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INSERM, U-369, Faculté de Médecine Lyon-RTH Laennec, 69372 Lyon Cedex 08, France; and INSERM, U-244, Département de Biologie Moléculaire et Structurale, Commissariat à l’Energie Atomìque-Grenoble (J.-J.F.), 38054 Grenoble Cedex 09, France
Address all correspondence and requests for reprints to: Prof. Bernard Rousset, INSERM U-369, Faculté de Médecine, Lyon-RTH Laennec, 69372 Lyon Cedex 08, France. E-mail: u369{at}laennec.univ-lyon1.fr
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Abstract |
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TSH promoted follicle formation and inhibited TSP1 production. On the contrary, the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (1–100 nM) prevented TSH-induced follicle formation and strongly increased the synthesis of TSP1. Activation of TSP1 synthesis was dependent upon messenger RNA synthesis. Transforming growth factor-ß, like 12-O-tetradecanoyl-phorbol 13-acetate, increased TSP1 synthesis and prevented TSH-induced follicle formation. Thus, signaling molecules that depressed or conversely activated TSP1 production, respectively promoted or prevented thyroid folliculogenesis.
TSP1, purified from platelets, was devoid of effect on cell substratum attachment, but exerted a concentration-dependent inhibition of the TSH-activated reconstitution of thyroid follicles (half-inhibition at 40 µg/ml). TSP1 exhibited the same effect when added to thyroid cell aggregates representing primitive follicle structures.
Our data suggest that the control of thyroid follicle formation may operate at least in part through regulation of the production of the matricellular protein TSP1, which acts as a negative modulator of the cell-cell adhesion process involved in thyroid follicle morphogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TSP is a large trimeric glycoprotein; it was originally identified as a
component of 
-granules in human platelets and was later found to be
produced by a variety of cells in culture (19). Each TSP subunit has a
molecular mass of about 180 kDa. TSP influences multiple biological
processes such as cell attachment, migration, and proliferation as well
as angiogenesis and neurogenesis (20). More recently, it has been shown
that TSP belongs to a family of five secreted calcium-binding
glycoproteins (21, 22). TSP was then renamed TSP1; the other members of
the family were TSP2, TSP3, TSP4, and COMP. TSP1 and TSP2 are both
homotrimeric, and their multimodular subunits contain an N-terminal
heparin-binding domain, a procollagen homology domain, three type I
repeats, three type II repeats, seven type III repeats (calcium-binding
sites), and a C-terminal globular domain. TSP3, TSP4, and COMP are
pentameric, and their subunits lack the procollagen and type I domains
present in TSP1 and TSP2. TSPs possess multiple types of cell surface
receptors that recognize discrete domains of the molecule. The
specificity of their function on a given cell type thus seems to be
dictated by the combinatorial arrangement of available cell surface
receptors (23). In the present study, we first determined to which
member of the family TSP secreted by thyrocytes corresponds. We then
studied how signaling molecules that influence the in vitro
reconstitution of thyroid follicles or alter the structural integrity
of preformed follicles act on the synthesis and secretion of TSP. The
resulting data led us to conduct a direct analysis of the effects of
purified TSP1 on the ability of thyroid epithelial cells to reorganize
into three-dimensional follicle structures.
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Materials and Methods |
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Cell culture
Thyroid cells were isolated from pig thyroid glands by
discontinuous trypsin treatment (25). The dissociation procedure
resulted in a cell suspension composed of isolated cells and clusters
of few cells. Cells were cultured in tissue culture-treated petri
dishes in Ham’s F-12 medium supplemented with 10% calf serum in the
absence or presence of TSH (1 mU/ml), to obtain thyroid cell monolayers
or RTF, respectively, as previously reported (5, 6, 26). Cells were
seeded at a high density of 0.5 x 106
cells/cm2 and cultured at 37 C in an air-CO2
(95/5%) atmosphere for 3–5 days.
To assess TSP secretion, cultured cells were washed and preincubated in serum-free Ham’s F-12 medium for 2 h. Then the medium was replaced with fresh serum-free medium containing TSH, TPA, or growth factors. Culture media were collected after 2–24 h for subsequent analyses.
Gel electrophoresis and Western blot
Proteins present in culture media were precipitated by addition
of trichloroacetic acid (TCA) to a final concentration of 10%
(wt/vol). After centrifugation at 10,000 x g, the pellets
were solubilized in Laemmli’s sample buffer, and proteins were
separated by SDS-PAGE (on 6% gels). Molecular mass markers, including
myosin (Mr, 200,000), phosphorylase b
(Mr, 97,400), BSA (Mr,
68,000), ovalbumin (Mr, 43,000), and carbonic
anhydrase (Mr, 29,000) from Sigma Chemical Co. or Life Technologies (Gaithersburg, MD) were
run on a parallel lane. Purified human TSP1 and bovine
corticotropin-induced secreted protein/TSP2 (24) were used as internal
references. Proteins were either stained with Coomassie blue or
electrophoretically transferred onto nitrocellulose sheets for Western
blotting.
After the transfer, the nitrocellulose sheets were preincubated for 1 h in PBS containing 0.05% Tween-20 (PBS/Tween) and incubated with rabbit polyclonal antibovine TSP2 antibodies (diluted to 1/1000 in PBS/Tween) or the anti-TSP1 mouse monoclonal antibody A6.1 (diluted to 1/2200 in PBS/Tween) for 1 h at room temperature. Antigen-antibody complexes were detected using biotinylated antirabbit IgG or antimouse IgG antibodies, streptavidin conjugated to alkaline phosphatase and bromochloroindolyl phosphate/nitroblue tetrazolium as substrate.
Metabolic labeling with
[35S]methionine and
immunoprecipitation
Cells cultured in Ham’s F-12 medium supplemented with 10% calf
serum with or without 1 mU/ml TSH for 3 days were washed and then
incubated in the same medium without serum in the absence or presence
of TSH, TPA, or TGFß for various periods of time. During the last
2 h of the incubation period, cells were placed in a
methionine-free medium containing [35S]methionine (50
µCi/ml). The medium was then collected and centrifuged at 15,000
x g for 10 min to remove cell debris.
Radiolabeled media (1 ml) were incubated for 30 min at 4 C in the presence of nonimmune rabbit serum at a final concentration of 2% and with 50 µl of a 50% (vol/vol) suspension of protein A-Sepharose CL-4B and centrifuged at 10,000 x g for 5 min. The supernatants were incubated with 1 µg/ml anti-TSP1 monoclonal IgG for 1 h at 4 C. Then, 50 µl of the protein A-Sepharose suspension were added, and after a 30-min incubation at 4 C, the beads were washed three times by centrifugation at 10,000 x g for 5 min and resuspension in 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0) and once with 0.1% SDS. Proteins bound to protein A-Sepharose beads were released by boiling in Laemmli’s sample buffer and fractionated by SDS-PAGE. Radiolabeled proteins were visualized by fluorography.
Immunofluorescence and video microscopy
Cells were fixed with 4% paraformaldehyde in PBS for 30 min at
room temperature and permeabilized with 0.05% Triton X-100 in PBS for
30 min. After washing in PBS-BSA, fixed and permeabilized cells were
incubated with the monoclonal anti-TSP1 antibody (10 µg/ml) or normal
mouse serum at a 1:50 dilution in PBS-BSA overnight at 4 C. Immune
complexes were detected using a sheep antimouse IgG antibody conjugated
to fluorescein. Observations were made using a Zeiss Axiophot
fluorescence microscope (Zeiss, New York, NY). Fluorescence images
taken with a silicon intensified target video camera (Lhesa, Cergy
Pontoise, France) were numerized using the Crystal Sapphire image
processor from Quantel (Montigny le Bretonneux, France).
Photomicrographs were obtained using a UP-5000 P video printer from
Sony (Tokyo, Japan).
Phase contrast images of cells cultured in different conditions were made on a Zeiss Axiovert 35M inverted microscope using either a 24 x 36 Contax camera or a CCD video camera coupled to the image processor and the video printer mentioned above. For quantitative analysis of the formation of follicles, images of microscope fields (using the objective x5) taken at random were numerized and stored on 35-megabyte Bernoulli boxes (Iomega Corp., Roy, UT). Follicles were counted on the monitor screen; under basal culture conditions, there were about 100 follicles/field.
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Results |
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) exhibiting
an internal cavity, the follicle lumen. The tightness of the lumen of
in vitro RTF has been demonstrated by microinjection of
fluorescent molecules (6). The organization of thyroid cells into
follicles was clearly visible after 2–3 days. The size of the follicle
structures increased with the time of culture; this was mostly due to
the enlargement of the lumen. In the absence of TSH, thyroid cells
formed monolayers (Fig. 1A), which gave rise to domes after 4–5 days
in culture (27). The presence of serum was required for cell adhesion.
Serum could be removed after 24 h of culture without any
detectable change in the morphology of either monolayer cells or RTF
for up to 5 days. Addition of 0.1 µM TPA at the time of
seeding did not modify cell-substrate adhesion, but accelerated the
establishment of the cell monolayer (Fig. 1C). In contrast, 0.1
µM TPA completely inhibited the TSH-induced formation of
RTF (Fig. 1D).
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, E and F) and a total
disappearance of RTF after 24 h leading to a cell monolayer.
Addition of 0.1 µM TPA to 3-day-old RTF induced within
6–10 h the total disruption of preformed RTF and the development of a
continuous cell monolayer. TPA exerted the same action regardless of
the presence or absence of TSH (Fig. 1, G and H).
Protein secretion by RTF: differential actions of TSH and
TPA
We investigated whether modifications of thyroid cell organization
in response to TSH or TPA treatments were accompanied by changes in
protein secretion, i.e. protein composition of culture
medium. Preformed RTF (resulting from thyroid cells cultured for 3 days
in the presence of TSH) were placed in serum-free medium and incubated
without or with TSH or TPA for 24 h. Coomassie blue-stained
proteins from culture medium fractionated by SDS-PAGE under reducing
conditions are presented in Fig. 2A. One
major band migrating at the top of the gel was present in all cases; it
corresponded to the monomer of thyroglobulin, the thyroid hormone
precursor protein. When RTF were incubated in the presence of TPA, a
second band migrating as a 180-kDa protein was easily identifiable. The
size of this protein was consistent with that of TSP described in
thyrocyte-conditioned medium by Prabarakan et al. (14) and
Bellon et al. (17). To identify this protein as a TSP and to
determine whether it corresponded to TSP1 or TSP2, we performed Western
blot analyses using a monoclonal antibody (A6–1) recognizing the type
II repeats of human TSP1 or a rabbit polyclonal antibody raised against
bovine TSP2 (24). The anti-TSP1 antibody strongly labeled the 180-kDa
band in the culture medium from TPA-treated RTF. The labeled protein
had the same mobility as authentic TSP1 purified from human platelets
(Fig. 2A). The anti-TSP1 antibody also detected TSP1 in medium in which
the 180-kDa band was not visible after Coomassie blue staining. The
amount of medium immunoreactive TSP1 was decreased in response to TSH
and strongly increased in response to TPA. Anti-TSP2 antibodies that
specifically labeled purified TSP2 (used as a positive control) did not
detect any band in RTF-conditioned media.
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). De
novo synthesized TSP1 was identified in the culture media after
immunoprecipitation, but was also easily detectable after a simple TCA
precipitation. Two other components, the thyroglobulin monomer
migrating as a 280-kDa protein (28) and an unidentified protein of
100–110 kDa, were also detected as neosynthesized
35S-labeled proteins in TCA precipitates. TPA markedly
reduced medium 35S-labeled thyroglobulin.
35S-Labeled TSP1 was barely detectable in cell lysates
(data not shown); intracellular neosynthesized TSP1 represented a small
fraction of the total neosynthesized TSP1. Thus, medium TSP1
measurements gave a good account of TSP1 production.
The data presented in Fig. 3A show the
time-dependent changes in TSP1 synthesis when RTF cultured in complete
medium (Ham’s F-12 medium, 10% calf serum, and 1 mU/ml TSH) were
exposed to serum-free Ham’s F-12 medium (none) or serum-free Ham’s
F-12 medium supplemented with either TSH (1 mU/ml) or TPA (0.1
µM). First, it must be noticed that the replacement of
complete medium with serum-free medium led to an increase in the
rate of TSP1 synthesis; this change could due in part to the removal of
serum and in part to the withdrawal of TSH. Indeed, the rate of TSP1
synthesis by RTF was significantly reduced when TSH was added to the
serum-free medium. These observations indicate that serum probably
contains factors affecting TSP1 production and that in normal culture
conditions of RTF, the presence of serum could contribute, together
with TSH, to a lowering of TSP1 production. The inhibitory effect of
TSH on TSP1 synthesis was apparent after 6 h. Addition of TPA
caused a rapid increase (within 4 h) of the rate of TSP1
synthesis. The inhibitory effect of TSH was maximum at a concentration
of 1 mU/ml (Fig. 3B), which corresponded to the hormone concentration
normally used to obtain the reconstitution of thyroid follicles. The
effect of TSH (1 mU/ml) was reproduced by the dibutyryl derivative of
cAMP (1 mM; result not shown). TPA exerted a
concentration-dependent activation between 1–100 nM. At
the maximum, TPA caused a 2-fold increase and TSH caused a 2-fold
decrease in the rate of TSP1 synthesis. The TPA-induced increase in
TSP1 production by RTF required messenger RNA (mRNA) synthesis; it was
inhibited more than 90% by DRB, an inhibitor of RNA polymerases (29)
(Fig. 4). This is consistent with a TSP1
mRNA half-life of about 3 h (30).
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). After 8 h of TPA
treatment, RTF were disassembled, and the resulting cell monolayer
(Fig. 5, C and D) was strongly TSP1 positive. Immunoreactive TSP1 was
located in vesicular structures and intracellular compartments,
probably corresponding to secretory compartments. In some regions of
the petri dishes where cell density was higher, we could observe
fluorescent deposits between cells (data not shown). Contrary to
TPA-treated cells, cells in the form of RTF were not or were only
slightly labeled (Fig. 5A).
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demonstrate that TGFß1 actually
stimulated TSP1 synthesis and secretion. Three-day-old RTF incubated in
the presence of TGFß1 (10 ng/ml) for 24 h exhibited a higher
rate of TSP1 synthesis than untreated cells regardless of the presence
or absence of TSH. The TGFß1-induced activation of TSP1 synthesis was
concentration dependent; a 2-fold increase was obtained at a
concentration of 5–10 ng/ml. Phase contrast images in Fig. 7 show that TGFß1 (10 ng/ml) added at
the outset of culture prevented TSH-dependent folliculogenesis. The
adverse effects of TGFß1 were clearly apparent after 1–2 days of
culture (Fig. 7, A and C); TGFß1-treated cells did not form the cell
aggregates that correspond to prefollicle structures. On day 4 in the
absence of TGFß1, cells were mostly organized as follicles (Fig. 7B)
whereas in the presence of TGFß1, they formed a confluent monolayer
(Fig. 7D).
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). Cells cultured in
the presence of TSP1 at concentrations up to 25 µg/ml were also
organized in follicles; however, the proportion of cells at the
periphery of RTF not participating in follicle organization
progressively increased with TSP1 concentration (Fig. 8, B–D). In
addition, the size of the follicle lumena was smaller as the TSP1
concentration increased. At a concentration of 50 µg/ml, the number
of follicles was markedly reduced, and the remaining follicles appeared
to be formed from a few cells and had a very small lumen (Fig. 8E); the
majority of cells spread on the culture substratum. There was no
follicle structure when cells were cultured in the presence of 100
µg/ml TSP1 (Fig. 8F). The inhibitory effect of TSP1 on
folliculogenesis was assessed by counting follicles (whatever their
size) on microscope fields taken at random in each experimental
condition. The results are presented in Fig. 9. The TSP1 concentration that caused a
50% reduction of follicle formation was about 40 µg/ml.
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Discussion |
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The in vitro thyroid follicle morphogenesis has been shown
to be dependent on two main parameters: cell density (threshold,
0.25 x 105 cells/cm2) and the presence
of a hormone, TSH. Both factors appear equally important and to act in
synergy for promoting cell-cell interactions and follicle
reconstitution; if one of these two conditions is not fulfilled, cells
spread to form a monolayer (2, 4, 32). Cell-cell adhesion and
aggregation (which is favored by increasing cell density) are essential
for folliculogenesis and represent the step at which TSH influences the
morphogenic event. Recent studies (8, 9, 10, 33) indicate that follicular
differentiation is probably determined by the balance between adverse
adhesive forces acting upon the cells: cell-cell adhesion
vs. cell-substrate adhesion. These researchers found that
TSH has no effect on the attachment and spreading of cells to the
substratum, but inhibits spreading of cells from aggregates. This
effect was documented by analyzing the cell behavior when TSH was
withdrawn from the culture medium of reconstituted follicles (33).
Removal of TSH caused established follicles to reorganize into a
confluent two-dimensional epithelioid monolayer within 24 h. The
earliest change was the appearance of cells with broad lamellipodia at
the periphery of follicular structures. These cells became locomotile
as they spread from follicles and then formed the monolayer. Thus, TSH
probably contributes to thyroid follicular differentiation by exerting
a control on cell-cell interactions within three-dimensional cell
aggregates. Manley and co-workers (8) came to the conclusion that a
TSH-sensitive cell adhesion system might be involved in thyroid
folliculogenesis. Such a system would bring into play plasma membrane
proteins forming the complex network of adhesion structures and
intercellular junctions as well as secreted proteins that could
regulate the formation and/or the stability of the adhesion
structures.
TSP1 as a secreted protein could represent one of the intervening
regulatory proteins. Indeed, TSP1 is secreted at the basolateral pole
of polarized thyroid cells (14), where it could interfere with
cell-substratum and/or cell-cell adhesion processes. TSP1 does not seem
to act on the attachment of thyroid cells to tissue culture petri
dishes. Using cell attachment and cell spreading assays (34, 35), we
have been unable to detect any effect of purified TSP1, either in a
soluble form or coated to the dish, on the ability of dispersed thyroid
cells to form a monolayer. In contrast, purified TSP1 exerted a
definite inhibitory action on the TSH-induced three-dimensional
organization of thyroid cells. The TSP1 effect was highly specific, as
it was observed at concentrations ranging from 4 x
10-8 to 2 x 10-7 M in the
presence of 10% serum, i.e. about 5 mg/ml protein. Under
the same conditions, another extracellular matrix component,
fibronectin, was devoid of effect even at higher concentrations.
Interestingly, TSP1 had the same effect when added at the outset of
culture or 24 h later when thyroid cells were in the form of
aggregates representing prefollicular structures. It is thus reasonable
to think that TSP1 could inhibit follicle formation by interacting with
and thus inactivating a protein(s) belonging to the TSH-sensitive
adhesion system mentioned above. The multimodular structure of TSP1
implies that it can trigger its biological effects through interaction
with a variety of cell surface receptors. Interaction of TSP1 with
heparin or heparan sulfate proteoglycans is a prerequisite for binding
to the low density lipoprotein receptor-related protein (36).
Lipoprotein receptor-related protein is a multiligand receptor present
at the surface of numerous cell types that functions as a scavenger
receptor (37). TSP1 can also interact with CD36 through a peptide motif
located in the type I repeats. This interaction was recently shown to
mediate the antiangiogenic function of TSP1 (38). The RDG sequence
present in the seventh type III repeat of TSP1 is the putative binding
site to integrins vß3 and
IIbß3. In addition, the C-terminal domain
of TSP1 contains a binding site to the membrane receptor CD47, also
known as integrin-associated protein (39). To date, the identity of the
receptor(s) mediating the adhesive or antiadhesive properties of TSP1
is not known precisely. The presence of CD36 or CD47 at the surface of
thyroid cells has not been reported, and data dealing with the
expression of integrins are still scarce (40). It is therefore
difficult to predict which of these receptors could mediate the
preventing effect of TSP1 on thyroid follicle formation. Future studies
using recombinant fragments of TSP1 should help to decipher the
repertoire of TSP receptors present at the surface of thyrocytes and
the signaling pathway(s) involved in the antifolliculogenic action of
TSP1.
Data dealing with the endogenous TSP1 production and its regulation give further support to and extend the biological relevance of the observations made with exogenous TSP1. TSH, TPA, and TGFß that activate distinct intracellular signaling pathways were found to exert opposite regulatory actions on TSP1 synthesis and follicular differentiation. The relationship between down- or up-regulation of TSP1 synthesis and induction or prevention of folliculogenesis is sustained by different experimental data.
First, the inhibition by TSH or the activation by TPA or TGFß of TSP1 synthesis that was apparent within 4–6 h preceded the first detectable effects of these agents on follicle morphogenesis. The rapid changes in TSP1 expression are in keeping with data obtained on different cell types showing that TSP1 mRNA levels are regulated in a manner similar to immediate early genes (41, 42, 43).
Second, the alterations of TSP1 synthesis and follicular differentiation in response to a given signaling molecule were obtained within the same concentration range. TSH probably controls TSP1 synthesis through activation of the cAMP cascade; we have found that the TSH-induced inhibition of TSP1 synthesis was reproduced by the dibutyryl derivative of cAMP (1 mM), which mimics the action of TSH on folliculogenesis (1). Other pituitary hormones also regulate TSP1 synthesis in their target tissues. In ovarian granulosa cells, FSH down-regulates TSP1 synthesis, and it has been proposed that this phenomenon could play a role in the maturation of the tissue (44). The involvement of TSP1 in folliculogenesis in the ovary has not been investigated, but it may be worthwhile to examine this question in view of our results on thyroid follicle formation. In adrenocortical cells, TSP1 synthesis is down-regulated by ACTH, whereas TSP2 synthesis is simultaneously increased (45). In each of these cell types, the negative regulation of TSP1 synthesis occurs at the mRNA level and is cAMP mediated. In the thyroid cell system, we have found that both TPA and TGFß overcome the TSH-induced down-regulation of TSP1 and annihilated the TSH-induced follicle formation. The phorbol ester TPA, through the activation of protein kinase C, is known to inhibit the differentiation of thyroid cells, including TSH-induced follicle formation (reviewed in Ref. 46). The regulatory actions of TGFß on thyroid cell functions are less well characterized. On pig thyroid cells in culture, TGFß was reported to be a potent inhibitor of iodine metabolism activated by TSH (47). We report here another dedifferentiating effect of TGFß that was capable of inhibiting the TSH-induced folliculogenesis. Being produced by thyroid cells (48, 49), TGFß might represent a physiological modulator of follicle morphogenesis.
Third, in all of the experimental situations leading to up-regulation of TSP1 synthesis: 1) dispersed thyroid cells treated with either TPA or TGFß (in the presence of TSH) from the outset of culture, 2) preformed follicles treated with TPA (in the presence or absence of TSH), and 3) preformed follicles deprived of TSH, follicle structures were absent; either they did not form, or they disassembled.
Fourth, TSP1 has been identified by indirect immunofluorescence in thyroid cells in the course of establishing a monolayer after addition of TPA on preformed follicles. In contrast, TSP1 was not detectable in thyroid cells organized into follicles. Taken altogether, these data indicate that endogenously produced TSP1 could exert the same inhibitory effect on folliculogenesis as exogenous TSP1. It is worth noticing that purified TSP1, at a concentration that prevented TSH-induced folliculogenesis, was unable to cause the conversion of preformed follicles to a cell monolayer. Due to limitations in the preparation of purified TSP1, we could not check the effect of higher concentrations of TSP1. Nevertheless, this observation might indicate that to be active, TSP1 must be secreted in the intercellular spaces, close to the cell-cell adhesion system on which TSP1 is expected to act; on preformed follicles, the potential site of action of TSP1 could be far less accessible to exogenous TSP1 than to endogenous TSP1.
When available, anti-TSP1-neutralizing antibodies will be very useful to further analyze the functional role of TSP1 in the process of folliculogenesis and to determine to which extent down-regulation of TSP1 expression is a prerequisite for the formation and stability of follicles. Finally, the present work, by exploiting the rather unique property of polarized epithelial cells from the thyroid gland to undergo histiotypic morphogenesis in vitro, brings new information on the modulating effects of TSP1 on tissue differentiation.
Received March 6, 1998.
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