0013-7227/97/$03.00/0
Endocrinology
Copyright © 1997 by The Endocrine Society
Vol. 138, No. 3
Printed in U.S.A.
Differential Effect of Age on Transforming Growth
Factor-b1 Inhibition of Prolactin Gene Expression
Versus Secretion in Rat Anterior Pituitary Cells*
SAI-KOONG TAN, FUNG-FANG WANG, HSIAO-FUNG PU,
AND
TSUEI-CHU LIU
Faculty of Medical Technology (S.-K.T., T.-C.L.), Institute of Biochemistry (S.-K.T., F.-F.W.), Institute
of Physiology (H.-F.P., T.-C.L.), and Institute of Biotechnology in Medicine (T.-C.L.), National YangMing University, Shih-Pai, Taipei, Taiwan, Republic of China
ABSTRACT
Transforming growth factor-b1 (TGF-b1) synthesized in the pituitary may act as an autocrine/paracrine regulator of lactotrope function. We examined the effects of TGF-b1 on PRL messenger RNA
(mRNA), PRL synthesis, and PRL secretion in cultured anterior pituitary (AP) cells from rats at different ages. APs excised from ovariectomized female Sprague-Dawley rats, either young (2–3 months old;
average serum PRL: 9 ng/ml), middle-aged (11–12 months old; average serum PRL: 133 ng/ml), or old (24 months old; average serum
PRL: 159 ng/ml), were dispersed and cultured for 5 days. Then, cells
were washed and challenged with increasing doses of TGF-b1 (0 –100
ng/ml) for 1– 48 h in serum-free medium. Northern blot analysis
showed an increase in basal PRL mRNA levels, and a decrease in
responsiveness to TGF-b1 with age. TGF-b1 suppressed PRL mRNA
in a dose- and time-dependent manner in cells from young rats. Maximum inhibition was observed at 0.5–1 ng/ml of TGF-b1. At 0.5 ng/ml
TGF-b1, significant reduction in PRL mRNA was detected at 6 h, and
maximum inhibition was observed at 12– 48 h post TGF-b1 incubation. Cells from middle-aged rats were less responsive to TGF-b1,
whereas cells from old rats did not seem to respond under our experimental conditions. In addition to its effect on PRL mRNA in young
AP cells, TGF-b1 dose dependently inhibited the rate of PRL synthesis, as indicated by reduced [35S]methionine incorporation into
immunoprecipitated PRL. Responsiveness of PRL synthesis to
TGF-b1 inhibition also decreased with age; however, significant inhibition by TGF-b1 on PRL synthesis could still be observed in old AP
cells. Analysis by RIA demonstrated that young AP cells produced
lower levels (15 mg/106 cellsz24 h) of PRL in culture medium than old
AP cells (32 mg/106 cellsz24 h). TGF-b1 decreased medium PRL levels
in old AP cells as efficaciously as in young AP cells. Significant reduction in medium PRL secreted by young AP cells was observed at
3 h when changes in both PRL mRNA and PRL synthesis were not
evident. Taken together, our data suggest that TGF-b1 affects PRL
production at multiple levels. Moreover, its inhibition on PRL synthesis and mRNA expression, but not on PRL secretion, is age-related.
Thus, TGF-b1 may play an important role in regulating lactotrope
function during aging. (Endocrinology 138: 878 – 885, 1997)
P
TGF-b belongs to a superfamily of structurally related
dimeric proteins. These are multifunctional proteins regulating the growth and differentiation of many cell types. In
mammals, three homodimeric isoforms of TGF-b (25 kDa)
denoted TGF-b1, TGF-b2, TGF-b3, and at least two heterodimeric isoforms, TGF-b1.2 and TGF-b2.3, exist (15–18).
The cellular action of TGF-b1 is mediated through binding to
its cell surface receptors, type I and type II, which belong to
a transmembrane serine-threonine kinase receptor family
(18). Following binding of type II receptor to TGF-b1, type I
receptor is recruited into the complex and then phosphorylated by type II receptor. Phosphorylation allows receptors
to propagate the signal to downstream substrates, initiating
a signaling cascade (19). The involvement of TGF-b1 in pituitary hormone secretion was first demonstrated by Ying et
al. (20). TGF-b1 stimulates basal secretion of FSH and GH but
inhibits basal PRL secretion in rat AP cells (13, 20). Studies
from rat pituitary tumor cell lines further demonstrated inhibition by TGF-b1 of PRL gene transcription in GH3 cells
(14) and of newly translated PRL in GH4 cells (21). In addition, the estrogen-induced lactotrope growth was found to be
suppressed by TGF-b1 (13). The presence of TGF-b1 messenger RNA (mRNA) and protein in rat pituitary (13, 22) and
TGF-b1 bioactivity in human pituitary (22) suggests that
TGF-b1 is synthesized in the pituitary. Available evidence
further indicates that lactotropes and melanotropes are
RL IS A single polypeptide hormone produced in the
anterior pituitary (AP) gland by lactotropes. PRL acts
on a variety of target tissues via specific membrane receptors
and mediates a wide-ranging physiological processes involved in growth, development, reproduction, osmoregulation, and immune function (1–3). PRL production, usually
reflected by circulating PRL levels or secretion of PRL from
cultured pituitary cells, represents a composite of rates of
PRL synthesis and degradation by lactotropes and the rate of
its secretion into the systemic circulation. All of these processes appear to be independently regulated. The lactotrope
function is regulated by many inhibitory and stimulatory
factors released from the hypothalamus and synthesized in
the pituitary and also by peripheral hormones (2–11). Among
these factors, transforming growth factor beta (TGF-b) has
been reported to affect AP cell function (12–14).
Received September 30, 1996.
Address all correspondence and requests for reprints to: Dr. TsueiChu Liu, Faculty of Medical Technology/Institute of Biotechnology in
Medicine, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan,
Republic of China. E-mail: [email protected].
* This work was supported by grants from National Science Council
of the Republic of China (NSC-83-0412-B010-068 and NSC-84-2331-B010091) and Medical Research and Advancement Foundation in Memory of
Dr. Chi-Shuen Tsou (ROC) (to T.-C.L., F.-F.W., and S.-K.T). It was presented in part at the 76th Annual Meeting of the Endocrine Society,
Anaheim, CA, USA, June 15–18, 1994, p 426 (Abstract 904).
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AGING AND TGF-b1 ACTION ON PITUITARY PRL
TGF-b1 immunopositive cells, and the production of TGF-b1
in lactotropes can be negatively influenced by estrogen (22).
Collectively, these results suggest that TGF-b1 produced in
the AP may act in a paracrine/autocrine fashion in regulating
pituitary function.
There are age-related increases in both PRL secretion and
the percent of lactotropes in the AP (23, 24). Plasma and
pituitary concentrations of PRL are higher in old than in
young rats (25, 26). Increases in plasma PRL concentrations
first become detectable in middle-aged cycling and noncycling animals (27–29) and continue to increase with age. The
mechanism by which aging augments secretion and/or synthesis of PRL has not been fully elucidated. The objective of
this study was to determine if the action of TGF-b1 on lactotrope function changes with age. We examined the effects
of TGF-b1 on various aspects of PRL production in AP cells
from rats at different ages. Our data suggest that TGF-b1
inhibits PRL production via multiple sites and plays an important role in regulating lactotrope function during aging.
Materials and Methods
Animals and pituitary cell cultures
Young (2–3 months old), middle-aged (11–12 months old), and old (24
months old) female Sprague-Dawley rats were maintained at a room
temperature of 22 C 6 2 C on a 14 h-light (0600 –2000 h), 10 h-dark
schedule. They were fed food and tap water ad libitum. Rats were bilaterally ovariectomized under ether anesthesia and used 4 days later.
They were rapidly decapitated between 0800 – 0900 h. Trunk blood was
obtained, and serum separated by centrifugation and stored at 220 C
until assayed for PRL. Old rats that had visible, enlarged, and hemorrhagic pituitary tumors were not included in the studies. APs were
excised and dispersed into single cell suspensions by the method of Liu
et al. (30). Briefly, sliced tissue fragments were dissociated by collagenase
and hyaluronidase after exposure to trypsin. For studies of PRL mRNA
and PRL synthesis, 3 3 106 cells in 3 ml were seeded in each 60 3 15-mm
tissue culture dish (Corning Glass Works, Corning, NY). For studies of
time course and age effects on PRL secretion, the dispersed AP cells were
cultured at 2 3 105 cells/ml/well (Falcon, Taiwan Ivy Corp., Taipei,
Taiwan; 2 cm2/well). All AP cells were incubated at 37 C under moist
5% CO2 and 95% O2. The culture medium contained 2.5% FBS (Hyclone
Laboratories, Logan, UT) and 10% bovine calf serum (Hyclone) in supplemented medium 199 without phenol red (weak estrogen). All sera
were pretreated with dextran-coated charcoal to minimize steroid modulation of PRL production. After 24 h of culture, 5 ml fresh culture
medium was added to each dish and cells were cultured for another 2
days. Then the medium was replaced by 4 ml fresh medium, and the cells
were cultured for additional 2 days.
879
10 mm Tris-HCl, pH 7.0, containing 1 mm EDTA and 10 mm vanadylribonucleoside complexes. Cells were chilled on ice and, while vortexing, 5% NP-40 was added to a final concentration of 1% and the reaction
was proceeded at 4 C for 10 min. The mixture was centrifuged (35,000 3
g for 3 min), and the cell extract was extracted with equal volume of
phenol. For Northern blotting, RNA was denatured with formamide/
formaldehyde, and 5 mg RNA per lane was applied to a 1.2% agarose
gel containing formaldehyde for electrophoresis. Then, RNA was transferred to a nitrocellulose paper. The complementary DNA (cDNA)
probes for rat PRL mRNA (32, 33) and 18S rRNA were radiolabeled with
a32P-ATP by random priming. Hybridization was performed following
the procedure of Meinkoth and Wahl (34). Rat 18S rRNA was used as
an internal standard for the correction of RNA loading. The radioactivity
was detected by exposing the nitrocellulose paper to a Kodak x-ray film
for a suitable length of time and the extent of hybridization quantitated
by scanning with a densitometer (Molecular Dynamics, Sunnyvale, CA,
Personal Densitometer, model PD-120).
[35S]methionine incorporation into immunoprecipitable PRL
Cultured cells pretreated with or without TGF-b1 for various time
periods were washed three times and incubated in methionine-free
DMEM at 37 C for 30 min, then pulse-labeled with [35S]methionine (100
mCi/ml) in the same medium containing 1% BSA with or without
TGF-b1 for 2–3 h. The medium and cells were collected separately for
PRL immunoprecipitation (35). Briefly, phenylmethyl-sulfonylfluoride
(PMSF) was added at a final concentration of 0.1 mm to the medium.
Then, the medium was concentrated in Microcon microconcentrator
(Amicon, Beverly, MA, mw 10,000). Cells were washed three times with
PBS containing 0.1 mm PMSF and lysed with buffer (0.01 m Na2HPO4,
0.15 m NaCl, 0.01% (vol/vol) Triton X-100, 0.5% SDS, 0.2% NaN3, pH
7.25), the lysates were centrifuged at 30,000 3 g for 30 min. Aliquots of
supernatant containing either similar amount of 10% TCA precipitable
radioactivity (36) or equal amount of protein (35) were used for PRL
immunoprecipitation in the time course and aging experiments, respectively. Protein A-coupled Sepharose was added and the precipitate
discarded to eliminate the nonspecific binding. Then, anti-PRL serum
(NIDDK-anti-rPRL-S-9) was added to the supernatant. After continuous
shaking for 16 –18 h at 4 C, protein A-Sepharose was added, and the
mixture reacted for 2 h. The immunoprecipitates were then analyzed by
electrophoresis on a 10% SDS polyacrylamide gel and protein bands
visualized by autoradiography.
RIA for PRL
At the end of incubation, cells and medium were collected separately.
Cells were solubilized as described previously (36). PRL in the medium,
cell extracts, and serum was measured by RIA with protocol supplied
by the NIDDK Hormone Distribution Program. NIDDK-rPRL-RP-3,
NIDDK-rPRL-I-6, and NIDDK-anti-rPRL-IC-5 were used in the PRL
RIA. The sensitivity of the assay was 30 pg/tube. The intra- and interassay coefficients of variation were 9.0% and 14.5%, respectively.
Incubation of cultured AP cells
Experimental design and data analysis
Cultured AP cells were washed and then incubated at 37 C in the
culture medium without sera but with 1% BSA and increasing doses of
TGF-b1 (0 –100 ng/ml) for various time periods (1–24 h). In time course
studies of PRL mRNA and PRL synthesis, TGF-b1 was added at different
intervals such that all cells were exposed to a same period of serum-free
condition. At the end of incubation, the medium was separated from
cells, centrifuged, and stored at 220 C for measuring PRL by RIA. Cells
were extracted for cytosolic RNA and monitored for PRL mRNA levels
by Northern blot. TGF-b1 was a generous gift from Dr. R. C. Chang
(Celtrix Pharmaceuticals, Santa Clara, CA). It was prepared as an 1
mg/ml stock solution in 4 mm HCl containing 1 mg/ml BSA and diluted
in the challenge medium before use.
In each experiment, one batch of AP cells was prepared from approximately 20 APs. Aliquots of each cell batch were placed in separate
dishes or wells, and drug treatments were randomly assigned to each
vessel. Each experiment was reproduced at least three times. Data were
processed by variance analysis followed by the Duncan’s multiple range
test for comparison of individual means. In the time course study of
TGF-b1 inhibition of PRL secretion by young AP cells, data were subjected to logarithmic transformation before statistical analysis due to
heterogeneity of error (30). Student’s t test was also used when appropriate. P , 0.05 was considered to be significant, and P , 0.01 was highly
significant.
Preparation of cell extract, cytosolic RNA, and Northern
blot analysis
Age increases serum PRL
Cytosolic RNA was isolated by the method of White and Bancroft
(31). Cells were harvested, washed twice with PBS, and suspended in
To confirm that in vivo PRL secretion in female SpragueDawley rats increases with age, serum levels of PRL in ovari-
Results
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AGING AND TGF-b1 ACTION ON PITUITARY PRL
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Vol 138 • No 3
ectomized either young (2–3 months old), middle-aged
(11–12 months old), or old (25 months old) rats were monitored by RIA. Serum PRL levels were significantly (P , 0.01)
higher in both middle-aged (132.5 6 21.4 ng/ml, n 5 36 rats)
and old rats (158.9 6 23.1 ng/ml, n 5 29 rats) than in young
rats (9.0 6 1.0 ng/ml, n 5 57 rats).
Age increases basal and decreases TGF-b1- suppressed PRL
mRNA expression
The effects of TGF-b1 on PRL mRNA levels (measured by
Northern blot and expressed as PRL mRNA relative to 18S
rRNA) were first examined in AP cells from young rats.
TGF-b1 at 0.1–100 ng/ml for 24 h suppressed PRL mRNA
levels in a dose- (Fig. 1) and time-dependent manner (Fig. 2).
Maximum inhibition (64 6 9%) on PRL mRNA was obtained
at 0.5–1 ng/ml of TGF-b1. Doses of TGF-b1 greater than 1
FIG. 2. Time course for TGF-b1 inhibition of PRL mRNA levels in
young rat AP cells. Day 5 cultured AP cells (3 3 106 cells/dish) from
ovariectomized young rats were washed and transferred to serumfree medium. Diluent or TGF-b1 (0.5 ng/ml) was then added at different intervals such that all cells were subjected to serum-free culture for 48 h. Cytosolic PRL mRNA was measured by Northern blot.
A, Representative autoradiograms. B, Percent relative expression of
PRL mRNA. Data are expressed as percentage of control cells which
were treated with diluent for 48 h (0 h exposure to TGF-b1). Each bar
represents the mean 6 SEM of three separate batches of AP cells. See
legend to Fig. 1 for details.
FIG. 1. Dose dependency for TGF-b1 inhibition of PRL mRNA levels
in young rat AP cells. AP cells (3 3 106 cells/dish) from ovariectomized
young rats were cultured in serum (dextran-charcoal treated)-containing medium for 5 days. Then AP cells were washed and challenged
in serum-free medium containing 1% BSA with increasing doses of
TGF-b1 at 37 C for 24 h. AP cells were extracted for cytosolic RNA and
PRL mRNA analyzed by Northern blot followed by autoradiography.
A, Representative autoradiograms for PRL mRNA and 18S rRNA
(18S). B, Percent relative expression of PRL mRNA. Extent of hybridization with 32P labeled cDNA probes for PRL mRNA and 18S
rRNA was quantitated by scanning with a densitometer. The ratios
of PRL mRNA to 18S rRNA hybridization at different TGF-b1 doses
were calculated and then divided by control cells (0 dose of TGF-b1)
to obtain percent relative expression of PRL mRNA. Each bar represents the mean 6 SEM of three to nine separate batches of AP cells.
Bars not labeled with the same alphabetical letters are significantly
different at P , 0.05 (Duncan’s).
ng/ml tended to be less effective than 0.5–1 ng/ml TGF-b1.
Significant suppression of PRL mRNA by TGF-b1 at 0.5
ng/ml was first detected (P , 0.05) at 6 h and reached the
maximum value (P , 0.01) following 12 h incubation. Levels
of PRL mRNA remained maximally suppressed by TGF-b1
after 24 – 48 h incubation. In contrast, during the 24-h incubation period, the suppression by TGF-b1 at 0.25, 0.5, or 1
ng/ml on PRL mRNA was less (P , 0.05) in AP cells from
middle-aged rats than that from young rats (Fig. 3). Moreover, similar doses of TGF-b1 did not seem to alter PRL
mRNA in AP cells from old rats (Fig. 4).
To examine if basal PRL mRNA expression in AP cells
changes with age, PRL mRNA levels in diluent-treated AP
cells from rats at different ages were compared. AP cells from
young rats expressed significantly lower (P , 0.01) basal
levels of PRL mRNA than that from either middle-aged or
old rats. Ratios of PRL mRNA/18S rRNA from three individual experiments were 1.7 6 0.2, 2.34 6 0.3, and 3.1 6 0.3,
for young, middle-aged, and old AP cells, respectively.
AGING AND TGF-b1 ACTION ON PITUITARY PRL
FIG. 3. Decline in TGF-b1 inhibition of PRL mRNA levels in AP cells
from middle-aged rats. Day 5 cultured AP cells from young (2–3
months old) and middle-aged (12 months old) ovariectomized rats
were washed and challenged in serum-free medium containing 1%
BSA with TGF-b1 (0 –1 ng/ml) at 37 C for 24 h. Cytosolic PRL mRNA
was measured by Northern blot. A, Representative autoradiograms.
B, Percent relative expression of PRL mRNA. Data are expressed as
percentage of respective control cells (0 dose TGF-b1 for either young
or middle-aged AP cells). Each bar represents the mean 6 SEM of three
separate batches of AP cells. See legend to Fig. 1 for details.
Age decreases both basal and TGF-b1- suppressed PRL
synthesis
Effect of TGF-b1 on protein synthesis was next examined
on young AP cells. Vehicle or 0.5 ng/ml of TGF-b1 was
added to cells at different intervals so that all cells were
exposed to serum-free medium for 24 h. [35S]methionine
labeling was carried out during the last 2-h incubation period. Cell lysates were precipitated with anti-PRL antiserum
and analyzed by electrophoresis on a 10% SDS polyacrylamide gel. The cellular [35S]methionine-labeled PRL, identified as a 23-kDa protein and absent when immunoprecipited with normal rabbit serum, was suppressed (P , 0.01) by
TGF-b1 in a time-dependent manner (Fig. 5). Significant inhibition (62.2 6 7.3%) was observed after 24 h incubation. In
contrast, total TCA-precipitable [35S]methionine-protein was
not affected by TGF-b1 treatment in either young or old AP
cells (data not shown). [35S]methionine-labeled PRL was undetectable in the medium samples.
Similar to AP cells from young rats, treatment with TGF-b1
(0.25–1 ng/ml) for 24 h also significantly inhibited (P , 0.05)
881
FIG. 4. Lack of TGF-b1 inhibition on PRL mRNA levels in AP cells
from old rats. Day 5 cultured AP cells from young (2–3 months old)
and old (24 months old) ovariectomized rats were washed and challenged in serum-free medium containing 1% BSA and TGF-b1 (0 –1
ng/ml) at 37 C for 24 h. Cytosolic PRL mRNA was measured by
Northern blot. A, Representative autoradiogram. B, Percent relative
expression of PRL mRNA. Data are expressed as percentage of respective control cells (0 dose TGF-b1 for either young or old AP cells).
Each bar represents the mean 6 SEM of three separate batches of AP
cells. See legend to Fig. 1 for details.
the [35S]methionine-labeled-PRL in a dose-related manner in
AP cells from old rat (Fig. 6). However, maximum inhibition
by TGF-b1 was attenuated in old AP cells; TGF-b1 at maximum doses of both 0.5 ng/ml (P , 0.01) and 1.0 ng/ml (P ,
0.05) caused significantly greater inhibition in young AP cells
(76 – 88% decrease) than that in old AP cells (55–59% decrease). Despite elevated basal levels of PRL mRNA expression, old AP cells exhibited a reduced rate of PRL synthesis,
as suggested by a decreased (P , 0.05) level of [35S]methionine-labeled PRL when compared with young ones (76.66 6
4.71% of young, n 5 3).
Age increases basal medium PRL without attenuating TGFb1-inhibited PRL secretion
To examine if both basal and TGF-b1-suppressed PRL
secretion in vitro change with age, PRL secreted into the
culture medium in the absence and presence of TGF-b1 by
AP cells from rats at different ages was monitored by RIA.
In young AP cells, TGF-b1 decreased the medium accumulation of PRL during the 24 h incubation (Fig. 7). Maximum
inhibition (67 6 5.5%) was obtained at 0.5 ng/ml of TGF-b1
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AGING AND TGF-b1 ACTION ON PITUITARY PRL
FIG. 5. Time course for TGF-b1 inhibition of [35S]methionine-labeled
PRL in young rat AP cells. Day 5 cultured AP cells from young
ovariectomized rats were washed, and then challenged in serum-free
medium containing 1% BSA with or without TGF-b1 (0.5 ng/ml) at 37
C for various time intervals. Before harvesting, cells were incubated
with [35S]methionine (100 mCi/ml) for 2 h. All cells were subjected to
serum-free culture for a total length of 24 h. Cells were then collected
and lysed before immunoprecipitation with anti-PRL antiserum or
normal rabbit serum (NRS). The immune complexes from cell extracts
were analyzed on 10% SDS-PAGE. A, Radiolabeled PRL bands revealed by autoradiography. Numbers at the right are molecular mass
markers (M) in kDa. B, Quantitation of [35S]methionine-labeled PRL
by scanning with a densitometer. Values are expressed as fold of
control cells (0 h exposure to TGF-b1). Each bar represents the
mean 6 SEM of three separate batches of AP cells. Groups not labeled
with the same alphabetical letters are significantly different at P ,
0.05 (Duncan’s).
(P , 0.01). The inhibition could also be observed at 1–100
ng/ml of TGF-b1 (P , 0.05); however, higher doses (30, 100
ng/ml) were less effective (P , 0.01) than the 0.5 ng/ml dose.
Suppression on medium PRL by TGF-b1 was time-related
(Fig. 8); significant inhibition was detected after 3– 48 h exposure to 0.5 ng/ml TGF-b1.
Medium PRL level of cultured old AP cells (32 6 5 mg/106
cellsz24 h) was significantly higher (P , 0.01) than that of
young AP cells (15 6 1 mg/106 cellsz24 h) (Fig. 9). It remained
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Vol 138 • No 3
FIG. 6. Decline in TGF-b1 inhibition of [35S]methionine-labeled PRL
in AP cells from old rats. All day 5 cultured AP cells from young (2–3
months old) or old (24 months old) ovariectomized rats were washed
and then challenged in serum-free medium containing 1% BSA and
TGF-b1 (0, 0.25, 0.5, 1 ng/ml) at 37 C for a total length of 24 h. Before
harvesting, cells were incubated with [35S]methionine (100 mCi/ml)
for 3 h. A, Radiolabelled PRL bands revealed by autoradiography. B,
Quantitation of [35S]methionine-labeled PRL by scanning with a densitometer. Values are expressed as fold of respective control cells (0
ng/ml TGF-b1 for young or old AP cells). Each point represents the
mean 6 SEM of three separate batches of AP cells. See legend to Fig.
5 for details.
significantly higher (P , 0.01) in the presence of 0.25–1
ng/ml TGF-b1 (Fig. 9). Under such conditions, TGF-b1 induced maximum inhibition (P , 0.01) by 65– 69% in old AP
cells and by 55–58% in young AP cells. The percent suppression by TGF-b1 on medium PRL was not changed significantly with age (P . 0.05).
Discussion
We have demonstrated that TGF-b1 exerted multiple actions on PRL production in normal rat AP cells. Most importantly, we have provided, to our best knowledge, the first
evidence that aging specifically reduced the efficacy of
TGF-b1 on PRL synthesis and mRNA expression, but not on
PRL secretion. These results suggest an autocrine/paracrine
role of TGF-b1 in regulating lactotrope function during
aging.
Previously, TGF-b1 has been shown to be a potent inhib-
AGING AND TGF-b1 ACTION ON PITUITARY PRL
FIG. 7. Dose dependency of TGF-b1 inhibition of medium PRL levels
in young rat AP cells. See legend to Fig. 1. At the end of the 24-h
incubation with increasing doses of TGF-b1, medium was collected
and measured for PRL by RIA, using rPRL-RP-3 as the standard.
Each bar represents the mean 6 SEM of three batches of AP cells.
FIG. 8. Time course for TGF-b1 inhibition of medium PRL levels in
young rat AP cells. Day 5 cultured AP cells (2 3 105 cells/well) from
ovariectomized young rats were washed and then incubated in serumfree medium with diluent for TGF-b1 (vehicle) or TGF-b1 (0.5 ng/ml)
at 37 C for 1– 48 h. At the end of incubation, the medium and cells were
collected separately and PRL was determined by RIA. Each bar represents the mean 6 SEM of three separate batches of cells, each performed with triplicate wells per treatment. Groups not labeled with
the same alphabetical letters are significantly different at P , 0.05
(Duncan’s).
itor of pituitary cell proliferation (13, 21, 37), and it inhibited
PRL mRNA in pituitary tumor cells (14, 21) while stimulating
FSHb subunit mRNA in sheep pituitary cells (37). Using rat
AP cells as a model system, we also found that TGF-b1
inhibited PRL mRNA in AP cells from young rats. Moreover,
this inhibition declined with age. Decline in sensitivity to
TGF-b1 may be attributed to at least four factors, i.e. the
amount of endogenously produced TGF-b1, TGF-b receptor
levels, postreceptor signaling mechanisms, and/or production of other autocrine or paracrine factors. Recently, Pas-
883
FIG. 9. Effect of TGF-b1 on medium PRL secreted by AP cells from
young vs. old rats. Day 5 cultured AP cells (2 3 105 cells/well) from
ovariectomized young (2–3 months old) or old rats (24 months old)
were washed and then incubated in serum-free medium with TGF-b1
(0, 0.25, 0.5, 1 ng/ml) at 37 C for 24 h. At the end of incubation, the
medium and cells were collected separately and PRL was determined
by RIA. Each bar represents the mean 6 SEM of three separate batches
of cells, each performed with triplicate wells per treatment. Groups
not labeled with the same alphabetical letters are significantly different at P , 0.05 (Duncan’s).
torcic et al. (38) have shown that TGF-b1 protein and mRNA
levels were higher in normal lactotropes than in GH3 tumor
cells, and the reduced sensitivity of GH3 cells to the antiproliferative effect of TGF-b1 correlated with levels of
TGF-b1 and TGF-b type II receptor in these cells. The mechanisms by which TGF-b1 exerts an age-related action on PRL
mRNA in AP cells remain to be determined.
Consistent with its effect on PRL mRNA expression in
young AP cells, TGF-b1 dose dependently inhibited the levels of [35S]methionine-labeled PRL, an effect similarly observed in GH4 pituitary tumor cells (21). By contrast, in old
AP cells while TGF-b1 was ineffective on suppressing PRL
mRNA levels, it inhibited the newly translated PRL, suggesting a direct effect of TGF-b1 on PRL synthesis unrelated
to its inhibition of PRL mRNA. Also, this effect was PRL
specific, as evidenced by unaltered incorporation of [35S]methionine into total TCA-precipitable protein in either young
or old AP cells. Interestingly, our data further revealed that
although TGF-b1 inhibited PRL synthesis in old AP cells, the
efficacy of inhibition was reduced as compared with young
AP cells. The inhibition by TGF-b1 of [35S]methionine-labeled PRL was decreased significantly from 76 – 88% in
young AP cells to 55–59% in old AP cells. Thus, it appears
that TGF-b1 may independently suppress PRL gene expression at the levels of PRL mRNA expression and PRL synthesis, and both actions are age dependent.
In addition to its effects on PRL mRNA and PRL synthesis,
TGF-b1 also dose dependently suppressed secretion of PRL
in young AP cells, a finding originally observed by Ying et
al. (20) and confirmed by others (12, 13). However, we further
demonstrated that TGF-b1 induced a two-phase suppression
of PRL secretion, and its action was not age dependent.
Maximum inhibition was obtained at 0.5 ng/ml of TGF-b1,
and doses greater than 0.5 ng/ml were less effective. Similar
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AGING AND TGF-b1 ACTION ON PITUITARY PRL
dose effect was noted on PRL mRNA inhibition. Because
TGF-b1 action requires formation of a ternary complex containing receptors type I, II, and TGF-b1, it is possible that at
supramaximal doses of TGF-b1, dimerization between type
I and type II receptors (19) necessary for signal transduction
may be decreased, due to binding of each receptor type to
excess TGF-b1. In addition, signals positively affecting PRL
mRNA levels or PRL secretion may be activated at high doses
of TGF-b1. Another interesting observation of this study is
that despite the ineffectiveness of TGF-b1 in suppressing
PRL mRNA in old AP cells, its inhibitory action on PRL
secretion was as efficacious as that in young AP cells. These
results suggest that the old AP cells retain functional TGF-b1
receptors, and the intracellular mechanisms responsible for
TGF-b1-suppressed PRL mRNA vs. PRL secretion appear to
be differentially affected by aging. In addition, these data
further support a primary action of TGF-b1 on PRL secretion
which is independent of its effect on PRL mRNA.
Time-course studies of TGF-b1 action in young rat AP cells
demonstrated that PRL secretion was inhibited before the
inhibition of either PRL mRNA or PRL synthesis. Significant
inhibition of PRL secretion by TGF-b1 was observed after 3 h
exposure, when changes in both PRL mRNA and PRL translation were not evident. These data again support that
TGF-b1 exerts a primary action on PRL secretion. Taken
together, TGF-b1 affects PRL production at multiple levels in
AP cells.
Pituitary PRL mRNA level has been reported to be either
unaltered or decreased with aging (23, 39). However, we
found that PRL mRNA expression increased 1.8-fold in old
AP cells as compared with young ones. Differences in methodology may lead to the divergent results. Our observations
on augmented PRL mRNA levels, together with elevated
PRL secretion, and declined responsiveness of PRL mRNA
and PRL synthesis to TGF-b1 inhibition in old AP cells may
all, in part, contribute to the increased PRL secretion in aged
rats noted by us and others (23–29). In addition, we found
that PRL translation was somewhat reduced, rather than
enhanced, in old AP cells. To account for the elevated pituitary PRL concentration observed during aging (25, 26),
changes at the posttranslational level such as molecular modification and intracellular degradation of PRL (40 – 44), which
may affect cellular pool of PRL, appear to be additional
factors contributing to the age-related increase in PRL production. One interesting phenomenon is that plasma PRL
elevated more than 15-fold in aged rats, whereas PRL secreted into culture medium by old AP cells increased only
2-fold. Higher in vivo vs. in vitro PRL secretion may reflect the
age-related alterations in responsiveness to endogenous PRL
inhibitory and stimulatory factors (45, 46), hypothalamic input to the pituitary affecting PRL secretion (47, 48), and
production of PRL from sources other than the pituitary (49).
In summary, we have demonstrated that in the rat AP cells,
TGF-b1 suppressed PRL production at multiple sites along
the biosynthetic and secretory pathways of PRL. These actions of TGF-b1 appear to be differentially affected by the
aging process. Whether changes in TGF-b1 receptor number/affinity, postreceptor signal transduction, PRL stability,
and/or PRL isoforms may be responsible factors merit further investigation.
Endo • 1997
Vol 138 • No 3
Acknowledgments
We thank Dr. R. C. Chang for the generous gift of TGF-b1, Dr. R. A.
Maurer for kindly providing us the PRL cDNA probe, and the National
Hormone and Pituitary Distribution Program (NIDDK) for the supply
of rat PRL RIA kit.
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