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Endocrinology Vol. 139, No. 12 5174-5181
Copyright © 1998 by The Endocrine Society

ARTICLES

Transforming Growth Factor ß1 Is a Paracrine Inhibitor of Prolactin Gene Expression1

Elizabeth J. Abraham, William J. Faught and L. Stephen Frawley

Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425

Address all correspondence and requests for reprints to: Dr. L. Stephen Frawley, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425. E-mail: frawleys{at}musc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have shown previously that rat mammotropes produce an activity that suppresses PRL gene expression by neighboring mammotropes. Here, we tested the hypothesis that this mammotrope-derived inhibitor is transforming growth factor-ß1 (TGFß1). To this end, we pursued a two-pronged strategy wherein we added exogenous TGFß1 to primary cultures of anterior pituitary cells transfected with a rat PRL-luc construct. Measurement of luciferase activity by luminometry of extracts revealed that administration of TGFß1, over a range of doses shown by others to be secreted by cultures of pituitary cells, caused a significant (P < 0.05) suppression of PRL gene expression. In contrast, immunoremoval of secreted TGFß1 led to an elevation of PRL promoter-driven reporter activity in these cultures. In a subsequent study, we repeated these experiments with a single cell model in an attempt to determine the demographics of the cellular responses. Accordingly, we transfected (via microinjection) individual mammotropes with the rat PRL-luc construct; exposed them to TGFß1, its neutralizing antibody, or respective controls; and then assessed PRL gene expression in "real-time" by quantification of photons emitted by the living cells after exposure to the substrate luciferin. Our results revealed that 1) TGFß1 inhibited PRL gene expression in all mammotrope studied; 2) only a subgroup of mammotropes (~23%) was relieved of TGFß1 inhibition by antibody treatment; and 3) the growth factor exerted its inhibitory effect via a paracrine, as opposed to an autocrine, mechanism. These findings identify TGFß1 as the paracrine agent that exerts a tonic inhibitory influence over PRL gene expression in mammotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERCELLULAR communication can have a dramatic influence on the secretion and gene expression of several anterior pituitary hormones (1, 2). Indeed, numerous studies aimed at delineating autocrine, juxtacrine, and paracrine interactions within this gland demonstrate the existence of intercellular factors that are proposed to exert local control (3, 4). Transforming growth factor-ß1 (TGFß1) is one such putative autocrine/paracrine agent and is secreted from normal as well as tumorous anterior pituitary cells (5, 6, 7, 8). This peptide exerts growth-inhibiting and differentiative influences on the pituitary as it does on many other tissues (5, 9, 10).

In a recent series of studies, we observed that expression of the PRL gene within mammotropes was particularly sensitive to intercellular signals emanating from neighboring mammotropes (11). To be more specific, we found that PRL gene expression (measured in "real-time" by photonic imaging of cells transfected with a PRL promoter-driven luciferase construct) was significantly suppressed when the living mammotrope under study was adjoined to another mammotrope. This inhibition of gene expression was not evident in mammotropes apposed to cells exhibiting a secretory phenotype (established post facto by immunocytochemistry) other than PRL. The present study was aimed at identifying this paracrine inhibitor of PRL promoter-driven gene expression. At the outset, we reasoned that TGFß1 was a strong candidate to subserve such a role for three reasons: it is secreted by mammotrope-enriched primary cultures (5); it is colocalized with PRL in the majority of rat mammotropes (12); and it is a potent inhibitor of PRL secretion and messenger RNA accumulation when administered to primary cultures of rat pituitary cells (13, 14, 15, 16). The results presented herein, which derive from studies on both single mammotropes and entire populations of anterior pituitary cells, confirm the hypothesis that TGFß1 functions in a paracrine manner to inhibit PRL gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell culture
Primiparous, lactating (days 5–12 postpartum) rats (Holtzman, Madison, WI) were housed with their littermates under a 12-h dark, 12-h light photoperiod, and food and water were provided ad libitum. All animals were handled and experiments were performed in accordance with NIH guidelines. Anterior pituitary glands were collected and enzymatically dispersed into single cells as described in detail previously (17). Briefly, cells were plated onto polylysine (0.025%)-coated, six-well plates or glass, grided coverslips in medium (an equivolume mixture of medium 199 and nutrient F-12) supplemented with 0.1% BSA, antibiotics, and insulin-transferrin-selenium Premix. One hour after attachment, medium with serum supplement was added, and the cell monolayers were placed in a humidified atmosphere of 5% CO2 and 95% air at 37 C.

Chemical transfection and measurement of luciferase activity in pituitary cultures
Cells were maintained in medium containing 10% FBS for 24 h after plating and then transiently transfected with a rat (r) PRL-luc reporter plasmid (containing 2.5-kb pairs of the 5'-flanking region and promoter of the rPRL gene fused to the firefly luciferase coding sequence) according to the lipofectamine method (18). This involved washing cultures once with DMEM supplemented with 2 mM L-glutamine and 10 mM HEPES. Then, 1 ml transfection mix consisting of the same medium with 2 µg DNA and 10 µl lipofectamine was added to each well. After 5 h, cells were washed and immersed in treatment medium (DMEM with 2 mM L-glutamine, 10 mM HEPES, 0.1% BSA, and antibiotics, with or without 10% FBS) containing test substances. After 48 h of treatment, medium was removed, and lysis buffer (200 µl/well) was added. Fifteen minutes later, a rubber policeman was used to remove cells, and extracts were collected, vortexed, and subjected to microcentrifugation. Supernatants were then tested for luciferase activity by use of reagents and according to guidelines provided with a kit from Promega Corp. (Madison, WI).

Real-time measurement of PRL-promoter driven luciferase activity in single, living mammotropes
Microinjection and photonic imaging. Cells were plated on glass coverslips (photoengraved with a numbered-lettered grid pattern) at 250,000 cells/90 µl medium and then cultured for 48 h in medium supplemented with 5% FBS. Cells within a single grid were microinjected with 0.2 µg/µl rPRL-luc plasmid as described previously (19, 20). The monolayers were then immersed for 24 or 48 h in medium containing 10% FBS. In selected experiments, this mixture was supplemented with test substances. For photonic imaging, cells transfected by microinjection were assembled into Sykes-Moore chambers and exposed to 3 mM luciferin (Sigma)-containing medium (DMEM-Ham’s F-12 medium supplemented with antibiotics) as described previously (19). Photonic emissions emanating from transfected cells were collected and quantified by a video-intensified photon-counting camera in series with an Argus-50 image processor.

Continuous monitoring of PRL gene expression. Forty-eight hours after transfection, cells were preincubated in 0.1 mM luciferin for 4 h in external medium (DMEM supplemented with 5 mM glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 5 mM sodium bicarbonate, 10 mM HEPES, 34 mM sodium chloride, 10% FBS, and insulin-transferrin-selenium Premix) before imaging. [This medium was selected for long term monitoring because of its superior buffering capacity. The preincubation with luciferin was conducted to ensure that steady state intracellular levels of the substrate were achieved before measurement commenced.] The coverslip bearing the cells was then assembled into a Sykes-Moore chamber, placed in the photon capture system, and perifused with 0.1 mM luciferin at a flow rate of 10 µl/min for an additional 17 h. Photons were accumulated continuously in 30-min bins for the entire period of measurement. Cells were imaged for 4 h before infusing medium containing porcine TGFß1 (1 ng/ml) or vehicle (4 mM HCl plus 0.1% BSA).

Immunocytochemistry
Cells were fixed with B5-buffered formalin for 45 min immediately after imaging. They were then rinsed and subjected to immunocytochemical identification of rPRL as detailed previously (17). In short, fixed cells were incubated with rabbit anti-rPRL (1:20,000) at 4 C overnight and then exposed to biotinylated goat antirabbit serum (Vectastain, Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:500. The preparations were then incubated in avidin-biotin complex, and the presence of immune complex was revealed by exposure to diaminobenzidine (Sigma).

Statistical analysis
Experiments with chemically transfected cells were repeated at least four times, and treatment differences were compared using predetermined, paired t tests (21). For cells transfected by microinjection, treatment differences were compared using an unpaired t test (21). Differences were considered significant at P < 0.05.

Unless indicated otherwise, all cell culture reagents were purchased from Life Technologies (Grand Island, NY). Porcine TGFß1 and chicken antihuman TGFß1 (catalog no. AF-NA-101) were purchased from R & D Systems (Minneapolis, MN), as was chicken IgG isolated from egg yolks.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment with TGFß1 inhibits PRL gene expression
Our initial efforts were focused on investigating whether exogenously supplied TGFß1 could affect PRL gene expression. To this end, we transfected entire cultures of pituitary cells with the rPRL-luc plasmid and treated them with various doses of TGFß1 in the presence or absence of FBS. As shown in Fig. 1Go (A and B), we found that doses of TGFß1 ranging from 0.1–10 ng/ml inhibited (by 60–70%; P < 0.05) PRL gene expression. Although this effect was not dose responsive over the range tested, there appeared to be a tendency toward escape from inhibition at the lowest dose. The presence/absence of serum had no influence on this response, indicating that the effect of TGFß1 on PRL gene expression occurred independent of possible interactions with serum factors. It is noteworthy, however, that the basal level of reporter activity was higher (7- to 12-fold) for serum-treated cultures (an effect obscured by normalization).



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  		Figure 1. TGFß1 inhibits PRL gene expression from primary anterior pituitary cultures. Cells were transfected with a rPRL-luc plasmid and treated with the indicated doses of TGFß1 in the presence or absence of FBS (10%). After 48 h of treatment, luciferase reporter activity was measured in cell extracts. Data are expressed as a percentage of the control (vehicle) values. Bars represent the mean + SEM of four independent experiments. *, P < 0.05 vs. VEH.

 
Having established that TGFß1 could decrease PRL gene expression at the population level, we then turned to a single cell model in an attempt to determine the fraction of mammotropes responsive to TGFß1 as well as the dynamics of the inhibitory response. Thus, we microinjected single pituitary cells with the rPRL-luc plasmid, waited 48 h, exposed them to luciferin, and then made continuous measurements of photonic emissions reflective of PRL gene expression over the subsequent 17-h period. Figure 2Go shows representative examples of single cells monitored in this manner. As illustrated in Fig. 2Go and by the averaged responses in Fig. 3Go, TGFß1 at a dose of 1 ng/ml caused a striking inhibition of PRL gene expression in individual mammotropes. Such inhibition first became significant within 4 h of initiating TGFß1 treatment (Fig. 3Go), whereas cells exposed to vehicle did not exhibit an average reduction of photonic emissions over the period monitored (Fig. 3Go). To evaluate the fraction of mammotropes responsive to the inhibitory effects of TGFß1, we pooled photonic activity for the 4 h immediately preceding and the last 4 h after commencement of TGFß1 infusion, and then plotted these values for each mammotrope studied. As shown in Fig. 4Go, TGFß1 reduced PRL gene expression in every mammotrope monitored, and the extent of this inhibition varied considerably from about 39% in some cells to 98% in others. In striking contrast, individual vehicle-treated cells exhibited random changes in PRL gene expression over the period of measurement; some cells showed an increase, whereas others displayed a decrease (Fig. 4Go).



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  		Figure 2. Photomicrographs depicting continuous monitoring of PRL gene expression in cells treated with TGFß1 or vehicle (VEH). Single pituitary cells within a grid on a photoengraved coverslip were microinjected with a rat PRL-luc plasmid and then immersed in serum-supplemented medium. Forty-eight hours later, transfected cells were preincubated in 0.1 mM luciferin-containing medium for 4 h. Then, the coverslip bearing the cells was mounted into a Sykes-Moore chamber and perifused with the same medium. Photons emitted from transfected cells were captured by a video-intensified photon camera and accumulated continuously in 30-min bins for a 17-h period. Treatments (1 ng/ml TGFß1 or VEH) were infused after an initial 4-h imaging period. Panels 1 (VEH-treated) and 5 (TGFß1-treated) show preexperiment, brightfield images of cells from representative experiments. The subsequent panels (2 3 4 6 7 8 ) represent photonic images captured from these same cells at the indicated times relative to treatment (TRT). Each individual amplified photonic signal is assigned a single pixel. Note, the progressive decline of photonic emissions in cells treated with TGFß1. Warmer colors here denote the convergence of multiple photonic signals consistent with the ascending color scale to the right of selected images.

 


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  		Figure 3. Dynamics of TGFß1 inhibition of PRL gene expression in individual mammotropes. Single pituitary cells that were transfected (via microinjection) with the rPRL-luc plasmid were subjected to continuous photonic imaging for 17 h as described in Fig. 2Go. This graph illustrates the average (mean + /or - SEM) responses of cells that were treated with 1 ng/ml TGFß1 (17 cells) or vehicle (VEH; 25 cells). The arrow represents the point at which infusions began. Data were normalized to a percentage of the basal level (which was calculated by averaging photonic emissions over the first 4 h before infusion). Note that there was a significant reduction (*, P < 0.05) of PRL gene expression within 4 h of infusion of TGFß1, and this decrease was even more pronounced as treatment progressed.

 


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  		Figure 4. TGFß1 decreases PRL gene expression in all mammotropes. To determine the percentage of mammotropes that responded to TGFß1, we averaged (for each cell) the photonic emissions accumulated (as in Fig. 2Go) for the 4 h immediately before and the last 4 h after initiation of treatment (1 ng/ml TGFß1 or VEH). As shown, all mammotropes treated with TGFß1 displayed a decrease in photonic activity (reflective of PRL-promoter driven luciferase activity). VEH-treated cells exhibited random changes in gene expression.

 
Immunoneutralization of TGFß1 increases PRL gene expression
Previous studies by others (5, 7) demonstrated that TGFß1 was secreted by primary cultures of anterior pituitary cells, and we found in the present study that superimposition of exogenous TGFß1 could reduce PRL gene expression. We reasoned that if secreted TGFß1 subserved a physiological role as an inhibitor of PRL gene expression, then removal of the peptide by immunoneutralization should increase the basal level of such activity. To test this line of reasoning, we transfected entire cultures of pituitary cells with rPRL-luc and later treated them with doses of a neutralizing antibody against TGFß1 that were found to be optimal in preliminary titration experiments. To be more specific, we selected doses of antibody (10 and 1 ng/ml) that yielded a maximal biological response and at which corresponding amounts of normal Ig-G (NIg-G) had effects indistinguishable from those in untreated controls. The results presented in Fig. 5Go reveal that exposure to anti-TGFß1 caused a significant augmentation of PRL gene expression at both doses of antibody tested (by 46% and 72% for the 1 and 10 ng/ml concentrations, respectively; P < 0.05).



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  		Figure 5. Immunoneutralization of endogenous TGFß1 increases PRL gene expression. Anterior pituitary cultures were transfected with the rPRL-luc reporter plasmid and treated with normal IgG (NIg-G) or one of two doses of an antibody against TGFß1 in the presence of serum. Luciferase reporter activity was measured after 48 h of treatment. Data were expressed as a percentage of NIg-G values. Bars represent the mean + SEM of five separate experiments. *, P < 0.05.

 
TGFß1 is a paracrine inhibitor of PRL gene expression
Data generated with entire cultures of pituitary cells identified secreted TGFß1 as an inhibitor of PRL gene expression. However, its precise route of action (autocrine vs. paracrine) remained to be established. In an attempt to distinguish between these possibilities, we returned again to our single cell paradigm for making real-time measurements of PRL gene expression from living mammotropes. The experimental protocol employed was to transfect cells as before (via microinjection) with rPRL-luc and to incubate them for the next 24 h in medium containing anti-TGFß1 or NIg-G. The next day, the cells were subjected to photonic imaging followed by immunocytochemical detection for PRL. Transfected cells identified as mammotropes at the end of the experiment were then categorized as singles (isolated from all other cells) or doublets (adjoined to just one other cell). The latter were further subgrouped as mammotropes attached to another mammotrope (M-M) or to a nonmammotrope (M-nonM). Interestingly, we found that PRL gene expression in M-M doublets treated with anti-TGFß1 was increased, on the average, by 57% (P < 0.05) compared with that in their NIg-G controls (Fig. 6Go). However, the level of PRL gene expression in M-nonM doublets exposed to anti-TGFß1 was not different (P > 0.05) from that in their respective controls (Fig. 6Go). Likewise, antibody treatment had no influence on the expression of the PRL gene by singles, indicating that TGFß1 does not exert its inhibitory influence in an autocrine manner.



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  		Figure 6. TGFß1 is a paracrine inhibitor of PRL gene expression. Anterior pituitary cells plated on grided glass coverslips and microinjected with the rPRL-luc plasmid were then bathed in serum-supplemented medium containing 10 ng/ml anti-TGFß1 or Normal Ig-G (NIg-G). Twenty-four hours later, the coverslips were assembled into Sykes-Moore chambers and exposed to 3 mM luciferin. After 15 min, chambers were mounted onto the stage of our photon capture system. Photonic emissions from transfected cells were captured and accumulated for a 10-min period. Then, cells were immediately fixed in B-5 buffered formalin for subsequent analysis by immunocytochemistry for rPRL. Transfected pituitary cells that were identified as mammotropes were grouped as singles (isolated from other cells in culture) or doublets (attached to one other cell). The latter group was further subcategorized as a mammotrope attached to another mammotrope (M-M) or to a nonmammotrope (M-nonM). Cells from 27 different dispersions were used for this study. The bars represent the mean ± SEM, and the numbers in parentheses denote the number of cells in that category. *, P < 0.05 vs. NIg-G-treated M-M doublets.

 
Inasmuch as averaged responses can mask the behavior of individual cells, we constructed scatterplots of M-M cells treated with anti-TGFß1 or NIg-G to determine whether all transfected mammotropes or just a subgroup were relieved from TGFß1 inhibition by antibody treatment. As shown in Fig. 7Go, only a subpopulation of cells in the M-M configuration displayed an increase in PRL gene expression after antibody treatment; the remainder were in the range exhibited by NIg-G-treated cells. Although it is difficult to quantify precisely the size of the responsive subpopulation, an arbitrary grouping of values above and below 3000 photonic emissions/10 min provides a convenient means for comparison. Using this demarcation, we found that the fraction of mammotropes exhibiting a high value of PRL gene expression was almost 5-fold greater in antibody-treated than in NIg-G-treated cultures (22.7% vs. 4.7%, respectively). When viewed as a whole, these findings indicate that TGFß1 acts in a paracrine manner to inhibit PRL gene expression by a distinct subpopulation of mammotropes.



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  		Figure 7. Scatterplots of mammotropes maintained as M-M doublets. For the sake of clarity and simplicity, we arbitrarily drew a line at 3000 photonic emissions/10 min to separate the high and low expressors. Raw data from Fig. 6Go were used to construct these plots. Note that a subpopulation of M-M cells that were treated with an antibody against TGFß1 expressed higher levels of photonic emissions than their respective controls (NIg-G).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented herein provide two lines of compelling evidence that TGFß1 is an intercellular mediator of PRL gene expression. Each line of evidence is supported by experiments with both population and single cell models. First, we showed that exogenous treatment with TGFß1 inhibited PRL promoter-driven reporter gene activity at the population level, and that it did so at a dose deemed to be physiological in that it mimicked the concentration (0.1–4 ng/ml) of TGFß1 measured by others in medium bathing primary cultures of pituitary cells (7, 22). In addition, exogenous TGFß1 had a similar overall effect on PRL gene expression within single mammotropes. This individual cell model, although more technically onerous than its population counterpart, was employed because of its increased power of analysis; it enabled us to determine not only whether all mammotropes responded to the inhibitory effects of TGFß1, but also whether there was heterogeneity among cells in the degree of response. Our results demonstrate clearly that all mammotropes exhibited a decrease in PRL gene expression after exposure to TGFß1. This finding was both interesting and unexpected, because we observed previously that the dopamine agonist bromocryptine inhibited PRL gene expression in only 88% of the single mammotropes tested; the remainder exhibited either no response or stimulation after dopamine treatment (19). Thus, the inhibitory action of TGFß1 on PRL gene expression rivals that of dopamine which is the consensus, primary, endogenous regulator of PRL release and gene expression. Another interesting point provided by single cell analysis in the present study was that the degree of inhibition evoked by TGFß1 was not homogeneous, a phenomenon also observed previously in our studies with the dopamine agonist bromocryptine. In the case of TGFß1, we found that the degree of inhibition within individual mammotropes ranged from 39–98% of the initial basal values.

The results of our immunoneutralization studies comprise the second line of evidence that TGFß1 functions as an intercellular regulator of PRL gene expression. Once again, this conclusion is supported by experiments conducted at the population as well as the single cell level. The antihuman TGFß1 antibody employed recognized the corresponding rat molecule with a high degree of specificity, in that it was shown by other investigators (23) and the distributor to have negligible cross-reaction (<2%) with the human TGFß2 or TGFß3 and by us (dot blot, data not shown) to not cross-react with human TGF{alpha} or ovine PRL. This antibody blocked inhibition of HT-2 (a murine cell line) cell proliferation imposed by addition of exogenous recombinant human TGFß1 in immunoneutralization studies conducted by the supplier. Moreover, it has also been successfully used as a neutralizing antibody in other in vitro assay systems (24, 25). When added to primary cultures of pituitary cells, this antibody induced a significant elevation of PRL gene expression as measured by PRL promoter-driven luciferase activity. This observation is entirely consistent with the hypothesis that TGFß1 exerts an inhibition of PRL gene expression that was relieved at least in part by antibody treatment. Our immunoneutralization experiments with the single cell model support the same conclusion. It is noteworthy that an identical dose of antibody (10 ng/ml) elevated PRL gene expression in populations of cells by almost exactly the same degree (72%) as it did in M-M doublets (57%). This is reassuring because the latter group, as shown here and previously (11), is proposed to be the target for most of the intercellular inhibition of PRL gene expression by a paracrine agent. Interestingly, removal of TGFß1 by immunoneutralization did not elevate PRL gene expression in all mammotropes but in only a subpopulation, accounting for about 23% of the total. At first glance, it is difficult to reconcile this observation with the aforementioned finding that all mammotropes are responsive to the inhibitory effects of TGFß1. A likely explanation for this apparent discrepancy is that the concentration of secreted TGFß1 in these cultures was not sufficient to maximally inhibit PRL gene expression in all cells. Therefore, superimposition of exogenous TGFß1 could cause an additional decrement in this parameter. An alternative possibility is that the amount of antiserum employed was insufficient to neutralize all of the TGFß1 produced locally, particularly in M-M microenvironments where the rate of TGFß1 secretion might be quite high. However, the likelihood of this possibility is diminished somewhat (but not discounted) by our observation in population studies (Fig. 5Go) that a 10-fold higher concentration of antibody (10 ng/ml) was no more effective in stimulating PRL gene expression than was the 1 ng/ml dose. The exact reason for this discrepancy notwithstanding, it is clear that locally produced TGFß1 exerts a tonic inhibitory influence over expression of the PRL gene.

Analysis of TGFß1 action and secretion at the single cell level also afforded us the opportunity to distinguish between putative autocrine and paracrine influences of the peptide. As is widely known, such distinctions are rarely attempted (thus the phrase autocrine/paracrine effect) because of the problems associated with evaluating whether a particular cell’s secretory product acted back on itself or on a neighbor. This problem was potentially more confusing in the present study in that virtually all primary mammotropes are reported to contain (and presumably secrete) TGFß1 as well as possess the appropriate receptors (26). Because we could monitor PRL gene expression in single transfected mammotropes maintained in both the single and M-M doublet configuration, we reasoned that the distribution of photonic values for singles and doublets should be very similar after antibody treatment if both autocrine and paracrine modes of inhibitory communication were operative. To the contrary, we found that removal of TGFß1 led to the appearance of a population of high expressors among M-M doublets that was not present in the singles. Thus, TGFß1 appears to exert its inhibitory effects on PRL gene expression almost exclusively via a paracrine route.

The intracellular mechanism(s) by which TGFß1 suppresses PRL gene expression remains to be established. It could operate secondary to activation of cytoplasmic signaling pathways through a TGFß1-inhibitory response element. This is a conserved 10-bp canonical sequence present in many TGFß1-inhibited genes such as stromelysin and hepatocyte growth factor (27, 28). This possibility is reinforced by the presence of two such putative TGFß1 inhibitory response elements in the 5'-flanking regions of the rat PRL gene, at positions -1016 and -1561, and both of these are included in the regulatory sequences of the reporter plasmid used in the present studies (16). The activation mechanism could also include nuclear proteins such as c-Fos or SMAD that are reported to mediate TGFß1 inhibition of gene expression in other systems (27, 29, 30, 31). Although identification of the relevant intracellular pathways is beyond the scope of the current investigation, their future elucidation will be critical to our understanding of how the paracrine signals described herein culminate in a biological response.

The negative effects of TGFß1 on various aspects of the PRL secretory pathway are strikingly similar to those reported for dopamine, the consensus PRL-inhibiting factor in mammals (32, 33). In this regard, both are reported to inhibit basal PRL release as well as that evoked by the stimulatory agents 17ß-estradiol (5, 12) and TRH (14). Likewise, both are reported to inhibit PRL gene expression as measured by steady state levels of PRL messenger RNA accumulation (16, 32) and real-time analysis of PRL promoter-driven reporter activity. These similarities invite speculation as to why seemingly redundant systems appear to operate in tandem to inhibit PRL secretion and gene expression. Although limited available evidence does not enable us to answer this intriguing question, the different mechanisms by which these agents are delivered to mammotropes may provide some insight into this phenomenon. TGFß1, of course, is produced largely by mammotropes and is believed to act locally within the anterior pituitary as a paracrine inhibitor of PRL release and gene expression. Dopamine, on the other hand, is secreted by hypothalamic neurons into blood that perfuses the vascular beds of the anterior pituitary. This "distant" inhibitor of mammotrope function derives from two distinct sources: the tuberoinfundibular dopaminergic neurons and the tubero-hypophyseal dopaminergic neurons. The former group of neurons terminate in the hypothalamic median eminence where they release their contents into capillaries associated with the long portal vessels that perfuse the outer zone of the anterior lobe (34). In contrast, the latter neurons terminate in the neurointermediate lobe, and their dopamine is transported to the inner zone of the anterior pituitary via the short portal vessels. Interestingly, these two zones are differentially responsive to dopamine inhibition; when tested in vitro, cells from the outer zone were quite unresponsive to dopamine action, whereas their inner zone counterparts were highly responsive (35). Given this scenario, it is tempting to speculate that hypothalamic dopamine may provide the primary inhibitory tone for inner zone mammotropes and that locally produced TGFß1 may subserve a comparable role for those cells of the outer zone. Resolution of this possibility must await the outcome of experiments aimed at establishing whether mammotropes from the different zones of the anterior pituitary gland are differentially responsive to TGFß1 in a manner opposite that of dopamine.

In summary, our results identify TGFß1 as a paracrine inhibitor of PRL gene expression. This conclusion is supported by our observations, with both population and single cell models, that exogenous treatment with TGFß1 suppresses PRL gene expression and that removal of the peptide by immunoneutralization augments the same parameter. Our findings about gene expression are entirely consistent with those of others who monitored TGFß1 action on PRL release (5, 14). Taken together, the local actions of TGFß1 bear a remarkable resemblance to those of hypothalamic dopamine delivered from a more distant site by hypophyseal portal blood. The physiological relevance and rationale (if any) for such regulatory redundancy will serve as the focus for some interesting future studies.


   Acknowledgments
 
The authors thank Dr. R. Mauer (Oregon Health Sciences) for the generous gift of the rPRL-luc plasmid, and D. C. Leaumont for assistance with imaging protocols.


   Footnotes
 
1 This work was supported by NIH Grant DK-38215 and USDA Competitive Research Grant 95–37206-2438. Back

Received June 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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