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Department of Veterinary and Comparative Anatomy (D.K.S., M.P., A.D., M.B.G.), Pharmacology and Physiology, Center for Reproductive Biology (D.K.S.), Washington State University, Pullman, Washington 99164-6520; and Department of Cell Biology (M.E., H.M.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6838
Address all correspondence and requests for reprints to: Dr. Dipak K. Sarkar, Professor, Department of VCAPP, Washington State University, Pullman, Washington 99164-6520. E-mail: sarkar{at}vetmed.wsu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100%) between species.
Many cells express one or more of the three isoforms. These isoforms of
TGF-ß inhibit or stimulate the growth and/or gene transcription of a
variety of cell types including epithelial, endothelial, lymphoid,
myeloid, and mesenchymal cells (1, 2, 3). The pituitary gland is also a
site where TGF-ß isoforms are produced and act locally to control
cell growth and function. Within the pituitary gland, melanotropes of
the intermediate lobe of the pituitary and lactotropes of the anterior
lobe of the pituitary gland produce TGF-ß1 and TGF-ß3 (4, 5, 6). While
the effects of TGF-ß3 on pituitary lactotropes are not well
characterized, several reports suggest an inhibitory action of TGF-ß1
on PRL secretion, PRL gene expression and lactotropic cell
proliferation (4, 7, 8, 9, 10). In lactotropes, a wide dose range (pg to
ng/ml) of TGF-ß1 inhibits cell proliferation and PRL secretion (4, 7, 9, 10), although very low doses of TGF-ß1 (<pg/ml) stimulate cell
proliferation (9). Because lactotropes are the primary source of
TGF-ß1 production and action in the pituitary, the polypeptide may
act on lactotropes by an autocrine and/or paracrine mechanism (5).
Using F344 rats, we have shown that the levels of TGF-ß1 protein and
mRNA decrease during anterior pituitary tumorigenesis (11). The
reduction in the amounts of TGF-ß1 protein and mRNA in the anterior
pituitary during tumorigenesis correlate with the low levels of
TGF-ß1 mRNA and protein observed in tumor cell lines. Furthermore,
the growth inhibitory response of TGF-ß1 is reduced in pituitary
tumor cell lines (11, 12). A reduction in the PRL-inhibitory response
of TGF-ß1 is also observed in the aged rat pituitary that produces
and secretes increased amounts of PRL (9). Hence, it appears that
TGF-ß1 may be a physiological regulator of lactotropic cell growth
and secretion. The role of cell surface proteins that mediate TGF-ß1 actions on lactotropes is not well understood. In many cell types, three binding protein complexes, known as types I-III, have been identified as TGF-ß-binding proteins (13, 14, 15). The TGF-ß type I (TßR-I) and TGF-ß type II (TßR-II) receptors have been shown to be transmembrane proteins containing cytoplasmic serine/threonine kinase domains. The heteromeric interaction of TßR-I and TßR-II is thought to mediate the action of TGF-ß1, whereas the TGF-ß type III-binding protein binds and presents TGF-ß1 to the TßR-II receptor. It has been shown that lactotropes are the major cell type in the anterior pituitary expressing TßR-II mRNA and protein, and that the level of TßR-II mRNA decreases in parallel with the levels of TGF-ß1 mRNA in the anterior pituitary of ovariectomized F344 rats following estrogen administration (16). Hence, it appears that TßR-II receptors may be involved in mediating the TGF-ß1 growth-inhibiting action on lactotropes. However, it is not known whether lactotropes express TßR-I and whether heteromeric interaction between TßR-I and TßR-II mediates ligand-dependent gene expression in these cells. In the present work, we determined the PRL-inhibitory response to TGF-ß1 in normal lactotropic cells and transformed lactotropic cells (PR1 cells; 11) that had no growth inhibitory response to TGF-ß1. The cell surface expression and the mRNA levels of TßR-I and TßR-II in primary lactotropes and in PR1 cells were compared. Additionally, TßR-I and TßR-II interaction in PR1 cells was investigated using a reporter assay. In this report, we provide evidence that TGF-ß1-regulated gene expression in lactotropes is mediated by the heteromeric interaction of TßR-I and TßR-II receptors. Furthermore, evidence is provided to support the notion that differential expression of TßR-I and TßR-II levels may cause variable biological responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary cultures of anterior pituitary cells
The methods for preparing anterior pituitary cell cultures were
previously described (11). Anterior pituitaries of estrogen-treated
rats were used to enrich lactotropes in cultures (7). Briefly, anterior
pituitary cells were enzymatically dissociated, suspended into DMEM:F12
(1:1; Sigma, St. Louis, MO) with 100 U/ml penicillin, 100 µg/ml
streptomycin and 200 µM ascorbic acid, 1 mg/ml BSA and
10% FCS (HyClone, Logan, UT) and seeded on
poly-L-lysin-coated 24-well (2.5 x 105)
or 6-well plates (5–10 x 105). Cultures used for
BrdUrd incorporation were seeded on poly-L-lysin-coated
coverslips in a 24-well plate. Cultures were maintained at 37 C in
7.5% CO2. On day 2, cultures received 2.5% FCS for 2 days
and then were maintained in serum supplement (human transferrin, 100
µM; insulin, 5 µM; and putrescine, 1
µM) until experimentation.
PR1 cell lines
This cell line was derived from a pituitary tumor of an F344
ovariectomized rat treated with estrogen for 3 months (11). The tumor
cells were grown in culture for 28 generations and a population of
cells showing PRL immunostaining and a similar shape was isolated.
These cells were maintained in DMEM:F12 with 2.5% FCS for 42–51
generations and used in this study.
Treatment of PR1 cells and primary pituitary cell cultures with
TGF-ß1
Experiments dealing with the measurement of PRL mRNA levels in
pituitary cells and PR1 cells were maintained in cultures for 5 days at
a 6 x 105/4 ml/plate cell density. On day 6, at the
onset of the experiment, cells were washed 3 times with 3 ml DMEM:F12
containing 1 mg/ml BSA, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
200 µM ascorbic acid, and plates were filled with 3 ml of
the serum-free medium. The 4 µg/ml stock of TGF-ß1 from porcine
platelets (R&D Systems, Minneapolis, MN) was diluted into 1 mg/ml BSA,
4 mM HCl, and 30 µl of each of the 1/10 serial dilutions
were added to the plates. Control plates received 30 µl BSA-HCl
buffer. Cells were incubated with TGF-ß1 for 24 h. At the end of
incubation time, the medium was removed, the cells were washed 3 times
with 3 ml PBS, harvested and homogenized in guanidinium
isothiocyanate-phenol solution, and total cellular RNA was isolated
(17).
Experiments involving the measurement of TGF-ß1 effects on cell growth were conducted with primary cultures of pituitary cells or PR1 cells (2.5 x 105) plated on poly-L-lysin-coated coverslips and maintained at 37 C in the presence of 7.5% CO2. On day 5, at the onset of experiment, cells were washed and then incubated with serum-free medium containing serum supplement. Cultures were treated with medium only, 10 nM estradiol 17ß and 10 µl BSA-HCl buffer or 10 nM estradiol 17ß and various doses of TGF-ß1. These treatments were repeated twice at 48-h intervals. At the end of the experiments, cells were harvested for determination of lactotropic cell proliferation.
Lactotropic cell proliferation
Lactotropes proliferation was determined by identifying the
cells with both bromodeoxyuridine (BrdUrd) and PRL immunoreactivities,
assuming that PRL cells that undergo DNA synthesis incorporate BrdUrd
(18, 19). The methods for double staining with BrdUrd and PRL were as
described by us previously (12). Briefly, cultures were treated with 30
µg/ml BrdUrd 2 h before harvesting, then fixed with 99% ethanol
and incubated at 4 C overnight in a humid chamber with BrdUrd
monoclonal mouse IgG (Becton Dickinson Immunocytometry Systems, San
Jose, CA) at a concentration of 1:200 in PBS or as a negative control
with 3% normal serum from the host species. The BrdUrd
immunoreactivity was stained using biotinylated goat antimouse IgG as
second antibody and diaminobenzidine as chromagen supplied by
Vectastain ABC kit (Vector Incorporated, Burlingame, CA). After DAB
precipitation, the slides were immersed in 3% normal goat serum for 30
min at room temperature and then incubated overnight at 4 C with the
PRL antibody (PRL-S9; a gift of NIDDK) at a concentration of 1:100,000
in PBS. The PRL immunoreactivity in cells was stained using Biogenex
supersensitive kit (Bigenex Laboratories, San Ramon, CA) containing
antirabbit biotinylated secondary antibody and fast red/nepthol
phosphate chromogen. Two investigators independently performed cell
counts, which involved counting five separate areas in each coverslip
and 500 cells/area.
Affinity-labeling of TGF-ß binding proteins in pituitary
lactotropes
Affinity labeling was performed as described by Takahashi
et al. (20). Assays were performed with 100 cm2
confluent plates containing monolayers of PR1 cell lines or primary
cultures of anterior pituitary cells (0.25–1 x 106
cells/ml). Cells were washed twice with PBS and incubated at 37 C in
binding buffer containing 125 mM NaCl, 5 mM
MgSO4, 5 mM KCl, 1.2 mM CaCl2, 50
mM HEPES, pH 7.4, and 1% BSA to allow the dissociation of
endogenously bound TGF-ß. The cells were washed with cold binding
buffer and placed at 4 C. The 125I-TGF-ß1 solution was
diluted appropriately just before addition to the cultures. Binding
reaction was for 4 h at 4 C with shaking at 120 rpm in binding
buffer containing 45 pM 125I-TGF-ß1 (100
µCi/ml; R&D) in the absence or presence of a 100-fold excess of
TGF-ß1 as a competitive inhibitor of receptor binding. Additionally,
we examined competition by TGF-ß2 and TGF-ß3, as well as inhibin,
another member of the TGF-ß superfamily. The receptor-bound ligand
was cross-linked with bis-(sulfosuccinimidyl)-suberate (Pierce Chemical
Co., Rockford, IL) in a total volume of 400 µl for 20 min at 22 C and
followed by termination through the addition of excess glycine (28
mM) for 5 min. The receptors were solubilized by
homogenization (Brinkmann polytron, setting 5, 30 sec) in 10
mM 2-(4-hydroxymethyl) propane-sulfonic acid (pH 7.4), 1%
Triton X-100, 1 mM EDTA, 10 µg/ml leupeptin, and 100
Kallekrein units (KU) aprotonin and centrifugation at 40,000 rpm at 4 C
to remove insoluble cell debris. The supernatants were collected and
protein contents were assayed using BCA protein reagents (Pierce). The
125I-TGF-ß1-labeled cellular protein (200 µg/sample)
and molecular weight protein markers (Sigma) were separated by 6%
SDS-PAGE, stained with Coomassie Brilliant Blue R-250 to detect
proteins, dried and exposed to Kodak XAR film with enhancing screens.
The results were analyzed by autoradiography.
PRL mRNA quantification: RNase protection assay
Total cellular RNA from anterior pituitary cells and PR1 cells
was prepared and hybridized for 16–20 h with labeled
antisense RNA probes for PRL (5 x 104 cpm) and
cyclophilin (CYC) mRNA (2 x 105 cpm) together in
conditions described previously (11). The PRL probe was synthesized
from PRL/KS, which includes the entire PRL cDNA. The plasmid was
linearized using the unique BglII site present within the
PRL coding sequence. Transcription from the T3 promoter yielded a
305-nucleotide (nt) full-length probe that included 185-nt PRL
antisense sequence. The CYC probe was transcribed from prPXCYC/BS
linearized with EcoRI. T7 RNA polymerase (Promega Corp.,
Madison, WI) produced a 170 nt full-length probe including 111 nt
cyclophilin antisense sequence. Hybridization mixtures (30 µl)
contained 60% deionized formamide, 10 mM HEPES, pH 7.5,
600 mM NaCl, 2 mM EDTA, and transfer RNA to
equalize the total amount of RNA to 20 µg in all samples.
Hybridizations were carried out at 56 C for 16–20 h. Nonhybridized RNA
was digested with 2.5 µg/ml RNase A as described previously (21).
After phenol extraction and ethanol precipitation, samples were
analyzed on 5% polyacrylamide-7 M urea gels. RNA was
quantified by laser scanning of the autoradiograms. In this assay, the
protected PRL fragment was a 185-nt fragment hybridized product of PRL
mRNA and PRL probe, whereas the protected cyclophilin mRNA hybridized
fragment corresponded to the 117-nt fragment (see arrow in
Fig. 2
). The faint band seen at 170 nt corresponds to full-length
cyclophilin probe protected by very small amounts of the plasmid DNA
template used to synthesize the probe in vitro. The
intensity of this fragment appears to remain constant in all samples
and is not scanned.
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). Subsequently,
cells were maintained in fresh media for 42 h in the presence or
absence of 400 pM TGF-ß1. Cells were washed extensively
with 1 x PBS and lysed at 4 C with agitation for 15 min in
luciferase lysis buffer (100 mM Na2H2 (PO4), 1% Triton
X-100, 1 mM ß-mercaptoethanol, 2 mM EDTA, pH
7.8). Crude lysates were subjected to pulse sonication on ice and
subsequently clarified by microcentrifugation (1 min at 15,000 rpm, 4
C). Luciferase activity in duplicate lysates was determined in 30
mM tricine, 3 mM ATP, 15 mM
MgSO4, 10 mM ß-mercaptoethanol, pH 7.8, after
injection of 1 mM luciferin. ß-glactosidase activity was
determined using a galacton-Plus Kit assay as described by the
manufacturer (Tropix, Bedford, MA) Both assays were performed using an
Analytical Luminescence Laboratories Monolight 2010 luminometer.
Luciferase results were normalized to ß-glactosidase output (Tropix)
to control for transfection efficiency. Mammalian expression plasmids
[pCMV5-TßR-I:HA, pRK5-TßR-II:flag and p3TP-Luciferase; gifts of
Drs. J Massagué (Memorial Sloan-Kettering Cancer Center, New
York, NY) and R. Derynck (University of California at San Francisco,
San Francisco, CA)] were used.
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PCR product detection and quantification. A 15-µl aliquot of PCR reaction was analyzed by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and documented with black and white instant Polaroid film 665. The negative was used to measure band intensities using a laser scanning densitometer (Molecular Dynamics, Sunnyvale, CA). RT-PCR was calibrated by reverse transcription of different amounts of tissue RNA in the presence of 5 x 105 copies of control RNA pAW 109 RNA. The ratio of the intensity of the TßR-I or TßR-II band to the PAW109 band was used as a relative measure for mRNA abundance. RT- (-) and RNAse-treated control reactions were also performed to ensure that the amplified fragments originated from RNA rather than contaminant DNA.
Immunocytochemical method
Anterior pituitary cell cultures and PR1 cell cultures were
double stained for PRL and TßR-I or TßR-II using the
double-immunocytochemical methods as described previously (12, 16).
Briefly, cultures grown on coverslips were fixed with 99% ethanol,
treated with hydrogen peroxide and normal goat serum to block
nonspecific bindings, and then incubated at 4 C overnight with a
polyclonal primary antibody directed against either TßR-II receptor
or TßR-I receptor (Upstate Biotechnology, Lake Placid, NY; 5 µg/ml;
these antibodies are specific for each antigen and do not cross-react
with other known TGF-ß receptors according to the manufacturer’s
specifications). Negative control cultures, incubated with normal serum
from the host species, were also run in parallel. Additional control
experiments involving preincubation of the antisera with excess
antigens TßR-II or TßR-I (10–50 µg; UBI) were also conducted.
After overnight incubation, cultures were copiously rinsed in PBS and
stained using Vectastain ABC kit (Vector Incorporated, Burlingame, CA).
To confirm the colocalization of TßR-I or TßR-II and PRL, a
double-labeling technique was employed. After DAB precipitation, the
cultures were immersed in 10% normal goat serum in PBS for 30 min at
room temperature. The cultures were then incubated overnight at 4 C
with the PRL antiserum (PRL-S9; 1:100,000). Cultures were copiously
rinsed in PBS and incubated with goat antirabbit biotinylated secondary
antibody (Biogenex Laboratories, San Ramon, CA), then with
streptavidin-alkaline phosphatase conjugate (Biogenex), followed by
immersion in fast red/nephthol phosphate chromogen (Biogenex). Finally,
the cultures were counterstained with hematoxylin and mounted with
crystal mount.
Statistics
The data shown in the figures and text are mean ±
SE. The PRL mRNA data obtained from the RNase protection
assay varied between experiments. We approached the problem of the
between-experiment variability by transforming the data to a percentage
of control value. We found that the percentage change normalization
yield data fit the normality assumption, whereas the raw data did not.
Additionally, normalization effectively minimized the
between-experiment variability. All the data presented were analyzed
using one-way ANOVA. Posthoc test involved Student-Newmann-Keuls test.
A value of P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that
TGF-ß1 markedly inhibited lactotropic cell proliferation in primary
cultures of anterior pituitary cells, but it failed to inhibit
proliferation of transformed lactotropes (PR1 cells). However, TGF-ß1
inhibited PRL mRNA levels both in anterior pituitary cell cultures and
PR1 cell cultures (Fig. 2
). The minimum
effective dose of TGF-ß1 that inhibited PRL mRNA levels in PR1 cells
(0.2 ng/ml) was 10-fold lower than that in primary pituitary cells (2
ng/ml). Hence, it appears that PR1 cells that show no growth-inhibitory
response to TGF-ß1 show marked PRL mRNA-inhibitory response to
TGF-ß1.
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Affinity labeling studies indicated that anterior pituitary cells in
primary cultures contain several TGF-ß-binding protein complexes; two
major complexes are identified as 65 kDa size TßR-I and 95 kDa size
TßR-II. Both of these protein complexes competed efficiently with
TGF-ß1 and to a lesser extent with TGF-ß2 and TGF-ß3 (Fig. 3A). These complexes did not compete
efficiently with inhibin (not shown). In contrast to the pituitary
primary cells, PR1 cells contained primarily a 65-kDa size protein
complex, which was a size similar to the TßR-I receptor complex (Fig. 3B).
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) and primarily TßR-I protein
immunoreactivity in PR1 cells, although a very faint RII protein
immunoreactivity was noted in PR1 cells. The TßR-I and TßR-II
staining in PR1 cells was inhibited by excess antigen, suggesting that
the immunoreactivity for TßR-I and TßR-II receptor proteins is
specific for each antigen.
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).
Within each cell type, the relative abundance of TßR-I mRNA was
higher than the TßR-II mRNA. Between cell types, TßR-I and TßR-II
mRNA levels in PR1 cells were respectively 21% and 47% lower than the
levels of mRNA in pituitary primary cells.
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). PR1 cells displayed TGF-ß-dependent
reporter transactivation in the absence of cotransfected type I or type
II receptors. Consistent with results described above, these data
suggest that PR1 cells retain TGF-ß-mediated transcriptional
responses but lose their growth responses. Introduction of the type II
receptor confers ligand-independent reporter transactivation in these
cells. TßR-I alone had no potentiating effect on p3TP-lux activity.
When TßR-I and TßR-II were introduced concurrently,
ligand-independent transactivation was again observed, which was
further enhanced by TGF-ß. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the loss of the growth- but not the gene-regulating response of TGF-ß1 in PR1 cells, one question that arose was whether growth- and gene-regulating responses of lactotropes were regulated by different types of transmembrane TGF-ß receptors. Determination of TGF-ß receptor complexes using affinity labeling with 125I-TGF-ß1 indicated that the pituitary tissue contains three major TGF-ß-binding protein complexes similar to the 65-kDa size of TßR-I, 95 kDa size of TßR-II and approximately 200 kDa size of type III. In many other cell types, TßR-I and TßR-II receptors have been shown to be cell surface receptors. Like other cells, lactotropes also appear to express TßR-I and TßR-II receptors. These are documented by the observation that lactotropes showed immunoreactive TßR-I and TßR-II proteins. TßR-I and TßR-II mRNA transcripts were detectable in the anterior pituitary cells. Additionally, 125I-TGF-ß1 binding (as determined by autoradiography) and TßR-II mRNA transcripts (as determined by double immuno-in situ hybridization techniques), have been identified in lactotropes of the anterior pituitary (16). Hence, TßR-I and TßR-II receptors are present on the lactotropes in the pituitary. A protein complex similar to type III receptor is identified by affinity labeling with 125I-TGF-ß1 in the mixed pituitary cell cultures. The expression of this protein in lactotropes is not characterized because TßR-III is a betaglycan and its role in cell signaling is not established (13, 14, 15). In PR1 cells, the three major TGF-ß-binding protein complexes were not identified by affinity labeling with 125I-TGF-ß1. These cells produced a primarily TßR-I protein complex following affinity labeling with 125I-TGF-ß1. However, very low levels of immunoreactive TßR-II protein and TßR-II mRNA transcript were detected in these cells. Hence, it appears that PR1 cells have TßR-I receptors and express a very low amount, if any, of functional TßR-II or TßR-III receptors.
In many cell types, TßR-I and TßR-II have been
shown to be transmembrane proteins containing cytoplasmic
serine/threonine kinase domains, and the heteromeric interaction of
these transmembrane proteins is thought to mediate the action of
TGF-ß1 (13, 14, 23). Because lactotropes appear to produce both
TßR-I and TßR-II receptor proteins, the possibility was raised that
the heteromeric interaction of these transmembrane proteins may also be
involved in mediation of TGF-ß1 signaling in lactotropes. Indeed,
data of reporter assays support that TGF-ß-regulated gene expression
in transformed lactotropes is mediated by the heteromeric interaction
of TßR-I and TßR-II receptors (Fig. 6). In the reporter assay, when
PR1 cells were transfected with TßR-I alone, the gene transactivation
could be initiated by the ligand. When PR1 cells were transfected with
both TßR-I and TßR-II, a ligand-independent gene transactivation
was observed. Hence it appears that even in low expression of TßR-II
receptor proteins, TGF-ß1 can activate gene transcription in
lactotropes.
In PR1 cells, TGF-ß1 inhibited PRL mRNA levels and activated gene transactivation but failed to affect lactotropic cell proliferation. PR1 cells have low expression of TßR-II receptor proteins. Like PR1 cells, tumor pituitary lactotropes, which are under the influence of reduced TGF-ß1-growth inhibition, also show reduced production of TßR-II receptors (6, 11). Hence, it is possible that the TGF-ß1 growth response is more dependent on the amount of TßR-II receptor expression than it is on the TGF-ß1-PRL response.
There are several proposed mechanisms that could explain the differential regulation of lactotropic cell functions by TßR-I and TßR-II receptors. It has been suggested that variable ratios of TßR-I and TßR-II expression cause differential binding of TGF-ß ligands, thus causing variable biological responses (26). Chen et al. (27) have proposed that type I and type II receptors either work together to signal, or type I receptors signal independently without signaling through type II receptors. This dual signaling pathway produces distinct cellular events. Finally, receptor and ligand-receptor interactions can be regulated by association with other proteins in the extracellular matrix or at the cellular membrane (28, 29). These examples provide a possible scenario for the multiple actions of TGF-ß1 through TßR-I and TßR-II receptor interaction.
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Acknowledgments |
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Footnotes |
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Received December 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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parallel neurogenesis in
the rat spinal cord. J Neurosci 15:2575–2591[Abstract]
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