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Endocrinology Vol. 144, No. 5 2164-2171
Copyright © 2003 by The Endocrine Society

Antiproliferative Action of Calcitonin on Lactotrophs of the Rat Anterior Pituitary Gland: Evidence for the Involvement of Transforming Growth Factor ß1 in Calcitonin Action

Yong Qing Wang, Ren Yuan, Ya-Ping Sun, Tae-Jin Lee and Girish V. Shah

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas 79106

Address all correspondence and requests for reprints to: Girish V. Shah, Ph.D., Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, 1300 Coulter, Amarillo, Texas 79106. E-mail: girish.shah{at}ttuhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcitonin-like pituitary peptide, which is synthesized and secreted by gonadotrophs of the rat anterior pituitary (AP) gland, is a potent inhibitor of prolactin biosynthesis and lactotroph cell proliferation. Because TGF-ß1 is an autocrine inhibitor of lactotroph cell proliferation, we investigated a possibility that calcitonin (CT) interacts with TGF-ß1 to inhibit lactotroph cell proliferation.

The actions of CT on GGH3 cell proliferation were examined in the absence or presence of anti-TGF-ß1 serum. Subsequent experiments tested the effects of CT on TGF-ß1 mRNA abundance as well as TGF-ß1 synthesis. The studies also tested whether the stimulatory action of CT on TGF-ß1 mRNA expression involves stabilization of TGF-ß1 mRNA. Finally, the experiments investigated in vivo actions of CT on TGF-ß1 synthesis in the AP gland. This was accomplished by studying the changes induced by iv administered CT in TGF-ß1-immunopositive cell populations of adult female rat AP glands.

The results have shown that the inhibitory action of CT on proliferation of GGH3 cells was attenuated by rabbit anti-TGF-ß1 serum. Moreover, CT stimulated TGF-ß1 mRNA expression, as well as TGF-ß1 synthesis, in a dose-dependent fashion. Stimulatory action of CT on TGF-ß1 expression may be posttranscriptional, because it significantly increased TGF-ß1 mRNA stability. When administered in vivo, CT significantly increased TGF-ß1-immunopositive cell populations of adult female rat AP gland. Colocalization studies for prolactin and TGF-ß1 suggest that CT increased TGF-ß1 synthesis in lactotrophs, and possibly in nonlactotroph cell populations. These results suggest that antiproliferative action of CT on lactotrophs may, at least in part, be mediated by CT-induced TGF-ß1 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS KNOWN THAT the anterior lobe of the pituitary (AP) gland contains several cell types, each of which is present in different proportions (1, 2, 3). Moreover, the proportions of these cell types vary with changes in the hormonal milieu and physiological status. For example, the proportion of lactotrophs in the AP gland increases significantly, from 20% or less to as high as 50–70%, during pregnancy and lactation. This is followed by a rapid and massive loss of lactotrophs in the last few days of lactation, which brings the populations to prepregnancy levels (4, 5). These large changes in lactotroph cell density are managed predominantly by modulation of their rates of proliferation and apoptosis (5, 6, 7).

There is evidence to suggest that paracrine/autocrine factors secreted by lactotrophs or their neighboring cells play an important role in the regulation of lactotroph density of the AP gland (8, 9, 10, 11, 12). For example, autocrine peptides, such as galanin and vasoactive intestinal polypeptide, are shown to induce proliferation of lactotrophs and may mediate estrogen-induced increase in these cell populations (7, 13). In contrast, gonadotroph-derived pituitary-calcitonin (CT)-like peptide (pit-CT) and lactotroph-derived TGF-ß inhibit lactotroph cell proliferation, and they both are down-regulated by estrogens (14, 15, 16, 17, 18). This suggests that pit-CT, which not only inhibits lactotroph cell proliferation but also attenuates basal and TRH-induced prolactin (PRL) gene transcription, plays a critical role in the regulation of lactotroph cell populations. This possibility is supported by the previous findings that pit-CT expression varies significantly with the changes in endocrine status of the AP gland (11).

Because pit-CT and TGF-ß1 exert inhibitory actions on PRL secretion, PRL mRNA expression, and lactotroph proliferation, the objective of the present study was to test whether pit-CT and TGF-ß1 interact with each other or act in concert to modulate lactotroph cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
32P-Uridine triphosphate and 3H-methyl thymidine were purchased from NEN Life Science Products (Boston, MA). DMEM, penicillin G-streptomycin mixture, and horse and fetal calf sera were purchased from Life Technologies, Inc. (Grand Island, NY). TGF-ß was purchased from R & D laboratories (Minneapolis, MN), and anti-TGF-ß serum was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Synthetic salmon CT was purchased from Peninsula Laboratories, Inc. (Thousand Oaks, CA). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Animals
Sixty-day-old Fisher 344 rats were purchased from Harlan Sprague Dawley, Inc. (Milwaukee, WI), and housed two per cage. The animals were maintained under the conditions of 12 h of light, 12 h of darkness (lights on at 0600 h), with ad libitum access to tap water and rat chow (Ralston Purina Co., St. Louis, MO). The rats were killed by decapitation under ketamine anesthesia. All animal procedures were conducted according to the protocol approved by the institutional animal care and use committee of the Texas Tech University Health Sciences Center.

Cell line
GGH3 cell line, a variant of GH3 cell line stably expressing GnRH-receptor, was kindly provided by Dr. Michael Conn of Oregon Health Sciences University. The cells were thawed and grown in the complete medium (DMEM supplemented with 10 mM HEPES, 15% horse serum, 5% fetal calf serum, 280 µg/ml bacitracin, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate).

GGH3 cell culture and cell proliferation assay
3H-thymidine incorporation.
GGH3 cells were plated at the density of 10,000 cells per well in 24-well culture plates and allowed to attach in 500 µl complete medium for 48 h. The cells were rinsed with basal DMEM (supplemented with 10 mM HEPES, 0.3% BSA, 280 µg/ml bacitracin, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate), and treated with either CT and/or rabbit anti-TGF-ß1 serum (1:100), as well as 0.5 µCi 3H-thymidine for 24 h. At the end of incubation, the cells were rinsed with chilled PBS-1 µM thymidine, and incorporated radioactivity was determined in trichloracetic acid-precipitable fraction (16).

Proliferating cell nuclear antigen (PCNA) immunocytochemistry (ICC).
In a separate experiment, GGH3 cells were plated at the density of 5000 cells per well in polylysine-coated glass chamber slides and allowed to attach in 100 µl complete medium for 48 h. The cells were rinsed with basal DMEM (supplemented with 10 mM HEPES, 0.3% BSA, 280 µg/ml bacitracin, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate), treated with either CT and/or rabbit anti-TGF-ß1 serum (1:100), and incubated for 24 h. At the end of incubation, the cells were rinsed with chilled PBS, fixed in Zamboni’s solution for 1 h, washed three times with PBS, and incubated with the blocking solution (10% goat serum in 0.4% Triton X-100-PBS). This was followed by an overnight incubation with mouse anti-PCNA antibody (1:40). Subsequently, the cells were washed and incubated with biotinylated antimouse second antibody (1:200) for 3 h and processed for horse radish peroxidase staining using an ABC kit (Vector Laboratories, Inc., Burlingame, CA). The manufacturer’s instructions were followed. The brown color, indicative of PCNA staining, was observed in a Nikon (Melville, NY) Optiphot Microscope connected with Spot digital camera to a Power PC computer. Total PCNA-immunopositive cells were counted in 20 different high-power fields (x400). At least 1000 cells per each treatment group were counted by two researchers independently. The data are expressed as percent PCNA-immunopositive cells per field.

TGF-ß1 immunohistochemistry (IHC)
Adult cycling female rats (in diestrus) were iv injected with 50 µg CT in 100 µl saline and killed after 8 h. The AP glands were quickly removed, fixed in Zamboni’s solution for 2 h, and frozen by submersion in an isopentane-dry CO2 bath after mounting in the embedding medium (OCT compound, Tissu-Tek; Miles Laboratories, Elkhart, IN) as previously described (18). The frozen tissues were sliced to 5- to 10-µm-thick sections and thaw-mounted on Superfrost plus glass slides (Fisher Scientific, Pittsburgh, PA). The sections were processed for TGF-ß1 IHC using rabbit anti-TGF-ß1 serum (1:50, Santa Cruz Biotechnology, Inc.). The secondary antibody was tetramethylrhodamine isothiocyanate-conjugated rabbit anti-IgG serum (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The experiments were repeated two more times. Three animals per group were used for these experiments. Sections from all animals were processed simultaneously. Two researchers independently evaluated the slides, scoring all slides at the same time to avoid comparing preparations that had been stored or exposed to UV-light for different periods of time. The sections (at least 12/experiment from 3 different animals) were observed under a Nikon Optiphot microscope with epifluorescence attachment. The digital images were captured on a G3 Power PC computer by a Spot camera attached to the microscope. TGF-ß1-immunopositive cells per x400 field were counted in at least 5 fields for each control and treatment group. A total of 2000 cells from each group were counted. The data are expressed as number of TGF-ß1-immunopositive cells per x400 field.

For PRL and TGF-ß1 double immunochemistry, the procedure was similar except that two primary antisera (mouse antirat PRL kindly provided by Dr. Schammel, 1:3500; and rabbit anti-TGF-ß1 serum, 1:50) were used during the first overnight incubation at 4 C. After removal of primary antisera, the sections were treated with fluorescein isothiocyanate-conjugated antimouse and tetramethylrhodamine isothiocyanate-conjugated antirabbit secondary antibodies (1:50) for 3 h at room temperature in a light-sealed moist chamber. The slides were observed for PRL (green) and TGF-ß1 (red) staining as described before.

Plasmid constructs and riboprobe preparation
Plasmid pTGF-ß1 was 1.04 kbp rat TGF-ß1 cDNA, which was kindly provided by Dr. A. Singh (Genentech, Inc., San Francisco, CA). A 360-bp-long Bam HI-Eco RI fragment of this cDNA was subcloned in pBSK vector. The template for antisense TGF-ß1 riboprobe was obtained by linearizing the plasmid with SacII. The pTRI-ß-actin mouse antisense control template contained a 245-bp fragment of mouse cytoplasmic ß-actin gene, which extends from codon 220 to codon 303 (Ambion, Inc., Austin, TX). 32P-labeled antisense riboprobes for TGF-ß1 and ß-actin were transcribed using either T7 or T3 RNA polymerase. After transcription, the reaction mixtures were digested with ribonuclease-free deoxyribonuclease (Roche Molecular Biochemicals, Indianapolis, IN) and, after the riboprobes were extracted with phenol/chloroform, were precipitated in ethanol. The quality of riboprobes was tested by running a small aliquot in 8 M urea and 5% polyacrylamide gel.

RNA isolation and S1 nuclease protection assay
Total RNA from GGH3 cells was extracted by the modified method of Chomczynski and Sacchi as described by Xie and Rothblum (19). In brief, the cells were rinsed with prechilled PBS and lysed using a single step acid-guanidinium thiocyanate-phenol-chloroform extraction. Total RNA was precipitated in isopropanol. The precipitates were washed in 70% ethanol and dissolved in diethylpyrocarbonate-treated water.

Total RNA samples from the AP glands were analyzed for the abundance of TGF-ß1 and ß-actin separately, but in the same samples, by S1 nuclease protection assays as previously described (12, 16). In brief, 20 µg total RNA were incubated with appropriate antisense riboprobes (approximately 500,000 cpm) for 18 h at 42 C. Sense riboprobes served as negative controls. The samples were then digested with 50 U S1-nuclease for 30 min at 37 C. The protected RNA was fractionated on 8 M urea 5% polyacrylamide gel. The gel was then dried and autoradiographed. The autoradiograms were scanned on a GS-700 image analysis densitometer (Bio-Rad Laboratories, Inc., Hercules, CA). Each experiment was repeated three separate times.

Effect of CT on half-life (t1/2) of TGF-ß1 mRNA
GGH3 cells were plated at a density of 0.5 x 106 cells per 10 cm culture dish and cultured for 48 h. The cells were then rinsed with serum-free basal medium and incubated with 0.5 µg/ml actinomycin D, with/without 100 nM CT, for various incubation periods. At the end of incubation, the cells were rinsed with chilled PBS and lysed, and total RNA was extracted. The RNA samples were then processed to determine TGF-ß1 mRNA levels as described above.

The data points of TGF-ß1 mRNA degradation curves were fitted in a straight line as defined by the relationship: ln RT/R0 = -KD·t, where R0 is the level of TGF-ß1 mRNA before exposure to actinomycin D, RT is the level of TGF-ß1 mRNA at time t of actinomycin D treatment, and KD is the apparent decay constant. The t1/2 of TGF-ß1 mRNA was taken as 0.693/k, where k is the slope of ln RT/R0 vs. time t calculated by linear regression (20).

TGF-ß1 immunoreactivity in GGH3 cells: Western blotting
Cell extracts from GGH3 cells were prepared with Nonidet P-40 extraction buffer containing 25 mM Tris (pH 7.4), 10% glycerol, 1% Nonidet P-40, and 50 mM NaF and freshly supplemented with 10 mM sodium pyrophosphate, 1 mM sodium vanadate, and protease inhibitors (leupeptin at 10 µg/ml, aprotinin at 5 µg/ml, and phenylmethylsulfonyl fluoride at 1 mM). The cell lysate (50 µg proteins per lane for TGF-ß1; 5 µg per lane for ß-actin) was size-fractionated on 12.5% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. After rinsing with Western blot solution (100 mM Tris-HCl (pH 7.4), 100 mM MgCl2, 0.5% Tween20, 1% Triton-X-100, 1% BSA, 5% FCS), the membrane was incubated with 1:500 rabbit anti-TGF-ß serum (Santa Cruz Biotechnology, Inc.) or anti-ß-actin IgG (1:3000; Oncogene, San Francisco, CA), respectively, for 18 h at 4 C. The membranes were then incubated with 1:1000 antirabbit IgG-HRP and 1:1000 antimouse IgG-HRP, respectively, for 1 h at room temperature and then washed 3 times with Western blot solution and one time with PBS. The staining was visualized on the film, using an ECL Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ). The autoradiograms were scanned on a GS-700 imaging densitometer (Bio-Rad Laboratories, Inc.), and density of each band was determined using an NIH image analysis program. Each experiment was repeated three separate times.

Statistical analysis
The results are presented as mean ± SEM and were statistically evaluated by one-way ANOVA, and the significance was derived using Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CT attenuates GGH3 cell proliferation: possible role of TGF-ß in CT action
Our previous studies have shown that CT attenuates proliferation of lactotrophs and GH3 cells, and this can be reliably assessed by PCNA ICC (12). Because TGF-ß1 is an autocrine inhibitor of lactotroph proliferation (15), we tested whether CT-induced inhibition of GGH3 cells persists even when TGF-ß1 is depleted. The effect of CT and rabbit anti-TGF-ß1 serum on PCNA ICC, as well as 3H-thymidine incorporation in GGH3 cells, was examined. Controls received equivalent amounts of nonimmune rabbit serum. The results presented in Fig. 1Go, A and B, demonstrate that 1 µM CT caused a dramatic decrease in PCNA-immunopositive cell populations as well as DNA synthesis of GH3 cells. The similarity of results in these two systems suggests that 3H-thymidine incorporation can be used as an index of cell proliferation of GGH3 cells. The results presented in Fig. 1BGo show that CT decreased 3H-thymidine incorporation by almost 76%. As expected, anti-TGF-ß1 serum induced a significant, 19%, increase in 3H-thymidine incorporation. However, anti-TGF-ß1 serum significantly attenuated the inhibitory action of CT on GGH3 cell proliferation. This is indicated by the result that CT could reduce 3H-thymidine incorporation by only 25% in the presence of anti-TGF-ß1 serum.



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  		Figure 1. Effect of CT on proliferation of GGH3 cells. A, GGH3 cells cultures were incubated with CT (1 µM) and/or anti-TGF-ß1 (1:100) serum for 24 h at 37 C. The data are expressed as number of PCNA-immunopositive GGH3 cells per high power field (x400). The results were statistically evaluated by two-way ANOVA. Levels of significance: P < 0.0001 for CT; P < 0.0002 for anti-TGF-ß1; P < 0.0134 for interaction between two groups. B, GGH3 cells cultures were incubated with CT and/or anti-TGF-ß serum for 24 h at 37 C. Percentage of proliferating cells was determined by 3H-thymidine incorporation as described in Materials and Methods. The results are presented as mean DPM ± SEM 3H-thymidine incorporated. The results were statistically evaluated by two-way ANOVA. Levels of significance: P < 0.0001 for CT; P < 0.0001 for anti-TGF-ß1; P < 0.0001 for interaction between two groups. C, Control.

 
Effect of CT on TGF-ß1 expression in GGH3 cells: time course
Because the inhibitory action of CT on GGH3 cell proliferation was attenuated by anti-TGF-ß1 serum, we tested the effect of CT on TGF-ß1 mRNA expression. The results presented in Fig. 2Go demonstrate that 100 nM CT induced a marked increase in steady-state TGF-ß1 mRNA levels, but it took almost 4 h to induce an observable increase. A larger increase was observed after 8 h.



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  		Figure 2. Effect of CT on steady-state TGF-ß1 mRNA levels: time course. GGH3 cells were treated with 100 nM CT, for various time periods, at 37 C. The cells were then lysed, and total RNA was extracted. TGF-ß1 and ß-actin mRNA levels in these samples were determined by S1-nuclease protection assay. The experiment was repeated two more times. The results were digitized, normalized with ß-actin mRNA levels, and pooled. The results are expressed as mean ± SEM (n = 3) of normalized mRNA abundance. A representative autoradiogram is presented.

 
Increase of TGF-ß1 mRNA abundance by CT: dose response
The results presented in Fig. 3Go show that the stimulatory action of CT on TGF-ß1 mRNA abundance was dose-related, and a maximal increase of approximately 7-fold over the control was observed when treated with 1 µM CT for 8 h. The CT-induced increase in TGF-ß1 mRNA abundance was significant even at the lowest tested concentration of 0.1 nM. The calculated IC50 of CT was approximately 0.89 nM. This IC50 value is in the range of KD for high-affinity CT receptors that are in the range of 0.1–10 nM range (22).



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  		Figure 3. Effect of steady-state TGF-ß1 mRNA levels in GGH3 cells: dose response. GGH3 cells were treated with various concentrations CT for 8 h at 37 C. TGF-ß1 and ß-actin mRNA levels were determined by S1-nuclease protection assay. The cells were then lysed, and total RNA was extracted. TGF-ß1 (in 20 µg total RNA) and ß-actin mRNA levels (2 µg total RNA) in these samples were determined by S1-nuclease protection assays. The experiment was repeated two more times. The results were digitized, normalized with ß-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean ± SEM (n = 12) of normalized mRNA abundance. A representative autoradiogram is presented. *, Significantly different from control, P < 0.05.

 
CT increases stability of TGF-ß1 mRNA
The degradation of existing TGF-ß1 mRNA in the presence of actinomycin D was observed over a period of time (up to 24 h), and the t1/2 value of the mRNA was determined. We used 0.5 µg/ml actinomycin D to block TGF-ß1 transcription, and this also inhibited the 3H-thymidine uptake by more than 95% (data not shown). The results presented in Fig. 4Go suggest that TGF-ß1 mRNA levels of vehicle-treated GGH3 cells were relatively stable through the experimental period (top panel). In the presence of actinomycin D, the steady-state TGF-ß1 mRNA levels declined progressively over a period of 24 h, and only 15% of the initial mRNA levels remained at the end of the experiment (middle panel of Fig. 4Go). The presence of 100 nM CT significantly attenuated the rate of degradation of TGF-ß1 mRNA (bottom panel of Fig. 4Go). Approximately 55% of the initial mRNA remained at the end of incubation period, compared with 15% in the absence of CT. The degradation curves were analyzed for curve-fit straight line (r = 0.95, Act. D, P < 0.0001; r = 0.94, CT + Act. D; P < 0.0001; Fig. 5Go). The KD for TGF-ß1 mRNA degradation in actinomycin D-treated cells was 0.031. However, the addition of CT markedly decreased this value to 0.012. This curve yielded a t1/2 value of approximately 5.61 h for TGF-ß1 mRNA degradation in the absence of CT. However, the t1/2 value significantly increased (to 13.89 h) in the presence of 100 nM CT.



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  		Figure 4. Effect of CT on t1/2 of TGF-ß1 mRNA. A, GGH3 cells were treated with either CT (100 nM) and/or actinomycin D (AD; 0.5 µg/ml) for various time periods. The mRNA was extracted, and TGF-ß1 mRNA levels were determined by S1-nuclease protection assays. The experiment was repeated two more times. The results were digitized, normalized with ß-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean ± SEM (n = 12) of normalized mRNA abundance. A representative autoradiogram is presented. The TGF-ß1 mRNA levels (ln Rt/R0) were plotted against time t to determine the t1/2, as described in Results.

 


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  		Figure 5. Effect of CT on TGF-ß immunoreactivity. GGH3 cells were incubated with various concentrations of CT for 24 h, and cell lysates (50 µg proteins for TGF-ß1; 5 µg proteins for ß-actin) were fractionated by SDS-PAGE. The proteins were then electrically transferred to nitrocellulose, and TGF-ß immunoreactivity was determined using rabbit TGF-ß1 antiserum. Immunoreactive bands were visualized using the ECL Western Blot detection system. The experiment was repeated two more times. The results were digitized, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean ± SEM (n = 12) densitometric units. A representative autoradiogram is presented. *, Significantly different from 0.0, P < 0.05.

 
CT stimulates the synthesis of TGF-ß1 immunoreactive protein
This experiment examined the effect of CT on the synthesis of TGF-ß1 by GGH3 cells. The results presented in Fig. 5Go show that CT induced a dose-dependent increase in TGF-ß1 immunoreactivity, and a maximal increase of more than 10-fold over the control was observed when treated with 100 nM CT. ß-Actin immunoreactivity was tested as a control protein in those lysates, and its synthesis was not affected by CT.

CT increases TGF-ß1 immunoreactive cell populations of rat AP gland
To examine whether CT exerts a similar effect on TGF-ß1 synthesis in vivo, we injected 50 µg CT iv to cycling female rats on the day of diestrus. Our previous studies have shown that this CT treatment significantly decreased the proliferating lactotroph cell populations in the AP glands of diestrus rats (12). Eight hours after CT administration, the AP glands were obtained, and TGF-ß immunoreactivity in AP sections was examined by IHC. The representative micrographs presented in Fig. 6AGo show a marked increase in the intensity as well as the number of TGF-ß1 immunoreactive cell populations. A 3-fold increase in the number of TGF-ß1 immunoreactive cell population was observed in response to CT treatment. Further analysis of these sections for PRL (green) and TGF-ß1 (red) suggests that the predominant populations of TGF-ß1-immunopositive cells in the AP gland of vehicle-treated rats colocalized with PRL (Fig. 6BGo). However, in the CT-treated group, there was an observable increase in cell populations that expressed TGF-ß1 but not PRL.



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  		Figure 6. Effect of CT on TGF-ß1-immunopositive cell populations of rat AP gland. A, TGF-ß1 IHC. Three cycling female rats, in diestrus, were injected with 50 µg CT iv, and the AP glands were collected after 8 h. Three concurrent control rats received equivalent amounts of saline. The micrographs A (control) and B (CT-treated) represent typical profiles of TGF-ß1-immunopositive cells in the AP gland (x400). Total immunopositive cells per x400 field were counted in at least 12 different sections. The results (mean ± number of cells per high power field, x400) are presented in C. The results were statistically evaluated by one-way ANOVA and Newman-Keuls test (*, P < 0.05). B, Double IHC for PRL and TGF-ß1. The sections from A were simultaneously probed for PRL and TGF-ß1 with mouse antirat PRL and rabbit anti-TGF-ß1 sera as described in Materials and Methods. The micrographs (+CT and -CT) represent typical profiles of TGF-ß1 (red)- and PRL (green)-immunopositive cell populations in the AP gland (x400). The arrows indicate predominant colocalization of TGF-ß1 and PRL in same cells of control (-CT) AP sections. In contrast, the existence of numerous TGF-ß1-positive but PRL-negative cells can be seen in the AP sections of CT-treated rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because pit-CT and TGF-ß are both inhibitors of lactotroph cell proliferation (12, 15), the primary objective of the present study was to examine the action of CT on lactotroph cell proliferation when TGF-ß is depleted. The present results are consistent with our earlier results that CT is a potent inhibitor of lactotroph cell proliferation (12). Moreover, the present findings, that the inhibitory action of CT on GGH3 cell proliferation was comparable with that on primary rat AP cells, suggest that GGH3 cells are also responsive to CT. Although GGH3 cells express recombinant GnRH receptor, it has no relevance in the present study because we did not use GnRH in our experiments and used this cell line as an in vitro model for CT-responsive PRL cells.

The present studies report a new finding, that the inhibitory action of CT on lactotroph cell proliferation was significantly diminished in the presence of TGF-ß1 antiserum. This raises a new possibility that the inhibitory action of CT on lactotroph proliferation may, at least in part, be mediated by TGF-ß1. To evaluate the significance of this finding, we examined the effect of CT on steady-state levels of TGF-ß1 mRNA and protein. The results have shown that CT is a potent stimulator of steady-state TGF-ß1 mRNA levels. In vivo studies also demonstrated potent effect of CT on TGF-ß1 synthesis, as indicated by a dramatic increase in TGF-ß1-immunopositive cell populations. It is unlikely that this increase reflects an actual increase in the proliferation of TGF-ß1-immunopositive cells. It is more likely that the cell populations that were invisible by TGF-ß1 IHC before the CT treatment became visible because of increase in their TGF-ß1 content. Moreover, it is conceivable that the stronger in vivo effect of CT on TGF-ß1 synthesis in lactotrophs may be a combination of its direct effect on lactotrophs and an indirect action mediated through its action on hypothalamic dopamine synthesis (23, 24, 25). The results from double IHC are qualitative but present an interesting finding. Predominant populations of lactotrophs in the AP glands of vehicle-treated rats displayed colocalization of TGF-ß1 with PRL. However, CT treatment significantly altered this profile. There was an apparent increase in PRL-negative TGF-ß1-immunopositive cells. This may have occurred because of two possibilities: 1) It is likely that the CT treatment would have reduced PRL content of some lactotrophs to undetectable levels, while increasing their TGF-ß1 content. 2) Alternatively, CT may have increased TGF-ß1 content in nonlactotroph AP cell populations. There is evidence for the synthesis of TGF-ß1 by several AP cell types, including gonadotrophs and thyrotrophs (26). Additional studies will be necessary to examine the role of CT in regulation of TGF-ß1 synthesis in nonlactotroph AP cell populations.

In consistence with CT-induced increase in TGF-ß1 immunoreactivity in AP cells, CT also increased TGF-ß1 mRNA abundance in GGH3 cell populations. The increase in steady-state mRNA levels could occur as a consequence of either an increase in transcription or the decrease in mRNA stability. We first investigated the latter possibility. This is because stabilization represents an important posttranscriptional mechanism to regulate steady-state levels of various mRNAs, including TGF-ß mRNAs (20, 21). This phenomenon has been well characterized for a number of protooncogenes with short t1/2 periods and occurs via the binding of a trans-acting factor to the 3'-untranslated region of the transcript (28). To investigate the effect of CT on TGF-ß1 mRNA stability, the kinetics of TGF-ß1 mRNA degradation was examined in the presence of actinomycin D, a potent inhibitor of gene transcription (20, 21). The treatment with actinomycin D caused a progressive decline of TGF-ß1 mRNA levels, with a t1/2 of 5.6 h. CT attenuated this decline and increased the t1/2 of the mRNA to 13.9 h. On the basis of these observations, it seems that CT increases TGF-ß1 mRNA stability. However, signaling mechanisms associated with this CT action remain to be elucidated. Previous evidence from this and other laboratories suggest that CT inhibits PRL secretion by inhibiting TRH-induced inositol phosphate generation, attenuating mobilization of intracellular calcium, and inhibiting p42/44 MAPK activity (22, 29, 30, 31). It is conceivable that either of these mechanisms could increase TGF-ß1 mRNA stability. However, additional studies will be necessary to investigate this possibility.

Previous studies from this and other laboratories have reported several effects of CT on lactotroph function. For example, CT inhibits basal and TRH-induced PRL secretion from lactotrophs in a matter of minutes, and this may be mediated by inhibitory actions of CT on inositol phosphates generation and biphasic increases in cytosolic calcium. Similarly, CT also inhibits PRL gene transcription in GH3 cells. However, both these actions are acute and can be observed in a period of a few minutes to half an hour. In contrast, the inhibitory actions of CT on TGF-ß1 expression, as well as GGH3 cell proliferation, required several hours. These results raise a possibility that the long-term actions of CT on lactotroph cell proliferation may be mediated by pit-CT-inducible factors, such as TGF-ß1.

It has been known that the AP gland is a heterogeneous gland consisting of various distinct cell types secreting specific pituitary hormone. In addition, AP cells secrete numerous paracrine/autocrine peptides, which include activin, inhibin, TGF-ß, galanin, vasoactive intestinal polypeptide, and pit-CT (7, 15, 32, 33, 34). Although specific role(s) for these peptides has not yet been defined, they have been shown to produce multiple effects on their target cells, which include changes in secretory activity, in responsiveness to their tropic hormones, and in rates of proliferation and/or apoptosis. It is conceivable that autocrine/paracrine factors like pit-CT play a prominent role in determining proportion of their target cells in the AP gland.

In brief, our present results, that CT inhibits lactotroph cell proliferation and also stimulates TGF-ß1 synthesis in lactotrophs as well as GGH3 cells, raise a possibility that CT may amplify its own action by stimulating TGF-ß1 synthesis. Significance of this CT action in pituitary remodeling will have to be investigated.


   Acknowledgments
 
The authors gratefully acknowledge Drs. A. Singh and Michael Conn for providing plasmid TGF-ß1 cDNA and GGH3 cell line, respectively. We also thank Dr. Jonathan Schammel for mouse antirat PRL serum.


   Footnotes
 
This work was supported by NIH Grants DK-45044 and CA-96534.

Abbreviations: AP, Anterior pituitary; CT, calcitonin; ICC, immunocytochemistry; IHC, immunohistochemistry; KD, dissociation constant; PCNA, proliferating cell nuclear antigen; pit-CT, pituitary-calcitonin-like peptide; PRL, prolactin; t1/2, half-life.

Received July 22, 2002.

Accepted for publication January 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aoki A, Pasolli HA, Torres AI 1994 Pituitary plasticity as correlate of secretory activity. Exp Toxicol Pathol 46:435–436[Medline]
  2. Orgnero de Gaisan E, Maldonado C, Diaz Gavier MF, Aoki A 1997 Diversity of pituitary cells in primary cell culture. An immunocytochemical study. Anat Anz 179:453–460[Medline]
  3. Pasolli HA, Torres AI, Aoki A 1992 Influence of lactotroph cell density on prolactin secretion in rats. J Endocrinol 134:241–246[Abstract]
  4. Aoki A, de Gaisan EO, Pasolli HA, Torres AI 1996 Disposal of cell debris from surplus lactotrophs of pituitary gland. Exp Clin Endocrinol Diabetes 104:256–262[Medline]
  5. Aoki MP, Maldonado CA, Aoki A 1998 Apoptotic and non-apoptotic cell death in hormone-dependent glands. Cell Tissue Res 291:571–574[CrossRef][Medline]
  6. Perez RL, Machiavelli GA, Romano MI, Burdman JA 1986 Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats. J Endocrinol 108:399–403[Abstract]
  7. Wynick D, Small CJ, Bacon A, Holmes FE, Norman M, Ormandy CJ, Kilic E, Kerr NC, Ghatei M, Talamantes F, Bloom SR, Pachnis V 1998 Galanin regulates prolactin release and lactotroph proliferation. Proc Natl Acad Sci USA 95:12671–12676[Abstract/Free Full Text]
  8. Allaerts W, Mignon A, Denef C 1991 Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture. Cell Tissue Res 263:217–225[CrossRef][Medline]
  9. Baes M, Allaerts W, Denef C 1987 Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates. Endocrinology 120:685–691[Abstract]
  10. Seuntjens E, Vankelecom H, Quaegebeur A, Vande Vijver V, Denef C 1999 Targeted ablation of gonadotrophs in transgenic mice affects embryonic development of lactotrophs. Mol Cell Endocrinol 150:129–139[CrossRef][Medline]
  11. Chronwall BM, Sands SA, Li Z, Shah GV 1996 Calcitonin-like peptide containing gonadotrophs are juxtaposed to cup-shaped lactotrophs. Endocr J 4:27–33
  12. Shah GV, Chien J, Sun YP, Puri S, Ravindra R 1999 Calcitonin inhibits anterior pituitary cell proliferation in the adult female rats. Endocrinology 140:4281–4291[Abstract/Free Full Text]
  13. Cai A, Hayes JD, Patel N, Hyde JF 1999 Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia. Endocrinology 140:4955–4964[Abstract/Free Full Text]
  14. De A, Morgan TE, Speth RC, Boyadjieva N, Sarkar DK 1996 Pituitary lactotrope expresses transforming growth factor ß (TGF ß) type II receptor mRNA and protein and contains 125I-TGF ß 1 binding sites. J Endocrinol 149:19–27[Abstract]
  15. Minami S, Sarkar DK 1997 Transforming growth factor-beta 1 inhibits prolactin secretion and lactotropic cell proliferation in the pituitary of oestrogen-treated Fischer 344 rats. Neurochem Int 30:499–506[CrossRef][Medline]
  16. Ren Y, Chien J, Sun YP, Shah GV 2001 Calcitonin is expressed in gonadotropes of the anterior pituitary gland: its possible role in paracrine regulation of lactotrope function. J Endocrinol 171:217–228[Abstract]
  17. Shah GV, Deftos LJ, Crowley WR 1993 Synthesis and release of calcitonin-like immunoreactivity by anterior pituitary cells: evidence for a role in paracrine regulation of prolactin secretion. Endocrinology 132:1367–1372[Abstract]
  18. Li Z, Shah GV 1995 Estrogen attenuates expression of calcitonin-like immunoreactivity in the anterior pituitary gland. Endocr J 3:452–459
  19. Xie WQ, Rothblum LI 1991 Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques 11:324–327[Medline]
  20. Abdelaziz N, Colombo F, Mercier I, Calderone A 2001 Nitric oxide attenuates the expression of transforming growth factor-ß3 mRNA in rat cardiac fibroblasts via destabilization. Hypertension 38:261–266[Abstract/Free Full Text]
  21. Lee N, Earle-Hughes J, Fraser C 1994 Agonist-mediated destabilization of m1 muscarinic acetylcholine receptor mRNA. Elements involved in mRNA stability are localized in the 3'-untranslated region. J Biol Chem 269:4291–4298[Abstract/Free Full Text]
  22. Judd AM, Kubota T, Kuan SI, Jarvis WD, Spangelo BL, MacLeod RM 1990 Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phosphates production. Endocrinology 127:191–199[Abstract]
  23. Arbogast LA, Shah GV, Voogt JL 1999 3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity. Endocrinology 140:3273–3281[Abstract/Free Full Text]
  24. Tohei A, VandeGarde B, Arbogast LA, Voogt JL 2000 Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action. Neuroendocrinology 71:327–332[CrossRef][Medline]
  25. Pagani F, Netti C, Guidobono F, Lattuada N, Ticozzi C, Sibilia V 1998 Effects of amylin and salmon calcitonin on ß-endorphin-induced growth hormone and prolactin secretion in the rat. Neuroendocrinology 68:220–228[CrossRef][Medline]
  26. Qian X, Jin L, Grande JP, Llyod RV 1996 Transforming growth factor-ß and p27 expression in pituitary cells. Endocrinology 137:3051–3060[Abstract]
  27. Izzo Jr N, Tulenko T, Colucci W 1994 Phorbol esters and norepinephrine destabilize alpha 1B-adrenergic receptor mRNA in vascular smooth muscle cells. J Biol Chem 269:1705–1710[Abstract/Free Full Text]
  28. Alberta J, Rundell K, Stiles C 1994 Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation. J Biol Chem 269:4532–4538[Abstract/Free Full Text]
  29. Shah GV, Wang W, Grosvenor CE, Crowley WR 1990 Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretogogue-induced increases in cytosolic Ca2+. Endocrinology 132:621–628
  30. Zhang Q, Stanley SM, Shah GV 1995 Calcitonin inhibits prolactin gene transcription in rat pituitary cells. Endocr J 3:445–451
  31. Ren Y, Sun Y-P, Shah GV, Calcitonin inhibits prolactin promoter activity in rat pituitary GGH3 cells: evidence for the involvement of p42/44 mitogen-activated protein kinase in calcitonin action. Endocrine, in press
  32. Schwartz J 2001 Intrapituitary interactions: another level of endocrine regulation. Clin Exp Pharmacol Physiol 28:237–238[CrossRef][Medline]
  33. Maiter DM, Hooi SC, Koenig JI, Martin JB 1990 Galanin is a physiological regulator of spontaneous pulsatile secretion of growth hormone in the male rat. Endocrinology 126:1216–1222[Abstract]
  34. Todd JF, Small CJ, Akinsanya KO, Stanley SA, Smith DM, Bloom SR 1998 Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 139:4222–4229[Abstract/Free Full Text]




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