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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2533-2542
Characterization of Bone Marrow Laminins and Identification of
 5-Containing Laminins as Adhesive Proteins for Multipotent
Hematopoietic FDCP-Mix Cells
By
Yuchen Gu,
Lydia Sorokin,
Madeleine Durbeej,
Tord Hjalt,
Jan-Ingvar Jönsson, and
Marja Ekblom
From the Department of Animal Physiology, Biomedical Center, Uppsala,
Sweden; the Department of Experimental Medicine, University of
Erlangen, Erlangen, Germany; the Department of Laboratory Medicine,
Lund University Hospital, Malmö, Sweden; and the Department of
Internal Medicine, Uppsala Academic Hospital, Uppsala, Sweden.
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ABSTRACT |
Laminins are extracellular matrix glycoproteins that influence the
phenotype and functions of many types of cells. Laminins are
heterotrimers composed of  ,  , and  polypeptides. So far five
, three , and two polypeptide chains, and 11 variants of
laminins have been proposed. Laminins interact in vitro with mature
blood cells and malignant hematopoietic cells. Most studies have been
performed with laminin-1 (111), and its expression in bone
marrow is unclear. Employing an antiserum reacting with most laminin
isoforms, we found laminins widely expressed in mouse bone marrow.
However, no laminin 1 chain but rather laminin 2, 4, and 5
polypeptides were found in bone marrow. Our data suggest presence of
laminin-2 (211), laminin-8 (411), and laminin-10 (511) in bone marrow. Northern blot analysis showed expression of laminin 1, 2, 4, and 5 chains in long-term bone marrow cultures, indicating upregulation of laminin 1 chain expression in
vitro. Laminins containing 5 chain, in contrast to laminin-1, were
strongly adhesive for multipotent hematopoietic FDCP-mix cells.
Integrin 6 and 1 chains mediated this adhesion, as shown by
antibody perturbation experiments. Our findings indicate that laminins
other than laminin-1 are functional in adhesive interactions in bone marrow.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
STROMAL MICROENVIRONMENTS in the bone
marrow regulate proliferation and differentiation of hematopoietic stem
and progenitor cells. Extracellular matrix molecules play an important
role in this regulation by binding and stabilizing growth factors, and by colocalizing primitive stem cells and developing hematopoietic cells
to specific microenvironmental niches.1,2 Adhesion to
stromal cells and extracellular matrix molecules also directly influences functions of hematopoietic cells by signaling via cell surface receptors.3-5
Laminins are extracellular matrix proteins found in all basement
membranes, but also in the embryonic mesenchyme and loose connective
tissue. Laminins interact with other extracellular proteins and adhere
to cells via integrins and other receptors, thereby influencing cell
motility, proliferation, and differentiation.6,7 Laminins
are heterotrimers composed of variants of  , , and polypeptide
chains. So far five , three , and 2 chains have been
characterized, and 11 different heterotrimers have been
proposed.7 Laminin-1, the first characterized laminin,
composed of 1, 1, and 1 chains, is easily isolated from a
transplantable mouse tumor.8 Therefore cellular
interactions of laminin-1 in vitro are well known. Less is known about
the functions of other laminin isoforms. However, in vitro studies,
gene targeting experiments, and studies of mutated genes indicate
different functional roles for different laminins.7
Different laminins show varying binding specificity to cellular
receptors, also indicating that laminin isoform diversity is
functionally significant.9,10 Dystroglycan, for example, is
a major receptor for laminin 1 and 2 chains.9 Several
integrins (11, 21, 31, 61, 64, 71,
91, v3) bind to laminins, but with varying
affinities.7,10 The cellular interactions of the newly
characterized laminins containing 5 and 4 polypeptides are still
largely unknown.
Blood cells of several lineages have been found to interact with
laminins. Mature granulocytes,11-12
lymphocytes,13 mononuclear phagocytes,14
activated macrophages,15 and eosinophils16 adhere to laminins in vitro. Adhesion to laminins influences survival and maturation of eosinophils17 and proliferation of
macrophages,15 and facilitates CD3-mediated T-cell
proliferation.5 Bone marrow progenitor cells have not been
found to adhere to laminin-1,18 even though laminin binding
integrins have found on murine stem cells.19 Malignant
progenitor cells from chronic myelogenous leukemia have been reported
to adhere less to fibronectin and more to laminin-1 and have increased
integrin 6 chain expression, as compared with normal progenitor
cells.18 It has been suggested that such altered adhesive
interactions may render the leukemic cells insensitive to the normal
regulatory stimuli from the stroma,20 and facilitate
premature release of the leukemic cells into the circulation.18,21 Laminins have been found to promote
differentiation of a promyelocytic leukemia cell line with all-trans
retinoic acid22 and to promote chemotaxis of malignant
plasma cell lines mediated by integrin 3 and 6
subunits.23 It is therefore of interest to study which
laminin isoforms are expressed in the bone marrow.
Immunofluorescence staining using antisera against laminin-1 has shown
that laminins are abundantly expressed in long-term bone marrow
cultures24 and in the native bone marrow.25
However, 10 out of 11 of the laminin isoforms so far characterized
contain at least one of the three chains present in
laminin-17 and are recognized by anti-laminin-1 antisera.
Our previous findings showed the absence of laminin 1 polypeptide
and consequently laminin-1 in the bone marrow.25 Here, we
have analyzed the expression of other laminin isoforms in the bone
marrow. The myeloid long-term bone marrow culture system is a widely
used model for hematopoiesis,26 and therefore expression of
laminin isoforms was analyzed both in authentic bone marrow and in
culture conditions. Furthermore, we studied whether some of the laminin
isoforms expressed in the bone marrow are functional in adhesive
interactions with hematopoetic cells.
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MATERIALS AND METHODS |
Cell cultures.
The C57Bl/6 mouse strain (BK Stockholm, Sweden) was used for long-term
bone marrow cultures. Myeloid long-term bone marrow cultures were
generated essentially as described.26 Bone marrow cells
derived from 3-month-old mice were cultured at a density of 1.3 to 2 × 106 cells/mL in 6 mL in 25 cm2 Falcon
tissue culture flasks in Fischer's medium (GIBCO BRL, Täby,
Sweden), supplemented with 20% horse serum (GIBCO) with or without 1 × 10 6 mol/L hydrocortisone sodium succinate
(Sigma Chemical Co, Stockholm Sweden). The cells were grown at 33°C
in 5% CO2 in humidified air. After 1 week 4 mL culture
medium was added, and thereafter half of the medium was replaced with
fresh medium weekly. Human bone marrow obtained during orthopedic
surgery was a gift from Dr Östen Ljunggren (Uppsala, Sweden).
For human myeloid long-term bone marrow culture, mononuclear cells were
separated by density gradient centrifugation through Ficoll (Pharmacia,
Uppsala, Sweden). The cells were grown in Iscove's
modified Dulbecco's medium (IMDM) (GIBCO) with 10% horse serum, 10%
fetal calf serum (FCS) and 5 × 107 M
hydrocortisone sodium succinate at 33°C in 5% CO2
humidified air. MC3T3-G2/PA6 preadipocyte37 and 3T3 cells
were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO)
supplemented with 10% FCS at 37°C in 5% CO2. The
mouse multipotent hematopoietic FDCP-mix cell line28 was
maintained in IMDM (GIBCO) supplemented with 20% horse serum and
recombinant interleukin-3 (PeproTech EC, London, UK).
Antisera and antibodies.
Polyclonal rabbit antiserum against laminin-1,29 from mouse
transplantable Engelbreth-Holm-Swarm (EHS) tumor,8 or
affinity purified polyclonal antilaminin antiserum against EHS-laminin (Sigma) were used to detect laminin 1, 1, and 1 polypeptide chains. Rat monoclonal antibodies (MoAb) employed were 198 and 200 against the E3 carboxyterminal fragment of laminin 1
polypeptide,30 4H8-2 and 8G11-D10 against laminin 2
polypeptide,31 4G6 against the laminin 5
chain,32 and MECA-32 against an endothelial cell surface
marker.33 Mouse MoAb D5 was used to study expression of
laminin 2 chain34 in rat bone marrow. Rabbit antiserum
against the human laminin 4 chain was from Dr Robert Burgeson, and
rabbit antiserum raised against a synthetic peptide KPPVKRPELT located at the beginning of the carboxyterminal G-domain of human laminin 4
chain35 was from Dr Alan Richards. Monoclonal rat antibody GoH3 (IgG2a,  ) against integrin 6 chain36
was from Dr Arnoud Sonnenberg, monoclonal rat antimouse antibody 9EG7
(IgG2a, ) and monoclonal hamster antirat antibody Ha2/5
(IgM) against integrin 1 chain were from Pharmingen. Mouse MoAb 4C7,
previously thought to detect human laminin 1 chain,37
but now shown to be specific for human laminin 5
chain,38 was also employed. Cy3 and fluorescein-conjugated sheep antibodies against rat, rabbit, or mouse
immunoglobulins were purchased from Jackson Immunoresearch Laboratory
(West Grove, PA).
Immunofluorescence.
Tissues were frozen in Tissue Tek (Miles, Naperville, IL). 5 to 7 µm
cryostat sections from adult and newborn mouse (NMRI and C57 Bl/6) bone
marrow, newborn rat bone marrow, and human bone marrow were fixed in
methanol at  20°C for 2 to 5 minutes or 4% paraformaldehyde
at room temperature for 10 minutes. Adherent cells from myeloid
long-term bone marrow cultures grown on coverslips were fixed with
methanol at 20°C. The cells or tissue sections were treated
with 2% to 5% bovine serum albumin (BSA) (Sigma) or 5% goat serum in
phosphate-buffered saline (PBS), and thereafter incubated with antisera
or antibodies diluted in 2% goat serum in PBS.
Immunoblotting.
Adherent cells from bone marrow cultures were washed two times with
PBS. Proteins were extracted in Tris-buffered saline (TBS), pH 7.4 containing 10 mmol/L EDTA (TBS-EDTA), or sequentially in triple
detergent buffer39 followed by TBS-EDTA and 6 mol/L urea in
TBS pH 7.4, all containing protease inhibitors. Proteins from 4-weeks-old rat bone marrow and newborn rat kidney were extracted in
TBS-EDTA. For immunoblots, the samples were reduced by boiling 5 minutes in Laemmli buffer containing dithiotreitol or
-mercaptoethanol (Sigma) and separated on 6% or 3% to 12%
(wt/vol) continuous sodium dodecyl sulfate (SDS)-polyacrylamide gel.
EHS tumor extract or laminin-1 purified from the EHS
tumor40 were used as controls. Prestained Rainbow (BioRad,
Hercules, CA) or Caleidoscope (Amersham, Buckinghamshire, UK) high
molecular weight markers were run in parallel. Nonspecific binding
was blocked with 5% goat serum in PBS, 0.1% Tween-20 (USB) (PBS-T).
Membranes were incubated with the antibodies in 2% goat serum, PBS-T.
Control stainings were performed by omitting the first antibody. Bound
antibodies were detected with a peroxidase-conjugated goat antirabbit,
or antirat or antimouse antiserum (Amersham) at a dilution of 1:3000,
and by 4-chloro-1-naphthol and H2O2, or by
chemiluminescence using the ECL Western blotting protocol (Amersham
Int, Buckinghamshire, England) and Hyperfilm ECL (Amersham).
Immunoprecipitation.
Bone marrow cells from tibiae and femora of 1-week-old NMRI mice were
incubated overnight in cysteine- and methionine-free RPMI, 5% FCS, 30 µL/mL 35S-cysteine-methionine (Pro-mix, Amersham) at
37°C. The culture medium was collected after centrifugation.
C57Bl/6 mouse bone marrow cells were cultured for 3 weeks in myeloid
long-term culture conditions. Thereafter, adherent cells were washed
free of nonadherent cells and 4 mL of culture medium consisting of
cysteine- and methionine-free RPMI 1640 (GIBCO), 10% horse serum and 1 × 106 mol/L hydrocortisone sodium succinate
and 25 µL 35S-cysteine-methionine was added. After
24-hours incubation in 33°C 5% CO2 in a humidified
atmosphere the nonadherent cells were removed by centrifugation and the
culture medium was collected. In case of long-term bone marrow
cultures, the same volume of labeled medium was used for each
immunoprecipitation. For immunoprecipitation, the labeled culture media
were precleared by incubation with rabbit serum and Protein A Sepharose
(Pharmacia). Thereafter they were incubated with the primary antiserum
and Protein A Sepharose alone or, in case of monoclonal rat antibody,
together with rabbit antirat antiserum. Control immunoprecipitations
were performed by using rat IgG and nonimmune rabbit serum instead of
the primary antibodies. After washing three times with a high salt
buffer (0.5 mol/L NaCl, 1 mmol/L MgCl2, 1mmol/L
CaCl2, 20 mmol/L TRIS-HCl pH 7.4, 0.1% SDS), two times
with a low-salt buffer (the same as above except for 0.15 mol/L NaCl)
and once with TBS, the immunoprecipitated proteins were incubated 5 minutes in 95°C in Laemmli loading buffer containing dithiotreitol
(Sigma) and electrophoresed on a 6% or 3% to 12% SDS polyacrylamide
gel. 14C labeled or prestained molecular mass markers
(Bio-Rad, Amersham) were run in parallel. After electrophoresis the
gels were fixed, treated with Amplify (Amersham), dried, and exposed to
a Kodak XAR film (Rochester, NY).
Complementary DNA (cDNA) probes.
A 1.5 kb Sph1-Sma1 fragment of a cDNA clone corresponding to the
3' end of the coding region of mouse laminin 1 messenger RNA
(mRNA)41 was used. To detect laminin 2 mRNA, a 765 bp
cDNA probe corresponding to a BamHI/KpnI restriction
fragment (position 5969-6732)42 was used. A 601 bp cDNA
fragment43 corresponding to laminin 5
mRNA,44 a 795 bp cDNA corresponding to laminin 4
mRNA,45 and a 564 bp cDNA fragment46
corresponding to laminin 3A and 3B mRNAs were used. A 1.1 kb
human G3PDH cDNA probe (Clontech, Palo Alto, CA) was used as a control
for the amount of mRNA loaded.
Northern blot analysis.
Total RNA from bone marrow cells of 1-week-old and adult mice, adherent
layers of long-term bone marrow cultures grown without hydrocortisone,
3T3, and MC3T3-G2/PA6 cells were isolated by ultracentrifugation or by
phenol extraction.39 RNA was denatured with glyoxal,
electrophoresed on a 1% agarose gel, transferred to a Zeta-Probe
membrane (BioRad, Solna, Sweden) and fixed by UV cross-linking with a
Stratalinker (Stratagene, AH Diagnostics AB, Skarholmen, Sweden).
Membranes were prehybridized in 0.25 mol/L
Na2HPO4, 7% SDS, for 60 minutes at 65°C
and hybridized overnight in the same solution at 65°C. cDNA probes
were labeled with 32P-dCTP (Amersham) using RediPrime
labeling system (Amersham). Free nucleotides were removed by Push
Columns (Stratagene). After hybridization membranes were washed two
times for 1 hour in 20 mmol/L Na2HPO4, 5% SDS
at 65°C, and two times for 1 hour in 20 mmol/L
Na2HPO4, 1% SDS at 65°C. Membranes were
exposed to a Kodak XAR film.
Cell attachment assay.
96-well nontissue culture microtiter plates (Greiner, Sigma) were
coated for 1 hour at 37°C with extracellular matrix proteins diluted in Dulbeccos's PBS (GIBCO) at concentrations of 10 to 50 µg/mL in volumes of 50 to 100 µL. As negative controls, wells were
incubated with Dulbecco's PBS only. After coating, wells were washed
three times with PBS and incubated with 2% heat-denatured fatty acid
free BSA (Sigma) in PBS for 1 hour at 37°C. After 3 washes with
PBS, cells (1.5 to 3.1 × 105 cells/mL) in Dulbecco's
minimal essential medium (DMEM, GIBCO) in a volume of 50 to 100 µL/well were added and incubated at 37°C for 1 hour. After two to
three washes with PBS, adherent cells were fixed with 96% ethanol for
10 minutes, washed two times with PBS and stained with 0.1% chrystal
violet in H2O for 30 minutes. Thereafter, plates were
washed three times with large volumes of deionized water, and adherent
cells were lyzed with 0.2% Triton X-100. Absorbance was measured at
595 nm with a microtiter plate reader (Multiskan PLUS, Labsystems,
Stockholm, Sweden). All samples were analyzed in triplicates. Proteins
employed in adhesion assays were plasma fibronectin (from Dr Staffan
Johansson, Uppsala, Sweden), laminin-1 purified from the EHS
tumor40 (from Dr Mats Paulsson, Cologne, FRG), and laminin
from human placenta, affinity purified with the MoAb 4C7 (GIBCO). For
the antibody perturbation assays, the plates were coated with 30 µg/mL protein as described above. The FDCP-mix cells were resuspended
at +4°C in DMEM containing 2% BSA and the antibodies.
The cells were incubated in the wells for 1 hour on ice. Thereafter,
the plates were warmed to +37°C for 10 minutes, washed,
fixed, stained, and analyzed as described above.
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RESULTS |
Widespread distribution of laminins but absence of laminin-1 in the
bone marrow.
The expression of laminin 1, 1 and 1 polypeptides in the bone
marrow was studied using an antiserum against laminin-1 from the EHS
tumor. This antiserum reacts well with both the 400 kD 1 polypeptide
and the 200 to 210 kD 1 and 1 polypeptides, as shown in
immunoblotting of protein extracts from the EHS tumor (Fig 1). Immunostaining of adult mouse bone
marrow with the antilaminin-1 antiserum showed widespread distribution
of laminins in the arteriolar walls and in the sinusoidal
subendothelial basement membranes, but also in the intersinusoidal
interstitial connective tissue. This was shown by double
immunofluorescence for laminin, and an endothelial-specific antigen
identified with the MoAb MECA-32 (Fig 2a
and 2b).
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| Fig 1.
Immunoblotting of laminin polypeptides in the bone
marrow stroma. Immunoblotting of protein extracts from an EHS tumor
(EHS), adherent cells from mouse long-term bone marrow cultures
(LTBMC), adult mouse bone marrow (BM), human long-term bone marrow
cultures (HuLTBMC), and laminin-1 isolated from EHS tumor (EHS-laminin)
was performed. Antibodies used were: (111), a polyclonal
antiserum against the three chains of laminin-1; 1, MoAb 200 against
laminin 1 chain; 2, MoAb 8G11-D10 against laminin 2 chain;
4, two polyclonal antibodies against a fragment of human laminin
4 protein (left), or a synthetic peptide corresponding to human
laminin 4 chain (right) as an immunogen. C, control immunoblotting
in the absence of primary antibody. The positions of a 200 and 70 kD
molecular mass markers run in parallel are shown to the left of each
blot.
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| Fig 2.
Expression of laminin polypeptides in the adult mouse
bone marrow (a-f), and human bone marrow (g-j). Double
immunofluorescence staining with an antiserum reacting with laminin
1, 1, and 1 polypeptides (a) and with an endothelial cell
marker MECA-32 (b) shows expression of either laminin 1, 1, or
1 chains in the artery, sinusoidal subendothelial basement
membranes, and in the intersinusoidal spaces. Immunostaining with MoAb
4G6 (d) shows expression of laminin 5 chain in the arteriolar walls
and in the subendothelial basement membrane of the sinusoids (arrows).
Immunostaining with antibody 200 specific for laminin 1 chain (e) is
negative. Laminin 2 chain, identified with antibody 8G11-D10 (f) is
present only in the arteriolar walls. Laminin 4, identified with a
polyclonal antiserum against a synthetic peptide corresponding to
laminin 4 chain, is localized in the intersinusoidal spaces (h,i),
and in the walls of arteries (i). Double immunofluorescence staining
with antibody 4C7 against human laminin 5 chain (g) and with the
antiserum against synthetic peptide of laminin 4 chain (h) shows
expression of 5 and 4 chains in the arteriolar walls (arrows),
and 4 chain also in the intersinusoidal connective tissue. Control
immunostainings with nonimmune rabbit serum (NRS) (c, j) are negative.
Laminin polypeptide chains detected by each antiserum or antibody are
indicated in the lower left corners of the figures.
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In immunoblotting of protein lysates of adult mouse bone marrow cells,
laminin 1, and 1 polypeptide chains were well expressed, but the
400 kD laminin 1 polypeptide was not detectable. This was further
analyzed by immunofluorescent staining using MoAb 200 specific for
laminin 1 polypeptide (Fig 1). This antibody did not stain any
structures in adult bone marrow (Fig 2e), indicating that the laminin
1 polypeptide is not expressed in the bone marrow. In the adherent
stromal cells of myeloid long-term bone marrow cultures a very weak
reaction for the 1 chain was detected in immunoblotting after a long
exposure (not shown), suggesting an upregulation of laminin 1 in
stromal cells in culture.
Expression of laminin polypeptides other than
laminin 1 chain in bone marrow.
The low level of laminin 1 chain expression in the bone marrow
stroma implies that other chains are assembled to bone marrow laminins. In immunoblotting of the adherent layer of bone marrow cultures the MoAb 8G11-D10 against 2 chain reacted with a 150 kD
proteolytic fragment of laminin 2 polypeptide31 (Fig 1). In immunofluorescent staining laminin 2 polypeptides were found in
the arteriolar walls in adult bone marrow (Fig 2f). Laminin 5
polypeptides were localized by immunofluorescent staining with MoAb 4G6
also in arteriolar walls in adult mouse bone marrow, but in addition in
subendothelial basement membranes in the sinusoids (Fig 2d). However,
in the intersinusoidal connective tissue neither laminin 2 nor 5
chains were expressed. Polyclonal antibodies against either the human
laminin 4 polypeptide or against a synthetic peptide corresponding
to laminin 4 polypeptide sequences were tested. The specificity of
the antibodies was shown by immunoblotting. Both antibodies reacted
with a polypeptide of slightly over 200 kD in adherent stromal layer of
human bone marrow cultures (Fig 1), corresponding to the described
molecular mass for laminin 4 chain.47 In
immunostainings, laminin 4 chain was localized in the
intersinusoidal spaces, in large arteries, and in smaller arterioles in
adult human bone marrow (Fig 2h,i). In agreement with the findings from
mouse bone marrow, the vasculature in the human bone marrow expressed
laminin 5 chain, as shown by immunostaining with antibody 4C7
(Fig 2g).
Developmentally regulated expression of laminin polypeptides in the bone marrow.
Laminins were found well expressed in newborn mouse bone marrow and
showed a widespread distribution, similar to that observed in adult
bone marrow. Double immunofluorescence staining with the antilaminin-1
antiserum and MECA-32 showed laminins in sinusoidal basement membranes,
arteriolar walls, and in the intersinusoidal spaces
(Fig 3a,b). As in adult bone marrow, in
newborn bone marrow laminin 1 polypeptide was not found (Fig 3e),
and faint immunostaining for laminin 2 chain was observed in some
arterioles (Fig 3f). Laminin 5 polypeptide was also immunolocalized
in arteriolar walls, but in contrast to adult bone marrow, laminin 5
chain was not expressed in sinusoidal basement membranes in newborn bone marrow (Fig 3d), indicating a postnatal shift in the expression of
laminin isoforms in the sinusoids. The sinusoidal basement membranes
and the intersinusoidal connective tissue in newborn mouse bone marrow,
therefore, do not contain laminin 1, 2, or 5 polypeptides.
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| Fig 3.
Expression of laminin polypeptides in newborn mouse
bone marrow. Immunostaining with an antiserum reacting with laminin
1, 1, and 1 chains (a) shows widespread expression of some of
these chains in the arteriolar walls (arrow), in the sinusoids, and in
the intersinusoidal spaces, as shown by a double immunofluorescence
with MECA-32 to stain endothelial cells in the sinusoids and in the
arteriole (arrow) (b). Antibodies 4G6 reacting with laminin 5
polypeptide (d) and 8G11-D10 recognizing laminin 2 chain (f) and
show a reaction in some arterioles. Laminin 1 chain, identified with
MoAb 198, (e) is not expressed in the newborn bone marrow. Control
staining (c) with nonimmune rabbit serum (NRS) is negative. Laminin
polypeptide chains detected by each antiserum or antibody are indicated
in the lower left corners of the figures.
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The molecular masses of laminin polypeptides expressed in the early
postnatal bone marrow was studied by immunoprecipitation with the
antiserum against laminin-1 (Fig 4),
resulting in a precipitation of proteins with an apparent molecular
mass slightly above 200 kD, corresponding to the sizes of 4, 1,
and 1 polypeptides. No polypeptides of higher molecular mass were
detected. Because laminin 1, 2, 3B, and 5 chains have
molecular masses of 300 to 400 kD,7 the finding is in
agreement with the observed absence of laminin 1 chain and low level
of expression of laminin 2 and 5 chains in the immunostainings.
It suggests that laminin 4 chain is the major laminin chain in
the early postnatal bone marrow. This was supported by Northern blot
analysis that showed expression of laminin 4 mRNA in total RNA from
bone marrow of 1-week-old mice, whereas mRNAs for laminin 2, 5
(Fig 5), 3A, or 3B chains (not shown)
could not be detected.
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| Fig 4.
Immunoprecipitation of the proteins synthesized in the
culture medium by adherent cells of mouse long-term bone marrow culture
(LTBMC) or cells from 1 week postnatal mouse bone marrow (BM) with an
antiserum against laminin-1 (111), laminin 1 chain (1;
MoAb 198) or with a nonimmune rabbit serum (NRS). Positions of a
prestained 200 and 70 kD molecular mass markers and 400 kD laminin 1
polypeptide from EHS extract stained by Coomassie blue after
electrophoresis are shown to the left. LTBMC: 6% gel; BM: 3% to 12%
gradient gel.
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| Fig 5.
Expression of mRNAs for laminin 1, 2, 4, and
5 chains in 1-week postnatal mouse bone marrow (BM),
adherent cells from mouse long-term bone marrow cultures (LTBMC), 3T3,
and MC3T3-G2/PA6 (PA6M) cell lines. Northern hybridization of total RNA
was performed with the labeled cDNA probes corresponding to laminin
1, 2, 4, and 5 mRNA. Hybridizations with cDNA fragments
corresponding to laminin 2, 4, and 5 mRNAs were performed
consecutively on the same membrane. The amount of mRNA loaded was
analyzed by hybridizations of the membranes with a cDNA probe
corresponding to G3PDH mRNA (G3PDH). 28S; localization of 28S rRNA on
the membranes. The blots were exposed to radiograph films; exposure
time for a cDNA fragment corresponding to laminin 1 mRNA was 17 days, to laminin 2 mRNA 19 days, to laminin 4 mRNA 21 days, and
to laminin 5 mRNA 28 days.
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Expression of laminin chain mRNAs in the adherent stroma of
long-term bone marrow cultures, and in stromal cell lines.
Laminin polypeptides with a molecular mass of about 400 kD were
expressed in the adherent stromal layer of long-term bone marrow
cultures (Fig 4), as shown by immunoprecipitation with the
antilaminin-1 antiserum. The proteins precipitated have a molecular
mass of slightly above 200 kD, corresponding to laminin 4, 1, and
1 polypeptides (Fig 4), whereas the precipitated 150 kD band has an
electrophoretic mobility identical to the laminin-associated polypeptide nidogen, as judged from immunoprecipitations of bone marrow
culture media with an antiserum against nidogen (not shown). Immunoprecipitation with MoAb 200 (not shown) and 198 against the
laminin 1 polypeptide showed a very faint band of about 400 kD (Fig
4). Northern blot analysis showed that mRNAs for laminin 1, 2,
4, and 5 chains were expressed in adherent cells from bone marrow
cultures (Fig 5), whereas mRNAs for laminin 3A or 3B chains were
not detected (not shown).
In adult bone marrow no signals for laminin mRNAs were obtained in
Northern hybridization to total RNA (not shown). In the studied cell
lines, mRNA for laminin 5 chain was detected in 3T3 cells, whereas
mRNAs for laminin 2 and 4 chains were detected in preadipocytic
MC3T3-G2/PA6 cells (Fig 5).
Absence of laminin 2 polypeptide in bone marrow.
Laminin 2 polypeptide was not found in rat bone marrow in
immunoblotting and immunofluorescence by using MoAb D5, even though the
antibody detected 2 chains in other anatomical areas
(Fig 6).
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| Fig 6.
Absence of laminin 2 polypeptide in the bone marrow.
(a); immunoblotting of proteins from 4-weeks-old rat bone marrow (BM)
and newborn rat kidney with the MoAb, D5, shows a band of about 190 kD
corresponding to laminin 2 in kidney lysates but not in bone marrow.
Position of 200 and 97 kD molecular mass markers run in parallel are
shown on the left. Immunofluorescent staining with the antibody D5 does
not stain any structures in newborn rat bone marrow (b), whereas the
antibody stains an artery in the dermis in the same section (c).
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Adhesion of hematopoietic FDCP-mix and stromal cell lines to laminins
containing 1 and 5 polypeptides.
The FDCP-mix cells adhered to the laminin-containing 5 chain
(laminin-10/11) isolated from human placenta, but to a much lesser
degree to laminin-1 (Fig 7A). FDCP-mix
cells also adhere to fibronectin.57 Adhesion of FDCP-mix
cells to laminins containing the 5 chain was inhibited by antibody
GoH3 against 6 integrin subunit. Of two MoAbs against integrin 1
chains employed, Ha2/5 inhibited adhesion of FDCP-mix cells to
laminin-10/11, whereas 9EG7 did not (Fig 7B). The fibroblastic 3T3
cells and preadipocytic MC3T3-G2/PA6 cells adhered equally well to
laminin 10/11, laminin-1, and fibronectin, with the exception that
higher concentrations of laminin-1 were required
(Fig 8).
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| Fig 7.
(A) Adhesion of the FDCP-mix cells to fibronectin,
laminin-1 purified from the EHS-tumor, and human placental laminin
containing the 5 chain (Laminin-10/11). The results are from four
experiments performed in triplicate (Mean ± SD). The mean absorbance
values at 595 nm were 0.099 to 0.264 for laminin 10/11, 0.034 to 0.133 for fibronectin, and 0.028 to 0.055 for laminin-1, each coated at 30 µg/mL, and 0.012 to 0.023 for controls. (B) Effect of MoAbs GoH3
against integrin 6 chain, and Ha2/5 and 9EG7 against integrin 1
chain on the adhesion of FDCP-mix cells to laminins containing 5
chain. (Mean ± SD, GoH3, and 9EG7, two experiments performed in
triplicate; Ha2/5, two experiments with a total of nine measurements).
The mean absorbance values for controls without antibodies were 0.482 and 0.081 for the two experiments.
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| Fig 8.
Adhesion of MC3T3-G2/PA6 cells (A) and 3T3 cells (B) to
fibronectin, laminin-1, and laminin containing 5 chain
(Laminin-10/11) (Mean ± SD, 3 experiments).
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Expression of the integrin 6 chain in the cultured
bone marrow stromal cells.
We analyzed by immunofluorescence the expression of the integrin 6
subunit in the adherent cells from myeloid long-term bone marrow
cultures. The GoH3 antibody stained a subset of hematopoietic cells in
the adherent cell layer, but also some large stromal cells
(Fig 9).
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| Fig 9.
Immunofluorescent localization of integrin 6 chain in
the adherent cells of mouse myeloid long-term bone marrow culture,
studied with GoH3 antibody.
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DISCUSSION |
We show here that laminin 1 chain, and consequently laminin-1, is
not detectable in the bone marrow. Rather, we have identified laminin
2, 4, and 5 chains apparently assembled into laminins -2, -8, and -10 as the isoforms in the bone marrow. We also show that laminins
containing the 5 chain are more adhesive to multipotent hematopoietic FDCP-mix cells than laminin-1 and fibronectin, suggesting that bone marrow laminins are functional in adhesive interactions with
hematopoietic cells.
The finding that bone marrow does not contain laminin 1 chain is in
agreement with our previous findings that laminin 1 chain is
restricted mainly to epithelial basement membranes.48,49 Recent studies by us and others have confirmed this finding both in
mouse and human tissues.38,43,50,51 Other laminin isoforms have been localized in the mesenchyme and in nonepithelial basement membranes.43,50,51 We show here that laminin 2 chain is
present in the arteriolar walls of bone marrow. In other tissues, the laminin 2 polypeptide is expressed in the basement membranes of
muscle fibers and Schwann cells.31 It is likely that
laminin 2 chain in the bone marrow is also produced by smooth muscle cells in the arteriolar walls. Recently, laminin 5 polypeptide has
been found widely expressed in adult and embryonic
tissues,32,43,50,51 with expression increasing during
postnatal development. This chain has been suggested to be a major
laminin chain in adult blood vessels.32 We found
laminin 5 polypeptides in arteriolar walls and in sinusoidal
endothelial linings in the adult bone marrow, but only in the
arteriolar walls in newborn bone marrow, indicating a postnatal shift
in the laminins in the sinusoidal basement membranes. Laminin 4
chain was expressed in the intersinusoidal loose connective tissue and
in arteries in adult bone marrow.
Because laminin polypeptides have considerable size variations, it
was to some degree possible to analyze by immunoprecipitation followed
by gel electrophoresis, which polypeptides form authentic laminin
heterotrimers with the 1 and 1 polypeptides in the bone marrow
stroma. Immunoprecipitation of laminins synthesized by 1-week postnatal
bone marrow cells showed polypeptides with a molecular mass slightly
above 200 kD, corresponding to the molecular mass of laminin 4,
1, and 1 polypeptides, but no proteins of higher molecular mass.
The data indicate that laminin 4 chain, assembled with 1 and 1
chains to form laminin-8, is the major laminin isoform in the
developing bone marrow. This finding was further supported by Northern
blot analysis, which showed expression of laminin 4 chain but no
other chain mRNAs in bone marrow from 1-week-old mice.
Immunoprecipitation from long-term bone marrow cultures with polyclonal
antilaminin-1 antiserum showed a 400 kD polypeptide and 200 kD
polypeptides. The 400 kD polypeptide band could consist of laminin
1, 2, and 5 polypeptides, because their mRNA was found by
Northern blot analysis. Expression of laminin 1 chain in adherent
cells from long-term bone marrow cultures but not in bone marrow in
vivo suggests that the expression of laminin chains is altered in
bone marrow cells during in vitro culture. As shown by immunoblots and
by Northern hybridization, laminin 4 chain was likewise well
expressed in the stromal layer of human and mouse bone marrow cultures.
Laminin 2 chain, which forms laminin heterotrimers with chains
and 1 chain,7 was not found in bone marrow. Accordingly,
our findings indicate that laminin-2 (211), laminin-8
(411), and laminin-10 (511) are present in the
hematopoietic tissue. The laminin isoforms in arteriolar walls are
laminin-2, laminin-8, and laminin-10, whereas in sinusoidal endothelial
linings laminin-10 is the only isoform expressed, and the laminin in
the intersinusoidal spaces is laminin-8.
Northern hybridization to total RNA did not show any expression of
laminin chain mRNA in adult bone marrow. Because the laminin
polypeptides were nevertheless well expressed in adult bone marrow as
shown by immunofluorescence and immunoblotting, the result indicates a
low level of synthesis and a low turnover of the laminins in the fully
differentiated bone marrow. This low rate of synthesis despite abundant
expression of the proteins has also been noted in other adult
organs.49
Laminin 3 and 2 chains, the other known laminin and polypeptide variants, are associated with laminin 3 to form
laminin-5, which is present in epithelia.52 Because mRNAs
for the 3 splice variants 3A and 3B were not detectable in the
early postnatal or adult bone marrow or in bone marrow cultures, it is
unlikely that laminin-5 is expressed in the bone marrow. However, low
expression of mRNA at some developmental stage does not necessarily
exclude expression of the corresponding protein, because the turnover and rate of protein synthesis may be low. Therefore, expression of
laminins containing 3A and 3B polypeptides in the bone marrow should be studied with specific antibodies.
Because of its localization in the adult bone marrow adjacent to the
hematopoietic cells, laminin containing the 4 chain is the most
likely isoform to have biologically relevant interactions with
developing hematopoietic cells, whereas laminin containing the 5
polypeptide might be involved both in adhesive interactions with the
bone marrow cells within the intersinusoidal spaces and during the
trafficking of the mature blood cells across the sinusoidal linings.53 Indeed, we have here shown that laminins
containing the 5 chain are more adhe |
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ABSTRACT
INTRODUCTION