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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2884-2897
Heparan Sulfate Proteoglycan Expression Is Induced During Early
Erythroid Differentiation of Multipotent Hematopoietic Stem Cells
By
Zofia Drzeniek,
Georg Stöcker,
Barbara Siebertz,
Ursula Just,
Timm Schroeder,
Wolfram Ostertag, and
Hans-Dieter Haubeck
From the Institute for Clinical Chemistry and Pathobiochemistry,
Medical Faculty, University of Technology, Aachen, Germany; Institute
for Clinical Molecular Biology-GSF Research Center, München,
Germany; and Heinrich-Pette-Institute for Experimental Virology and
Immunology, Hamburg, Germany.
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ABSTRACT |
Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells
and their extracellular matrix are important components of the
microenvironment of hematopoietic tissues and are involved in the
interaction of hematopoietic stem and stromal cells. Although previous
studies have emphasized the role of HS proteoglycan synthesis by BM
stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan.
Immunochemical, reverse transcriptase-polymerase chain reaction
(RT-PCR), and Northern blot analysis of this HS proteoglycan showed
that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS
proteoglycans is associated with the differentiation state of
hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell
lines. Proteoglycans were isolated from metabolically labeled cells and
purified by several chromatographic steps. Isolation and
characterization of proteoglycans from the cell lines HEL and ELM-D,
which like TF-1 cells have an immature erythroid phenotype, showed that
these cells synthesize the same HS proteoglycan, previously detected in
TF-1 cells, as a major proteoglycan. In contrast, cell lines of the
myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and
B2, do not express HS proteoglycans. Taken together, our data strongly
suggest that expression of this HS proteoglycan in hematopoietic
progenitor cell lines is associated with the erythroid lineage. To
prove this association we have analyzed the proteoglycan expression in
the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction
of erythroid or granulocytic differentiation. Our data show that HS
proteoglycan expression is induced during early erythroid
differentiation of multipotent hematopoietic stem cells. In contrast,
during granulocytic differentiation, no expression of HS proteoglycans
was observed.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE ESSENTIAL ROLE of the hematopoietic
microenvironment, eg, the bone marrow (BM) stromal cells and their
extracellular matrix (ECM), for the control of growth and
differentiation in the hematopoietic system has been shown in long-term
BM cultures, where stromal cells are necessary to support the
proliferation and differentiation of hematopoietic stem and progenitor
cells in vitro.1-3 In these cultures hematopoietic stem
cell self-renewal, differentiation, and development into mature cells
do not depend on added hematopoietic growth factors but requires direct
physical contact between the stromal cells and the hematopoietic
cells.1-5 However, the molecular mechanisms of the
interaction of stromal cells and hematopoietic cells are not fully
understood. Whereas stromal cells have been shown to produce a variety
of growth factors, either constitutively or after activation, the
requirement of a direct cell-cell contact between stromal cells and
hematopoietic cells indicates that additional signals are needed.
Recently it has been shown that proteoglycans might be involved in the
interaction of primitive hematopoietic progenitor cells and stromal
cells.6-12 Roberts et al7 have shown that
interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) can be bound by heparan sulfate (HS) proteoglycans from
BM stromal cells or their ECM and can be presented in a biologically
active form to hematopoietic cells. These data indicate that binding of
growth factors by HS proteoglycans might be an important mechanism for
the interaction of hematopoietic stem and stromal cells. Binding of
growth factors to heparin and HS proteoglycans has also been shown in
other systems.13-18
Although these studies have shown that proteoglycans are involved in
the interaction of hematopoietic stem and stromal cells, a detailed
analysis of these proteoglycans is, because of the limited availability
of primitive hematopoietic progenitor and stromal cell lines, still
lacking. In fact, there are only a few reports on proteoglycans from
hematopoietic cell lines.10,19-26 In the earlier studies
either whole BM or long-term BM cultures have been
used.4,26 However, this approach does not allow either the
direct identification of the cells that synthesize the proteoglycans or
a localization of the proteoglycans in the microenvironment. Most of
the studies have pointed to stromal cells rather than hematopoietic
stem cells or progenitor cells as the source of HS
proteoglycans.10,19-22,24,26 However, recently we have
analyzed the proteoglycans from the murine multipotent hematopoietic
progenitor cell line FDCP-Mix A4 and the human erythroleukemia-derived cell line TF-1. Whereas in the murine cell line FDCP-Mix A4 only a
chondroitin-4-sulfate proteoglycan was observed, in the human progenitor cell line TF-1 a novel HS proteoglycan was found as a major
proteoglycan.25 However, an important question was whether these differences were due to the differentiation state of the hematopoietic progenitor cell lines. To address this question, in the
present study we have analyzed the proteoglycan synthesis of several
murine and human hematopoietic progenitor cell lines and in the
nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of
erythroid differentiation. Here we present evidence that HS
proteoglycan expression is induced during early erythroid differentiation.
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MATERIALS AND METHODS |
Materials.
Carrier-free H2[35S]O4 and the
[14C]-labeled amino acid mixture were obtained from New
England Nuclear (NEN; Dreieich, Germany). The Pro-Mix cell labeling
mixture ([35S]-methionine and
[35S]-cysteine) was obtained from Amersham (Braunschweig,
Germany). TSK G 3000 SW, TSK G 4000 SW columns equipped with guard
columns, and Sepharose Q columns were obtained from Pharmacia/LKB
(Freiburg, Germany). [14C]-labeled molecular-weight
standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) were obtained from Amersham Buchler. 3-[3-(cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), benzamidine hydrochloride, 6-amino-hexanoic acid, phenylmethylsulfonyl fluoride (PMSF), and N-ethyl-maleinimide were purchased from Sigma (Deisenhofen, Germany). Guanidinium chloride was obtained from Pierce
(Cologne, Germany).
Chondroitin sulfate lyase AC [EC 4.2.2.5], chondroitin sulfate lyase
ABC [EC 4.2.2.4], heparinase (heparin lyase [EC 4.2.2.7]), and
heparitinase (HS lyase [EC 4.2.2.8]) were purchased from Seikagaku
(Tokyo, Japan).
Iscove's modified Dulbecco's medium (IMDM), RPMI 1640, and
 -modified Eagle medium (-MEM) were obtained from GIBCO
(Eggenstein, Germany). Preselected batches of horse serum and fetal
calf serum were obtained from Sigma (Deisenhofen, Germany), PAN-Systems
GmbH (Eidenbach, Germany), and Harlan-Seralab (UK).
Recombinant murine (rm) IL-3, GM-CSF, erythropoietin (Epo), G-CSF, and
human GM-CSF were from Boehringer (Mannheim, Germany). X63/Ag8-653-mIL-3-conditioned medium was used as a source of IL-3 during maintenance of FDCP-Mix-A4 cells.27
Reverse transcriptase (RT) (Superscript II) was obtained from GIBCO-BRL
(Eggenstein, Germany), and Taq-polymerase (Ampli-Taq) from Perkin-Elmer
Cetus (Überlingen, Germany). Oligo-dT was purchased from
Boehringer (Mannheim, Germany). The Qiex gel extraction kit was
obtained from Qiagen (Hilden, Germany). The pCRTM
II plasmid cloning kit was purchased from Invitrogen
(Leek, The Netherlands). XL1-Blue cells were obtained from Stratagene
GmbH (Heidelberg, Germany). FMC-Seakem LE agarose was obtained from Biozym (Hameln, Germany), and positively charged nylon membranes were
from Boehringer. Oligonucleotide primers were synthesized by MWG
Biotech (Ebersberg, Germany).
All remaining reagents were of analytical grade (Merck, Darmstadt, Germany).
Culture and radiolabeling of cell lines.
The human hematopoietic progenitor cell line TF-1 was established from
a patient with erythroleukemia.28 These cells are strictly
dependent on IL-3 or GM-CSF for their survival and proliferation. Alternatively, TF-1 cells can be maintained in close contact with stromal cells in the absence of added growth factors (M. Bögel, U. Bergholz, U. Just, and W. Ostertag, unpublished
results). TF-1 cells show many characteristics of immature
erythroid cells, such as immature erythroblast morphology, positive
periodic acid-Schiff (PAS) staining, and constitutive expression of
globin genes. They can be induced to differentiate into more mature
erythroid cells, but also to some extent into macrophage-like cells,
and therefore might be regarded as an immature erythroid cell line with
bipotent differentiation potential. HEL cells, originally established
from a patient with erythroleukemia,29 were obtained from
the American Type Culture Collection (Rockville, MD). HEL cells are
similar to the TF-1 cell line, are of immature erythroid origin, and
have a similar differentiation potential.29 The murine
committed erythroid progenitor cell line ELM-D has been established
from a murine erythroblastic leukemia and grows strictly dependent on
stromal cells.30 The murine myeloblastic/promyelocytic B1 and B2 cell lines have been established from a murine myeloid leukemia.31 The multipotent hematopoietic cell line
FDCP-Mix A4 was isolated from murine long-term BM
cultures.1 These cells are nonleukemic and retain a normal
karyotype. In the presence of horse serum and IL-3, FDCP-Mix A4 cells
show self-renewal. When cultured in fetal calf serum in the presence of
appropriate hematopoietic cytokines or when cocultured with stromal
cells without added growth factors, FDCP-Mix A4 cells can undergo
multilineage differentiation.1 The fibroblast cell line,
used as a control, was isolated from a human cervix
sample.32
TF-1 cells were routinely grown in RPMI 1640 supplemented with 10%
fetal calf serum (FCS) and recombinant human GM-CSF (5 × 102 U/mL). HEL cells were routinely kept in RPMI-1640
supplemented with 10% FCS. ELM-D cells were kept in -MEM
supplemented with 20% HS. B1 and B2 cell lines were kept in IMDM with
20% HS. FDCP-Mix A4 cells were routinely grown in IMDM supplemented
with 20% horse serum and X63Ag8-653-mIL-3-conditioned medium as a
source of IL-3 at a concentration that stimulated optimal cell growth.
Cells were metabolically radiolabeled by 25 µCi/mL
[35S]-sulfate in sulfate-reduced medium at a cell density
of 3 × 105/mL for 24 hours (TF-1: sulfate-free RPMI
1640 with 10% FCS and 5 × 102 U/mL recombinant human
[rh] GM-CSF; HEL: sulfate-free IMDM with 10% FCS; ELM-D in
sulfate-free IMDM and 20% HS; B1 and B2 cell lines in
sulfate-free IMDM with 10% HS).
For metabolic labeling of HS proteoglycan core proteins expressed by
the HEL cell line, cells were incubated with 25 µCi/mL of a
[14C]-labeled amino acid mixture (NEN) and 50 µCi/mL of
[35S]-methionine/[35S]-cysteine
(Amersham) for up to 24 hours at a cell density of 5 × 105/mL.
Induction of erythroid differentiation of FDCP-Mix A4 cells.
To induce erythroid differentiation, the FDCP-Mix A4 cells were washed
in IMDM and were seeded in IMDM with 20% FCS and 5 U/mL epo and 2 U/mL
rmIL-3.33 At different time points after induction, cells
were metabolically radiolabeled by 25 µCi/mL [35S]-sulfate at a cell density of 2.5 × 105/mL for 24 hours. In parallel, cultures were set up for
mRNA preparation. In a control experiment, granulocytic differentiation
was induced in FDCP-Mix A4 cells by culturing the cells in IMDM
supplemented with 20% FCS, 1,000 U/mL G-CSF, 50 U/mL GM-CSF, and 2 U/mL IL-3 for up to 5 days.34 At different time points
after induction, cells were metabolically labeled with 25 µCi/mL
[35S]-sulfate for 24 hours. To monitor differentiation,
cytospin preparations were made daily and stained with
May-Grünwald-Giemsa.
Proteoglycans from the supernatant and/or the cell extract of
FDCP-Mix-A4 cells obtained at different time points after induction of
erythroid or granulocytic differentiation were analyzed as described below.
Isolation of proteoglycans.
Proteoglycans were isolated as described elsewhere.25,35
Briefly, proteoglycans were extracted from metabolically labeled cells
(cell layer, "Ex") by 4 mol/L guanidinium chloride (pH 5.8) containing 50 mmol/L sodium acetate, 50 mmol/L EDTA, 0.1 mol/L 6-aminohexanoic acid, 50 mmol/L benzamidine hydrochloride, 5 mmol/L PMSF, 10 mmol/L N-ethylmaleimide (NEM), and 0.5 % (wt/vol) CHAPS. Proteoglycans were also isolated from the culture medium (supernatant, "SN"). Extracts and supernatants were cleared by centrifugation at 2,000g for 20 minutes.
The total incorporation of [35S] sulfate in proteoglycans
was calculated from size exclusion chromatography on TSK 3000 column for supernatants and extracts. Elution was performed by 20 mmol/L sodium phosphate containing 4 mol/L guanidinium chloride and 0.05 % (wt/vol) CHAPS (pH 6.0). The elution profile was monitored by liquid
scintillation counting.
Purification of proteoglycans was performed by anion-exchange
chromatography and size exclusion chromatography. Briefly, to remove
the guanidinium chloride from the cell extracts, extracts were
precipitated with 9 vol of 95% ethanol (EtOH). Precipitate was
reconstituted in 7 mol/L urea buffer containing 0.1 mol/L LiCl/0.1
mol/L NaCl, 0.05% CHAPS, and protease inhibitors as described above.
Ion-exchange chromatography was performed on a Sepharose Q column
(Pharmacia). The column was equilibrated with 20 mmol/L sodium
phosphate containing 0.1 mol/L LiCl/0.1 mol/L NaCl 0.05% CHAPS (pH
4.5). Proteoglycans were eluted by a one-step procedure using 4 mol/L
guanidinium chloride in 20 mmol/L sodium phosphate containing 0.1 mol/L
LiCl/0.1 mol/L NaCl, 0.05% CHAPS (pH 4.5) or, alternatively, using a
salt gradient as described.25,32 The elution was monitored
by liquid scintillation counting.
Size exclusion chromatography was performed on a TSK 4000 column using
20 mmol/L sodium phosphate containing 4 mol/L guanidinium chloride and
0.05% CHAPS (pH 6.0) as eluent. The elution profile was monitored by
liquid scintillation counting.
For further purification of HS proteoglycans, samples from the
supernatants or cell extracts were diluted with digestion
buffer36 and digested with chondroitinase AC/ABC (2 hours,
37°C). Digested samples were diluted to  0.15 mol/L NaCl by 50 mmol/L Tris buffer (pH 7.4) and applied to a DEAE-sephacel-column (2 mL), equilibrated with 50 mmol/L Tris-HCl, 2 mol/L urea, 0.1% Triton
X-100 (Sigma), 0.15 mol/L NaCl, and 1 mmol/L PMSF (pH
7.4). Elution was performed with 6 mL 1 mol/L NaCl in 50 mmol/L
Tris-HCl, 2 mol/l urea, 0.1% Triton X-100, and 1 mmol/L PMSF (pH 7.4).
HS proteoglycans were precipitated by 90% ethanol in the presence of
50 µg bovine serum albumin (BSA) at  20°C.37
For analysis of HS proteoglycan induction during erythroid and
granulocytic differentiation, samples from the supernatants or cell
extracts were treated as described above. After removal of guanidinium
chloride, proteoglycans were precipitated by 90% ethanol, digested
with chondroitinase AC/ABC (2 hours, 37°C), purified by Sepharose Q
chromatography, and analyzed on a TSK 4000 column before and after
treatment with heparinase/heparitinase.
Biochemical characterization of proteoglycans.
Biochemical characterization of isolated proteoglycans was performed by
electrophoresis before and after digestion with glycan-specific enzymes
using fluorography. Enzymic digestion with heparinase (heparin
lyase [EC 4.2.2.7]) and heparitinase (HS lyase [EC 4.2.2.8]),
chondroitin sulfate lyase AC (EC 4.2.2.5), and chondroitin sulfate
lyase ABC (EC 4.2.2.4) was performed in the presence of proteinase
inhibitors as described.36,38 Electrophoresis was performed
according to Laemmli39 using gradient gels (4% to 15% or
8% to 25% T, 3% C). After electrophoresis, gels were treated with
En[3H]ance (NEN) according to the manufacturer's
instructions. Kodak XAR-5 film (Eastman Kodak, Rochester,
NY) was used for fluorographic detection. Exposure times
ranged from 5 to 14 days. In addition, molecular-weight distributions
of native and HNO2-treated proteoglycans were estimated by
size exclusion chromatography on a TSK 4000 column.
 -Elimination and HNO2-degradation.
The size of the glycosaminoglycan chains was determined after alkaline
-elimination40 by gel permeation chromatography on a TSK
4000 column. HNO2-degradation of HS was performed according to the method described by Shively and Conrad.41 Briefly,
proteoglycans were treated with 0.6 mol/L nitrous acid (pH 1.5) for 10 minute at room temperature. Thereafter, samples were neutralized by the addition of 1 mol/L sodium carbonate (pH 8.0).
Immunochemical analysis of proteoglycans.
Immunochemical analysis of proteoglycans was performed either by
dot-blot analysis and/or Western blot analysis. The following monoclonal antibodies (MoAbs) against cell-surface HS proteoglycans were used: S1 (anti-glypican), 2E9 (anti-syndecan 1 + 3), 1C7 (anti-syndecan 3), and 8G3 (anti-syndecan 4). In addition, the MoAb 3 G
10 against an epitope generated in heparitinase-digested HS
proteoglycans was used (MoAbs were kindly provided by G. David, University of Leuven, Leuven, Belgium).
For dot-blot analysis, samples were immobilized on Biodyne B membrane
(Pall, Dreieich, Germany). Blocking was performed by 5% BSA/1% dry
milk in 0.02 mol/L Tris/0.3 mol/L NaCl/pH 7.5 for 1 hour at room
temperature. Incubation with specific MoAbs (1 to 10 µg/mL) was
performed overnight at 4°C. Detection of bound antibodies was
performed according to standard procedures using either
peroxidase-conjugated second antibodies and diaminobenzidine or a
chemiluminescence detection system (ECL; Amersham, Braunschweig, Germany). Western blot analysis was performed according to standard procedures. Detection was done by chemiluminescence (ECL).
RNA isolation and cDNA synthesis.
Total RNA was isolated according to the method of Chomczynski and
Sacchi.42 Two microliters (1 µg/µL) of total RNA was
used to synthesize cDNA by RT (Superscript II; GIBCO-BRL) starting with
oligo-dT (Boehringer). cDNA was synthesized in 20 mmol/L Tris-HCl pH
8.4, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 10 mmol/L
dithiothreitol (DTT), 500 µmol/L each dNTP, 500 nmol/L
oligo-dT, and 200 U of RT (Superscript II) at 42°C for 50 minutes.
The reaction was terminated by incubation at 70°C for 15 minutes
and degradation of RNA by RNAse H at 37°C for 20 minutes. If
necessary, poly-A+ RNA was isolated by the use of Dynabeads
Oligo (dT)25 (Dynal, Hamburg, Germany).
Northern blot.
Approximately 1 µg of poly-A+ RNA was run on a 1.0%
denaturing formaldehyde gel and transferred onto positively charged
nylon membranes (Boehringer). RNA blots were hybridized against
DIG-labeled (Boehringer) polymerase chain reaction (PCR)-synthesized
probes in DIG-Easy Hyb at 50°C for 14 hours. Washing was done two
times in 2X SSC (SSC = 3 mol/L sodium chloride, 0.3 mol/L sodium
citrate, pH 7.2), 0.1% sodium dodecyl sulfate (SDS) at room
temperature for 5 minutes and twice in 0.1X SSC, 0.1% SDS at 50°C
for 15 minutes. Chemiluminescence detection of RNA was performed
according to the manufacturer's instruction (Boehringer).
RT-PCR.
PCR conditions for amplification of the different members of the
syndecan family, betaglycan and glypican, were optimized using the
Optiprime kit (Stratagene, Heidelberg, Germany). Two microliters of
synthesized cDNA was used for PCR reactions. The reactions were
performed in 67 mmol/L TrisHCl pH 8.8, 6.7 mmol/L MgCl2,
170 µg/mL BSA, 16.6 mmol/L
(NH4)2SO4, 100 µmol/L each dNTP, 2.5 U of Taq-polymerase (Ampli-Taq; Perkin-Elmer Cetus), and 50 pmol of
the respective 5'-primers and 3'-primers for Syndecan 2 and 4. Oligonucleotides for Syndecan 2 were
(5'-ATGAGACGCGCGGCGCTCTGGC-3') as 5'-primer and
(5'-GGCGTA-GAACTCCTCCTGCTTGGT-3') as 3'-primer and
for syndecan 4 were (5'-CGAGAGACTGAGGTCATCGAC-3') as
5'-primer and (5'-CGCGTAGAACTCATTGGTGG-3') as
3'-primer. Primers for betaglycan were
(5'-GAGCTGTATAACACAGACCTC-3') as 5'-primer and
(5'-CGTCGTCAGGAGTCACACAC-3') as 3'-primer.
Amplification of betaglycan was performed in 10 mmol/L Tris HCl pH 9.2, 3.5 mmol/L MgCl2, 75 mmol/L KCl. The primer pair for
glypican was (5'-CGCCAGATCTACGGAGCCAAG-3') as
5'-primer and (5'-GAACTTGTCGGTGATGAGCAC-3') as
3'-primer. Amplification of glypican was performed in 10 mmol/L
Tris HCl pH 8.3, 3.5 mmol/L MgCl2, 75 mmol/L KCl, and 4%
dimethyl sulfoxide (DMSO) (Optiprime; Stratagene,
Heidelberg, Germany). Primers for syndecan-3 were (5'-CACGGCTGACATAAGGACC-3') as 5'-primer and
(5'-CTC-TAGTATGCTCTTCTGAG-3') as 3'-primer.
Amplification of syndecan-3 was performed in 10 mmol/L Tris HCl pH 8.3, 1.5 mmol/L MgCl2, 25 mmol/L KCl.
For analysis of the -globin expression, a probe was generated using
the following primers: 5'-primer:
5'-ATGGTGCACCTGACTGATGC-3', 5' nested primer:
5'-TGAGAAGGCTGCTGTCTCTT-3' and 3'-primer:
5'-CAGTGCAGCTCACTGAGATG-3'. The PCR product of 261 bp was
cloned into pGEM-T easy (Promega, Heidelberg, Germany).
Oligonucleotide primers were synthesized by MWG Biotech (Ebersberg,
Germany). PCR was performed in a thermal cycler (Trioblock; Biometra,
Göttingen, Germany). Denaturation was at 94°C for 30 seconds,
annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds. The number of cycles was 30. PCR amplification products were
analyzed on a 1.2% agarose gel (Seakem LE; FMC, Biozym), visualized by
ethidium bromide staining.
Cloning of PCR products.
PCR-amplified DNA fragments were extracted from agarose gels (Quiex;
Quiagen, Hilden, Germany), ligated in pCRTM II (Invitrogen)
or, for -globin, in pGEM-T easy (Promega) for 16 hours at 14°C
and transformed in competent XL1-Blue cells (Stratagene). Positive
clones were checked for the right insert by sequence analysis.43
| |
RESULTS |
Characterization of proteoglycans from the hematopoietic progenitor
cell lines.
Proteoglycans were isolated from metabolically labeled murine and human
hematopoietic cell lines and purified by several chromatographic steps.
The content of metabolically labeled proteoglycans ([35S]
incorporation) of supernatants (SN) and extracts (Ex) was calculated from size exclusion chromatography on the TSK 3000 column. Whereas for
the human factor-independent erythroleukemia cell line HEL about 50%
of the proteoglycans were found both in the supernatant and cell
extract, for the human factor-dependent erythroleukemia cell line TF-1
and the murine stromal cell-dependent erythroid progenitor cell line
ELM-D the majority of proteoglycans was found in the supernatant (80%
and 62%, respectively).
Purification of proteoglycans was performed by an initial anion
exchange chromatography of supernatants and cell extracts on a
Sepharose Q column. The molecular-weight distribution of proteoglycans,
purified by anion exchange chromatography, was analyzed by size
exclusion chromatography on a TSK 4000 column and SDS-PAGE. The
relative proportion of HS proteoglycans was calculated by
size-exclusion chromatography of native, HNO2-treated samples and/or heparinase/heparitinase-treated samples
(Figs 1, 2, and
3). By this procedure about 30% of proteoglycans (34% in the cell extract and 27% in the supernatant) from the HEL cell line
could be identified as HS proteoglycans (Figs 1 and 2), compared with
about 39% in the TF-1 cell line.25 In the ELM-D cell line the relative content of HS proteoglycan was even higher (about 80% in
the cell extract and about 50% in the supernatant) (Fig 3).
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| Fig 1.
Size exclusion chromatography of proteoglycans from the
supernatant (A) and cell extract (B) of the immature erythroid
progenitor cell line HEL. Proteoglycans isolated by anion exchange
chromatography were submitted to size exclusion chromatography on a TSK
4000 column. Elution profiles for the proteoglycans from the
supernatant (A) and cell extract (B) are shown for the native ( )
and HNO2-treated (- - -) samples. Elution profiles were
monitored by liquid scintillation counting.
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| Fig 2.
Size exclusion chromatography of proteoglycans after
digestion with glycosaminoglycan-specific enzymes from the supernatant
(A) and cell extract (B) of the immature erythroid progenitor cell line
HEL. Proteoglycans isolated by anion exchange chromatography from the
supernatant (A) and cell extract (B) of HEL cells were submitted to
size exclusion chromatography on a TSK 4000 column. Elution profiles
are shown for the native, HNO2-treated,
heparinase/heparitinase (Hep/Hept), and chondroitinase AC/ABC-treated
(AC/ABC) samples. Elution profiles were monitored by liquid
scintillation counting.
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| Fig 3.
Size exclusion chromatography of proteoglycans from the
supernatant (A) and cell extract (B) of the murine erythroid progenitor
cell line ELM-D. Proteoglycans isolated by anion exchange
chromatography from the supernatant (A) and cell extract (B) of ELM-D
cells were submitted to size exclusion chromatography on a TSK 4000 column. Elution profiles are shown for the native,
HNO2-treated, heparinase/heparitinase (Hep/Hept), and
chondroitinase AC/ABC-treated (AC/ABC) samples. Elution profiles were
monitored by liquid scintillation counting.
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|
Size exclusion chromatography of proteoglycans from the supernatant and
cell extract of HEL cells is shown in Figs 1 and 2. For the
native sample proteoglycans in the range of Kav 0.05 to Kav 0.50 were detected. HNO2 treatment of this
sample showed that it contained chondroitin sulfate and HS
proteoglycans with comparable molecular-weight distributions (Figs 1
and 2). SDS-PAGE analysis of the samples before and after digestion
with glycosaminoglycan-specific enzymes using fluorographic detection
showed a molecular-weight distribution of about >300,000 to 150,000 daltons for the chondroitin sulfate proteoglycan and >200,000 to
100,000 daltons for the HS proteoglycan
(Fig 4), calculated from the
electrophoretic mobility compared to [14C]-labeled
standard proteins. The molecular-weight distribution of the HS
proteoglycan was comparable to that isolated from the TF-1 cell
line.25 In addition, in the cell extract of the HEL cell
line a second smaller HS population was observed (Fig 2B).
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| Fig 4.
SDS-PAGE of proteoglycans isolated from the supernatant
of the immature erythroid progenitor cell line HEL. HS proteoglycans
from HEL cells were metabolically radiolabeled with
[35S]-sulfate. In addition, core proteins were
metabolically labeled by a mixture of [35S]-methionine
[35S]-cysteine and a [14C]-labeled amino
acid mixture. Proteoglycans isolated from the supernatant of the
immature erythroid progenitor cell line HEL were digested by
chondroitinases AC/ABC (A), heparinase/heparitinase (H), or a
combination of chondroitinases AC/ABC and heparinase/heparitinase (A/H)
and submitted to SDS-PAGE on a 4% to 15% gel. N, native sample.
|
|
Size exclusion chromatography of proteoglycans from the supernatant and
cell extract of ELM-D cells is shown in Fig 3. For the native samples
from the supernatant and cell extract proteoglycans in the range of
Kav 0.05 to 0.45 were detected. HNO2 treatment of these samples showed that they contained chondroitin sulfate and HS
proteoglycans with slightly different molecular-weight distributions
(Fig 3). SDS-PAGE analysis of the samples before and after digestion
with glycosaminoglycan-specific enzymes showed a molecular-weight
distribution of about >300,000 to 100,000 daltons for the HS
proteoglycans and about >250,000 to 100,000 daltons for the
chondroitin sulfate proteoglycan (data not shown).
In addition to the analysis of the native proteoglycans, the
molecular-weight distribution of their glycosaminoglycan moieties was
analyzed after -elimination (Figs 5 and
6). Proteoglycans from the supernatant of
HEL cells had HS glycosaminoglycan chains with a Kav of
0.38 to 0.81, corresponding to a molecular weight (Mr) of
38,000 to 5,800 daltons when compared with glycosaminoglycan chains
of known molecular-weight distribution44 (and data not shown) (Fig 5A), whereas the size of the chondroitin sulfate chains was
larger with a Kav of 0.13 to 0.50 (Fig 5A), corresponding to an Mr of 65,000 to 25,000 daltons. Comparable data were obtained for the glycosaminoglycan chains of proteoglycans isolated from the
cell extract (Fig 5B). In contrast, HS glycosaminoglycan chains from
the proteoglycans in the supernatant of ELM-D cells were larger with a
Kav of 0.13 to 0.54, corresponding to an Mr of 65,000 to
21,000 daltons (Fig 6A), whereas the size of the chondroitin sulfate
chains with a Kav of 0.18 to 0.81, corresponding to an Mr
of 60,000 to 5,800 daltons, was similar to those found in the HEL
cell line (Fig 3). Glycosaminoglycan chains of proteoglycans isolated
from the cell extract (Fig 6B) showed a somewhat different distribution. In addition to the large HS chains with a Kav
of 0.13 to 0.54 corresponding to an Mr of 65,000 to 21,000, which may be attributed to betaglycan, a second HS population with a Kav of 0.54 to 0.81, corresponding to an Mr of 21,000 to
5,800, was observed, which was comparable to the smaller HS chains
observed in HEL and TF-1 cells and therefore most probably can be
attributed to the 59-kD core protein HS proteoglycan.
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| Fig 5.
Size exclusion chromatography of glycosaminoglycan chains
obtained after -elimination of proteoglycans from the supernatant
(A) and cell extract (B) of the immature erythroid progenitor cell line
HEL. Proteoglycans from the supernatant (A) and cell extract (B) of HEL
cells were treated first either by heparinase/heparitinase or
chondroitinase AC/ABC and thereafter submitted to -elimination. Size
exclusion chromatography was performed on a TSK 4000 column. Elution
profiles were monitored by liquid scintillation counting.
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|
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| Fig 6.
Size exclusion chromatography of glycosaminoglycan chains
obtained after -elimination of proteoglycans from the supernatant
(A) and cell extract (B) of the murine erythroid progenitor cell line
ELM-D. Proteoglycans from the supernatant (A) and cell extract (B) of
ELM-D cells were treated first either by heparinase/heparitinase or
chondroitinase AC/ABC and thereafter submitted to -elimination.
Size-exclusion chromatography was performed on a TSK 4000 column.
Elution profiles were monitored by liquid scintillation counting.
|
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Characterization of proteoglycans from the promyelocytic/myeloblastic
hematopoietic progenitor cell lines B1 and B2.
Analysis of metabolically labeled proteoglycans from the murine
promyelocytic/myeloblastic cell lines B1 and B2 by SDS-PAGE, before and
after treatment with different glycosaminoglycan-specific enzymes,
showed a chondroitin sulfate proteoglycan with a molecular-weight distribution of about Mr >200,000 to 100,000 daltons as the only proteoglycan (data not shown).
RT-PCR and Northern blot analysis of proteoglycans from the
hematopoietic progenitor cell lines.
To address the question of whether the HS proteoglycans expressed by
the hematopoietic progenitor cell lines TF-1, HEL, and ELM-D are
related to known cell-surface-associated HS proteoglycans, RT-PCR
analysis was performed using specific primer pairs for the different
syndecans (1-4), glypican and betaglycan. Specificity of
PCR-amplification products was confirmed by sequencing of cloned amplification products. RT-PCR analysis showed that in the HEL cell
line syndecan-2 and syndecan-4, and in TF-1 cells only syndecan-4, were
detectable on the mRNA level. In contrast, in ELM-D cells syndecan-1,
syndecan-2, syndecan-4, betaglycan, and glypican could be detected on
the mRNA level (data not shown). To confirm expression of the different
HS proteoglycans shown by RT-PCR, poly(A+) RNA from TF-1
and HEL cells was analyzed in Northern blots using DIG-labeled probes
for syndecan-2, syndecan-4, and betaglycan. The probe for syndecan-4
detected one band at 2.6 kb in HEL and TF-1 cells
(Fig 7). The probe for syndecan-2 detected
one band at 2 kb and a double band at 2.9 kb in the HEL but not in
the TF-1 cell line (data not shown). For betaglycan no message was detectable in HEL and TF-1 cells even after prolonged exposure time.
However, in control experiments using the BM stromal cell line MS-5, a
message for betaglycan with the expected size of 6 kb was
observed.45
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| Fig 7.
Northern blot and Western blot analysis of syndecan-4
expression in TF-1 and HEL cells. Northern blot analysis was performed
using a digoxigenin-labeled hybridization probe for syndecan-4. A
specific signal could be detected for syndecan-4 in TF-1 (A) and HEL
(C) cells. GAPDH was used as a control (B and D). For Western blot
analysis (E), HS proteoglycans from the supernatant of HEL cells were
isolated and digested by heparinase/heparitinase (H) or chondroitinases
AC/ABC (A) and submitted to SDS-PAGE on a 4% to 15% gel. The
untreated sample is shown on lane N. Detection was performed using the
syndecan-4 specific MoAb 8G3. The core protein of syndecan-4 is
indicated by an arrowhead. It might be noted that in HEL cells a
substantial amount of free core protein of syndecan-4 is detectable.
|
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Immunochemical analysis of proteoglycan core proteins from
hematopoietic progenitor cell lines.
Immunochemical analysis of HS proteoglycans core proteins was performed
using MoAbs against cell-surface HS proteoglycans of the syndecan
family and against glypican and the MoAb 3 G 10 against an epitope
generated in heparitinase-digested HS proteoglycans.46 Dot-blot analysis with the specific antibodies was used instead of
Western blot analysis because of the higher sensitivity of detection.
HS proteoglycans isolated from the cell extract of a human cervix
fibroblast cell line were used as positive controls. The reactivity of
the proteoglycans from the supernatant and cell extract of HEL and
ELM-D cells with the different MoAbs in the semiquantitative dot-blot
analysis is summarized in Table 1. These data indicate expression of syndecan-4 in the HEL and ELM-D cell
lines on the protein level.
In Western blot analysis, using the MoAb 3 G 10, a strong staining was
observed for the core proteins of HS proteoglycans from the human
cervix fibroblast cell line (Fig 8,
control). This staining could be attributed, according to the size of
the core proteins detected and by semiquantitative dot-blot analysis
using the specific MoAbs, to syndecan-1 (70 kD),
syndecan-2 (48 kD), syndecan-3 (125 kD), syndecan-4 (35 kD), glypican
(64 kD), and betaglycan (120 kD). In the supernatant and cell
extract of HEL cells only one signal in the range of 60 kD
was detected (Fig 8), and like the HS proteoglycan core protein of the
same size that has been isolated from TF-1 cells,25 could
not be assigned to known members of the syndecan family, nor to
glypican or betaglycan. In contrast to a positive staining for
syndecan-4 in the dot-blot analysis (Table 1), the Western blot using
the MoAb 3G10 did not show a core protein with the expected size of 35 kD in HEL cells, indicating that syndecan-4 is expressed at a low
level. This was confirmed by Western blot analysis of a concentrated sample (by a factor of about 40), using the
syndecan-4-specific MoAb 8G3. Figure 7 shows that syndecan-4 is
expressed as a HS proteoglycan in the HEL cell line. It might be noted
that, in contrast to the cell extract, in the supernatant a
considerable amount of free core protein was observed. A decreased
staining of the native proteoglycan after treatment with
chondroitinases AC/ABC indicated that at least part of the syndecan-4
core protein is substituted with chondroitin sulfate chains. In the
supernatant and cell extract of ELM-D cells, in addition to the signal
in the range of 60 kD, a strongly stained band was observed at
110 to 145 kD (Fig 8) and could be assigned to betaglycan. This was confirmed by RT-PCR analysis (data not shown). Although in ELM-D cells
glypican was detectable by RT-PCR (data not shown) and the sensitive
dot-blot analysis, it could not be detected by the less sensitive
Western blot using the glypican-specific antibody S1, indicating that
glypican is expressed at a low level.
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| Fig 8.
Immunochemical detection of HS proteoglycan core proteins
isolated from hematopoietic progenitor cell lines. Proteoglycans from
the human immature erythroid progenitor cell lines TF-1 and HEL and the
more mature murine erythroid progenitor cell line ELM-D were treated by
heparitinase, submitted to SDS-PAGE on a 4.5% to 15% gradient gel,
and subsequently blotted to Biodyne B membrane. Detection was performed
using MoAb 3G10 and the ECL-detection system. The MoAb 3G10 recognizes
a common neoepitope, generated after heparitinase-digestion of HS
proteoglycans.46 This MoAb has been shown to detect most,
if not all, HS proteoglycan core proteins after heparitinase-digestion.
HS proteoglycans from cervix fibroblasts (cell extract) are shown as
positive control (Co, lane 1); supernatant (SN) of TF-1 cells (lane 2),
supernatant (lane 3), and cell extract (Ex) (lane 4) of HEL cells;
supernatant (lane 5) and cell extract of ELM-D (lane 6). The arrow
indicates the HS proteoglycan core protein detected by the MoAb 3G10 in
the erythroid progenitor cell lines TF-1, HEL, and ELM-D.
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Analysis of metabolically labeled HS proteoglycan core proteins
expressed by the immature erythroid progenitor cell line HEL.
HS proteoglycan core proteins expressed by the HEL cell line were
metabolically labeled, isolated as described,25 and
submitted to SDS-PAGE before and after digestion with
glycosaminoglycan-specific enzymes. Figure 4 shows a molecular-weight
distribution of about >300,000 to 150,000 daltons for the native
chondroitin sulfate proteoglycan and >200,000 to 100,000 daltons for
the native HS proteoglycan. For the HS proteoglycan a labeled core
protein with a size of 59 kD was detected. Core protein labeling was
hampered by the fact that expression of HS proteoglycan was
downregulated under serum-free conditions. As dialysis of serum did not
improve the results, labeling was performed in the presence of serum. Because of the presence of nonlabeled competing compounds, labeling efficiency was rather low. The metabolically labeled core protein of 59 kD was comparable to the core protein detected by the MoAb 3G10 in the
HEL cell line (see above) and to that previously described for the TF-1
cell line.25
Induction of HS proteoglycan expression during early erythroid
differentiation in the murine multipotent cell line FDCP-Mix-A4.
To determine if HS proteoglycan expression is associated with erythroid
differentiation, we analyzed the proteoglycan production of FDCP-Mix-A4
cells after induction of erythroid differentiation or as a control
after induction of granulocytic differentiation. In two separate
experiments, FDCP-Mix-A4 cells were cultured in the presence of FCS,
Epo, and low IL-3. After 3 to 4 days, consistent with the appearance of
normoblasts in culture (data not shown), -globin expression was
observed in both experiments (Fig 9A). In a
separate control experiment, granulocytic differentiation was induced
by culturing FDCP-Mix-A4 cells in FCS, GM-CSF, G-CSF, and low IL-3.
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| Fig 9.
Induction of HS proteoglycan expression during early
erythroid differentiation in the murine multipotent cell line FDCP-Mix
A4. Erythroid differentiation of the murine multipotent hematopoietic
cell line FDCP-Mix A4 was induced by culturing the cells in FCS, Epo,
and low IL-3. As a control, FDCP-Mix A4 cells were induced to
differentiate along the granulocytic lineage by culturing the cells in
FCS, GM-CSF, G-CSF, and low IL-3. Cells were metabolically radiolabeled
by [35S]-sulfate for 24 hours. In parallel, cultures were
set up for mRNA isolation. Proteoglycans from the supernantant of
FDCP-Mix-A4 cells obtained at different time points after induction of
erythroid differentiation were digested by chondroitinase AC/ABC,
submitted to Sepharose Q anion exchange chromatography, and
subsequently analyzed on a TSK 4000 column before () and after
heparinase/heparitinase treatment (- - -). (A) The corresponding
Northern blot analysis of -globin mRNA expression (-actin was
used as control). (B) HS proteoglycan expression (analyzed as shown in
part [D] as percent HS proteoglycan) in FDCP-Mix-A4 cells at
different time points after induction of erythroid differentiation. (C)
HS proteoglycan expression in FDCP-Mix-A4 cells at different time
points after induction of granulocytic differentiation. (D) The
respective chromatogram is given for day 2; the inset shows the
corresponding Western blot of HS proteoglycan core protein using the
MoAb 3G10 (see legend to Fig 8), and the 59-kD core protein is
indicated by an arrowhead.
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Cells were metabolically radiolabeled at different time points during
erythroid differentiation by 35S-sulfate for 24-hour
intervals. Proteoglycans isolated from the supernantant and cell
extract of FDCP-Mix-A4 cells were digested by chondroitinase AC/ABC,
submitted to Sepharose Q anion exchange chromatography, and
subsequently analyzed on a TSK 4000 column before and after
heparinase/heparitinase treatment (Fig 9D). Whereas no HS proteoglycan
was detectable in the cell extract (data not shown), the HS
proteoglycan content in the supernatant was upregulated between day 1 and day 3 after induction of erythroid differentiation (Fig 9B).
Because these samples were predigested with chondroitinase AC/ABC, the
relative maximum value of 32% of HS proteoglycans corresponds to
7% of total proteoglycans in the supernatant. Analysis of the HS
proteoglycan core proteins, using the MoAb 3G10, showed a core protein
of 59 kD (Fig 9D, inset). In contrast to previously published
data19,25 and the control experiment shown in Fig 9C, a low
expression of HS proteoglycan (up to 6%, corresponding to 1.3% of
total proteoglycans) was observed in the supernatant of FDCP-Mix-A4
cells in experiments I and II on day 0 (Fig 9B). Morphological analysis
showed a relatively high degree of spontaneous differentiation along
all lineages in these experiments. This has been observed previously
depending on the batches of horse serum used. The remaining
proteoglycans after digestion with heparinase/heparitinase (Fig 9D,
dashed line) were submitted to an additional treatment with
chondroitinases AC/ABC, resulting in a complete degradation of
proteoglycan material (data not shown).
As a control, proteoglycan expression was analyzed during granulocytic
differentiation of FDCP-Mix-A4 cells. After granulocytic differentiation by culturing cells in FCS, GM-CSF, G-CSF, and low IL-3,
FDCP-Mix-A4 cells were metabolically labeled with
[35S]-sulfate for 24 hours at different stages of
granulocytic differentiation. Proteoglycans from the supernatant and
cell extract were analyzed as described above. As shown in Fig 9C, HS
proteoglycans were not expressed during granulocytic differentiation.
Granulocytic differentiation was confirmed by analysis of total
cellular myeloperoxidase activity in cell extracts (data not shown).
| |
DISCUSSION |
HS proteoglycans represent a heterogeneous family of macromolecules
that are involved in fundamental biological processes like cell-cell
interaction and control of cell growth and
differentiation.8,18,47-56 Recently, it has been shown that
HS proteoglycans also play an important role in the interaction of
hematopoietic stem and stromal cells.6-10,12,25,57 Most of
these studies have pointed to stromal cells rather than hematopoietic
stem cells or progenitor cells as the source of HS
proteoglycans.10,19,20,21-25,57 The recent establishment of
factor-dependent multipotent progenitor cell lines1,29 that
are able to adhere and grow on stromal cells in the absence of added
growth factors has made a detailed analysis of their proteoglycans
possible. Studies on the murine committed myeloid progenitor cell lines
FDCP-1 and FDCP-2,20,23 the multipotent hematopoietic
progenitor cell line FDCP-Mix-A4,19,25 and the multilineage
cell line Myl-D-7 (Drzeniek Z, Stöcker G, Just U, Siebertz B, and
Haubeck H-D, unpublished results) have shown a chondroitin-4-sulfate proteoglycan to be the only proteoglycan in these
murine hematopoietic cell lines. However, thus far the number of cell
lines analyzed is rather limited and the question remains as to whether
these results are representative for all hematopoietic progenitor cells.
Our results for the human hematopoietic progenitor cell line TF-1
clearly show that this is not the case.25 Analysis of proteoglycans from the TF-1 cell line showed that TF-1 cells expressed an HS proteoglycan as a major proteoglycan. The molecular-weight distribution of this HS proteoglycan was determined to be about >200
to 100 kD for the native molecule and the size of the core protein to
59 kD. To answer the question of whether this HS proteoglycan is
related to known cell-surface-associated HS proteoglycans, immunochemical analysis of proteoglycans was performed by dot-blot and
Western blot analysis using MoAbs against the different syndecan isoforms and glypican. In contrast to HS proteoglycans isolated from a
human cervix fibroblast cell line, which were used as a control, the HS
proteoglycan from the TF-1 cell line did not react with any of these
MoAbs.25 Taken together, these data clearly show that this
HS proteoglycan is distinct from known HS proteoglycans.
However, an important question was whether the difference between these
results and previous results, obtained for the multipotent and
committed myeloid progenitor cell lines, was due to the differentiation state of the hematopoietic progenitor cell lines (erythroid v myeloid). To address this question, in this study we have analyzed the
proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Isolation and characterization of metabolically labeled proteoglycans from the cell lines HEL and ELM-D, which like
TF-1 cells have an immature erythroid phenotype and the potential to
differentiate along the erythroid lineage,29,30 showed that these cells synthesize the same HS proteoglycan, previously detected in
TF-1 cells, as a major proteoglycan. Expression of HS proteoglycans in
HEL cells was indicated by the previous studies of Schick et al.58,59 Although these investigators have focused on
chondroitin sulfate proteoglycans and have not analyzed the HS
proteoglycans in detail, it can be concluded from their data that about
25% of proteoglycans in the HEL cell line were HS proteoglycans. The investigators claimed that serglycin, a chondroitin sulfate
proteoglycan, that is expressed in most hematopoietic progenitor cell
lines,8,60,61 and betaglycan are the major proteoglycans in
HEL cells.58,59 The chondroitin sulfate proteoglycan
observed in our studies in TF-1, HEL, and ELM-D cells can most probably
be attributed to serglycin, as evidenced by RT-PCR for HEL, TF-1, and
ELM-D cells (data not shown). Although for serglycin our results are in
accordance with the data of Schick et al,58,59 for
betaglycan their interpretation could not be confirmed. In our hands
betaglycan was not detectable by Western blot, RT-PCR, and Northern
blot analysis in the HEL cell line. However, betaglycan was detected in
the more mature murine committed erythroid progenitor cell line ELM-D
(see below).
Immunochemical analysis using the MoAb 3G10 indicated that the HS
proteoglycan with a core protein of 59 kD was the major HS proteoglycan
in the HEL cell line. This was confirmed by metabolic labeling of the
core protein, even though the labeling efficiency was low due to the
presence of serum in the culture medium. To answer the question of
whether the HS proteoglycans isolated from the hematopoietic progenitor
cell lines TF-1, HEL, and ELM-D are related to known
cell-surface-associated HS proteoglycans, RT-PCR, Northern blot,
dot-blot and/or Western blot analysis was performed. These data clearly
show that the HS proteoglycan with a 59-kD core |
TOP
ABSTRACT
INTRODUCTION