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Proteoglycan
Synthesis
in Two
Murine
By Suzanne
There
is evidence
are
an
poietic
stromal
support
vitro
with
HCI
(CS/DS)
medium.
H
cells
The
were
respective
dependent
STEM
CELL
culture3
has
for study of the hematopoietic
cellular
and the molecular
level.
types
as potential
poietic
been
implicated
microenvironmental
hematopoietic
at
K,,
least
0.68
=
in
function
the
in vivo
is
is evidence
indicating
mediators
of hemato-
and
that
a
row
of
demon-
consist
ester,
of an amino
sugar
of proteoglycan
bone marrow
culture
p-nitrophenyl-fl-D-xyboside’7
effects
increase
a tenfold
cells
increased
levels
Only
increase
of CS
in the
medium.’8
major
study
thesis
were demonstrated
both of which were shown
CS/dermatan
are
closely
acid/N-acetyl
sulfate
has
was
primarily
in the
cell
layer
extracts.
whereas
the
smaller
wt
of K,., 0.78
18
kd
and
was
average
present
in
glycosaminoroughly
equal
& Stratton.
Inc.
material.
Intact
long-term
bone
to
marrow
proteoglycans.
the
It
synthesis
classes
is
not
of particular
therefore
possible
proteoglycan
to
species
to
of cells.
we investigated
MS3-2A20’2’
are
and
summarized
respect:
D2XRII
competent
and
when
cocultivated
support
with
MS3-2A
cells
to
are
of alkaline
not
function
dihydroxyvitamin
found
two
such
stromal
cell
lines,
namely,
D2XRII,22a3
the major
properties
of which
in Table
1. These
cell lines differ
in one
cells
are
long-term
hematopoietic
microenvironmentally
in
vitro
hematopoiesis
progenitor
cells.23
microenvironmentally
as
D stimulation
phosphatase
competent
preosteoblasts
responding
with
greatly
increased
but
to
1:3levels
activity.24
MATERIALS
Materials.
Guanidine
(optical
grade),
Sephadex
AND
HCI
METHODS
(practical
G-50,
grade),
diethyl
cesium
aminoethyl
chloride
(DEAE)-
culture
medium
of
to have
profound
to
a fivefold
(CFU-G/
From
sity
hematopoietic
were
associated
addressed
the
with
question
of
marrow
syn-
layer and the medium,
at least three species
of
proteoglycans.
related
glycosaminoglycans,
galactosamine
disaccharide
A heparan
contain
a variety
of cellular
phenotypes,
both strohematopoietic,
many of which
have been shown
to
appear
long-term
in long-term
bone
patterns
of proteoglycan
in the cell
to contain
(CS/DS)
Vol 70. No 6 (December),
(CS)
in pluripotentiab
synthesis
similar
are
to a carboxylate,
and lead
progenitors
changes
a single
of
subunits
in murine
to the
shown
These
intact
proteoglycan
culture.’9
Essentially
Blood.
synthesis
by addition
was
(CFU-S).
number
in the medium
and
gbycosaminoglycan
in the
bone
marrow
cultures,’6
on hematopoietic
function’8
in granulocyte/macrophage
M) and
stem
linked
sulfate
to be the major
glycosaminoglycan
heparan
sulfate
(HS),
the major
cell layer
of murine
long-term
Perturbation
a variable
Chondroitin
kd.
glycosaminogly-
An alternative
approach
for the investigation
of stromal
proteoglycan
synthesis
is the study
of stromal
cell lines
derived
from long-term
bone marrow
cultures.
In the present
matrix
chains.
Glycosaminoglycans
of repeated
disaccharide
or both.’4
40
and
elements.
particular
core
in turn
layer
species
synthesize
central
that
present
and
0.58
synthesized
cell
by Grune
cultures
mal and
essential
a sulfate
kd
=
only in the degree
of epimerization
of glucuronic
acid
iduronic
acid. HS proteoglycan
accounted
for approximately
regulatory
functions
in a number
of systems.’5
are complex
macromolecules
consisting
of a
side
consisting
was
cells
mol
stromal
to have growth
Proteoglycans
glycosaminoglycan
pobysaccharides
38
of K1,
glycosaminoglycan
mel wt. 38 kd) was dense
mainly in the culture medium.
A single CS/DS
of
ascribe
variety
been
extracelbubar
is attached
kd
in the
(K,, - 0.68;
and present
study,
to which
were
two HS proteoglycans.
larger (K.,
0.45; glycosaminoglycan
mol wt. 30 kd)
of low density
on gradient
centrifugation
and more
components
play a significant
role in supporting
hematopoiesis in long-term
bone
marrow
culture.
Proteoglycans
are
important
components
of extracelbular
matrices’
and appear
protein
36
10% of the labeled
strated,’#{176}’3
There
wt
D2XRII
V 1987
with a complex
of long-term
in vitro model
have
values
amounts
in the medium
and in the cell layer. MS3-2A
and
D2XRII thus appear phenotypically
distinct with respect
to
proteoglycan
synthesis.
These differences
are discussed
in
relation
to the microenvironmental
function
of bone mar-
enzymatic
microenvironment
at both the
A variety
of cellular
pheno-
regulators
wt)
proteoglycan
proteoglycan
molecular
function,’9
growth
(HS)
by
and
interaction
The
technique
provided
a valuable
Lines
glycan
primarily
on close-range
cellular
bone marrow
have
0.40
=
(mol
sulfate
The
was
Cell
Bentley
weight
mol
Stromal
chromatogra-
glycosaminoglycan
EMATOPOIETIC
by
density
produced
present
A.
prominent
chondroitin/dermatan
K,,
proteoglycans.
CL-2B.
were
guanid-
analyzed
sieve
Two
in
Cells
chloride
examined
MS3-2A
species.
not.
4 mol/L
were
molecular
further
digestions.
Sepharose
does
and
media
capacity
Marrow
Stuart
extract.
supporting
cesium
were
proteoglycan
sulfate
and
and
Proteoglycans
chemical
on
cells
centrifugation.
three
MS3-2A
and
can
was
hemato-
in their
D2XRII
as precursor.
of
hemato-
synthesis
differ
vitro.
chromatography.
the
and D2XRII
lines
in
whereas
extracts
gradient
phy.
These
MS-sulfate
ion-exchange
and
lines.
hematopoiesis
hematopoiesis.
labeled
proteoglycans
of
MS3-2A
in the
cell
stromal
component
Proteoglycan
investigated
poietic
me
that
functional
microenvironment.
therefore
to
indicating
important
L. Kirby
Bone
CS
and
with
glucuronic
subunits
differing
1987: pp 1777-1783
DS
the Department
ofNorth
ofPathology,
Carolina
at Chapel
School
ofMedicine,
Univer-
Hill.
Submitted
May 5, 1987; accepted July 30. 1987.
Supported
by Nationallnstitutes
ofHealth
Grant No. AM 35743
and by a Charles E. Culpeper
Fellowship
award (S.L.K.).
Address
reprint requests to Stuart A. Bentley. MD, PhD, Department of Pathology,
Brinkhouse-Bullitt
Bldg 228H, University
of
North
Carolina,
The publication
charge payment.
“advertisement”
indicate
a-; J 987
Chapel
Hill,
costs ofthis
This article
in
accordance
NC
27514.
article
must
with
were defrayed
in part by page
therefore
be hereby
marked
18
U.S.C.
§1 734
solely
to
this fact.
by Grune
0006-4971/87/7006-001
& Stratton,
Inc.
I$3.OO/O
1777
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1778
KIRBY
Table
1 . Major
Characteristics
D2XRII
Stromal
of the MS3-2A
collagens
Basement
membrane
MS3-2A
(types
I. Ill)
D2XRII
+ +
+
linear
-
+
Fibronectin
+
+
Laminin
-
+
M-CSF
+
+
Multi-CSF
-
-
+
+
+ +
-
-
+
mL. 35S-labeled peaks were pooled and then concentrated
on DEAESephacel
columns of 1.5-mL bed volume.25 Recoveries
of 35S-labeled
material
were >80%.
Analytic columns ofSepharose
CL-2B and CL-4B (1 .6 x 100 cm)
were prepared
in and eluted with 4 mol/L
guanidine
HC1, 0.05
mol/L Tris, 0.05 mol/L sodium acetate, and 0.2% Triton X-l00, pH
7.0, at a flow rate of 12 mL/h.
Analytic
columns
of Sepharose
CL-6B (1.6 x 100 cm) were prepared
in and eluted with 0.1 mol/L
sodium acetate
and 0.1 mol/L Tris, pH 7.3 (flow rate, 12 mL/h).
The void and total column volumes were marked with blue dextran
and free 35S-sulfate,
respectively.
Recoveries
of labeled macromolecules were >80%.
Isopyknic
cesium
chloride
centrifugation.
Solid cesium chloride, 0.5 g/g, was added
to medium
and cellular
proteoglycan
fractions
in 4 mol/L
guanidine
HCI buffer after ion-exchange
chromatography
and concentration
to give an initial density of I .40
g/mL.
Dissociative
density
gradients
were formed
by using a
Beckman
60 SW rotor at 37,000
rpm at 10#{176}C
for 48 hours in a
Beckman
L8-70 ultracentrifuge
(Beckman
Instruments,
Inc., Fullerton, CA). Fifteen 0.25-mL
fractions
were collected
after piercing
the bottom
of the tube. These
fractions
were pooled into four
approximately
equal fractions,
designated
Dl through
D4 from
bottom to top, for subsequent
gel-filtration
chromatography.
Chemical
analyses.
Alkaline
borohydride
treatment
was performed
in 0.05 mol/L
sodium
hydroxide
and 1 mol/L
sodium
borohydride
at 45#{176}C
for 24 hours, which resulted in a-elimination
of
collagen
Hydrocortisone-induced
Preosteoblast
(type
IV)
adipogenesis
properties
Microenvironmental
competence
Sephacel,
Sepharose
(CL-2B,
CL-4B,
and CL-6B),
6-aminohexanoic acid, benzamidine
HCI, N-ethylmaleimide
(NEM),
phenybmethylsulfonyl
fluoride (PMSF),
CS (whale cartilage),
chondroitinase
ABC (Proteus
vulgaris),
and chondroitinase
AC (Arthrobacter
aurescens)
were purchased
from Sigma Chemical
Co. St Louis;
heparitinase
(Flavobacterium
heparinum)
from ICN Immunobiologicals,
Lisle, IL; sodium “S-sulfate
from ICN Radiochemicals,
Irvine, CA; Safety-Solve
scintillation
cocktail from Research
Products International
Corp. Mount Prospect,
IL; fetal calf serum and
sulfate-free
RPMI 1640 medium from GIBCO,
Grand Island, NY;
L-glutamine,
l000x
penicillin/streptomycin,
HEPES,
RPMI
1640,
and medium
199 from the Tissue Culture
Facility,
University
of
North Carolina
at Chapel Hill.
Cell culture techniques.
The MS3-2A
cell line was generously
provided
by Dr TM. Dexter, Manchester,
England.
The D2XRII
cell line was generously
provided by Dr iS. Greenberger,
Worcester, MA. Cultures
were maintained
in 25-cm2 tissue culture flasks at
34#{176}C
in a humidified
atmosphere
of 5% CO2. 95% air. MS3-2A
cells
were cultured
in medium
199, supplemented
with 15% fetal calf
serum,
1% 200 mmol/L
L-glutamine,
and penicillin/streptomycin
l000x.
D2XRII
cells were grown in RPMI 1640 containing
10%
fetalcalfserum,
1% 1 mol/L HEPES,
l%200mmol/LL-glutamine,
and l000x
penicillin/streptomycin,
under similar conditions.
The
cells reached confluence
in four to five days and were subcultured
by
trypsinization,
with a split ratio of 1:5.
Radioisotope
labeling.
Labeling
was initiated
as the cell cubtures approached
confluence
by adding
fresh medium
containing
either 50 jzCi/mL
or 200 zCi/mL
sodium
355-sulfate
as precursor.
Sulfate-free
RPMI 1640 was used in the labeling experiments.
Isolation
of proteoglycans.
After
labeling,
the cells were
removed
from the bottom of the flask with a cell scraper and the
contents transferred
to a centrifuge
tube. The medium and cells were
centrifuged
at I ,000 g for 15 minutes
at 5#{176}C.
The medium
was
decanted,
and 0.53 g/mL
solid guanidine
HCI, 10 tL/mL
lOOx
NEM, and 10 ML/mL lOOx PMSF were added to make the solution
4 mmol/L
in guanidine
HCI, 10 mmol/L
in NEM, and I mmol/L
in
PMSF.
Cell pellets were extracted
at room temperature
with 4
mmol/L
guanidine
HCI, 0.1 mol/L
6-aminohexanoic
acid, 10
mmol/L
NEM, 1 mmol/L
PMSF, 5 mmol/L
benzamidine
HCI, 50
mmol/L
sodium
EDTA,
50 mmol/L
sodium
acetate,
and 0.5%
Triton X-lOO, pH 5.8, 1 mL/25-cm2
flask culture.
The cell extracts
and media were stored at - 20#{176}C
for subsequent
analysis.
Unincorporated
isotope was removed
from the medium
fractions
and cell
extracts
by Sephadex
0-50 chromatography
(bed volume of 8 mL
for each I .5-mL sample).
Columns
were eluted with 8 mol/L urea,
0.05 mol/L sodium acetate,
0.15 mol/L sodium chloride,
and 0.5%
Triton X- I 00 for subsequent
ion-exchange
chromatography.
Recovcries
BENTLEY
acetate,
and 0.5% Triton X-100 were applied
to DEAE-Sephacel
columns
(1.6 x 4 cm) equilibrated
with the same 8 mol/L
urea
buffer. After sample application,
the columns
were washed
with
approximately
20 mL 8 mol/L urea buffer and then eluted with a
Cell lines
Characteristic
Interstitial
and
AND
of macromolecular
material
were
unincorporated
material
was complete.
Column
chromatography.
Sephadex
in 8 mol/L
urea,
0.15
mol/L
sodium
>85%,
and
separation
G-50-excluded
chloride,
0.5
from
fractions
mol/L
sodium
NaCI
gradient
glycosaminoglycan
(0. 1 5 to 1 .0 mol/L)
chains
from
the
using
a total
protein
core.26
volume
Excess
of 100
borohy-
dride was destroyed
by neutralization
with glacial
acetic
acid.
Nitrous
acid treatment
was performed
at room temperature
in 0.18
mol/L acetic acid with 0.24 mol/L sodium nitrite for 80 minutes.27
The reaction
was stopped by the addition
of an equal volume of 2
mol/L ammonium
sulfamate.
Enzymatic
treatments.
Samples
were digested
for four hours at
37#{176}C
with chondroitinase
ABC (0.05 to 0.1 U/mg
carrier)
and
chondroitinase
AC (0.25U/mg
carrier)
in a 0.1 mol/L
Tris, 0.1
mol/L sodium acetate buffer, pH l.3.
Enzyme concentrations
were
adjusted
for the content of CS carrier.
Heparitinase
digestion
was
carried out in a 0.1 mol/L Tris, 0.1 mol/L sodium acetate, and 0.005
mol/L
calcium
acetate
buffer,
pH 7.0, containing
0.5 mg/mL
ovalbumin,
I mmol/L
PMSF, and 0.1 U/mL heparitinase.
Digestion was continued
for three hours at 37#{176}C
and fresh enzyme added
for one additional
hour.
RESULTS
Kinetics
accumulation
was linear
ofmacromolecular
incorporation
of 35S-labebed
for up to 96 hours
layer
raphy
extracts
indicated
stable
lation
for 96 hours,
although
of smaller
molecules,
of/abel.
The
macromolecules
in the media
but tended
to plateau
in the cell
(Fig 1). Preliminary
that proteoglycan
gel-filtration
synthesis
was
there was
presumably
chromatogqualitatively
progressive
proteoglycan
accumudegra-
dation
products,
during
the later stages
of the observation
period
(data
not shown).
A 48-hour
labeling
period
gave
maximum
incorporation
of isotope
with acceptable
bevels of
proteoglycan
degradation
and
mab for subsequent
experiments.
the ratio of macromolecular
355
in the cell
approximately
Analysis
layer
of
extracts
culture
was
was
media
therefore
considered
optiAt the 48-hour
time point,
activity
in the media
to that
proteoglycans.
2: 1.
DEAE-
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HEMATOPOIETIC
STROMAL
PROTEOGLYCANS
1779
Table
2.
Analysis
From
of CsCI
MS3-2A
Ea
Density
and D2XRII
Pe;c:t
Gradient
Culture
CS/DS
U
Fractions
Medium
P roteoglycan
HS Prot eoglycan
4)
0
Fraction
Density
(gIL)
Gradient
Activity
Percent of
Fraction
1.59
25
Fraction
K,
(Xl
K,,
46
0.36,0.70
47
0.62
28
0.60
>-
F>
FU
MS3-2AD1
1 .48
34
64
0.42,
MS3-2AD3
1.39
28
73
0.38,0.67
37
0.62
U)
MS3-2AD4
1.33
13
62
0.60
43
0.69
MS3-2A
D2
D2XRIID1
0.69
1.58
22
20
0.85
80
0.66
Ui
D2XRII
D2
1 .48
33
40
0.82
60
0.69
a:
D2XRII
D3
1.39
24
65
0.78
30
0.62
D2XRII
D4/I
1.31
9
42
0.64
66
0.55
0.80
20
0
00
D2XRIID4/ll
0
z
1.31
CS/DS
12
proteoglycans
sensitive
70
were
quantitated
and HS proteoglycans,
activity
as
chondroitinase
ABC-
as heparitinase-sensitive
activi-
ty.
Scanty
LABELLING
PERIOD
residual
material
with
no clearly
defined
peak.
(hr)
Fig 1 .
Accumulation
of S-Iabeled
macromolecules
medium
(#{149})
and cell layer
(A)
of MS3-2A
cultures.
identical results were obtained
in D2XRII cultures.
in the
Virtually
D2XRII
the
medium
range
0.65
fractions
to 0.75
contained
a major
Kay, whereas
the
also contained
a clearly
defined
second
peak
The percentages
of 35S-labeled
material
Sephacel
chromatography
both MS3-2A
and
broad
35S-labeled
mol/L
NaC1
(data
peaks
was
ugation.
fractions
Fig 2A.
further
of macromolecular
material
analyzed
by CsCb
Sepharose
CL-2B
from the MS3-2A
All the gradient
density
gradient
chromatograms
culture
medium
fractions
contained
to 0.45,
resolved
also
contained
CL-2B chromatograms
of the gradient
D2XRII
culture
medium
are illustrated
subsequent
Sepharose
Table
2 together
with
Digestion
centrif-
data
indicate
that
from
of
a tendency
density.
to
The
gradient.
D2 and
of Kay 0.35
polydisperse
the
All
HS
also
2
4
recovered
E
4
.5
D2
80
at
>-
>-
F>
F0
.5 80
I>
F0
.5
U)
D3
U)
Ui
>
Ui
>
20
-I
-j
D4
Ui 80
a:
Ui
a:
20
C
A
#{149}‘
60
80
‘
0
ELUTION
20
40
VOLUME(mI)
60
80
proteo-
accounted
for 20% of the
all fractions
contained
a
(Kay, 0.61)
from
the
that
accounted
gradient.
K0
a
Fig 2.
Sepharose
Cl-2B chromatograms
of
CsCI
gradient
fractions
from
MS3-2A
and
D2XRII
culture
media.
All MS3-2A
gradient
fractions
(A) contained
proteoglycans
of K,
0.65 to 0.70 in substantial
quantities.
The Dl to
D3 fractions
also contained
proteoglycans
of
K,,, 0.35
to 0.45.
whereas
the D4 fraction
contained
an incompletely
resolved
peak
of
intermediate
hydrodynamic
size.
All D2XRII
medium
fractions
(B) contained
a major labeled
peak in the range 0.65 to 0.75 K,,.,. In addition.
the D4 fraction
contained
a clearly
defined
second peak of K,,, 0.55 (B).
CS/DS
for D2XRII
culture
medium
indicate
that
in all fractions
(Kay, 0.65
to 0.75)
contained
80
.5
Ui
0.
fractions
a larger
data
peaks
A
fractions
of Kay, 0.68
recovered
of the
60
.
medium
35%
6
and heparvolume)
on
intermediate-density
to 0.45)
that
In addition,
K0
0
ABC
column
all MS3-2A
contained
proteoglycan
activity
in
of Kay 0.55.
in each culture
CS/DS
proteoglycan
of the total radioactivity
The
D3)
glycan
(Kay,
0.35
recovered
activity.
an incompletely
size. Sepharose
fractions
from
in Fig 2B.
the
(mainly
peak
D4 fraction
CL-2B
chromatography
are shown
in
K,. values
for the resistant
material.
proteoglycans
proteoglycans
whereas
the D4 fractions
contained
peak of intermediate
hydrodynamic
sensitive
to chondroitinase
and eluting
at Vt (total
contained
a polydisperse
that accounted
for 45%
in substantial
quantities,
with
hydrodynamic
size with decreasing
Dl to D3 fractions
fraction
digestion
of the gradient
are illustrated
in
0.65 to 0.70
decreasing
medium
itinase
from
D2XRII
culture
media
revealed
single,
peaks
eluting
between
0.40
and
0.65
not shown).
Pooled
material
from these
labeled
medium
for
Digestion
the
major
varying
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
KIRBY
1780
somewhat
less dense polydisperse
CS/DS
proteoglycan
(Kay
0.78).
These
proteoglycans
accounted,
respectively,
for 45%
mol/L
material
and 0.65 mol/L
NaC1
from
these
peaks
was
(data
further
density
gradient
Sepharose
and 49%
Analysis
tograms
extract
of the gradient
are illustrated
proportions
of a dense
of the total
of the
D4
presence
that
of a larger,
accounted
I-IS proteoglycan
activity
gradient
of Kay 0.67
recovered
fraction
low-density
from
also
a
the gradient.
revealed
the
HS proteogbycan
for approximately
and
(Key, 0.55)
6% of the
total
fractions
recovered
D3
radioactivity.
Proteoglycans
medium
were
nase
and
CL-2B.
trated
from
MS3-2A
and
digested
with chondroitinase
the
resistant
material
The resulting
by ultrafiltration,
dride
treatment
peaks
and
for subsequent
that
corresponded
kd. The elution
noglycan
showed
profile
showed
to a molecular
Both
which
confirmed
material
in both
digestion,
ABC-resistant
HS proteoglycan.
The
was
purified
separately
fraction
and subjected
subsequent
Sepharose
included
noglycan
(mob
wt)
peaks
were
fully
the
original
chromatogram
extract
tinase
to
chondroitisamples
larger,
bow-density
HS
from
the
D2XRII
to alkaline
borohydride
CL-6B
The
susceptible
that
showed
CL-2B
was
isolated
from the
were
virtually
single
included
peaks
to a glycosaminoglycan
chains
from the D2XRII
D4
A
a single
of Kay 0.36
that
corresponded
K0,
0.60.
tO
chroma-
resolved
layer
of all
Dl
to
species
in the
Fraction
peak
cell
Fractions
of a proteoglycan
Pooled
CsC1
of
equivalent
Dl also
of Kay 0.73.
Sepha-
two major
peaks of K,,, 0.55
The K, 0.78 peak predom-
whereas
the K.jy 0.55
D3. The D4 fraction
to 0.60
showed
pik
peaks
of 35S-labeled
material
in each
sensitive
to chondroitinase
ABC
and eluting
at Vt on subsequent
are
shown
for the resistant
cell layer
extract
in Table
material.
indicate
cell
layer
and hepariSepharose
3 together
Digestion
that all
with
data for the
fractions
con-
tamed
a HS proteoglycan
majority
of this proteoglycan
of K,, 0.55 to 0.60, although
the
was present
in the low-density
fractions
HS
(D3
and
D4).
45%
The
fractions
Dl
to that
accounted
found
for
teoglycan
considerably
smaller
and gave a single
included
peak of K,,,
0.47 that corresponded
to a mob wt of 22 kd.
Analysis
of cell layer
extract
proteoglycans.
DEAESephacel
elution
profiles
of macromolecubar
material
obtained
from
MS3-2A
and
D2XRII
cell layer
extracts
showed
single,
broad
35S-labeled
peaks eluting
between
0.40
CL-2B
size to that
seen
0.35 to 0.45).
chromatography
radioactivity.
were
by
0.62.
fraction
digestion
approximately
the gradient.
two MS3-2A
medium
CS/DS
identical
to each
other
and
mob wt of 38 kd. Glycosaminoglycan
medium
CS/DS
proteoglycan
and
Kay values
not shown).
analyzed
chromatograms
of the CsCl gradient
fractions
cell layer extract
are illustrated
in Fig 3B. The
percentages
MS3-2A
proteoglycan
medium
treatment.
(K,,
mated
in fraction
Dl,
predominated
in fraction
of Kay 0.45
of 36
peak of Kay 0.35 that corresponded
to a glycosamimob wt of 40 kd. The elution
profiles
of glycosami-
noglycans
proteoglycans
showed
for MS3-2A
culture
a single
peak of Kay
weight
of Kay 0.55
amount
Dl to D3 fractions
each showed
to 0.60 and K,,, 0.78, respectively.
analysis.
profile
for D2XRII
medium
HS glycosamia single peak of Kay 0.36 that corresponded
nitrous
nase
concenborohy-
peaks
an incompletely
rose CL-2B
from D2XRII
BENTLEY
fractions
from
MS3-2A
in Fig 3A. Chromatograms
a small
hydrodynamic
fractions
contained
Sepharose
glycosaminoglycan
to a mob wt of 38 kd.
acid
on
(see Fig 2) were pooled,
subjected
to alkaline
The Sepharose
CL-6B
elution
medium
1-IS glycosaminoglycan
0.37
separated
showed
contained
similar
medium
D2XRII
culture
ABC or hepariti-
centnifugation.
AND
to D3 was
a large
in MS3-2A
approximately
Fractions
of K,,, 0.65
D2
to 0.70
total recovered
radioactivity.
fraction
Dl was fully sensitive
tion and accounted
ty. On Sepharose
alkaline
borohydride
0.36 and 0.44,
proteoglycan
accounted
for
of the total radioactivity
recovered
from
Kay 0.35
to 0.45
material
in gradient
CS/DS
proteoglycan
culture
10% of
medium,
the total
to D4
that
contained
similar
and
this
recovered
a CS/DS
accounted
for about
The K,y 0.73 ik
to chondroitinase
pro35%
of
in gradient
ABC diges-
for 10% of the total recovered
radioactiviCL-6B
chromatography
before
and after
treatment,
the observed
K,, values
were
respectively.
This
suggests
that
the material
in
K0,
80
60
40
-
20
:
0
.5
I80
8860
;40
I-
20
>
F-
80
U)
60
40
0
Ui
>
20
-j
80
60
40
20
Fig 3.
Sepharose
CL-2B chromatograms
of
CsCI
gradient
fractions
from
MS3-2A
and
D2XRII
cell layer extracts.
MS3-2A
cell layer
(A) fractions
Dl to D3 contained
proteoglycans
in the range of K,, 0.35 to 0.45. All fractions
contained
peaks of K,, 0.55 to 0.60. Fraction
Dl
also
contained
an incompletely
resolved
peak
of K,,, 0.73. The D2XRII
cell layer (B) Dl to D3
fractions
each showed
two
major
peaks
of K.
0.55 to 0.60 and K, 0.78. respectively,
the K
0.78 peak predominating
in fraction
Dl and the
Kr, 0.55 to 0.60 peak. in fraction
D3. The 04
fraction
showed
peaks of K,,, 0.45 and 0.62.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
HEMATOPOIETIC
Table
STROMAL
3.
Analysis
of CsCl
From
MS3-2A
and D2XRII
Fraction
MS3-2A
Density
Gradient
Density
(9/LI
Percent of
Fraction
1.60
30
76
0.35,
1.49
32
68
1.40
21
43
1.35
17
40
MS3-2AD2
D3
MS3-2AD4
CS/DS
Table
Fractions
Cell layer
Percent of
Total
Gradient
Activity
Dl
MS3-2A
1781
PROTEOGLYCANS
Proteoglycan
HS Proteolycan
Fraction
(%)
K,,
0.75
K,,
MS3-2A
19
MS3-2A
45
0.68
0.78
Int
DS
36
0.61
Poly
HS
36
0.65
70
0.55
D2X-M-HS1
D2XRII
45
0.67
High
HS
38
75
0.55
D2X-M-HS2
D2XRII
Low
HS
12
13
0.66
85
0.58
D2XRII
D2/ll
1.49
20
85
0.81
20
0.85
D2XRII
D3/l
1.42
13
19
0.69
79
0.59
D2XRII
D3/ll
1.42
8
80
0.82
35
0.82
D2XRII
D4/l
1.33
5
20
85
0.55
D2XRII
D4/ll
#{149}
60
0.73
quantitated
as
35
Abbreviations:
mediate;
0.67
chondroitinase
Poly,
activi-
peak.
CS/DS
CS/DS
and
proteoglycan
that
glycan
mob wt was 26 kd.
Digestion
data for the D2XRII
the
presence
rather
than
the corresponding
a free
of an intermediate-
extract
to low-density
indicate
HS
from
the
Kay 0.58
approximately
8% of
diateto high-density
accounted
ered
for approximately
from
Cell
nase
rated
proteo-
glycan
MS3-2A
accounted
An
of
for
activity
recov-
digested
with
chondroiti-
2B-CL.
by ultrafiltration,
treatment
and
for subsequent
analysis.
The Sepharose
CL-6B
cell layer
HS glycosaminoglycan
thus
equal
in size
inoglycan,
although
somewhat
larger
to the
the
and
glycosaminoglycans
CS/DS
proteoglycans
MS3-2A
intact
sepaFig 3)
subjected
to
glycosamino-
elution
profile
for
showed
a single
more
medium
cell
layer
buoyant.
isolated
were
HS glycosamproteoglycan
The
elution
profiles
proteoglycans
profile
for
D2XRII
from
cell
layer
MS3-2A
HS
of
subsequent
nitrous
acid
digestion.
The
larger,
low-density
of the
D2XRII
secreted
culture
two
differing
secreted
medium
mob
layer
of Kay
wt
of
IS
in
proteoglycans
media
clearly
size
and
identified
are summarized
in
identifiable
CS/DS
buoyant
density.
In
relatively
than
did
more
HS proteoglycan
into the culture
MS3-2A
cells.
In further
contrast,
the
medium
HS
a small,
larger,
single,
proteoglycans
high-density
low-density
polydisperse
in MS3-2A
in Table
CS/DS
and
species
HS
of the
cell
layer
5. The D2XRII
cell
proteoglycan,
closely
the culture
medium.
In contrast,
were
identified
in the MS3-2A
MS3-C-DS2)
readily
resolved
(D2X-M-HS1)
(D2X-M-HS2),
proteoglycan
D2XRII
and
were
species
MS3-2A
culture
medium.
The major
characteristics
Table
5.
Summary
Identified
and
proteoglycans
extracts
identified
are summarized
layer contained
resembling
that
three CS/DS
cell layer.
were
of Major
in MS3-2A
similar
Characteristics
and D2XRII
me-
to
Designation
Source
(Cell)
Percent
Source
of
PG
only a single
identified
in
proteoglycans
Two of these
to correspond-
of Proteoglycans
Cell layer
K,, CL-2B
a
whereas
only a
was identifiable
in
Extracts
GAG
glycosami-
noglycan
showed
a single peak of Kay 0.41 that corresponded
to a mob wt of 30 kd. This material
was fully susceptible
cell
peak
to a glycosaminoglycan
characteristics
(MS3-C-DS1
from the two MS3-2A
cell layer
virtually
identical
to each other
isolated
D2XRII
included
was
and showed
single
included
peaks
of Kay 0.36 that corresponded
to a glycosaminoglycan
mob wt of 38 kd. These
results
were
in turn
identical
to those
obtained
for the
corresponding
dium.
The elution
the
a single
contrast,
D2XRII
cells
produced
only
a single
CS/DS
proteoglycan
that
was smaller
and more
dense
than
the
corresponding
MS3-2A
proteoglycan
(MS3-M-DS2),
with
smaller
glycosaminoglycan
side
chains.
D2XRII
cells
into
peak of Ky 0.37 that corresponded
to a mob wt of 36 kd. This
material
was fully
susceptible
to subsequent
nitrous
acid
digestion.
The
MS3-2A
cell layer
HS glycosaminoglycan
was
major
proteoglycans
intermeKay 0.78
and the resistant
material
The resulting
peaks
(see
concentrated
borohydride
corresponded
D2XRII
were
heparitinase
on Sepharose
pooled,
alkaline
This
of the total
58%
proteoglycans
or
that
from
showed
in MS3-2A
and
Table
4. MS3-2A
the gradient.
layer
ABC
were
material.
the recovered
activity.
CS/DS
proteoglycan
inter-
DISCUSSION
glycan
of Kay 0.58 that accounted
for 34% of the “S activity
recovered
from the gradient.
The D4 fraction
also contained
a low-density
HS proteoglycan
of Kay 0.45 that appeared
distinct
isolated
glycosamino-
layer
Int,
kd.
The
cell
40
glycosaminoglycan;
peak of Kay 0.41 that corresponded
to a
mob wt of 30 kd. The elution
profile
of
proteoglycan
0.52,
a small
GAG,
polydisperse.
glycosaminoglycan
defined
0.55
proteoglycan;
a single
included
glycosaminoglycan
ABC-
as heparitinase-sensitive
6
PG.
HS proteoglycan
was purified
from the D2XRII
cell layer
D4 fraction
and subjected
to alkaline
borohydride
treatment.
A subsequent
Sepharose
CL-6B chromatogram
also showed
ty.
was
22
49
1.49
glycosaminoglycan
40
D2XRII
D2/I
question
38
DS
MS3-2A
D2XRII
no clearly
DS
Poly
MS3-M-HS
20
with
GAG
Int
D2X-M-DS
0.82
material
Density
0.61
85
#{149}Scantyresidual
K,, CL-2B
0.35-0.40
0.52
33
HS proteoglycans,
MS3-M-DS1
GAG
mol wt
(kd)
67
5
and
of
PG
51
0.69
25
activity
Percent
Source
0.35,
1.59
sensitive
Designation
Source
(Medium)
Media
0.36,0.70
1.59
12
of Proteoglycans
Culture
MS3-M-DS2
Di/l
were
Characteristics
and D2XRII
0.56
D1/ll
1.33
of Major
in MS3-2A
35
D2XRII
proteoglycans
Summary
Identified
D2XRII
CS/DS
4.
Extracts
Density
GAG
mol WI
(kd)
MS3-C-DS1
MS2-2A
10
0.35-0.40
lnt
DS
38
MS3-C-DS2
MS3-2A
25
0.65-0.70
Poly
DS
40
26
MS3-C-DS3
MS3-2A
10
0.73
High
DS
D2X-C-DS
D2XRII
58
0.78
High
DS
15
MS3-C-HS
MS3-2A
55
0.55-0.60
Low
HS
36
D2X-C-HS1
D2XRII
34
0.58
Int
HS
30
D2X-C-HS2
D2XRII
8
0.45
Low
HS
30
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
KIRBY
1782
ing proteoglycans
MS3-M-DS2),
C-DS3)
more
CS/DS
in the culture
medium
whereas
a third CS/DS
closely
resembled
the
proteoglycan.
Both
HS proteoglycans.
The
were
further
resolved
fractions
whether
The
ences
present
studies
the
MS3-2A
and
pattern
low-density
have
D2XRII
of
cells.
cells
in a variety
The
had
of
substantial
proteoglycan
much
cell
on
Sepharose
The major
a low-density
CL-2B.
glucuronic
lines.
cells
contained
DS
D2XRII
mainly
cells
cells.
more
Bovine
these
cells
proteoglycan,
from the
synthesis
resembled
that
aortic
endothelial
cells
buoyant
HS proteoglycan,
of Kay 0.43 on Sepharose
of K,y
associate
differences
in
differences
to represent
to develop
of so-called
and it has
do not provide
of hematopoiesis
in
Because
the proteoglycan
marrow
been
a microenvironment
long-term
synthesis
in IMR-90
two CS/DS
Kay 0.68 on
large,
idu-
proteogbycan
medium
pattern
and
of
in endobeen
active
human
proteoglycan
fibroblasts
demonstrated
is
material
bone
from
in this
pattern
species
cells,
with
contributors
species
smaller,
iduronic
small quantities
cells,
in terms
a Sepharose
of
cells
Kay 0.30
cells,
D2XRII
musclelike
cells
tainly a possibility,
can heterogeneity
It was
previously
on
been
Sepharose
suggested
that
proposed
of
as
to produce
glucuronic
CL-2B
and
a
of Kay 0.69,
with only
Thus smooth
muscle
synthesis,
more
which are incompetent
cells. Although
the
bone
it is probable
reflected
the
in
of
microenvironmental
acid-rich
species
of HS proteogbycan.35
in long-term
column
resembled
have been shown
including
a large,
of proteoglycan
ble MS3-2A
tally, than
CL-6B
system.
to hematopoietic
proteoglyglucuronic
CL-6B,
richer
for only 10%
synthesis
have
similar
the cell layer.’9
A large,
in the void volume
of a
culture
which
BENTLEY
of CS/DS
size,
on Sepharose
accounted
of proteoglycan
muscle
acid-rich
closely
resem-
microenvironmenpresence
of smooth
marrow
cultures
is cer-
that the observed
proteoglypresence
of multiple
stromal
and hematopoietic
phenotypes.
Synthesis
of proteoglycan,
especially
CS proteoglycan,
has been demonstrated
in lymphocytes,36’37
monocytes,37’38
neutrophils,39’#{176} eosinophibs,4’
basophils.42
Proteoglycans
derived
from
elements
may thus represent
a substantial
proteoglycan
synthesis
in hematopoietically
bone marrow
cultures.
There
is compelling
evidence
ute
functional
significantly
to the
that
hematopoietic
proportion
of total
active
long-term
proteoglycans
hematopoietic
contribmicroenvi-
ronment.’8
To evaluate
this contribution,
it will be necessary
to identify
and
characterize
the
specific
proteoglycans
involved.
Proteogbycans
are extremely
complex
structures
and
supportive
bone
marrow
culture.5
pattern
of D2XRII
cells
bong-term
this
and
shown
a smaller
CL-4B,
and
has more in common
with the endothelial
cell phenotype,
the
present
findings
are in accordance
with the current
consensus
that the hematopoietic
microenvironment
in long-term
bone
marrow
culture
is provided
by an endothelial-like
cell.
Hematopoieticably
labeled
function.9
Smooth
muscle
two CS/DS
proteoglycans,
between
these cell lines.
a fibroblast-like
phenointo osteoblasts.24
The
bone
further
the
that
potential
observed
have
excluded
and
major
species
in
fibroblasts.
The
a DS
proteoglycan
proteoglycan
fraction
that
a smaller,
three
and
eluted
and a smaller
species,
Kay 0.39
iduronic
acid.
HS proteoglycan
with
a
produce
of hydrodynamic
medium
species
Vogel
found
and
closely
synthesis
with functional
MS3-2A
cells appear
type
with the potential
that
proteoglycan
HS
to synthesize
a large
dense HS proteoglycan
a CS/DS
proteoglycan
It is tempting
to
osteoblastic
potential
well documented,34
al3’32
proteoglycan
to be the
from embryonic
skin
similar
to the large DS
free
DS chains.
The
thelial
et
in the
acid-rich
acid-rich
smooth
proteoglycan
species
found in
HS proteoglycan
of Kay 0.40
Carlstedt
acid-rich
ronic acid-rich
DS
the culture
medium
in
synthesis
in common
fibroblast
Peterson,3#{176} studying
proteoglycan
synthesis
human
embryonic
lung fibroblasts,
identified
proteoglycans
in the medium
of Kay 0.29 and
Sepharose
CL-2B.
the cell layer was
differ-
synthesis
proteoglycan
to
Sepharose
CL-4B
column.
The remaining
CS/DS
can appeared
to consist
of an intermediate-sized
HS proteoglycans
and low-density
demonstrated
shown
on the basis
heterogeneity
glucuronic
species.
patterns
of MS3-2A
observed
contained
were
proteoglycan
and D2X-C-HS2).
It is uncertain
distinct
proteoglycan
species
or
of a single
between
layers
D2XRII
cell layer
into intermediate-
(D2X-C-HSI
these
represent
polydispersity
cell
cultures
(MS3-M-DS1
and
proteoglycan
(MS3D2XRII
cell layer
AND
overlap
are
therefore
between
polydisperse.
different
species
There
with
is thus
respect
considerable
to chemical
and physical
properties,
and resolution
of individual
glycans
is difficult,
even
in pure
culture
systems.
heterogeneous
ture,
such
system
resolution
microenvironmentably
potentially
valuable
proteoIn a
such as long-term
bone marrow
cubis virtually
impossible.
The study
of
competent
alternative.
stromal
cell
lines
offers
a
marrow
REFERENCES
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TM: Stromal
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haemopoiesis.
J Cell
Physiol [Supplj
1:87, 1982
2. Bentley SA: Close range cell: cell interaction
required
for stem
cell maintenance
in continuous
bone marrow culture.
Exp Hematol
9:308, 1981
3. Dexter TM, Allen TD, Lajtha
LG: Conditions
controlling
the
proliferation
of haemopoietic
stem cells in vitro. J Cell Physiol
91:355,
1977
4. Allen TD, Dexter TM: Cellular
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during in
vitro granubopoiesis.
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6:191, 1976
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of marrow derived
adherent
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Blood 56:1006,
1980
6. Castro-Malaspina
H, Gay RE, Saletan
5, Oettgen
B, Gay 5,
Moore MAS: Phenotypic
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in long term mouse bone marrow cultures.
Blood 58:107, 1981 (suppl
1)
7. Keating
A, Singer
JW, Killen PD, Striker
GE, Sabo AC,
Sanders J, Thomas ED, Thorning
D, Fialkow PJ: Donor origin of the
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microenvironment
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in
man. Nature
298:280,
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1987 70: 1777-1783
Proteoglycan synthesis in two murine bone marrow stromal cell lines
SL Kirby and SA Bentley
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