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Centro di Endocrinologia ed Oncologia Sperimentale, C.N.R. (G.R.); Dipartimento di Biologia e Patologia Cellulare e Molecolare (M.V., A.C., G.R.), and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica (M.I., T.D., GF.F.), Università Federico II, Naples, Italy
Address all correspondence and requests for reprints to: Dr. Mario Vitale, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, 80131 Naples, Italy. E-mail: mavitale{at}CDSunina.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1–6 integrin subunits was determined by flow
cytofluorometry with specific antibodies. Follicular cells of
subconfluent monolayer cultures expressed
2ß1 and 3ß1
at high levels, while 1ß1 was only
slightly expressed, and 4ß1,
5ß1, and 6ß1
were never detected. Cell attachment assays were performed in
fibronectin-, type I collagen-, and laminin-coated microtiter plates.
Thyroid cells, while adherent to collagen and fibronectin, showed poor
attachment to laminin despite the abundance of their putative receptors
2ß1 and 3ß1.
In serum-free medium, collagen and fibronectin induced cytoskeletal
organization, change of cell shape from round to flat, and cell
spreading. [3H]Thymidine incorporation and proliferation
assays were used to evaluate the effects of collagen and fibronectin on
DNA synthesis and cell growth in the absence of a change in spreading
or cell shape. Both substrates, in low serum-containing medium, induced
a concentration-dependent increase in [3H]thymidine
incorporation partially inhibited by RGD-containing peptides that
blocked the cell attachment. Thyrocytes cultured in low serum-containing medium on immobilized fibronectin or collagen showed a dose-dependent stimulation of proliferation. These data indicate that fibronectin and collagen can regulate the cytoskeletal organization and cell shape and stimulate the proliferation of normal human thyroid cells in culture and that integrins mediate these effects of extracellular matrix proteins.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
heterodimers characterized by a common ß1 chain
noncovalently associated with one of the variant chains (5). The
-chain confers the binding specificity for ECM proteins such as
collagen (CoG), laminin (LM), fibronectin (FN), vitronectin, or ligands
expressed on the cell membrane such as the vascular cell adhesion
molecule 1 (6). Analysis of small synthetic peptides has shown that in
some ECM proteins the recognition sites for cells is carried by the
sequence Arg-Gly-Asp-X, where X is a flanking amino acid residue (7, 8). When X is a serine (RGDS), the peptide in a soluble form inhibits
the attachment of cells to FN, whereas the peptide RGDT, where T is a
threonine, inhibits the attachment to both FN and CoG. Each of the
ß1 receptors displays binding specificity for one or
more ECM components. This binding specificity is cell type related;
thus, a certain integrin may function as an adhesion structure in a
cell and may not function in another cell type (9, 10). The ß
complex has a large extracellular domain bearing the ligand binding
site and a short intracellular domain that interacts with cytoskeletal
proteins, such as -actinin and talin (11, 12). On the cytoplasmatic
side, several regulatory proteins, such as
pp60src, pp125FAK,
protein kinase C, and mitogen-activated protein kinase, colocalize with
integrins in a subcellular structure defined as focal adhesion (for
review, see Ref.13). The signal transduction from ECM to the cell
interior initiates from these subcellular sites. Many other potential
candidates have been identified as a components of the signal
transduction pathway originated from integrins, including protein
kinase C, intracellular cAMP, Ca2+, and pH (14, 15, 16).
Integrin activation by its ligand, through its own signal transduction
pathway, can contribute to determination of the biological behavior of
the cell, such as motility, spreading, differentiation, and
proliferation (1, 17, 18).
The profile of ß1 integrin expression is cell type
related and undergoes modification upon differentiation,
transformation, and cytokine induction (19, 20, 21). In normal thyroid
glands, the majority of follicular cells expresses only the
3ß1 integrin at the basal site of cell
membrane (22). In the papillary carcinoma, the most frequent malignant
thyroid neoplasm, integrin expression is changed (23). Both tumor
specimens and tumor cell lines express all -subunits from 1–6, and
their polarized distribution is lost. In thyroid cells in culture, cell
to cell contact down-regulates the expression of some integrins; in
nonconfluent cultures, the expression of
3ß1 integrin is up-regulated 30-fold, and
2ß1 is expressed de novo (24).
In this study, we investigated some possible biological functions
mediated by integrins in thyroid cells in monolayer culture. The
results of this study indicate that thyroid cells adhere to FN and CoG,
but not to LM; adhesion was mediated by integrins; and FN and CoG
stimulated cell proliferation through their integrin receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cultures were prepared as previously described (24). Briefly, tissues were chopped by scalpels in small pieces and digested by type IV collagenase (Sigma Chemical Co., St. Louis, MO; 1.25 mg/ml) in Ham’s F-12 medium and 0.5% BSA overnight at 4 C under rotation. Cells were pelleted by centrifugation at 150 x g for 5 min, washed twice in BSA-Ham’s F-12 medium (BSA/F12), seeded in petri dishes, and cultured in 5% CO2 atmosphere at 37 C in Ham’s F-12 medium supplemented with 10% FCS. Medium was changed every 3–4 days, and the cells to be examined were harvested by treatment with 0.5 mM EDTA in calcium- and magnesium-free PBS containing 0.05% trypsin (trypsin/PBS). The follicular origin of the cultures was confirmed by the presence of cytokeratin and thyroglobulin, as assessed by flow cytofluorometry.
Antibodies and flow cytometric analysis
Antihuman thyroglobulin serum was obtained in rabbits by
repeated immunization with purified human thyroglobulin (25).
Anticytokeratin monoclonal antibody (MoAb) was purchased from Ortho
(Raritan, NJ). MoAb against the ß1-chain and the various
-subunits were kindly donated, as indicated: A1A5
(anti-ß1) and B-5G10 (anti-4), Dr. M. E.
Hemler (Boston, MA); TS2/7 (anti-1), Dr. F.
Sanchez-Madrid (Madrid, Spain); 10G11 (anti-2), Dr. A.
E. G. Kr. von dem Borne (Amsterdam, The Netherlands); J143
(anti-3), Dr. L. J. Old (New York, NY); and GoH3
(anti-6), Dr. A. Sonnenberg (Amsterdam, The
Netherlands). MoAb against 5 was purchased from Telios
(San Diego, CA); fluorescein-conjugated antimouse IgG and sheep
antirabbit IgG were purchased from Ortho.
Cells harvested from cell cultures by trypsin/PBS were incubated with the specific monoclonal antibody for 1 h at 4 C in PBS and 0.5% BSA (BSA/PBS), washed in the same buffer, and incubated again with the second fluorescein-conjugated antibody for 30 min at 4 C. Cells were resuspended in BSA/PBS and analyzed by flow cytometry. For intracellular immunofluorescence (cytokeratin and thyroglobulin), cells were fixed in 70% ethanol for 30 min at room temperature, washed twice in PBS, and resuspended in BSA/PBS; immunostaining was then performed as described above, using rabbit antihuman thyroglobulin serum and sheep antirabbit IgG as a secondary antibody. Direct immunofluorescence was performed by incubating fixed, permeabilized cells with the fluorescein-conjugated anticytokeratin antibodies for 30 min at 4 C. Serum from nonimmunized rabbits or nonspecific fluoresceinated Igs of the same isotype were used as controls.
Cell attachment assay
The assay was performed in 96-well flat-bottomed microtiter
plates (Costar, Cambridge, MA). The wells were filled with 100 µl of
the appropriate dilution in PBS of type I collagen (Sigma),
fibronectin, or laminin (Collaborative Research, Bedford, MA). After
overnight incubation at 4 C, the plates were washed with PBS, filled
with 100 µl 1% heat-denatured BSA, and incubated for 1 h at
room temperature. Then, plates were washed and filled again with 100
µl/well PBS, 0.9 mM CaCl2, and 0.5
mM MgCl2 containing 7 x 104
cells. After 30 min at 37 C, plates were gently washed three times with
PBS, and the attached cells were fixed with 3% paraformaldehyde for 10
min followed by 2% methanol for 10 min and finally stained with 0.5%
crystal violet in 20% methanol. After 10 min, plates were washed with
tap water, the stain was eluted with a solution of 0.1 M
sodium citrate, pH 4.2, in 50% ethanol, and the absorbance at 540 nm
was measured by a spectrophotometer.
In the adhesion inhibition assay, 5 x 104 cells/well were coincubated with 500 µg/ml RGD (RGSP = Gly-Arg-Gly-Asp-Ser-Pro; RGTP = Gly-Arg-Gly-Asp-Thr-Pro)- or RGE (Gly-Arg-Gly-Glu-Ser-Pro)-containing peptides (Telios) in plates previously coated with 2 µg/ml FN or CoG. All experiments were performed in quadruplicate. Results were expressed as a percentage of the adhesion obtained in the absence of peptides.
Cytoskeletal organization and cell spreading
Cells were plated in 0.5% BSA/F12, and serum-free medium on
glass coverslips uncoated or coated with 10 µg/ml of FN or CoG. After
30 min, 1 day, and 3 days, cells were fixed and permeabilized with 3%
paraformaldehyde, 0.5% Tween-20, and actin was stained with
phycoerythrin-conjugated falloidin (Sigma). Cells were observed with a
fluorescence microscope (Zeiss, Oberkochen, Germany) and photographed
using Tri-X film (Eastman Kodak, Rochester, NY).
The effect of serum alone and with substrates on thyroid cell spreading was evaluated after 48 h of culture. Cells were cultured in six-well plates in the presence of various FCS concentrations or in plates coated with various concentration of CoG or FN in the presence of 0.4% FCS. Cells were observed by inverted phase contrast microscope at x400 magnification and photographed. For each experimental point, the surface of 50 cells was measured, and the results were expressed as the mean ± SD.
[3H]Thymidine incorporation and
proliferation assay
Thyroid cells in culture for 9–12 days were extensively washed
with PBS, harvested by trypsin/PBS, and seeded in 96-well flat bottom
plates previously coated with type I CoG or FN. Coated plates were
obtained as described above using substrate concentrations ranging from
0.08–10 µg/ml.
Cells were cultured in the presence of 0.4% FCS for 24–72 h. Basal and maximal [3H]thymidine incorporation were obtained in uncoated wells in the presence of 0.4% and 10% FCS, respectively. Cultures were pulsed with 1 µCi [3H]thymidine/well during the last 24 h of culture, then cells were harvested, absorbed onto nitrocellulose paper, and extensively washed, and radioactivity was counted in a ß-counter (Beckman LS1801). All experiments were performed in quadruplicate. Results were expressed as the fold increase [mean incorporation at the experimental point (counts per min)/mean incorporation at the basal point (counts per min)].
To investigate whether integrins mediate the mitogenic effect of FN and CoG, blocking experiments with RGD-containing peptides were performed. A total of 5 x 104 cells were plated on 10 µg/ml FN- or CoG-coated 96-well plates in 0.4% FCS/F12 medium, in the presence of 100–500 µg/ml RGDSP, RGDTP, or RGETP peptide. In the CoG-coated wells, FCS was omitted to avoid the RGDTP blocking effect on the FN present in the 0.4% serum. Cells were cultured for 72 h and pulsed with 1 µCi [3H]thymidine/well during the last 24 h of culture. Results were expressed as the percent (counts per min) incorporation obtained in the absence of peptides.
The proliferative response to ECM proteins was determined in cells cultured in 0.4% FCS in plates previously coated with 0.4, 2, and 10 µg/ml FN or type I CoG or cultured in uncoated plates in the presence of 0.4% or 10% FCS. After 1 h, 7 days, and 14 days, culture was stopped, and cells of triplicate plates were counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1–6 chains. Table 1
reports the
average expression of integrin subunits measured in four cultures of
different subjects at 75% confluence. Analysis was performed on 9- to
12-day-old cultures after two or three passages, and expression of the
integrin receptors 2ß1 and
3ß1 was determined. The expression of some
subunits changed during the culture under the regulatory effect of cell
to cell contact. The 1-chain was slightly expressed
throughout the culture, whereas 4, 5, and
6 remained constantly undetectable. Soon after the cells
were plated, 2 and 3 increased to a
remarkably high level until the cultures achieved a high cell density;
then, as previously shown (24), cell to cell contact down-regulated
their expression. In the following experiments, the cells were always
used at subconfluence after two or three passages.
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). Thyroid cells from subconfluent cultures showed poor
attachment to LM even though 2ß1 and
3ß1 integrins are their putative receptors
and are abundant on the cell membrane. The adhesion to CoG and FN was
concentration dependent, reaching its maximum at about 10 µg/ml. The
substrate concentration required to achieve 50% of the maximal cell
adhesion was about 2 µg/ml for both ECM components.
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). Cell adhesion to FN was inhibited by
RGD-containing, but not RGE-containing, peptides. The RGDSP peptide is
a specific inhibitor of integrin binding to FN, whereas RGDTP inhibits
the binding to both CoG and FN (7, 26). The inhibition of adhesion to
FN in the presence of 50 µg/ml peptide was 56% with RGDSP and 82%
with RGDTP. Cell adhesion to CoG was blocked only by RGDTP (54%
inhibition), whereas RGDSP and RGESP peptides had no effect.
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, A–C). In the cells
cultured on CoG (Fig. 3, D–F) and FN (Fig. 3, G–I), the cortical
actin initiated to organize itself and form fibers; then a progressive
cytoskeletal organization and cell spreading were observed. Cells were
photographed by inverted phase contrast microscope at x400
magnification. The surface of 50 cells for each experimental point was
measured, and the results were expressed as the mean ±
SD. An adequate concentration of FCS in the culture medium
was required for thyroid cells to spread in the absence of substrates
(Fig. 4A). Under these culture conditions, maximal
spreading was achieved with 0.4% FCS. At this serum concentration,
cell spreading was 2.7-fold higher than that in the absence of FCS.
Higher serum concentrations induced significantly less spreading. In
Fig. 4A all samples vs. 0.4% FCS were statistically
significant (P < 0.005). In the presence of 0.4% FCS,
the variations in cell spreading induced by CoG and FN (Fig. 4B) were
not significant.
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). The substrate concentration used to coat 96-well
flat bottom microtiter plates ranged from 0.08–10 µg/ml. In the
control samples, in the absence of substrate,
[3H]thymidine incorporation was very poor at any time
(401 ± 62 cpm at 48 h and 534 ± 70 cpm at 72 h).
The presence of 10% FCS in the culture medium induced a striking
increase in [3H]thymidine incorporation at 48 and 72
h (8.7- and 14.3-fold increases, respectively). In the first 24 h
of culture, substrates and serum did not induce any stimulation of
[3H]thymidine incorporation. After 48 and 72 h, both
CoG and FN induced a concentration-dependent increase in
[3H]thymidine incorporation, reaching a maximal increase
of about a 3.5-fold the radioactivity incorporated by the control
cells. As suggested by the adhesion blocking experiments,
RGD-containing, but not RGE-containing, peptides inhibited the
mitogenic effect of FN and CoG (Fig. 6). Inhibition was
peptide concentration dependent.
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). The number of adherent cells was determined after
1 h and then after 7 and 14 days. Although the cell number
remained unchanged in 0.4% FCS, 10% FCS induced a 2.93-fold increase,
and a dose-dependent proliferation of both FN and CoG was observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit. Follicular cells of normal thyroids have a restricted
integrin repertoire. Although many different integrin receptors are
expressed by thyrocytes in thyroid diseases such as nodular goiter and
tumors, only 3ß1 has been detected on the
cell membrane of normal thyrocytes in vivo (22, 23). In
these cells cultured in vitro, ß1 integrin
expression is regulated by cell to cell contact (24), and in
subconfluent cultures it is different from that observed in
vivo. In addition to 3ß1, follicular
cells of subconfluent cultures also expressed
2ß1 and 1ß1,
the latter at a very low level. Based on the presence on the cell
membrane of 2ß1 and
3ß1 receptors, cultured thyrocytes would
attach to several ECM components, such as FN
(3ß1) and CoG and LM
(2ß1 and
3ß1), known to be their putative ligands.
Surprisingly, although adhesion to type I CoG and FN was high and
concentration dependent, thyroid cells showed poor attachment to
laminin despite the fact that both 2ß1 and
3ß1 are their putative receptors, and LM
is a major component of the basal membrane surrounding the follicles
(27). The inhibition experiments with RGD peptides supported the
evidence that the adhesion to FN and CoG is mediated by
2ß1 and 3ß1
integrins, but could not define whether binding to CoG was mediated by
one or both receptors. Indeed, the RGDSP peptide inhibits only
3ß1 binding to FN, whereas RGDTP inhibits
the binding of both 2ß1 and
3ß1 to CoG (7, 26). Only antibody-blocking
experiments can better clarify which of these integrins mediates the
adhesion to CoG. It is interesting that the RGTP peptide gave a
stronger inhibition in FN-coated plates than in CoG-coated ones, and in
the same FN plates, it caused an inhibition of cell adhesion higher
than that caused by RGDSP. These observations could be explained on the basis of the binding affinities of integrin receptors for their ligands; unfortunately, few data are available in the literature on this regard. In addition, the efficacy of synthetic peptides to block cell attachment not only depends upon the presence of specific and seldom repeated motifs in the ligand primary structure, but also upon the surrounding sequences that may either enhance or suppress the activity of the cell attachment sequences. Folding of the protein and quaternary structure may make the sequence more or less available for the integrin receptors.
It is also known that certain integrins appear to undergo cell
type-specific regulation of adhesive capabilities. In some instances,
integrins can assume in the same cell multiple, rapidly triggered
functional states. In platelets and lymphocytes, ß1
integrin-mediated adhesion to CoG and FN is enhanced by phorbol esters
(9). This enhancement is independent from de novo protein
synthesis, suggesting that protein kinase C induces conformational
changes in preexisting receptors (21, 28). Additionally, certain
antibodies to the ß1 and ß3 (29, 30, 31)
integrin subunits modulate binding affinity by directly inducing a
conformational change in the receptor at the surface of the cell
membrane. Moreover, certain integrin receptors display a cell
type-dependent regulation of ligand specificity. Whereas
2ß1 is a collagen and laminin receptor in
melanoma and endothelial cells (10, 32, 33), it binds only collagen in
fibroblasts and platelets (10, 28, 34). Finally, the same
2 complementary DNA construct expressed in two different
cell types can yield three distinct patterns of binding specificity
(30). The mechanism for this cell-specific regulation of ligand
specificity is unknown, but it may be based on posttranslational
modification. More recently, urokinase-type plasminogen activator
receptor (uPAR) has been shown to colocalize with integrin receptors in
focal contacts (35). We have also shown the presence of uPAR in thyroid
cells (36). uPAR, which does not contain a transmembrane domain and
coprecipitates with integrins, inhibits native integrin adhesive
function with a still unknown mechanism, possibly by complex formation
or signaling (37).
The simple occupancy of integrin receptors is sufficient to activate a
signal transduction pathway, but cell shape and cytoskeleton
organization are also required to activate complex cellular phenomena,
including DNA synthesis and transcription. In anchorage-dependent cells
such as epithelial cells, attachment to ECM prevents apoptosis by
inducing cytoskeleton organization and cell spreading rather than by
simple integrin binding (38). Although some cellular events, such as
FAK phosphorylation or antiporter activation, can be induced by
integrin binding in the absence of a complete cytoskeleton
organization, cell growth of anchorage-dependent cells can be induced
by ECM only in the presence of cell spreading (39, 40, 41). The results of
our study indicate that FN and CoG can stimulate the proliferation of
human thyroid cells without changes of shape or spreading increase.
Maximal thyroid cell spreading with low thymidine incorporation and
absence of proliferation were obtained in the presence of 0.4% FCS.
The soluble FN present in the serum and/or other unknown serum factors
could be responsible for the adhesion and cell spreading observed in
low serum cultures. In these minimal culture conditions, immobilized FN
and CoG induced a concentration-dependent stimulation of DNA synthesis
without a change in cell shape or spreading. A number of ECM proteins
have binding sites for growth factors, such as transforming growth
factor-ß and insulin, that associate with FN and type V CoG,
respectively (42, 43). Inhibition experiments are needed to exclude
that some of the biological effects of ECM proteins are mediated by
associated growth factors. The inhibition experiments with
RGD-containing peptides supported the evidence that FN and CoG,
directly interacting with 2ß1 and
3ß1 integrins, are involved in ECM-induced
proliferation. Antibody-blocking or -activating experiments could
elucidate the specific roles of each of these two receptors.
The effect of integrin interaction with FN and CoG was not limited to only cytoskeletal organization and DNA synthesis; proliferation was also stimulated. The low proliferative response to 10% FCS, FN, and CoG obtained after 14 days of stimulation should be considered in the light of the limited growth capability of thyroid primary cultures. Normal thyroid cells fail to proliferate after three or four replications and quickly lose their differentiation markers (44). Cells were cultured for a few days before the proliferation experiments; thus, only a few more replications could be expected even in the presence of 10% FCS.
In conclusion, the results of our study indicate that FN and CoG can induce cytoskeletal organization, determine cell shape, and stimulate the proliferation of normal human thyroid cells in culture; these effects of ECM proteins are mediated by integrins. Further experiments are needed to clarify the relevance of the interaction of thyroid cells with ECM components in vivo and the possible significance for the growth of neoplastic cells.
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Acknowledgments |
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Footnotes |
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Received September 13, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 integrin-mediated cell spreading on
fibronectin. J Biol Chem 268:21459–214625ß1 integrin receptor in the proliferative
response of quiescent human melanoma cells to fibronectin. Cancer Res 52:4499–45062 cDNA clone:
interconversion of forms induced by an anti-ß1 antibody. J Cell Biol 120:537–5432ß1 integrin as a
laminin receptor. J Cell Biol 109:2455–24625ß1
independent of cell shape. Proc Natl Acad Sci USA 88:7849–7853
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