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Tumor Biology |
3ß1 Integrin-mediated Adhesion and Neurite-like Outgrowth and Inhibits Proliferation of Small Cell Lung Carcinoma Cells
Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [N-h. G., H. A-B., J. C., J. M. S., H. C. K., D. D. R.], and Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 [N. S. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 or ß1 integrin subunits, and by soluble
3ß1 integrin ligands. SCLC cells extended neurite-like processes
on a TSP1 substrate, which was also mediated by 3ß1 integrin.
Process formation on a TSP1 substrate was specifically stimulated by
epidermal growth factor and somatostatin. Adhesion on TSP1 weakly
inhibited SCLC cell proliferation, but this inhibition was strongly
enhanced in the presence of epidermal growth factor. TSP1 and an
3ß1 integrin-binding peptide from TSP1 also inhibited
proliferation when added in solution. High-affinity binding of
125I-labeled TSP1 to OH-1 cells was heparin-dependent and
may be mediated by sulfated glycolipids, which are the major sulfated
glycoconjugates synthesized by these cells. Synthesis or secretion of
TSP1 by SCLC cells could not be detected. On the basis of these
results, the 3ß1 integrin and sulfated glycolipids cooperate to
mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through
this integrin inhibits growth and induces neurotypic differentiation,
which suggests that this response to TSP1 may be exploited to inhibit
the progression of SCLC. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many SCLC cell lines have been established that are typically
nonadherent on tissue culture substrates and grow as tight aggregates
in suspension (4
, 5)
. Cell-cell adhesion is, therefore,
the dominant interaction for SCLC cells and is mediated by E-cadherin
(6)
and neural cell adhesion molecules. Expression of the
latter by SCLC correlates with poor prognosis (1
, 7)
.
Although SCLC cell lines generally fail to interact with the adhesive
proteins in serum and with most extracellular matrix components, some
SCLC lines can attach on laminin substrates (8, 9, 10)
. SCLC
cells express some ß1 integrins but not ß3, ß4, or ß5 integrins
(11, 12, 13, 14, 15)
and were reported to interact with laminins
through 3ß1 and 6ß1 integrins (12
, 16)
.
To better understand the role of cell-matrix interactions in the rapid
dissemination of SCLC, we have examined the possible role of TSP1 to
mediate interactions of SCLC cells with the extracellular matrix. TSP1
is a major component of the -granules of platelets and is a member
of the thrombospondin family of matricellular proteins that is
synthesized by many cell types in response to growth factor stimulation
(reviewed in Ref. 17
).
In common with other extracellular matrix proteins such as fibronectin and laminins, TSP1 plays important roles in regulating growth, motility, survival, and adhesion of cells (reviewed in Ref. 18 ) and modulating tumor growth and metastasis (reviewed in Ref. 19 ). TSP1 can directly influence adhesion, growth, and motility of some tumor cell lines in vitro (reviewed in Ref. 19 ), but its major inhibitory effect on tumor growth in vivo is thought to result from the inhibition of angiogenesis (20, 21, 22, 23, 24, 25) .
TSP1 is recognized by several cell surface receptors including ß3 and ß1 integrins, CD47, syndecan-1, sulfatides, and CD36 (reviewed in Ref. 19 ). TSP1 also binds to several extracellular matrix components (26) , which may in turn mediate its binding to cells through additional receptors. Distinct signaling pathways may be induced by the binding of TSP1 to each class of receptor (27, 28, 29, 30) ; therefore, identification of the specific TSP1 receptors used by each cell type is important for understanding the responses that result from these interactions.
We recently observed that OH-1 cells and other nonadherent SCLC cell
lines attached avidly to TSP1 substrates and grew as adherent cells on
this substrate. We have characterized the receptors that mediate this
response and demonstrate here that both sulfated glycolipids and
3ß1 integrin on SCLC cells function as TSP1 receptors. The
activity of this integrin is stimulated by EGF and somatostatin. In
addition to mediating adhesion, the 3ß1 integrin promotes
neurite-like outgrowth on TSP1 substrates and modulates SCLC cell
growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The function-blocking CD36 antibody OKM-5 was purchased from Ortho-mune
(Raritan, NJ). The integrin vß3 antibody LM609 was the gift of Dr.
David Cheresh (Scripps Research Institute, La Jolla, CA; Ref.
35
). Rat monoclonal antibodies to the human ß1 integrin
(mAb 13) and 5 subunits (mAb 16) were provided
by Dr. Kenneth Yamada (National Institute for Dental and Craniofacial
Research, Bethesda, MD; Ref. 36
). Integrin
function-blocking antibodies P1B5 (3ß1), P4C2 (4ß1), and P1D6
(5ß1) were obtained from Life Technologies, Inc. The ß1
integrin-activating antibody TS2/16 (37)
and the CD98
antibody 4F2 were prepared from hybridomas obtained from the American
Type Culture Collection (Rockville, MD).
The following oligonucleotides were synthesized (The Midland Certified Reagent Co., Midland, TX) and used as primers for the RT-PCR: upstream primer for human THBS1: 5'-CAA CCA CAA TGG AGA GCA CCG-3'; downstream primer sequence for THBS1: 5'-TAG TTG CAC TTG GCG TTC TTG TTG-3'; upstream primer for human THBS2: 5'-CTC CAC CAG CAA GGT GCC TCG CTG-3'; downstream primer for THBS2: 5'-CCG TCG CCC GCG TAG CCT GTC TGG-3'; upstream primer sequence for human THBS3: 5'-GAC ACA GTG CCT GAG GAC TTT GAG-3'; downstream primer for THBS3: 5'-TGG CAA TGT GCT GTC ATC TTT CC-3'; upstream primer for glyceraldehyde-3-phosphate dehydrogenase: 5'-GCT CTC CAG AAC ATC ATC CCT GCC-3'; downstream primer sequence of human glyceraldehyde-3-phosphate dehydrogenase: 5'-TCC TTG GAG GCC ATG TGG GCC ATG-3'.
Cell Culture.
The OH-1 cell line (38)
was provided by Dr. Joel Shaper
(Johns Hopkins University, Baltimore, MD). Variant OH-1 arose after
prolonged culture of OH-1 and had lost the tight aggregate
morphology. The H128, H69, H82, and H209 cell lines were purchased from
the American Type Culture Collection. These cell lines were established
from pleural fluids of SCLC patients (5)
. N417 and H345
cell lines (39)
were provided by Dr. A. Gazdar (University
of Texas Southwestern Medical Center, Dallas, TX). N417
originated from a lung metastasis and H345 from a bone marrow
metastasis. All of the cell lines were cultured suspended in RPMI 1640
with 15% FCS (Biofluids Inc., Rockville, MD) at 37°C in a 5%
CO2 incubator. The medium was changed every 5
days.
Cells were passaged every 9- 11 days. In brief, cells were centrifuged at 400 x g for 2 min, and the medium was aspirated. Cell pellets were washed once with RPMI 1640 containing 5 mM MgCl2 and treated for 5 min with 50 µg/ml DNase-1 (Biofluids Inc.) in RPMI 1640 containing 5 mM magnesium chloride. The cells were triturated three times, and 0.1 volume of trypsin (2.5%; Life Technologies, Inc.) was added for 5 min and triturated as above. The single cell suspension was washed once with the same medium, centrifuged, and suspended in fresh medium.
For adhesion assays, cell aggregates were washed once with RPMI 1640 and centrifuged at 200 x g for 2 min. The pellet was suspended in PBS (pH 7.4), containing 2.5 mM EDTA and incubated for 10 min at 37°C. After trituration three times and centrifugation for 2 min at 400 x g, the cells were resuspended in RPMI 1640 containing 0.1% BSA (Sigma). Trypan blue staining showed greater than 90% cell viability.
Adhesion of SCLC Cells to Extracellular Matrix Proteins.
Extracellular matrix proteins or peptides in Dulbecco’s PBS were
adsorbed onto polystyrene by incubating overnight at 4°C. Adsorption
isotherms of TSP1 on plastic have been reported previously
(40)
. The supernatant fluid was removed, and the dishes
were incubated with Dulbecco’s PBS with 1% BSA for 30 min to minimize
nonspecific adhesion. The dishes were washed twice with cold PBS (pH
7.2) and overlaid with dissociated SCLC cells, prepared as described
above, at a density of 5 x 104/cm2. For inhibition,
inhibitors or antibodies were added and incubated with SCLC cells at
the indicated concentrations. After incubation for 60–90 min at
37°C, the dishes were washed three times with PBS (pH 7.2), fixed
with 1% glutaraldehyde in PBS (pH 7.2), and stained with Diff-Quik.
Attached cells were counted microscopically.
Neurite outgrowth was assessed in SCLC cells after incubation for 75–90 min on a TSP1 substrate. Neurites extending more than one-cell-diameter from the central cell body were counted microscopically in four adjacent 0.25-mm2 fields for each triplicate analysis.
Immunoprecipitation and Western Analysis.
OH-1 cells were surface-labeled using EZ-Link Sulfo-NHS-LC-Biotin
(Pierce) as suggested by the manufacturer. The cells were then lysed in
50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40,
0.5% sodium deoxycholate, 1 mM EGTA, 1 mM NaF
supplemented with 10 µg/ml each of the following protease inhibitors:
antipain, pepstatin A, chymostatin, leupeptin, aprotinin, soybean
trypsin inhibitor, and 1 mM phenylmethylsulfonyl
fluoride. A total of 90 µg of proteins were immunoprecipitated
using 0.75 µg mouse monoclonal anti-EGF receptor antibody
(Transduction Laboratories) prebound to antimouse IgG agarose (Sigma).
After washing, the immune complex was eluted with sample buffer.
Immunoprecipitated proteins were fractionated on a 10% SDS gel along
with 5 µg of A431 carcinoma total proteins as a control. The proteins
were transferred to polyvinylidene difluoride membrane and
blotted with mouse anti-EGF receptor antibody. The membrane was washed
and incubated with a mixture of horseradish peroxidase-conjugated goat
antimouse IgG antibody (Kirkegaard and Perry, Gaithersburg, MD)
and horseradish peroxidase-streptavidin (Pierce) and was visualized
using chemiluminescent substrate (Pierce).
Proliferation.
Effects of soluble and substrate-bound TSP1 or TSP1 peptides on cell
proliferation were quantified using a tetrazolium proliferation assay
(CellTiter Assay, Promega). Treatment with soluble TSP1 was performed
in 96-well tissue culture plates, and proliferation was determined
after 72 h in RPMI containing 15% FCS. Proteins and peptides were
immobilized on Nunc Maxisorp 96-well plates by overnight incubation
with the proteins or peptides dissolved in 50 µl of sterile
Dulbecco’s PBS. The supernatant fluid was removed, and the wells were
incubated for 30 min. in Dulbecco’s PBS containing 1% BSA. OH-1 cells
(1 x 104/well) were added in RPMI
containing 15% FCS and incubated for 72 h at 37° in 5%
CO2. For assessing inhibition by soluble proteins
or peptides, OH-1 cells were grown in suspension in Nunclon 96-well
tissue culture plates using the same medium supplemented with the
indicated inhibitors and growth factors.
Cell-binding Assay.
A 0.2-ml cell suspension, dissociated using EDTA as above, was
transferred into 12 x 75-mm polypropylene tubes (PGC
Scientific Inc., Gaithersburg, MD). Iodinated proteins (final
concentration of 0.2 µg/ml) were added and incubated for 1 h on
ice with rotary shaking. Bound radioactivity was quantified after
centrifugation of the cells through oil as described previously
(32)
. For inhibition studies, inhibitors were added first
and incubated with the cells for 15 min. Iodinated proteins, peptides,
or fragments were then added and incubated as above. To test the effect
of divalent cations on binding of TSP1 to OH-1 cells, the cells were
treated and suspended in HBSS or HBSS containing EDTA or
divalent cations. After incubation for 1 h at 4°C, cells were
centrifuged and separated from the unbound ligand by centrifugation
through oil and counted in a gamma counter. Using the same procedure,
other media (including DMEM, RPMI 1640, or RPMI 1640 without phosphate) were
tested but had no effect on the binding of TSP1 to the cells.
Extraction of Sulfatide and Interaction with TSP1.
Sulfatides were extracted from SCLC cells, desalted by Sephadex G-25,
and separated into neutral and acidic fractions by DEAE-Sepharose
ion-exchange chromatography according to the previously described
method (41)
. Fractions were evaporated to dryness and
dissolved in chloroform-methanol (1:1) for analysis. The extracts were
chromatographed on aluminum-backed high-performance TLC plates and
incubated with 125I-labeled TSP1 according to the
previously described method (41)
.
RT/PCR.
Total RNAs from SCLC cell lines were extracted using RNAzol B according
to the supplier’s protocol. RNA pellets were washed with 75% ethanol,
briefly dried under vacuum for 2 min, and stored in -70°C. RT-PCR
was performed according to the manufacturer’s procedure (Perkin-Elmer
Cetus). Aliquots (2 µl) containing 250 ng of human SCLC total RNA
were brought to a final volume of 20 µl with reverse transcription
buffer containing rTth polymerase and downstream primer. The samples
were heated to 70° for 15 min. PCR buffer with the same amount of
sense primers was added to a final volume of 100 µl. The reaction
mixture was heated to 95° for 1 min and amplified for 30–50 cycles:
at 95° for 10 s and at 60° for 15 s. Finally, the sample
was incubated at 60° for 5 min. The PCR products were analyzed by
agarose gel electrophoresis.
Metabolic Labeling of Sulfatides and Proteoglycans.
SCLC cells were cultured in 5 ml of RPMI 1640 without methionine
containing 4% Ultroser HY (IBF Biotechnics) for 12 h in
25-cm2 culture flask before the addition of 125
µCi of [35S]sulfate (ICN Radiochemicals).
After 48 h, the cells were harvested, and glycolipids or
proteoglycans were extracted according to the previously described
method (41)
. For some experiments, cells were grown in
sulfate-depleted medium supplemented with sodium chlorate to inhibit
sulfation as described previously (42)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Table 1
). All of the SCLC lines tested grew as aggregates in suspension with no
adhesion to the substratum when cultured in serum-based media. The cell
lines H128 (Fig. 1A
), OH-1 (Fig. 1C
), and a
variant of OH-1 (Fig. 1D
) that had lost the classic tight
aggregate morphology (38)
, all attached avidly on TSP1 but
failed to attach on murine laminin-1 or human plasma fibronectin. All
three of these proteins were functional to promote comparable levels of
adhesion for A2058 melanoma cells (Fig. 1B
). OH-1 SCLC cells
also failed to attach on substrates coated with vitronectin,
fibrinogen, type IV collagen, or gelatin (data not shown). Thus, the
OH-1 SCLC line lacks adhesion receptors for all of the matrix proteins
tested except TSP1. Several additional SCLC cell lines attached on TSP1
but also exhibited some adhesion to laminin-1 or fibronectin (Table 1)
.
H345 and H69 cells attached on TSP1 and laminin-1 at higher
levels than on fibronectin substrates, whereas N417 cells
adhered preferentially on laminin-1.
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3ß1 Integrin.vß3 integrin (LM609) and CD36 (OKM5), had no
effect on adhesion to TSP1 (results not shown). A function-blocking
antibody recognizing ß1 integrin was a dose-dependent inhibitor
of OH-1 cell adhesion on TSP1 but inhibited adhesion only by half at
saturating concentrations (Fig. 2A
and data not shown). The residual integrin-independent
adhesion of OH-1 cells on TSP1 may be mediated by the heparin-binding
sites of TSP1 because heparin also partially inhibited adhesion of OH-1
cells on TSP1, and a combination of heparin with either the
ß1-blocking antibody or the ß1 integrin ligand invasin
(43)
completely inhibited adhesion (Fig. 2A
).
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subunit antibodies were used to define
the specific ß1 integrin that recognized TSP1 (Fig. 2B
).
An 3ß1 integrin function-blocking antibody (P1B5) but not
anti-4 or anti-5
integrin antibodies, which have been reported to recognize TSP1 in
other cell types (44
, 45)
, partially inhibited adhesion on
TSP1. The 3 and ß1 function-blocking antibodies also partially
inhibited adhesion of OH-1 cells on an immobilized TSP1 peptide
(peptide 678), which is recognized by the 3ß1 integrin on breast
carcinoma cells (33
, 45)
, and on immobilized
invasin (Fig. 2B
). Invasin binds to several ß1 integrins,
including 3ß1, 4ß1, and 5ß1 (43)
;
therefore. the failure of the 4ß1 and 5ß1 antibodies to
significantly inhibit adhesion to immobilized invasin combined with
their lack of adhesion on the well-characterized ligand for these
integrins, fibronectin (Fig. 1
), indicates that OH-1 cells do not
express functional 4ß1 nor 5ß1 integrins. The consistently
greater inhibition by the integrin antagonists of adhesion on invasin
relative to that on TSP1 is also consistent with involvement of a
heparin-sensitive receptor in adhesion to TSP1.
The ß1 integrin-activating antibody TS2/16 enhanced adhesion on TSP1
and on the TSP1 peptide 678 but not on a CD36-binding peptide (Mal II)
or a heparin-binding peptide (p246) from TSP1 (Fig. 2C
).
This further confirmed that the recognition of TSP1 peptide 678 by OH-1
cells is ß1 integrin-mediated and suggested that this integrin exists
in a partially inactive state on OH-1 cells.
TSP1 Promotes Neurite-like Outgrowth of SCLC.
The attached OH-1 cells generally retained rounded cell bodies, but
many cells rapidly extended neurite-like processes on the TSP1
substrate (Fig. 3 A
). 3ß1 integrin binding was necessary for neurite
outgrowth on TSP1 because function-blocking 3 and ß1 integrin
antibodies inhibited the response (Fig. 3, B and D
). In contrast, function-blocking antibodies that
recognized 4ß1 or 5ß1 integrins did not inhibit neurite
outgrowth (Fig. 3D
). The 3ß1 integrin-binding peptide
678 from TSP1 (Fig. 3, C and D
) and the 3ß1
ligand invasin also inhibited neurite outgrowth on a TSP1 substrate
(Fig. 3D
). Ligation of 3ß1 integrin also was sufficient
to promote formation of neurite-like processes, in that substrates
coated with immobilized TSP1 peptide 678 or invasin both promoted
neurite formation (Fig. 3E
). The control peptide 690, in
which the essential Arg residue was replaced by Ala, was
inactive, and an analogue with decreased integrin binding, peptide 686
(33)
, was a weaker stimulator of neurite outgrowth. The
TSP1 heparin-binding peptide 246 only weakly promoted neurite
outgrowth, which indicated that the heparin-binding activity of TSP1 is
not sufficient to stimulate the neurite outgrowth response. Activation
of the 3ß1 integrin by antibody TS2/16 increased neurite formation
on TSP1 (Fig. 4A
).
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). ß1 Integrin
staining was also prominent in the neurites formed on TSP1 (Fig. 3G
). The integrin antibody also visualized long processes
extending from the neurites that lacked detectable actin staining. This
localization of the ß1 integrin supports the evidence from antibody
blocking studies which indicates that the 3ß1 integrin binding
mediates neurite-like outgrowth on TSP1.
EGF and Somatostatin Stimulate Integrin-mediated Outgrowth of SCLC
on TSP1.
Although we previously demonstrated that insulin and insulin-like
growth factor-1 specifically stimulated the 3ß1 integrin-mediated
spreading of breast carcinoma cells on TSP1 (45)
, these
growth factors had no significant effect on the function of the same
integrin in SCLC cells to promote adhesion on TSP1 or neurite outgrowth
(Fig. 4A
and data not shown). Basic FGF also had no effect,
but EGF was a potent inducer of OH-1 neurite outgrowth on TSP1 and
moderately increased cell adhesion on TSP1 or peptide 678 (Fig. 4A
and data not shown). Expression of EGF receptor by OH-1
cells was verified by immunoprecipitation using an EGF receptor
antibody, which comigrated with the EGF receptor from A431 epidermoid
carcinoma cells (inset in Fig. 4A
).
EGF-stimulated outgrowth of neurite-like processes on a TSP1 substrate
required the 3ß1 integrin because both 3- and ß1-specific
function-blocking antibodies reversed the stimulation by EGF (Fig. 4B
). Blocking the heparin-binding sites of TSP1 using
soluble heparin, in contrast, had no effect on EGF-stimulated outgrowth
(Fig. 4B
). EGF also specifically induced neurite outgrowth
on the 3ß1 ligand invasin, confirming that the stimulation of
neurite outgrowth by EGF was ß1 integrin-dependent (Fig. 4C
).
Several neuropeptides have also been reported to promote neurite
outgrowth in other neurectoderm-derived cell lines (46
, 47)
. Representatives of two major neuropeptide families were
tested. Somatostatin-14, a member of the somatostatin family,
stimulated outgrowth on TSP1 (Fig. 4A
) and slightly
stimulated adhesion on TSP1 (results not shown). In contrast, bombesin,
a member of the bombesin/gastrin-releasing peptide family that are
secreted by many SCLC cell lines, was inactive (Fig. 4A
).
CD47-binding peptides from the carboxyl-terminal domain of TSP1
activate the function of several integrins in other cell types
(27
, 29 , 48)
but did not stimulate 3ß1 integrin
function in breast carcinoma cells (45)
. Consistent with
the latter results, the CD47-binding peptide 7N3 did not significantly
stimulate neurite outgrowth or adhesion of OH-1 cells on TSP1 (Fig. 4A
and results not shown). Therefore, TSP1 cannot stimulate
a neurite-like outgrowth response of SCLC cells to itself by binding to
CD47.
TSP1 and an 3ß1 Integrin-binding Peptide from TSP1 Inhibit
SCLC Proliferation.
TSP1 is known to modulate the growth of several cell types (reviewed in
Ref. 19
). The addition of soluble TSP1 to nonadherent OH-1
cells markedly inhibited their proliferation, with an
IC50 of 40 nM (Fig. 5A
). This inhibition may result from ligation of the 3ß1
integrin because two additional ligands for this integrin, MBP-invasin
(IC50 = 80 nM)
and the TSP1 peptide 678 (IC50 = 6
µM), also inhibited OH-1 cell proliferation
(Fig. 5A
). The activity of peptide 678 was specific in that
the analogue 686, in which the essential Asn residue was replaced
by Ala (33)
, was inactive. A heparin-binding
peptide from the type 1 repeats (peptide 246) only weakly inhibited
OH-1 cell proliferation at the same concentrations (data not shown),
further indicating that the inhibition by the integrin-binding peptide
from TSP1 is specific.
|
). On the basis of
the observation that EGF stimulated the neurite outgrowth response to
TSP1 (Fig. 4
), we also examined the effect of immobilized TSP1 on the
proliferation of OH-1 cells in the presence of this growth factor.
Surprisingly, OH-1 cell proliferation was much more sensitive to
inhibition by immobilized TSP1 in the presence of EGF (Fig. 5B
). The addition of EGF alone had no significant effect on
the proliferation of OH-1 cells, but, in the presence of immobilized
TSP1, it produced a dose-dependent inhibition of proliferation (Fig. 5C
). The inhibition of proliferation on a TSP1 substrate by
EGF was specific in that IGF1 and bombesin did not display synergism
with TSP1 to inhibit proliferation (data not shown). The inhibition of
proliferation by a TSP1 substrate in the presence of EGF may also be
mediated by the 3ß1 integrin because substrates coated with TSP1
peptide 678 or MBP-invasin showed similar cooperative effects with EGF
to inhibit OH-1 cell proliferation (Fig. 5D
). TSP1 peptides
that bind to CD47 (7N3) or heparin (p246) did not synergize with EGF,
which indicated that the activity of TSP1 peptide 678 is specific (Fig. 5D
). Thus, EGF specifically and synergistically suppressed
proliferation of SCLC cells attached on TSP1 or an 3ß1-binding
sequence from TSP1.
Sulfatides Mediate High-affinity Binding of Soluble TSP1 to SCLC
Cells.
Although integrins mediate these biological responses of OH-1 cells to
TSP1, heparin-inhibitable binding accounted for most of the
high-affinity binding of soluble TSP1 to OH-1 cells. TSP1 bound
saturably to OH-1 cells with a dissociation constant of 72 ± 16 nM and 2.6 x 105 sites/cell (Fig. 6A
). This is comparable to the binding constant of 50
nM for TSP1 reported for TSP1 binding to resting
platelets (49)
and of 22 nM for
keratinocytes (50)
. The dissociation constant for H128
cells was slightly higher than for OH-1 cells
(Kd = 92 ± 21 nM) with 2.1 x 105 binding sites/cell (data not shown).
Additional low-affinity sites may be present on both cell lines but
were not detected using the accessible TSP1 concentrations.
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). Of the TSP1 antibodies tested, only A2.5, which
blocks heparin and sulfatide binding to TSP1 (51)
,
significantly inhibited high-affinity TSP1 binding to OH-1 cells (Fig. 6B
). To confirm the role of sulfated glycoconjugates in
high-affinity binding of soluble TSP1 to OH-1 cells, the cells were
cultured in medium containing chlorate to inhibit sulfation.
Incorporation of [35S]sulfate in macromolecules
was inhibited by 86% at 10 mM chlorate, and
binding of TSP1 was inhibited by 55% for these cells (Fig. 6B
). Binding of soluble TSP1 was not significantly inhibited
in the presence of 2.5 mM EDTA (67.4 ± 25.0 of control; P, 0.19 by a 2-tailed t
test), which provided further evidence that most high-affinity binding
of TSP1 to OH-1 cells was not mediated by the 3ß1 integrin.
OH-1 cells incorporated
35SO4 into acidic
glycolipids, glycoproteins, and proteoglycans. In contrast to the
predominant labeling of proteoglycans in most cell types examined
previously (Ref. 41
and unpublished
results),3
the majority (89%) of [35S]-incorporation was
recovered in the lipid fraction extracted using chloroform/methanol. No
binding of 125I-labeled TSP1 was detected to the
glycoprotein or proteoglycan fractions (results not shown); therefore,
these fractions were not further characterized. The sulfated
glycolipids identified by [35S]-labeling in
extracts of OH-1 cells are shown in Fig. 7
, Lane a. Galactosyl sulfatide was the major
sulfated lipid based on comigration of the labeled glycolipid with
authentic bovine brain sulfatide in two developing solvents. When the
total acidic glycolipids separated on TLC were incubated with
125I-labeled TSP1 (Lane b), a major
band comigrating with authentic galactosyl sulfatide from bovine brain
(Lane c) was strongly labeled. Several more
complex glycolipids also incorporated
[35S]sulfate (Lane a) but
were not present in sufficient concentration to detect TSP1 binding
(Lane b).
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, Lane a, and data not shown), but the predicted
PCR product was readily detected using endothelial cell RNA
(Lane e). THBS2 expression was
detected at low levels in some SCLC cell lines but only after 50 cycles
of amplification (data not shown). However, all six of the human SCLC
cell lines examined expressed significant levels of THBS3
mRNA (Fig. 8
, Lane c, and data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3ß1 integrin and sulfated
glycolipids. Interaction of the 3ß1 integrin on SCLC cells with
immobilized TSP1 stimulates formation of neurite-like processes and
inhibits SCLC cell growth. Both the induction of neurite-like processes
and the inhibition of proliferation by TSP1 are enhanced in the
presence of EGF, which suggests that TSP1 and EGF cooperate to reduce
SCLC growth by inducing neurotypic differentiation.
These results demonstrate that the 3ß1 integrin is an important
receptor in SCLC cells for transducing signals from TSP1. On the basis
of the activity of a TSP1 peptide, these signals are initiated by the
binding of residues 190–201 in the NH2-terminal
pentraxin module of TSP1 to this integrin. The 3ß1 integrin also
plays a major role in the adhesion of breast carcinoma and endothelial
cells to TSP1
(45)
,4
promotes neurite outgrowth in rat neurons on TSP1 (52)
,
and was recently shown to mediate interactions of neuroblastoma cells
with TSP1 (53)
. Although the 4ß1 and 5ß1
integrins are TSP1 receptors on other cell types (44
, 45
, 53)
, these integrins do not play a significant role in SCLC cell
adhesion on TSP1. The high-affinity binding of TSP1 to SCLC cells,
however, is not mediated by this integrin. This observation is
consistent with previous reports that the affinity of TSP1 binding to
3ß1 integrin is relatively low (34)
and that
high-affinity binding of TSP1 to both breast carcinoma cells and SCLC
cells is mediated by sulfated glycoconjugates rather than the 3ß1
integrin (45)
.
Sulfated glycolipids on cell membranes play a role in the interactions of several cell types with TSP1 (54) . In melanoma (41) , carcinoma, and endothelial cells,5 sulfated glycolipids typically account for only 0.5–10% of the total 35SO4 incorporation at steady state, but these are the major sulfated glycoconjugate on OH-1 cells. These glycolipids mediate most high-affinity binding of soluble TSP1 to OH-1 cells and significantly contribute to their adhesion on immobilized TSP1 but do not mediate neurite outgrowth.
Formation of neurite-like processes has been observed previously
when SCLC cells were plated on substrates coated with laminin-1
(10)
, polyethyleneimine or the extracellular matrix
produced by PC-9 lung carcinoma cells (55)
. The present
results identify TSP1 as a matrix protein that also induces this
response in SCLC cells and is mediated by the 3ß1 integrin. This
is consistent with the observations that the 3ß1 integrin mediated
neurite outgrowth of rat sympathetic neurons on TSP1 (52)
and that central and peripheral neurons (56)
and
neuroblastoma cells (53)
formed neurites when plated on
TSP1. EGF-induced neurotypic differentiation of thymic epithelial cells
induced TSP1 expression, and TSP1 also induced neurite outgrowth in
these cells (57)
. Therefore, TSP1 may inhibit the growth
of SCLC cells by triggering signaling through the 3ß1 integrins
that induce the cells to differentiate along a neuronal pathway.
Enhancement of this differentiation signal by EGF may explain the
synergism of these two proteins to inhibit SCLC cell proliferation. EGF
is known to have both growth-stimulating and -inhibitory activities in
other cell types (58)
, but inhibition by EGF has not been
observed in SCLC.
As was observed in breast carcinoma and endothelial cells
(45)
,4
the activation state of 3ß1 integrin in
SCLC cells to recognize TSP1 is regulated. The signals that mediate
this regulation, however, seem to be different for each cell type. The
3ß1 integrin in SCLC cells is activated by EGF and somatostatin
but not by CD47 ligation or by FGF or IGF1 receptor ligands, whereas
IGF1 but not EGF activates that same integrin in breast carcinoma
cells. The specificity for EGF receptor signaling versus
that from two other tyrosine kinase receptors in SCLC cells is
interesting in light of the extensive overlap in signaling pathways
regulated by these receptors (reviewed in Ref. 59
). One
established outcome of EGF signaling is modulation of integrin
activation (60)
. Additional work is needed to determine
how the activation state of the 3ß1 integrin is differentially
regulated by IGF1 and EGF receptor signaling in SCLC and breast
carcinoma cells.
Extracellular matrix can be an important regulator of the
malignant phenotype. Blocking of ß1 integrin signaling in breast
carcinoma cells induced differentiation of the tumor cells with loss of
their malignant phenotype (61)
. TSP1 has been implicated
in suppressing growth or inducing differentiation of several tumor cell
types. TSP1 inhibits proliferation of melanoma cells (28)
and breast carcinoma cells (62)
. It also inhibits
proliferation of HL60 cells while inducing their differentiation
(63)
. Increased TSP1 expression is also associated with
retinoic acid-induced differentiation of neuroblastoma cells
(64)
. On the basis of the present data, TSP1 may
coordinately induce differentiation of SCLC and suppress its growth.
Attachment on murine laminin-1 induced increased expression of
differentiation markers on SCLC, although proliferation was not
inhibited (10)
. The laminin-1 effect may also be mediated
by 3ß1, although laminin-1 is not a high-affinity ligand for this
integrin (65)
.
SCLC cell lines fail to express TSP1 at the protein or mRNA level but consistently express TSP3 mRNA. p53 mutations, which are common in SCLC, may suppress TSP1 expression in these cells (23) . A minor population of lung cells was identified as a site of THBS3 gene expression in the mouse (66 , 67) . The cells that expressed TSP3 were not identified, but the present data suggest that neuroendocrine cells, from which SCLC may derive, are a source of TSP3 expression in lung.
Expression of receptors for TSP1 but not TSP1 may contribute to the tumor biology of SCLC. Because TSP1 is an inhibitor of angiogenesis (20, 21, 22, 23, 24, 25) , lack of TSP1 expression may increase tumor growth by permitting neovascularization in response to angiogenic signals. Loss of TSP1 expression also may release the SCLC cells from the autocrine differentiating and antiproliferative activities of this matrix component and, therefore, may create an additional selective pressure to suppress TSP1 expression in SCLC cells.
Signals from the extracellular matrix may provide both positive (68) and, as demonstrated here, negative signals to control SCLC growth and survival. We have identified direct effects of soluble TSP1 and a synergistic interaction between a TSP1 matrix and EGF to suppress growth and increase neurotypic differentiation of SCLC. Inducing neuroendocrine differentiation of SCLC by a combination of TSP1 (or the integrin-binding peptide from TSP1) and EGF may facilitate treatment of this cancer because neuroendocrine differentiation is correlated with increased sensitivity of SCLC to radiotherapy (38) .
ACKNOWLEDGMENTS
We thank Drs. Steven Akiyama (NIH, Research Triangle Park,
NC), David Cheresh, William Frazier, Adi Gazdar, Ralph Isberg
(Tufts University School of Medicine, Boston, MA), Lance Liotta,
Joel Shaper, and Kenneth Yamada for providing reagents.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at NIH, National Cancer Institute, Building 10, Room 2A33,
10 Center Drive, MSC 1500, Bethesda, MD 20892-1500. Phone: (301)
496-6264; Fax: (301) 402-0043; E-mail: droberts{at}helix.nih.gov 
2 The abbreviations used are: SCLC, small cell
lung carcinoma; EGF, epidermal growth factor; IGF1, insulin-like growth
factor-1; mAb, monoclonal antibody; FGF, fibroblast growth factor; MBP,
maltose-binding protein; peptide 678, FQGVLQNVRFVF (TSP1 residues
190–201); peptide 686, FQGVLQAVRFVF; peptide 690, FQGVLQNVAFVF;
peptide 246, KYRFKQDGGWSHWSPWSS (TSP1 residues 412–428); peptide 7N3,
FIRVVMYEGKK (TSP1 residues 1102–1112); TSP1, thrombospondin-1; RT-PCR,
reverse transcription-PCR.
3 Roberts, D. D., unpublished
observations.
4 Chandrasekaran, L., He, C-Z., Krutzsch, H. C., Iruela-Arispe, M. L., and Roberts, D. D. Modulation of
endothelial cell behavior and angiogenesis by an 3ß1
integrin-binding peptide from thrombospondin-1, submitted for
publication.
5 Roberts, D. D., unpublished
observations.
Received 7/15/99. Accepted 11/11/99.
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), or human plasma
fibronectin (
). SCLC lines H128 (A), OH-1
(C), OH-1 variant (D), and the A2058
melanoma (B) cell lines were allowed to attach on each substrate for 60
min. Adherent cells were counted microscopically and are presented as
the mean of triplicate determinations.
), 5
µM TSP1 peptide 678
(
), or 0.2
µg/ml MBP-invasin (
) was determined in the presence of 5 µg/ml
antibody P1B5 (anti-
). Results are presented as mean ± SD
(n = 3)
). Net proliferation
was determined by the CellTiter assay (Promega) and is presented as
mean ± SD (n = 3).
B, growth on immobilized TSP1 inhibits proliferation in
the presence of EGF. OH-1 cell proliferation in growth medium (•) or
medium supplemented with 10 ng/ml EGF (
174 HaeIII
digested markers. All of the primers amplified the expected size
products using RNA from the human breast carcinoma cell line
MDA-MB-435, which was used as a positive control (not shown).