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Endocrinology Vol. 138, No. 3 878-885
Copyright © 1997 by The Endocrine Society

Articles

Differential Effect of Age on Transforming Growth Factor-ß1 Inhibition of Prolactin Gene Expression Versus Secretion in Rat Anterior Pituitary Cells1

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 Yang-Ming University, Shih-Pai, Taipei, Taiwan, Republic of China

Address all correspondence and requests for reprints to: Dr. Tsuei-Chu Liu, Faculty of Medical Technology/Institute of Biotechnology in Medicine, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan, Republic of China. E-mail: tcliu{at}ym.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß1 (TGF-ß1) synthesized in the pituitary may act as an autocrine/paracrine regulator of lactotrope function. We examined the effects of TGF-ß1 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-ß1 (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-ß1 with age. TGF-ß1 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-ß1. At 0.5 ng/ml TGF-ß1, significant reduction in PRL mRNA was detected at 6 h, and maximum inhibition was observed at 12–48 h post TGF-ß1 incubation. Cells from middle-aged rats were less responsive to TGF-ß1, 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-ß1 dose dependently inhibited the rate of PRL synthesis, as indicated by reduced [35S]methionine incorporation into immunoprecipitated PRL. Responsiveness of PRL synthesis to TGF-ß1 inhibition also decreased with age; however, significant inhibition by TGF-ß1 on PRL synthesis could still be observed in old AP cells. Analysis by RIA demonstrated that young AP cells produced lower levels (15 µg/106 cells·24 h) of PRL in culture medium than old AP cells (32 µg/106 cells·24 h). TGF-ß1 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-ß1 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-ß1 may play an important role in regulating lactotrope function during aging.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL 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, 2, 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, 3, 4, 5, 6, 7, 8, 9, 10, 11). Among these factors, transforming growth factor beta (TGF-ß) has been reported to affect AP cell function (12, 13, 14).

TGF-ß 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-ß (25 kDa) denoted TGF-ß1, TGF-ß2, TGF-ß3, and at least two heterodimeric isoforms, TGF-ß1.2 and TGF-ß2.3, exist (15, 16, 17, 18). The cellular action of TGF-ß1 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-ß1, 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-ß1 in pituitary hormone secretion was first demonstrated by Ying et al. (20). TGF-ß1 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-ß1 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-ß1 (13). The presence of TGF-ß1 messenger RNA (mRNA) and protein in rat pituitary (13, 22) and TGF-ß1 bioactivity in human pituitary (22) suggests that TGF-ß1 is synthesized in the pituitary. Available evidence further indicates that lactotropes and melanotropes are TGF-ß1 immunopositive cells, and the production of TGF-ß1 in lactotropes can be negatively influenced by estrogen (22). Collectively, these results suggest that TGF-ß1 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, 28, 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-ß1 on lactotrope function changes with age. We examined the effects of TGF-ß1 on various aspects of PRL production in AP cells from rats at different ages. Our data suggest that TGF-ß1 inhibits PRL production via multiple sites and plays an important role in regulating lactotrope function during aging.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 ± 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 -20 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 x 106 cells in 3 ml were seeded in each 60 x 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 x 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.

Incubation of cultured AP cells
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-ß1 (0–100 ng/ml) for various time periods (1–24 h). In time course studies of PRL mRNA and PRL synthesis, TGF-ß1 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 -20 C for measuring PRL by RIA. Cells were extracted for cytosolic RNA and monitored for PRL mRNA levels by Northern blot. TGF-ß1 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.

Preparation of cell extract, cytosolic RNA, and Northern blot analysis
Cytosolic RNA was isolated by the method of White and Bancroft (31). Cells were harvested, washed twice with PBS, and suspended in 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 x 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 µg 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 {alpha}32P-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-ß1 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 µCi/ml) in the same medium containing 1% BSA with or without TGF-ß1 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 x 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.

Experimental design and data analysis
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-ß1 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Age increases serum PRL
To confirm that in vivo PRL secretion in female Sprague-Dawley rats increases with age, serum levels of PRL in ovariectomized 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 ± 21.4 ng/ml, n = 36 rats) and old rats (158.9 ± 23.1 ng/ml, n = 29 rats) than in young rats (9.0 ± 1.0 ng/ml, n = 57 rats).

Age increases basal and decreases TGF-ß1- suppressed PRL mRNA expression
The effects of TGF-ß1 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-ß1 at 0.1–100 ng/ml for 24 h suppressed PRL mRNA levels in a dose- (Fig. 1Go) and time-dependent manner (Fig. 2Go). Maximum inhibition (64 ± 9%) on PRL mRNA was obtained at 0.5–1 ng/ml of TGF-ß1. Doses of TGF-ß1 greater than 1 ng/ml tended to be less effective than 0.5–1 ng/ml TGF-ß1. Significant suppression of PRL mRNA by TGF-ß1 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-ß1 after 24–48 h incubation. In contrast, during the 24-h incubation period, the suppression by TGF-ß1 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. 3Go). Moreover, similar doses of TGF-ß1 did not seem to alter PRL mRNA in AP cells from old rats (Fig. 4Go).



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  		Figure 1. Dose dependency for TGF-ß1 inhibition of PRL mRNA levels in young rat AP cells. AP cells (3 x 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-ß1 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-ß1 doses were calculated and then divided by control cells (0 dose of TGF-ß1) to obtain percent relative expression of PRL mRNA. Each bar represents the mean ± 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).

 


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  		Figure 2. Time course for TGF-ß1 inhibition of PRL mRNA levels in young rat AP cells. Day 5 cultured AP cells (3 x 106 cells/dish) from ovariectomized young rats were washed and transferred to serum-free medium. Diluent or TGF-ß1 (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-ß1). Each bar represents the mean ± SEM of three separate batches of AP cells. See legend to Fig. 1Go for details.

 


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  		Figure 3. Decline in TGF-ß1 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-ß1 (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-ß1 for either young or middle-aged AP cells). Each bar represents the mean ± SEM of three separate batches of AP cells. See legend to Fig. 1Go for details.

 


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  		Figure 4. Lack of TGF-ß1 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-ß1 (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-ß1 for either young or old AP cells). Each bar represents the mean ± SEM of three separate batches of AP cells. See legend to Fig. 1Go for details.

 
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 ± 0.2, 2.34 ± 0.3, and 3.1 ± 0.3, for young, middle-aged, and old AP cells, respectively.

Age decreases both basal and TGF-ß1- suppressed PRL synthesis
Effect of TGF-ß1 on protein synthesis was next examined on young AP cells. Vehicle or 0.5 ng/ml of TGF-ß1 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-ß1 in a time-dependent manner (Fig. 5Go). Significant inhibition (62.2 ± 7.3%) was observed after 24 h incubation. In contrast, total TCA-precipitable [35S]methionine-protein was not affected by TGF-ß1 treatment in either young or old AP cells (data not shown). [35S]methionine-labeled PRL was undetectable in the medium samples.



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  		Figure 5. Time course for TGF-ß1 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-ß1 (0.5 ng/ml) at 37 C for various time intervals. Before harvesting, cells were incubated with [35S]methionine (100 µCi/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-ß1). Each bar represents the mean ± 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).

 
Similar to AP cells from young rats, treatment with TGF-ß1 (0.25–1 ng/ml) for 24 h also significantly inhibited (P < 0.05) the [35S]methionine-labeled-PRL in a dose-related manner in AP cells from old rat (Fig. 6Go). However, maximum inhibition by TGF-ß1 was attenuated in old AP cells; TGF-ß1 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 ± 4.71% of young, n = 3).



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  		Figure 6. Decline in TGF-ß1 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-ß1 (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 µCi/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-ß1 for young or old AP cells). Each point represents the mean ± SEM of three separate batches of AP cells. See legend to Fig. 5Go for details.

 
Age increases basal medium PRL without attenuating TGF-ß1-inhibited PRL secretion
To examine if both basal and TGF-ß1-suppressed PRL secretion in vitro change with age, PRL secreted into the culture medium in the absence and presence of TGF-ß1 by AP cells from rats at different ages was monitored by RIA. In young AP cells, TGF-ß1 decreased the medium accumulation of PRL during the 24 h incubation (Fig. 7Go). Maximum inhibition (67 ± 5.5%) was obtained at 0.5 ng/ml of TGF-ß1 (P < 0.01). The inhibition could also be observed at 1–100 ng/ml of TGF-ß1 (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-ß1 was time-related (Fig. 8Go); significant inhibition was detected after 3–48 h exposure to 0.5 ng/ml TGF-ß1.



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  		Figure 7. Dose dependency of TGF-ß1 inhibition of medium PRL levels in young rat AP cells. See legend to Fig. 1Go. At the end of the 24-h incubation with increasing doses of TGF-ß1, medium was collected and measured for PRL by RIA, using rPRL-RP-3 as the standard. Each bar represents the mean ± SEM of three batches of AP cells.

 


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  		Figure 8. Time course for TGF-ß1 inhibition of medium PRL levels in young rat AP cells. Day 5 cultured AP cells (2 x 105 cells/well) from ovariectomized young rats were washed and then incubated in serum-free medium with diluent for TGF-ß1 (vehicle) or TGF-ß1 (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 ± 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).

 
Medium PRL level of cultured old AP cells (32 ± 5 µg/106 cells·24 h) was significantly higher (P < 0.01) than that of young AP cells (15 ± 1 µg/106 cells·24 h) (Fig. 9Go). It remained significantly higher (P < 0.01) in the presence of 0.25–1 ng/ml TGF-ß1 (Fig. 9Go). Under such conditions, TGF-ß1 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-ß1 on medium PRL was not changed significantly with age (P > 0.05).



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  		Figure 9. Effect of TGF-ß1 on medium PRL secreted by AP cells from young vs. old rats. Day 5 cultured AP cells (2 x 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-ß1 (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 ± 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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that TGF-ß1 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-ß1 on PRL synthesis and mRNA expression, but not on PRL secretion. These results suggest an autocrine/paracrine role of TGF-ß1 in regulating lactotrope function during aging.

Previously, TGF-ß1 has been shown to be a potent inhibitor of pituitary cell proliferation (13, 21, 37), and it inhibited PRL mRNA in pituitary tumor cells (14, 21) while stimulating FSHß subunit mRNA in sheep pituitary cells (37). Using rat AP cells as a model system, we also found that TGF-ß1 inhibited PRL mRNA in AP cells from young rats. Moreover, this inhibition declined with age. Decline in sensitivity to TGF-ß1 may be attributed to at least four factors, i.e. the amount of endogenously produced TGF-ß1, TGF-ß receptor levels, postreceptor signaling mechanisms, and/or production of other autocrine or paracrine factors. Recently, Pastorcic et al. (38) have shown that TGF-ß1 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-ß1 correlated with levels of TGF-ß1 and TGF-ß type II receptor in these cells. The mechanisms by which TGF-ß1 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-ß1 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-ß1 was ineffective on suppressing PRL mRNA levels, it inhibited the newly translated PRL, suggesting a direct effect of TGF-ß1 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-ß1 inhibited PRL synthesis in old AP cells, the efficacy of inhibition was reduced as compared with young AP cells. The inhibition by TGF-ß1 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-ß1 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-ß1 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-ß1 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-ß1, and doses greater than 0.5 ng/ml were less effective. Similar dose effect was noted on PRL mRNA inhibition. Because TGF-ß1 action requires formation of a ternary complex containing receptors type I, II, and TGF-ß1, it is possible that at supramaximal doses of TGF-ß1, 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-ß1. In addition, signals positively affecting PRL mRNA levels or PRL secretion may be activated at high doses of TGF-ß1. Another interesting observation of this study is that despite the ineffectiveness of TGF-ß1 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-ß1 receptors, and the intracellular mechanisms responsible for TGF-ß1-suppressed PRL mRNA vs. PRL secretion appear to be differentially affected by aging. In addition, these data further support a primary action of TGF-ß1 on PRL secretion which is independent of its effect on PRL mRNA.

Time-course studies of TGF-ß1 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-ß1 was observed after 3 h exposure, when changes in both PRL mRNA and PRL translation were not evident. These data again support that TGF-ß1 exerts a primary action on PRL secretion. Taken together, TGF-ß1 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-ß1 inhibition in old AP cells may all, in part, contribute to the increased PRL secretion in aged rats noted by us and others (23, 24, 25, 26, 27, 28, 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, 41, 42, 43, 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-ß1 suppressed PRL production at multiple sites along the biosynthetic and secretory pathways of PRL. These actions of TGF-ß1 appear to be differentially affected by the aging process. Whether changes in TGF-ß1 receptor number/affinity, postreceptor signal transduction, PRL stability, and/or PRL isoforms may be responsible factors merit further investigation.


   Acknowledgments
 
We thank Dr. R. C. Chang for the generous gift of TGF-ß1, 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.


   Footnotes
 
1 This work was supported by grants from National Science Council of the Republic of China (NSC-83-0412-B010-068 and NSC-84-2331-B010-091) 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). Back

Received September 30, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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