is possible that newly synthesized decorin contains an intact amino-terminal propeptide which is removed with time in culture
by the action of proteinases present in the extracellular 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 into 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 the medium and we have previously shown that they represent catabolic prod-ucts of aggrecan and versican [1] It must be pointed out that the amount of35S-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 of35S-labelled small proteoglycans by tendon explant cultures Because it was shown that [35S]sulfate appeared in the culture medium throughout the culture period (Fig 1), experiments were performed to determine if this was due to intracellular degradation of 35S-labelled small proteogly-cans Bovine deep flexor tendon was maintained in culture
in DMEM for 5 days after incubation with [35S]sulfate to allow for the loss of the majority of the radiolabelled large proteoglycans (Fig 2B) Cultures were then maintained in DMEM at 37C or 4 C for a subsequent 10 days to determine the effect of reduced cellular activity on the appearance of free [35S]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 [35S]sulfate was reversible Figure 5A shows that in cultures maintained at
37C, there was a continuous rate of formation of free [35S]sulfate in the culture medium The rate of generation of [35S]sulfate in the medium was reduced by over 90% in cultures maintained at 4C, suggesting that metabolically active cells were required for this process This reduction was also demonstrated when cultures were switched from
37C to 4 C on day 10 of the culture period, whereas in cultures that were initially maintained at 4C, there was an
Fig 4 Analysis of35S-labelled proteoglycan core proteins present in the
matrix or medium of explant cultures of tendon Newly synthesized
35
S-labelled proteoglycans remaining in the matrix or released into the
medium of tendon explant cultures after 10 days in culture were
iso-lated as described in Experimental procedures and digested 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 tissue, (ii) tissue 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 peptides are given.
Trang 6apparent increase in the rate of [35S]sulfate appearance
when these cultures were switched to 37C, demonstrating
that this effect was reversible In contrast to the rate of
formation of [35S]sulfate, Fig 5B shows that there was an
increase by 40% of35S-labelled proteoglycans appearing in
the culture medium of tendon explants maintained at 4C
The percentage of35S-labelled proteoglycans remaining in
the matrix of cultures maintained at 37C (calculated from
both the release of 35S-labelled proteoglycans and the
appearance of [35S]sulfate with time in culture) was
approximately 80% by the end of the culture period on
day 15 as shown in Fig 5C However, the loss of
35S-labelled proteoglycans was reduced in cultures
main-tained at 4C, where about 95% of 35S-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 are taken up by the cells and digested intracellularly
To demonstrate that the lysosomal system is involved in the
appearance of free [35S]sulfate in the culture medium,
tendon cultures were maintained in DMEM containing
10 mMammonium chloride following 5 days in culture in
DMEM alone Ammonium chloride is a lysosomotropic
amine and acts by raising the intralysosomal 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 mM ammonium
chloride, the rate of [35S]sulfate appearing in the medium
was suppressed by approximately 78% compared with
control cultures This suppression was further demonstrated
when cultures were switched on day 10 from DMEM alone
to DMEM containing 10 mMammonium chloride When
cultures were switched on day 10 from DMEM containing
10 mMammonium chloride to DMEM alone, the rate of
[35S]sulfate appearance was restored, demonstrating that
this effect was reversible The rate of release of35S-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
35S-labelled proteoglycans remaining in the matrix in
DMEM alone was approximately 80% by the end of the
culture period on day 15 as shown in Fig 6C However, the
loss of35S-labelled proteoglycans was reduced in cultures
maintained in the presence of ammonium chloride, where
about 85% of35S-labelled proteoglycans remained in the
matrix by the end of the culture period on day 15
Discussion
This study showed that the loss of the large aggregating
proteoglycans (aggrecan and V0 and/or V1 splice-variants
of versican) that make up approximately 17% of the
35S-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 the majority of newly synthesized
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 reduced temperature on the rate of formation of [ 35 S]sulfate and release of 35 S-labelled proteoglycans from tendon 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), DMEM at 37 C which was switched to 4 C on day 10 (,), or DMEM at 4 C which was switched to 37 C on day 10 (.) The culture medium was collected daily and analyzed for the presence of [ 35 S]sulfate and 35 S-labelled proteoglycans From this data, (A) the rate of appearance of [35S]sulfate, (B) the rate of release of
35
S-labelled proteoglycans, and (C) the percentage of 35S-labelled proteoglycans remaining in the matrix were determined, as described in Experimental procedures The error bars represent the range of duplicate samples over the remaining 10 days.
Trang 7population 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 is applied to the present study, it is likely that newly synthesized aggrecan and versican 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 other 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 core 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 ADAMTS-4 and ADAMTS-5 are expressed in bovine tendon cells [2], but at different 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)
In contrast to the rapid rate of loss of newly synthesized large proteoglycans, the newly synthesized small proteo-glycans were lost slowly from the matrix of tendon cultures with a half-life of greater than 20 days, which is consistent with findings from earlier studies in explant cultures of 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 the 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 the35S-labelled decorin that was lost from the matrix was taken up by the tendon cells and degraded within the lysosomal system This was shown by the generation of free [35S]sulfate by tendon explant 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 endosomes of skin fibroblasts, osteosarcoma cells and chondrocytes [5,22] Upon entering the cell by endocytosis, decorin is subsequently transported to the lysosomes Previous work has shown that at least two intracellular pathways are involved in the catabolism of endogenously radiolabelled proteoglycans associated with the cell surface in rat ovarian granulosa cells [23] One pathway leads to a rapid and complete intralysosomal degradation resulting in the release of [35S]sulfate In the second pathway, the rate of degradation is slower and commences with extensive proteolysis, generating glycos-aminoglycan chains bound to peptides before final hydro-lysis takes place [23]
Fig 6 Effect of ammonium chloride on the rate of formation of
[35S]sulfate and release of 35S-labelled proteoglycans from tendon
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 alone (d), DMEM
containing 10 m M ammonium chloride (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 presence of [35S]sulfate and
35 S-labelled proteoglycans From this data, (A) the rate of appearance
of [ 35 S]sulfate, (B) the rate of release of 35 S-labelled proteoglycans, and
(C) the percentage of 35S-labelled proteoglycans remaining in the
matrix were determined, as described in Experimental procedures The
error bars represent the range of duplicate samples over the remaining
10 days.
Trang 8This study indicates that the intracellular degradation of
decorin requires metabolically active cells including a
functional lysosomal system because this process was
inhibited at 4C and in the presence of ammonium chloride
(Figs 5 and 6) In addition, when these treatments were
applied to tendon explant cultures there was an inhibition of
the intracellular degradation of decorin and a simultaneous
increase in the appearance of35S-labelled of decorin in the
medium throughout the culture period (Figs 5 and 6), albeit
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 that 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 matrix in strong interactions with other extraextra-cellular
matrix macromolecules where the core protein of this
proteoglycan can undergo proteolytic processing (Fig 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] the 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 small proteoglycans In the case of
tendon this pathway represents about 60% of the
radio-labelled pool of small proteoglycans and in the 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 studies investigating the catabolism of small
proteoglycans in dense 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 support.
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