Endocrinology Vol. 139, No. 8 3620-3628
Copyright © 1998 by The Endocrine Society
Role of Transforming Growth Factor (TGF)-ß Type I and TGF-ß Type II Receptors in the TGF-ß1-Regulated Gene Expression in Pituitary Prolactin-Secreting Lactotropes1
Dipak K. Sarkar,
Martine Pastorcic,
Alok De,
Mike Engel,
Harold Moses and
M. Behnam Ghasemzadeh
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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Abstract
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Transforming growth factor ß1 (TGF-ß1) inhibits pituitary
lactotrope proliferation and secretion of PRL in an autocrine/paracrine
manner. In this study, the role of TGF-ß1 type I (TßR-I) and
TGF-ß type II (TßR-II) receptors in TGF-ß1-regulated gene
expression in lactotropes was determined using anterior pituitary cells
known to be responsive to TGF-ß1 growth inhibition and using a
transformed PR1 cell line known to be nonresponsive to TGF-ß1 growth
inhibition. Treatment with TGF-ß1 inhibited cell proliferation and
decreased PRL mRNA levels in anterior pituitary cells, but in PR-1
cells, the treatment caused only decreased PRL mRNA levels. Affinity
labeling of TGF-ß binding proteins indicated that anterior pituitary
cells contain several TGF-ß-binding protein complexes, including the
65 kDa size TßR-I and 95 kDa size TßR-II. In the PR1 cells, the
major complex found was similar to the 65 kDa size of TßR-I.
Immunocytochemistry identified TßR-I and TßR-II receptor proteins
in lactotropes but detected primarily TßR-I receptor protein in PR1
cells. RT-PCR detection of TßR-I and TßR-II mRNA identified both
receptor mRNA transcripts in anterior pituitary cells and in PR1 cells
but the levels of TßR-II and TßR-I mRNA transcripts in PR1 cells
was much lower than that in anterior pituitary cells. Determination of
the TGF-ß1 gene responses in PR1 cells following TßR-I and TßR-II
gene transfection indicated that PR1 cells transactivate transcription
of the TGF-ß-responsive p3TP-Lux reporter in the absence of
cotransfected TßR-II receptor. The introduction of the TßR-II
receptor alone or in combination with TßR-I confer ligand-independent
reporter transactivation in these cells. When only TßR-I was
introduced along with reporter, a ligand-dependent transactivation was
observed. These data suggest for the first time that the
TGF-ß1-mediated transcriptional activation response can be
distinguished from the growth response in lactotropes. Furthermore, the
TGF-ß1 gene-transcription response is less dependent on TßR-II
receptor expression than is the TGF-ß1 growth-inhibitory response.
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Introduction
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TRANSFORMING growth factor ß (TGF-ß) is
a polypeptide of 25 kDa and has three isoforms (TGF-ß1–3) in
mammals. There is a 60–80% amino acid homology between the three
peptides and they each are highly conserved (
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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Animals
Fischer 344 female rats of 180–250 g body weight were obtained
from Simonsen Laboratory (Gilroy, CA), housed in a controlled
environment (temperature 22 C, lights on 0500–1900 h) and provided
with certified rodent chow meal (Purina Mills, Inc., St. Louis, MO) and
water ad libitum. The animals were anesthetized using
halothane vapor and underwent bilateral ovariectomy followed by
surgical implantation of an estradiol-17ß-laden 1 cm, sc, SILASTIC
brand capsule (7) (Dow Corning, Midland, MI). At 7–10 days after
surgery, animals were killed by rapid decapitation and anterior
pituitary tissues were obtained for preparation of cell cultures.
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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Figure 2. Comparison of the PRL mRNA-inhibitory effect of
TGF-ß1 in normal lactotropes and PR1 cells. Cultures of anterior
pituitary cells and PR1 cells were prepared and treated with various
doses of TGF-ß1 for 24 h. Total RNA was isolated and PRL gene
transcripts were quantified by the RNase protection assay. Total RNA
(2.5 µg) from each sample was analyzed for PR1 cells and 5 µg RNA
for primary cultures. Cyclophilin RNA was monitored as an internal
control. Representative autoradiograms showing the changes of PRL mRNA
levels in PR1 cells following treatment with various doses of TGF-ß1
(0–20 ng/ml) are shown at the bottom. PRL transcripts
are represented by a protected fragment 185 nt long (PRL), and
cyclophilin RNA appears at 111 nt (C). Mean ± SE PRL
mRNA levels in primary pituitary cells (A) and PR1 cells (B) after
treatment with TGF-ß1 are shown on the top and
middle figures. PRL mRNA levels were quantified by laser
scanning of autoradiograms. The ratio of the transcripts PRL/CYC was
calculated before determining percentage of control value for each
treatment group. The means ± SE (n = 6) are
represented in the histogram. a, P < 0.05 compared
with TGF-ß1 (0–0.002 ng/ml)-treated groups. b, P
< 0.05 compared with TGF-ß1 (0–0.2 ng/ml)-treated groups.
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Transfection assay
PR1 cells maintained in 100-mm dishes in Ham’s F12 + 2.5% FBS
and 10% horse serum were transiently transfected with 5 µg of
receptor expression plasmid, as well as TGF-ß inducible reporter
p3TP-Lux constitutive pRSV-ß-gal reporter and equal amounts of
sheared salmon sperm by calcium phosphate coprecipitation (22) for
24 h, followed by glycerol shock and 4 h recovery in fresh
medium. The heterologous reporter p3TP-lux has been widely used to
assess TGF-ß mediated transcriptional activation responses. The
reporter consists of the TGF-ß responsive region of the plasminogen
activator inhibitor-1 (PAI-1) promoter and three concatamerized TPA
response elements (TRE) from the human collagenase promoter. PR1 cells
were transiently transfected with p3TP-lux, as well as TßR-I and
TßR-II cDNAs in the combinations shown (see Fig. 6). 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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Figure 6. Ligand-dependent and ligand-independent reporter
transactivation in transformed lactotropes (PR1 cells) overexpressing
TGF-ß-type I receptor and TGF-ß-type II receptors, respectively.
Subconfluent cultures of PR1 cells were used for transient transfection
using a calcium-phosphate precipitation method and mammalian expression
plasmids. The responsiveness to TGF-ß1 was measured by a
p3TP-luciferase reporter output. All the data were corrected for
transfection efficiency by an RSVß-gal reporter. Values are mean
± SEM obtained from N = 4.
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RT-PCR
Primer design. The PCR primers for RI and II mRNAs were: RI
forward primer, 5'-ACGTTCATGGTTCCGAGAGG-3'; RI reverse primer,
5'-TCGCAAAGCTGTCAGCCTAG-3'; RII forward primer,
5'-AAGTCTTGCATGAGCAACTGC-3'; RII reverse primer,
5'-GATGTCAGAGAAGATGTCC-3' (24). The primers for the pAW 109 control RNA
were: 5'-CCAGCCATCCTTCGAGATTTCT-3' (sense),
5'-GTTGTTCCTCCA-GTTCTTTCTCACC-3'(antisense) (25). All the primers
were obtained from Life Technologies Inc. (Grand Island, NY). Hot-start
RT-PCR methods were employed as previously described (25). Briefly,
total tissue RNA (0.7 µg) and 5 x 105 copies of
control RNA pAW 109 in a final volume of 20 µl were subjected to
reverse transcription reaction using random hexanucleotide primers at
42 C for 60 min, followed by 15 min at 75 C to inactivate the enzyme.
The PCR mix contained 2.5 mM MgCl2, 2.5 U
Taq DNA Polymerase (Life Technology Inc., Grand Island, NY)
and 0.5 µM of each primer. All PCR reagents were combined
in tubes (50 µl) and heated to 95 C for 5 min and had
MgCl2 added to Hot Start the amplification reaction. The
amplification protocols were as follows: 28 cycles of 94 C for 1.5 min,
60 C for 1.5 min, 72 C for 1 min, and a final extension time of 15 min
at 72 C. The linear range for amplification was within 24–36 cycles
and for RNA was 0.4 to 1.5 µg.
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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TGF-ß1 actions on cell proliferation and on PRL mRNA levels in
normal and transformed lactotropes
We have previously shown that TGF-ß1 inhibits lactotrope
proliferation (as determined by the cell number and
3H-thymidine incorporation) in primary cultures of anterior
pituitary cells (4). We have also shown that TGF-ß1 failed to inhibit
the proliferation (as determined by the cell number), of PR1 cells that
were derived from estrogen-induced PRLomas from a female rat (11).
These cell growth-controlling effects of TGF-ß1 on lactotropes and
PR1 cells were verified by using a BrdUrd incorporation assay that
identifies positive cells with both BrdUrd and PRL immunoreactivities
and assumes that PRL cells that undergo DNA synthesis incorporate
BrdUrd (7, 12, 18). Figure 1 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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Figure 1. Comparison of the cell growth-inhibitory effect of
TGF-ß1 on normal lactotropes and PR1 cells. Primary cultures of
anterior pituitary cells and PR1 cells were plated on coverslips at a
density of 2–5 x 105/ml. Cells were plated in
culture for 4 days and then were incubated with or without 10
nM of estradiol for an additional 4 days in serum-free
defined medium. Estradiol-treated cultures additionally received
various doses of TGF-ß1 (1 pg/ml-10 ng/ml) or vehicle. The DNA
synthesis in lactotropes was determined by colocalizing BrdUrd and PRL
in a single cell using a double-labeling technique. Histograms show the
changes in cell proliferation in primary pituitary cultures (A) and PR1
cells (B) after treatment with various doses of TGF-ß1. The
means ± SE (n = 6) are represented in the
histogram. a, P < 0.05 compared with no estrogen.
b, P < 0.05 compared with 0-dose control. c,
P < 0.05 compared with TGF-ß1 (0.001–0.1
ng/ml)-treated groups.
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TßR-I and TßR-II receptor proteins and mRNA levels in primary
pituitary cells and in PRL-secreting PR1 cells
The majority of studies has indicated that a heteromeric
interaction between TßR-I and TßR-II receptors on the cell surface
mediates TGF-ß1 actions (13, 14). Because PR1 cells and lactotropes
appear to respond differently to TGF-ß1, we examined whether these
two cell types have differential expression of TßR-I and TßR-II
receptors. To examine this, TGF-ß receptor subtypes present in
primary pituitary cells and PR1 cells were characterized by: 1)
affinity labeling of TGF-ß-binding proteins; 2) immunocytochemically
localizing TßR-I and TßR-II protein immunoreactivities; and 3)
measuring TßR-I and TßR-II mRNA levels using a RT-PCR assay.
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).
Immunocytochemical procedures also identified TßR-I and TßR-II
protein immunoreactivity in primary lactotropes (Fig. 4) 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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Figure 4. Colocalization of TßR-I receptor protein and PRL
immunoreactivity and TßR-II receptor protein and PRL immunoreactivity
in primary pituitary cells and PR1 cells. Primary pituitary cell
cultures (A) and PR1 cell cultures (B) were grown on coverslips,
treated with 10 ng of TGF-ß1 for 6 h (to activate receptors),
fixed, and processed for immunocytochemical localization of PRL,
TßR-I, or TßR-II receptors. Single immunostaining procedures using
DAB as chromogen (which gave dark brown color) was employed to stain
pituitary cell cultures for control (treated with PRL antibodies
preincubated with excess antigen; Aa), PRL (Ab), TßR-I (Ac), and
TßR-II (Ad). Some of the pituitary cell cultures were double stained
using DAB as chromogen to stain for PRL and using
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as
chromogen (which gave blue color and
black color when overlapped with brown)
to stain for TßR-I (Ae) and TßR-II (Af). All the PR1 cell cultures
were stained by single immunostaining procedures using DAB as chromogen
and antibodies for TßR-I (Bb) and TßR-II (Bd). Control coverslips
containing PR1 cells were treated with TßR-I (Ba) or TßR-II (Bc)
antibodies preincubated with excess respective antigen.
Blue nuclear stain represent hematoxylin stain. Some of
the positive immunostained cells are indicated by
arrows. Bar, 20 µm.
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|
Measurement of TßR-I and TßR-II mRNA levels using RT-PCR revealed
that both TßR-I and TßR-II mRNA transcripts were present in primary
pituitary cells and PR1 cells (Fig. 5).
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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Figure 5. Determination of TßR-I and TßR-II receptor
mRNA levels in the primary pituitary cells and PR1 cells using a RT-PCR
technique. On the top is the ethidium bromide-stained
agarose gel showing TßR-I or TßR-II and PAW 109 amplified cDNA
bands of the template RNA obtained from primary pituitary cell cultures
(pituitary) and PR1 cells. Five micrograms of template RNA from primary
pituitary cell cultures and PR1 cells and a constant amount of PAW 109
RNA (500,000 copies) were used in this assay. The TßR-I- and
TßR-II-amplified cDNA bands are 745 bp and 698 bp, respectively. The
standard pAW 109 band is 149 bp. Bottom figures show the mean ±
SEM ratio of TßR/Std amplified bands derived after
scanning the gels using a laser scanner densitometer. *,
P < 0.05 compared with pituitary cells. N =
5.
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Determination of PR1 cell responsiveness to TGF-ß1 following
transient transfection with TßR-I and TßR-II receptor cDNAs
PR1 cells were transiently transfected with p3TP-lux, as well as
TßR-I and TßR-II cDNAs in the combinations shown (Fig. 6). 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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|---|
Recent studies have indicated that TGF-ß1 is
produced in PRL-secreting pituitary lactotropes and controls the
secretion and proliferation of these cells in an autocrine/paracrine
manner. Data presented here suggest that, unlike normal lactotropes,
transformed PRL-secreting PR1 cells have lost the TGF-ß1-growth
inhibitory response but have maintained the TGF-ß1-PRL
mRNA-inhibitory response. The differential responses of the primary
pituitary cells and PR1 cells appeared to be related to the
nonneoplastic and neoplastic nature of these cells, rather than to
previous exposure of the primary pituitary cells to estrogen.
Previously, it has been shown that both primary cultures of pituitary
cells of estrogen-treated (4, 11) and of pituitary cells of
nonestrogen-treated Fischer-344 rats (9, 10) have siginificant
growth-inhibitory and PRL-reducing action toTGF-ß1. Furthermore,
both primary pituitary cells and PR1 cells had previous exposure to
estrogen. Primary cultures of pituitary lactotropes were prepared using
pituitaries (nontumor) from animals treated with estrogen for 7–10
days, whereas the PR1 cell line was derived from a pituitary (tumor) of
a rat treated with estrogen for 3 months. Additionally, both primary
pituitary cells and PR1 cells were similarly treated with estrogen or
vehicle before determination of TGF-ß1 effects on cell proliferation
and gene expression. Because both of these cells had similar treatment
protocols during culture, it appears that the differential responses of
these cells are related to the nonneoplastic and neoplastic nature of
these cells.
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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The authors would like to thank NIDDK and Pituitary Hormones and
Antiserum Center for providing the PRL RIA kit, and Ms. Jeanne Jensen
for editorial assistance. Animal surgery and care were in accordance
with institutional guidelines and complied with the NIH policy governed
by The Principles for Use of Animals and The Guide for the Care and Use
of Laboratory Animals.
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Footnotes
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1 This investigation was supported by the National Institutes of Health
Grants CA-56056, AA-11591, and AA-00220 (to D.K.S.). 
Received December 12, 1997.
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Abstract
Introduction
