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Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, 28029 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Pilar Santisteban, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: psantisteban{at}iib.uam.es
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Somatostatin was initially identified as a potent GH inhibitor in the hypothalamus and soon after was found in many tissues and cell lines, exerting different biological actions, including inhibition of hormone secretion processes, modulation of cardiovascular activity, control of neural cognitive and locomotor functions, and regulation of cell proliferation (4, 5). Immunoreactive somatostatin has also been found in the thyroid, more precisely limited to the parafollicular C cells, leading to the speculation that this peptide could elicit local effects on thyroid hormone release acting locally, in a paracrine fashion (6). In the differentiated thyroid cell line FRTL-5, somatostatin inhibits TSH- and IGF-I-mediated cell proliferation (7). More recently, the effect of somatostatin on cell proliferation has also been characterized in the thyroid cell line PC Cl3. In these cells, somatostatin is able to inhibit insulin-dependent and insulin-plus TSH-dependent cell growth, causing a delay block in the G1-S transition of the cell cycle (8).
Somatostatin action is mediated by specific receptors, and to date, six different subtypes (designated SSTR-1, -2A, -2B, -3, -4, and -5) have been cloned and functionally characterized in various cell systems, including pancreas, adrenal cortex, and brain tissue (9). They belong to the seven-transmembrane domain superfamily and are coupled to different G proteins that link SSTR to distinct cellular effector systems, such as adenyl cyclase, K+, and Ca2+ channels. Different receptors may regulate a variety of cellular functions, thus providing a broad spectrum of somatostatin action. SSTR2 along with SSTR1 and -5 are responsible for the antiproliferative effect of somatostatin and are expressed in many tumor types (10, 11). Currently, the design of synthetic somatostatin analogs with high affinity for these SSTR is a clinical approach for a variety of tumor diseases.
Several reports indicate that the mechanisms responsible for the antiproliferative effect of somatostatin are related to its ability to modulate phosphotyrosine phosphatase activity (8, 10, 12, 13). In this study we show that somatostatin action is mediated by the cyclin-dependent kinase inhibitor p27kip1. This protein, initially identified by the ability to bind the complexes cyclin E-cyclin-dependent kinase-2 (cyclin E-CDK2) (14) and cyclin D-CDK4 (15), belongs to the Cip/Kip family of CDK inhibitors and plays a critical role in the regulation of G1-S transition (16).
In this report we provide evidence that somatostatin is expressed in FRTL-5 cells. Furthermore, transcription of the somatostatin gene is under hormonal control exerted by TSH, as determined by transient transfection assays. Experiments with conditioned medium from TSH-treated FRTL-5 cells and somatostatin antibodies revealed that this peptide exerts a cytostatic effect on cell growth. This circumstance is further supported by cytometric assays, showing that somatostatin blocks FRTL-5 cell cycle in the G1 phase. As mentioned above, we demonstrate that this effect is mediated by the capacity of somatostatin to reestablish the expression of the CDK inhibitor p27kip1, which is down-regulated by TSH to promote proliferative signals. In thyroid tumor and transformed cells, the ability of somatostatin to modulate cell growth is altered. The results suggest that within the thyroid follicular cell, somatostatin expression is used as a precise, local mechanism to regulate cell growth in an autocrine fashion.
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Materials and Methods |
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RT-PCR amplification
Total RNA was isolated as previously described (23). For first
strand complementary DNA synthesis, total RNA (1 µg) was treated with
deoxyribonuclease and reversely transcribed at 42 C for 30 min using
Moloney murine leukemia virus reverse transcriptase (Pharmacia Biotech, Piscataway, NJ). Aliquots of the first strand reactions
(10 µl) were used as templates for subsequent PCR using
Taq polymerase (Perkin-Elmer, Norwalk, CT). For
detection of somatostatin transcripts, the following primers were
used: forward, 5'-CAGACTCCGTCAGTTTCTGCA-3'; and reverse,
5'-GCTAACAGGATGTGAATGTC-3'. Amplification was performed in 35 cycles of
30 sec (2 min for the first cycle) at 95 C, 30 s at 55 C, and 1
min at 72 C (5 min for the last cycle). For the different somatostatin
receptors (24, 25, 26, 27), the primers for forward and reverse annealing were:
SSTR-1, 5'-GCTACGTGCTCATCATTGCTA-3' and 5'-GGACTCCAGGTTCTCAGGTTC-3';
SSTR-2, 5'-TTGGTACACAG-GGTTCACAT-3' and 5'-GTCTCCGTGGTCTCATT
CAGC-3'; SSTR-3, 5'-TCATCTGCCTCTGCT ACCTA-3' and
5'-GAGCCCAAAGAAGGCAGGCT-3'; SSTR-4, 5'-ATCTTCGCA GACACCAGACC-3' and
5'-ATCAAGGCTGGTCACGACGA-3'; and SSTR-5, 5'-GCCGGCCTCTACTTCTTCGTG-3' and
5'-CCGTGGCGTCAGCGTCCTTGG-3'. Con-ditions for PCR have been recently
described (28). PCR products were visualized in 2% agarose gels, and
the identities of positive bands were confirmed by direct sequencing of
the PCR products (automated DNA sequencer, Applied Biosystems,
Foster City, CA).
Plasmids and transfections
The reporter construct containing the complete somatostatin gene
promoter (SMS900), deleted constructs containing the cAMP response
element (CRE)-binding site with or without the upstream enhancer
promoter (SMS120 and SMS65, respectively), and the construct containing
the region of the TATA box (SMS42) have been used in this study and
were described previously (29). These constructs were transiently
transfected by the calcium phosphate precipitation technique (30) into
confluent FRTL-5 cells and then maintained in basal medium (3H) or
treated for 24 h with 1 nM TSH. The plasmid CMV-Luc
was used to correct for transfection efficiency. Forty-eight hours
after transfection, cell extracts were prepared, and chloramphenicol
acetyl transferase and luciferase activities were determined (30, 31).
Cell proliferation and flow cytometric analysis
Conditioned medium from FRTL-5 cells maintained in basal
conditions (3H) and treated with TSH (3H+TSH) was collected after
72 h for functional assays. The mitogenic activity of the medium
was tested on quiescent FRTL-5, by cell counting after 24, 48, and
72 h. To test any potential role of somatostatin, this conditioned
medium was preincubated with a specific somatostatin antibody (4
µg/ml) (32) for 1 h at room temperature before addition to the
FRTL-5 cultures. Growth curves in the presence of increasing
concentrations of somatostatin were also performed. FRTL-5 cells
(2 x 104) were maintained in basal medium for 72
h and then treated with 1 nM TSH, somatostatin, or TSH plus
somatostatin (10 nM, 1 µM, and 2
µM). The number of viable cells was determined by cell
counting every 24 h for 4 consecutive days, and the average of
three independent experiments is represented. Cell cycle distribution
from propidium iodide-stained samples was performed as previously
described (33), using a FACScan flow cytometer (Becton Dickinson Co., Mountain View, CA). At least 10,000 events were collected
and analyzed. Data were integrated and plotted with the software
CellQuest.
Immunoblotting analysis
Total protein extracts (40 µg) were subjected to SDS-PAGE.
Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) in a buffer containing 25
mM Tris, 200 mM glycine, and 20% methanol.
After blocking the membranes with 10% low fat dried milk in
Tris-buffered saline containing 0.05% Tween-20, immunodetection was
performed using 5 µg/ml of a commercial antibody for
p27kip1 and cyclin D1 (Oncogene Science, Inc., Cambridge, MA). After probing with the antibodies,
membranes were incubated with a streptavidin-conjugated antirabbit
specific secondary antibody. Immunoreactive bands were visualized by
enhanced chemiluminiscence (Amersham, Arlington Heights,
IL). To assess equal loading of the samples, the same blots were
reprobed with a specific antiactin antibody (Calbiochem,
La Jolla, CA).
Statistical analysis
Statistical significance between different treatments was
determined using Student’s t test. Differences are
considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In control experiments, the same
band was obtained from RNA samples extracted from the human medullary
carcinoma cell line MTT and the rat pancreas-derived cell line RIN,
whereas in agreement with previous reports (34, 35), somatostatin
transcripts were absent in RNA extracted from liver tissue. The
integrity of the RNA preparations was confirmed with specific primers
for ß-actin (Fig. 1A). To further assure that amplified DNA contained
somatostatin related sequences, the 271-bp PCR product was purified and
subjected to direct DNA sequencing. The nucleotide sequence obtained
was identical to that previously described for the rat somatostatin
gene (36).
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), failing to
detect the expression of SSTR-1, -3, -4, and -5. We also observed
expression of SSTR-2 in MTT cells, RIN cells, and the human thyroid
carcinoma cell line ARO, which also expressed SSTR-1. Positive
amplification of SSTR-3, SSTR-4, and SSTR-5 was found in the human
medullary carcinoma cell line MTT.
Transcription of the somatostatin gene in FRTL-5 cells is
hormonally regulated by TSH
The detection of somatostatin transcripts in FRTL-5 cells prompted
us to analyze whether transcription of this gene is hormonally
regulated by TSH, one of the major inductors of thyroid cell
proliferation. Due to the low levels of somatostatin produced by FRTL-5
cells and the difficulty in determining precisely by RT-PCR messenger
RNA variations in response to TSH, an indirect transfection approach
was used. For that purpose, we determined the ability of TSH to promote
transcription of a reporter construct containing the complete
somatostatin gene promoter (designated SMS900). Also, considering that
somatostatin expression in other cell systems is mainly controlled at
the transcriptional level by CRE (29), the participation of CRE in
TSH-mediated somatostatin gene expression was evaluated by using
deletion constructs carrying a CRE-binding site with or without the
upstream enhancer element (SMS120 and SMS65, respectively). These
constructs, along with a negative TATA control vector (SMS42) were
transiently transfected in FRTL-5 cells, using the plasmid CMV-Luc to
correct for transfection efficiency. After transfection, cells were
maintained in 3H medium and then treated with TSH for 24 h.
Control assays without TSH were carried out in parallel. Results showed
a significant increase in transcription activity from the complete
somatostatin promoter (SMS900) in the presence of TSH (Fig. 2). To a minor extent, transcription was
also increased with the SMS120 construct, whereas SMS65, which contains
a unique CRE-binding site, yielded a weak activation of gene
transcription. When the same set of experiments were performed in the
presence of the cAMP analog forskolin, we found a similar patter of
induction of gene transcription. The results demonstrated that the
somatostatin gene promoter is functional in FRTL-5 cells, and although
moderate, its expression is positively regulated by TSH through the
cAMP signaling pathway.
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3H-conditioned medium did not have any significant effect on FRTL-5
cell growth. As expected, conditioned medium from TSH-stimulated FRTL-5
clearly induced cell proliferation due to the presence of TSH in the
conditioned medium. These effects were evident after 24 h of the
addition of conditioned medium.
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Somatostatin exerts an inhibitory effect of TSH-mediated cell
proliferation resulting in cellular accumulation in the
G1 phase of the cell cycle
After demonstrating a biological role for somatostatin in the
FRTL-5 conditioned medium, the effects of the peptide on cell growth
and cell cycle distribution were analyzed in more detail. Growth curve
profiles of depleted cells treated with TSH (1 nM),
somatostatin, and the combination of TSH and somatostatin (10
nM, 1 µM, and 2 µM) were
determined every 24 h for 4 consecutive days. Control cells
maintained in 3H medium were also included in the assay (Fig. 3). In the latter, the number of viable
cells remained unaltered throughout the assay. TSH rapidly increased
cell number, with the induction of cell proliferation more evident
within the first 24 h of TSH exposure. To a minor extent,
proliferation was detected up to 72 h. When TSH was added in
combination with somatostatin, a detectable inhibition of cell
proliferation was observed even at a dose of 10 nM. Higher
concentrations of somatostatin increased growth inhibition, and at 1
µM, reduction of TSH-induced proliferation was nearly
40% after 24 h. Similar results were obtained at 2
µM, suggesting that although the cellular response is
dose dependent, a partial desensitization in response to somatostatin
is observed at high doses. It is important to remark that whereas cell
number stabilized after 72 h of TSH treatment, the presence of
somatostatin in the medium decreased cell viability after 3 days in
culture. We also found that somatostatin requires the presence of the
positive effector to alter cell proliferation, as in the absence of
TSH, somatostatin did not exert any effect on FRTL-5 cell growth,
demonstrating the connection between somatostatin and TSH signaling
pathways.
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). Cells maintained in 3H
medium for 72 h were stimulated with TSH and treated with
different concentrations of somatostatin. After 16 and 24 h,
samples were collected, fixed in ethanol, and stained with propidium
iodide for cell cycle analysis. Results are summarized in Table 2. Cell cycle distribution of 3H cells
remained unaltered throughout the assay, with S phase values
approximately 4% and G2-M values 16% of the total cell
population. It is important to mention that although these values seem
to indicate cellular transit through the cell cycle, growth curves
indicate that FRTL-5 cells do not proliferate under these conditions
(Fig. 3). As it will be discussed below, we believe that this
circumstance could be due to the absence of somatostatin and the
presence of 0.2% serum in the basal medium. TSH treatment of depleted
FRTL-5 cells clearly promoted G1 transition to S phase,
reaching maximum accumulation in G2-M (42.8%) after
24 h. However, when the cells were treated with TSH and
somatostatin, a dose-dependent accumulation in G1 was
detected, and at 2 µM, cell cycle profiles were similar
to those obtained in cells maintained in 3H medium (Fig. 4).
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). The
results demonstrate that the ability of TSH to promote cell
proliferation is associated with a strong inhibitory effect on
p27kip1, which becomes undetectable after 24 h of TSH
treatment. Somatostatin was able to block TSH-mediated down-regulation
of p27kip1, returning protein levels to almost those levels
observed in quiescent cells. The effect was dose dependent, and as
shown in Fig. 5B, increasing the concentration of somatostatin resulted
in higher levels of p27kip1 protein. The results are in
agreement with flow cytometric profiles, indicating that maximum
G1 arrest is achieved at a 2-mM dose of
somatostatin. These results were already evident after 16 h of TSH
treatment and extended up to 36 h (not shown).
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). This
observation indicated that rather than decreasing the activity of the
cyclin-CDK complexes, somatostatin causes cell cycle delay by
increasing the level of the CDK inhibitor p27kip1.
The ability of somatostatin to modulate cell growth is altered in
transformed and tumor thyroid cells
The results presented indicate that somatostatin, secreted by
FRTL-5 cells and acting through an autocrine loop, regulates thyroid
cell growth. We analyze whether this potential regulatory pathway could
be altered in the process of thyroid carcinogenesis, testing the
cellular response to somatostatin in a transformed cell line
(K-ras-FRTL-5) and a tumor-derived cell line (FRT).
First, we analyzed whether these transformed and tumor cells express
somatostatin receptors that could mediate a potential response to
somatostatin. RT-PCR analysis reveals that as observed for FRTL-5
cells, K-ras-FRTL-5 cells and FRT cells express SSTR-2
receptor (Fig. 6).
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) showed
that K-ras-FRTL-5 cells are still sensitive to somatostatin,
although higher doses (2 µM) are required to obtain a
detectable increment in G1 phase. Any dose over 2
µM did not result in greater inhibition of cell
proliferation (Table 3). We also detected
the presence of hypodiploid cells, presumably undergoing apoptosis,
which are absent in control cells and FRTL-5 cells, although this
observation requires further analysis. However, fully neoplastic
thyroid FRT cells were no longer sensitive to somatostatin, as cell
cycle distribution remained unchanged in the absence or presence of
somatostatin.
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). Unlike the addition of
TSH to normal FRTL-5 cells, serum did not result in any significant
decrease in p27kip1. When serum-treated cells were exposed
to somatostatin, p27kip1 levels recovered, although these
results were not significant either. These results indicate that in
these transformed cells, the effect of somatostatin is rather weak,
presumably indicating that within the process of carcinogenesis, the
loss of somatostatin sensitivity is a late event and drives cells to a
more undifferentiated status.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of somatostatin transcription by TSH is specially relevant in FRTL-5 cells. First, it is important to remark that somatostatin expression is increased when FRTL-5 cells are treated with TSH. Second, the results obtained in our transfection assays indicate that the somatostatin promoter is functional and positively regulated by TSH. The fact that a positive effector of cell growth such as TSH promotes transcription of an antiproliferative peptide is physiologically important, because the interaction and balance between both signals may be responsible for the precise growth rate of the thyroid cell. Somatostatin gene transcription has been well characterized in the pancreatic RIN cells (29, 38) and has been demonstrated to be mainly controlled at the transcriptional level by CRE, which is recognized by the CREB and several other nuclear proteins (36). In these cells, a CRE site and another element, designated UE (upstream enhancer), cooperate synergistically to promote transcription, and therefore, a deleted construct of the somatostatin promoter containing these two elements confers maximum activity to reporter constructs in the presence of cAMP activators (29, 38). With respect to FRTL-5 cells, we demonstrated that the somatostatin promoter is functional, and therefore, all the regulatory elements necessary for the transcription of this gene are present within these thyroid cells. It would be of interest to analyze which transcription factor(s) is involved in the control of somatostatin gene transcription and whether thyroid-specific transcription factors (39) play any role in this process.
It is generally accepted that TSH and IGF-I synergize to stimulate thyroid cell proliferation (1, 2). Whether TSH is sufficient to promote cell proliferation of FRTL-5 cells may depend on the culture conditions (37, 40). In our experiments, the addition of TSH to cells maintained in 3H medium clearly induces cell proliferation. The explanation for this different response of FRTL-5 cells to TSH may lie in the absence or presence of somatostatin. This peptide is routinely added to the FRTL-5 culture medium (17, 41, 42), whereas it is absent in our control assays. Somatostatin may be sufficient to keep cells arrested, and our cell cycle profiles in the absence or presence of somatostatin also support the idea that this peptide is a critical regulator of thyroid cell proliferation. These results are further supported by the fact that somatostatin, secreted by FRTL-5 cells in the presence of TSH, exert a cytostatic effect on thyroid cell growth.
The results presented indicate that the control of cell cycle regulation exerted by somatostatin specifically targets the G1 phase of the cell cycle. As described previously (43, 44), we also show that proliferative signals induced by TSH are associated with a down-regulation of the cyclin-dependent kinase inhibitor p27kip1. This down-regulation is prevented by somatostatin, which returns p27kip1 protein levels to those observed in quiescent cells. Two pieces of evidence support the idea that this effect is specific rather than a consequence of differences in cell growth. First, the effect is dose dependent, and second, other cell cycle regulatory proteins important for G1-S transition, such as cyclin D1, are not modified by somatostatin treatment. In this context, our results are in agreement with the description that TSH is able to up-regulate the expression of cyclin D1 (37) to favor cell proliferation. We are currently investigating possible links between changes in cell cycle and changes in tyrosine phosphatase activity, which is also mediated by somatostatin, as previously described in the literature (8, 10).
Our results indicate that the ability of some transformed and tumor thyroid cell lines to respond to somatostatin is altered. Moreover, as many of these cell lines proliferate independently of TSH, the idea that somatostatin requires the positive effect induced by TSH to modulate cell growth is further supported. Our observations are in agreement with those previously reported (8) showing that differentiated PC-Cl3 thyroid cells turned insensitive to somatostatin when transformed with the E1a oncogene. These cells retain the expression of SSTR4 (8), and in our assays we have detected the expression of SSTR2 in both FRT and K-ras-FRTL-5 cells. These data indicate that the expression of somatostatin receptors does not directly correlate with the capacity of a given cell line to respond to somatostatin. Rather, some other intracellular mechanism, currently under investigation, is responsible for the interference with the somatostatin effect in thyroid transformed and tumor cells.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from the Spanish Ministerio de
Educación y Cultura.
Received April 29, 1998.
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