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Endocrinology Vol. 143, No. 10 3759-3765
Copyright © 2002 by The Endocrine Society

ARTICLE

Transforming Growth Factor-ß Stimulates Vascular Endothelial Growth Factor Production by Folliculostellate Pituitary Cells

U. Renner, P. Lohrer, L. Schaaf, M. Feirer, K. Schmitt, C. Onofri, E. Arzt and G. K. Stalla

Max-Planck-Institute of Psychiatry (U.R., P.L., L.S., M.F., K.S., C.O., G.K.S.), Department of Endocrinology, Munich D-80804, Germany; and Laboratorio de Fisiologia y Biologia Molecular (E.A.), Department de Biologia, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EHA, Argentina

Address all correspondence and requests for reprints to: Ulrich Renner, Ph.D., Max Planck Institute of Psychiatry, Department of Endocrinology, Kraepelinstrasse 10, D-80804 Munich, Germany. E-mail: renner{at}mpipsykl.mpg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGF-ß isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-ß has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-ß. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-ß1, -ß2, and -ß3. They also express TGF-ß receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-ßbinding and signaling. Stimulation of TtT/GF cells with either TGF-ß1 or TGF-ß3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-ß isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-ß-induced VEGF production by cycloheximide suggest that TGF-ß induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-ß1 and -ß3 stimulated VEGF production. TGF-ß stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TGF-ß belongs to the TGF-ß protein superfamily, which at present consists of more than 30 different members, including activin, inhibin, and bone morphogenetic proteins (1). Three isoforms of TGF-ß (TGF-ß1, -ß2, -ß3) exist that are pleiotropic factors that exert multiple biological effects on epithelial, endothelial, lymphoid, myeloid, and mesenchymal cell types (1, 2). TGF-ß isoforms bind to heterodimers of TGF-ß type 1 and type 2 receptors (TGF-ßR1, TGF-ßR2) that then phosphorylate cytoplasmic, TGF-ß receptor-specific R-Smad proteins Smad2 and Smad3 (1, 3). The latter two form a complex with phosphorylated Smad4, which represents a so-called co-Smad because it is also involved in the signal transduction pathway of other members of the TGF-ß protein superfamily (1, 3, 4). The Smad2/3/4 protein complex is transported to the cell nucleus and interacts with different DNA-binding cofactors, as well as coactivators, or repressors, which induce or suppress the transcription of numerous target genes (1, 3, 4).

In the normal anterior pituitary, TGF-ß1 and TGF-ß3 expression were found in lactotroph cells (5, 6, 7). Recently, folliculostellate (FS) pituitary cells have also been identified as a source of TGF-ß1 (8). To our knowledge, no studies on the intrapituitary production of TGF-ß2 have been performed.

TGF-ß1 was found to inhibit prolactin (PRL) secretion as well as lactotroph cell proliferation (7, 9, 10). In contrast, TGF-ß3 stimulated lactotroph cell growth (7, 11) and could therefore possibly enhance PRL secretion by increasing lactotroph cell numbers. Recently, FS cells have been shown to be another intrapituitary target for TGF-ß, as basic fibroblast growth factor (bFGF) production was stimulated by TGF-ß3 (12), and FS cell proliferation was enhanced by TGF-ß1 (8). TGF-ß2 was shown to suppress PRL mRNA expression in lactosomatotroph GH3 tumor cells (13), but its role in the pituitary is not yet clear. However, interestingly, intrapituitary TGF-ß1 and TGF-ß3 production is regulated by estradiol (E2) in different manners (7, 14), suggesting that the intrapituitary effects of estrogens may in part be mediated by TGF-ß isoforms (15).

Vascular endothelial growth factor (VEGF) has been shown to be expressed in normal anterior pituitary exclusively in FS cells, whereas no VEGF was detected in hormone-producing cells (16). It seems that the latter cells acquire the ability to produce VEGF only after transformation to tumor cells, because lactosomatotroph GH3, corticotroph AtT20, and human pituitary tumor cells secrete VEGF (17). Expression of the two VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), exclusively on endothelial cells (18) indicates that this factor has no direct influence on endocrine cells. Production of VEGF by FS cells suggests that one of the different, and little understood functions of this cell type (19), could be to mediate VEGF-induced effects on the intrapituitary endothelium. VEGF is needed to maintain vascular integrity and represents the most potent stimulator of vascular permeability and endothelial cell proliferation (18). VEGF production by FS cells is stimulated by IL-6 and pituitary adenylate cyclase-activating peptide, whereas glucocorticoids represent potent inhibitors of VEGF secretion (20).

We have tested whether both TGF-ß1 and TGF-ß3 are also effectors of VEGF production in this cell type. We focused our interest on TGF-ß1 and -ß3 because these isoforms seem to be most relevant to the pituitary, as they exert opposing effects on lactotrophs (7), and they are differently regulated by E2 (7, 14). To study the effect of TGF-ß isoforms on VEGF production, the FS TtT/GF mouse pituitary tumor cell line (21), which exhibits most of the characteristics of normal FS cells (8, 20), was used as a model. The effect of TGF-ß on VEGF secretion was comparatively studied in VEGF-producing (17) and TGF-ß-responsive (22) lactosomatotroph GH3 rat pituitary tumor cells. The results obtained with TtT/GF cells were further proven in normal VEGF-producing FS cells of rat pituitary cell cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture materials and reagents were obtained from Life Technologies, Inc. (Karlsruhe, Germany), Falcon (Heidelberg, Germany), Nunc (Wiesbaden, Germany), Seromed (Berlin, Germany), Flow (Meckenheim, Germany), and Sigma (St. Louis, MO); dexamethasone and cycloheximide were also provided by the latter. TGF-ß1 and TGF-ß3 were purchased from R&D Systems (Wiesbaden, Germany).

Cell culture
TtT/GF and GH3 cells were grown in 48 multiwell plates at 37 C and 5% CO2 in DMEM (pH 7.3) supplemented with 2% FCS, 2.2 g/liter NaHCO3, 10 mM HEPES, 2 mM glutamine, 2.5 mg/liter Amphotericin B, 105 U/liter penicillin-streptomycin, 5 mg/liter insulin, 5 mg/liter transferrin, 20 mg/liter sodium selenite, and 30 pM T3.

For primary cell culture, pituitaries from male Sprague Dawley rats (Charles River Laboratories, Sulzfeld, Germany) (150–180 g) were enzymatically and mechanically dispersed as previously described (23). Dispersed cells were seeded at an initial density of 100,000 cells per well in 48-well plates and cultivated in D-Val-MEM (to suppress fibroblast growth) supplemented with 10% FCS and the additives indicated above for TtT/GF cell culture. After an initial attachment period of 48 h, the cells were used for VEGF stimulation experiments.

Stimulation and measurement of VEGF
TtT/GF cells were cultured until they had formed a confluent monolayer (approximately 70,000 cells per well in 48-well plates). At this stage, as previously reported, the cells did not grow further (24). GH3 cells were cultivated until they reached approximately 50% confluence. Rat pituitary cells were used after the attachment period.

TtT/GF, GH3, and rat pituitary cells were washed with PBS, and serum-free culture medium was added for 24 h to remove any remaining serum. The cells were then washed again with PBS and stimulation experiments with TGF-ß1 or -ß3 were performed in stimulation medium as indicated. The medium used for stimulation was without FCS but contained 1% BSA to prevent both TGF-ß and VEGF binding to the plastic surface of the wells. Whereas the cell numbers of TtT/GF cells and rat pituitary cells did not change significantly during VEGF stimulation, GH3 cells continued to proliferate in serum-free stimulation medium. Therefore, before and after the treatment period, cell numbers were routinely monitored. Cell numbers were determined with an adapted Coulter counter as previously described (24). In experiments, in which the effect of cycloheximide on TGF-ß-induced VEGF production by TtT/GF cells was studied, cycloheximide was added 1 h before TGF-ß. At the end of these experiments the cell viability was examined by acridine orange/ethidium bromide staining to exclude toxic effects of cycloheximide on TtT/GF cells.

Mouse and rat VEGF was measured according to the manufacturers’instructions by ELISA (R&D Systems) as previously reported (17). The detection limit of the assay was 3 pg/ml. The intraassay and interassay coefficients of variation were 3.4%, and 6.4%, respectively.

In supernatants of TGF-ß-stimulated GH3 cells, rat PRL (detection limit: 1 ng/ml) was measured by RIA (23) with material kindly provided by Dr. Parlow from the National Hormone & Pituitary Program (Torrance, CA).

RT-PCR for murine TGF-ß isoforms, TGF-ß receptors, and Smad proteins
For RT-PCR, total cellular RNA was isolated from TtT/GF cells and mouse anterior pituitary by guanidinium isothiocyanat followed by the phenol-chloroform method (25). RT of 1 µg RNA was performed with Superscript-II (Life Technologies, Inc.) for 1 h at 45 C followed by a denaturation step at 94 C for 1 min. With the cDNA template obtained, 35-cycle PCR was performed with specific primers (Table 1Go). Each cycle consisted of denaturation at 94 C for 1 min, annealing of primers at 60 C for 1 min, and chain extension at 72 C for 1 min. Amplified products were electrophoresed in 1.8% agarose gel and stained with ethidium bromide.


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  		Table 1. Primer sequences and cDNA fragment size

 
Previously published (26) specific primers were used to detect murine TGF-ß mRNA isoforms and TGF-ß receptors as well as Smad2, Smad3, and Smad4 mRNA (Table 1Go).

Detection of Smad2 protein in cell lysates and nuclear extracts
TtT/GF cells were cultivated in 6-well plates until they were confluent (approximately 600,000 cells per well). Serum washout was performed as described above. The cells were stimulated in serum-free stimulation medium with TGF-ß1 or TGF-ß3 for 5, 10, and 15 min and were then immediately placed on ice. After the cells had been washed twice with ice-cold TBS buffer (2.42 g/liter Tris base; 8 g/liter NaCl, pH 7.6) whole cell lysates were obtained as previously described (27).

Nuclear cell extracts were prepared at less than 4 C according to the method of Schreiber et al. (28). In brief, cells were scraped off from the wells and centrifuged in TBS at 1500 x g for 5 min. The cell pellet was resuspended in 1 ml TBS, transferred to an Eppendorf (Hamburg, Germany) tube and centrifuged in an Eppendorf microfuge for 15 sec. The pellet was resuspended in 400 µl ice-cold swelling buffer (pH 7.9) consisting of 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. After 15 min on ice, 25 µl of Igepal CA-630 (Sigma) solution (10% in water) was added. The suspension was vortexed for 10 sec and centrifuged for 30 sec in an Eppendorf microfuge. The supernatant was carefully removed and the nuclear pellet was washed by resuspension in swelling buffer and subsequent centrifugation. Finally the nuclear pellet was resuspended in sample buffer composed of 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The protein content of whole cell lysates and nuclear extracts was determined with the Bradford method. Equal amounts of cell lysates and nuclear extracts protein were fractionated on a 10% sodium dodecyl sulfate-polyacryalamide gel, and then electrotransferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Smad2 was detected on the membranes with a kit obtained from Transduction Laboratories, Inc. (Lexington, KY). In brief, the membranes were treated with 5% wt/vol nonfat milk protein in TBST [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween-20] for 1 h at room temperature, and then overnight at 4 C with the primary antibodies diluted 1:500 in 2.5% nonfat milk protein in TBST. Following a 20-min wash in TBST, the membranes were incubated at room temperature for 1 h in a 1:2000 dilution of the hydrogen peroxidase-conjugated secondary antibody in 2.5% nonfat milk protein in TBST. The membranes were washed in TBST and then incubated with LumiGLO for 1 min at room temperature and then immediately exposed to Lumi-Film chemiluminescent detection film (Roche, Mannheim, Germany).

Statistics
Each of the experiments was repeated at least three times. VEGF stimulation experiments were performed with quadruplicate wells. ANOVA in combination with Scheffé’s test was used for statistics. The data are expressed as mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TGF-ß isoform mRNA, TGF-ß receptors, and Smad proteins in mouse anterior pituitary and TtT/GF cells
With RT-PCR, using specific primers, all three isoforms of TGF-ßmRNA could be detected in RNA preparations of mouse anterior pituitary and TtT/GF cells (Fig. 1Go). Expression of TGF-ß receptors 1 and 2 mRNA, as well as Smad2, Smad3, and Smad4 mRNA, was also found. Expression of both TGF-ß and TGF-ß receptors in TtT/GF cells suggests that TGF-ß may regulate FS cell function in an autocrine manner.



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  		Figure 1. Detection of TGF-ß isoforms, TGF-ß receptors, Smad2, Smad3, and Smad 4 mRNA expression by RT-PCR in mouse anterior pituitary (lane 2) and TtT/GF cells (lane 3). Each of the different amplification products had the predicted size indicated in Table 1Go. Bands were absent in negative controls where RT was omitted from RNA extracts obtained from TtT/GF cells (lane 1) or mouse anterior pituitary (not shown).

 
Translocation of Smad2 to the nucleus after TGF-ßstimulation
Signaling of TGF-ß isoforms is known to be associated with translocation of the Smad2/Smad3/Smad4 protein complex to the cell nucleus (1, 3). We have therefore studied the appearance of Smad2, as a representative member of this protein complex, in the cell nuclei of TtT/GF cells. We observed a rapid increase in Smad2 protein expression in extracts of cell nuclei from TtT/GF cells after stimulation with TGF-ß1 or TGF-ß3 (Fig. 2Go, A and B). Maximum expression of Smad2 in nuclear extracts was observed after 15 min. Stimulation of TtT/GF cells with TGF-ß1 and -ß3 for 30 or 60 min did not further increase nuclear Smad2 expression (not shown). Smad2 expression in lysates of whole cells did not change in response to TGF-ß1 (Fig. 2CGo) or TGF-ß3 (not shown). Identical Smad2 bands in immunoblots of lysates of whole cells, indicate that alterations in Smad2 expression are specific for nuclear extracts and are not experimental artifacts, since after protein extraction of nuclei or whole cells, further experimental steps were basically identical. Moreover, the increase in nuclear Smad2 was observed in three independent experiments for each TGF-ßisoform.



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  		Figure 2. Detection of Smad2 protein in extracts of cell nuclei of TtT/GF cells after stimulation with 1 ng/ml TGF-ß1 (A) or TGF-ß3 (B). Before stimulation, cells had been cultivated for 24 h in serum-free medium. By immunoblotting, increasing expression of Smad2 was observed within minutes after stimulation of TtT/GF cells with TGF-ß isoforms, whereas little or no changes of Smad2 expression was observed in controls (co), in which no TGF-ß was added to the cells. Smad2 expression in lysates of whole TtT/GF cells did not change after TGF-ß1 (C) or TGF-ß3 (not shown) stimulation.

 
Effects of TGF-ß1 and TGF-ß3 on VEGF secretion by TtT/GF and GH3 cells
Both TGF-ß1 and TGF-ß3 dose-dependently stimulated VEGF secretion by TtT/GF cells (Fig. 3Go, A and B). The stimulatory pattern was identical for both TGF-ß isoforms. Significant VEGF stimulation was achieved at 100 pg/ml of TGF-ß1 and TGF-ß3. A maximum 5- to 10-fold stimulation of VEGF production (depending on the passage number of the cells) was achieved with 10 ng/ml TGF-ß, whereas higher concentrations (20, 50 ng/ml) of both TGF-ß1 and -ß3 did not further stimulate production (data not shown). Although the use of 10-fold increments is critical, calculation of ED50 values for the effect of TGF-ß1 and -ß3 on VEGF production was performed. Based on three independent experiments, the ED50 values for TGF-ß1 and TGF-ß3 were 0.73 ± 0.09 ng/ml and 0.69 ± 0.17 ng/ml, respectively. In contrast to TtT/GF cells, VEGF production by GH3 cells was not affected by TGF-ß1 and -ß3 (Fig. 3CGo). However, PRL secretion was significantly inhibited by TGF-ß1 and -ß3 in a dose-dependent manner (data not shown), indicating that the GH3 cells are responsive to TGF-ß.



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  		Figure 3. Effect of TGF-ß1 and TGF-ß3 on VEGF secretion in TtT/GF and GH3 cells. Both TGF-ß isoforms dose-dependently stimulated VEGF release in TtT/GF cells (A, B), whereas the basal VEGF production was not affected in GH3 cells (C). Cells were stimulated for 24 h. Cell numbers were determined at the end of the stimulation period to calculate VEGF production as picograms per 1000 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. untreated cells.

 
Time course studies showed no short-term effects for TGF-ß1 (Fig. 4Go) or TGF-ß3 on VEGF secretion (data not shown). Only prolonged TGF-ß treatment resulted in significant VEGF production. This indicates that TGF-ß stimulates de novo synthesis of VEGF as was previously discussed for other VEGF-regulating factors (20). This assumption was confirmed by the observation that the translation inhibitor cycloheximide (10 µg/ml) completely suppressed TGF-ß-induced VEGF release (Fig. 4Go). The inhibitory effect of cycloheximide was not due to a toxic effect because, even after a stimulation period of 24 h, the cell viability of cycloheximide-treated TtT/GF cells did not differ from untreated control cells.



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  		Figure 4. Time course of TGF-ß1-stimulated VEGF secretion in TtT/GF cells, and the influence of cycloheximide. Cells were stimulated with 10 ng/ml TGF-ß1 (horizontal striped bars) for the indicated times and VEGF production was calculated as picograms of VEGF per 1000 cells (see legend to Fig. 3Go). Within the first 4 h, no significant stimulation above basal VEGF secretion (open bars) could be observed. The translation inhibitor cycloheximide (10 µg/ml) completely blocked TGF-ß1-induced VEGF production (black bars). Time-course pattern and inhibitory effect of cycloheximide was similar when the effect of 10 ng/ml TGF-ß3 on VEGF release by TtT/GF cells was studied (data not shown). **, P < 0.01; ***, P < 0.001 vs. corresponding basal values.

 
Effects of TGF-ß isoforms on VEGF production in rat pituitary cell cultures
Rat pituitary cell cultures, where FS cells are the only source of VEGF (16), were used to confirm the VEGF-stimulatory effects of TGF-ß isoforms on normal FS cells. Both TGF-ß1 and TGF-ß3 dose-dependently stimulated basal VEGF release in rat pituitary cell cultures in an identical manner (Fig. 5Go, A and B). The ED50 values for TGF-ß1 and TGF-ß3 were 0.89 ± 0.2 and 0.65 ± 0.17 ng/ml, respectively, and are thus similar to the corresponding ED50 values obtained for TGF-ß-induced VEGF production in TtT/GF cells. However, in contrast to TtT/GF cells, only a doubling of the VEGF secretion could be achieved at the highest concentration of the TGF-ß isoforms. Concerning the net increase of VEGF, normal FS cells seem to be less responsive to TGF-ß in comparison to FS TtT/GF tumor cell.



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  		Figure 5. Effect of TGF-ß1 and TGF-ß3 on VEGF production in rat pituitary cell cultures. Both TGF-ß1 (A) and TGF-ß3 (B) dose-dependently stimulated VEGF release in rat pituitary cell cultures in which FS cells represent the only source of VEGF. Time-course studies (C) showed that only prolonged treatment with 10 ng/ml TGF-ß1 (horizontal striped bars) or TGF-ß3 (vertical striped bars) induced an increase of VEGF production vs. corresponding controls. *, P <0.05; **, P < 0.01 vs. untreated cell cultures.

 
Dexamethasone suppression of TGF-ß-induced VEGF secretion
Glucocorticoids have been shown to inhibit both basal and stimulated VEGF secretion (20); therefore, the potential inhibitory action of dexamethasone on TGF-ß-stimulated VEGF secretion was investigated. Dexamethasone dose-dependently suppressed TGF-ß1 and TGF-ß3 induced VEGF secretion (Fig. 6Go, A and B). Complete blockade of TGF-ß-induced VEGF release was achieved at 1 µM dexamethasone in both TtT/GF cells and in normal FS cells of rat pituitary cell cultures. Our observation emphasizes the importance of glucocorticoids as negative regulators of VEGF production in the pituitary.



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  		Figure 6. Effect of dexamethasone on TGF-ß-induced VEGF production in TtT/GF cells (A) and in rat pituitary cell cultures (B). As previously reported, dexamethasone (black bars) suppressed basal VEGF secretion (open bars) in TtT/GF cells (A) and in normal FS cells (B). TGF-ß1-induced VEGF production by TtT/GF cells was dose-dependently inhibited by dexamethasone;complete suppression was achieved at 1 µM dexamethasone (A). In normal FS cells, the TGF-ß3-induced VEGF release was also completely blocked by increasing doses of dexamethasone (B). Identical inhibitory effects of dexamethasone on TGF-ß3-induced VEGF production in TtT/GF cells and of TGF-ß1-stimulated VEGF secretion in normal FS cells were observed (data not shown). In all experiments, 10 ng/ml TGF-ß was used for stimulation and cells were treated for 24 h. **, P < 0.01 vs. untreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous growth factors and their receptors have been identified in the anterior pituitary. Because many of these factors affect hormone secretion and pituitary cell proliferation, it has been postulated that locally produced growth factors may modulate the growth and function of the anterior pituitary by auto-/paracrine mechanisms (29, 30). In the present work, we demonstrate for the first time, that different TGF-ß isoforms are potent stimulators of FS cell-derived VEGF in the anterior pituitary, and moreover that FS cells represent both a source and a target for TGF-ß isoforms.

The production of TGF-ß isoforms has been found in lactotrophs;furthermore, Jin et al. (8) have recently demonstrated that TGF-ß1 is also produced by normal FS cells and TtT/GF cells. Our observation that the FS TtT/GF cell line synthesizes TGF-ß1, -ß2, and -ß3 indicates that FS cells represent a source of all TGF-ß isoforms within the anterior pituitary, as TtT/GF cells and normal FS cells are characteristically very similar (19). Moreover, we observed synthesis of TGF-ß receptors types 1 and 2 in TtT/GF cells. This confirms previous functional observations that FS cells also represent a target for TGF-ß because it has already been shown that TGF-ß3 stimulates bFGF secretion (12) and TGF-ß1 regulates both proliferation and leptin mRNA expression in FS cells (8). We demonstrated that, in addition, both TGF-ß1 and TGF-ß3 enhance VEGF secretion in TtT/GF cells and in normal FS cells in rat pituitary cell cultures.

VEGF acts via two types of tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), which are almost exclusively expressed on endothelial cells (18, 30, 31). VEGF is not produced by endothelial cells (18), and therefore represents a classical paracrine-acting factor, which is known to maintain vascular integrity and to stimulate vascular permeability and endothelial cell proliferation (18, 30, 31). Exclusive production of VEGF in normal pituitary by FS cells suggests that one of the multiple functions of this cell type is to mediate the VEGF-associated effects on the endothelium of the anterior pituitary. Development and maintenance of a dense capillary network within the pituitary is essential for optimal function of this organ because hypothalamic and peripheral hormones, or other factors regulating endocrine homeostasis, are transported to endocrine pituitary cells within the blood. Therefore, basal production of VEGF by FS cells is essential to maintain the integrity of the intrapituitary vascular system (18). Moreover, VEGF is the most potent stimulator of vessel permeability by virtue of its affect on endothelial cell fenestration (18, 32). Thus, increased production of VEGF by FS cells in response to TGF-ß may enhance the flux of compounds such as hypothalamic factors, and in particular small molecules like dopamine, from intrapituitary vessels to the hormone-producing cells. This could then enhance the response of corresponding endocrine pituitary cells to hypothalamic stimulation or inhibition. In the same way, feedback mechanisms of peripheral hormones and growth factors (e.g. IGF-I) on endocrine pituitary cells may also be improved. Therefore, regulation of intrapituitary vascular permeability through VEGF could represent an option to modify the response of hormone-producing pituitary cells to endocrine stimulation or inhibition. However, alterations in vascular permeability are slow, since, despite the rapid onset of TGF-ß signaling indicated by translocation of Smad2 to the cell nuclei, it took more than 4 h until VEGF secretion significantly increased above basal levels. It is probably that only sustained TGF-ß up-regulation may induce consistent de novo synthesis of VEGF, and thus enhanced vascular permeability. However, it should be emphasized that TGF-ß is not the only regulator of VEGF production by FS cells. We have previously shown that pituitary adenylate cyclase-activating peptide and IL-6 also stimulated VEGF, whereas dexamethasone strongly inhibits VEGF production via glucocorticoid receptors expressed in FS cells (20). Complete blockade of TGF-ß-induced VEGF production by dexamethasone confirms the importance of glucocorticoids as negative regulators of VEGF synthesis in FS cells. Thus, TGF-ß isoforms belong to a group of different factors that regulate VEGF release and VEGF-associated effects on the endothelium of the anterior pituitary.

Interestingly, the effects of TGF-ß on VEGF production differ in different pituitary cell types. In contrast to TtT/GF cells, basal VEGF production by lactosomatotroph GH3 cells was not regulated by TGF-ß1 and -ß3, although these factors inhibited PRL secretion indicating that the cells were responsive to TGF-ß as has already been described (15, 22). It is known that the mode of action of TGF-ßat a transcriptional level is highly dependent on the interaction of the Smad2/Smad3/Smad4 complex with different DNA-binding cofactors and transcriptional coactivators or repressors (1, 3). In particular, Smad2 and Smad3 have been reported to account for the different effects of TGF-ß on various cellular mechanisms (3). To explain the distinct actions of the TGF-ßisoforms in TtT/GF and GH3 cells, more work is necessary to identify the interaction of Smad2 and Smad3 with the different transcriptional regulators of the various TGF-ß-induced effects in the individual pituitary cell types.

TGF-ß1 represents a potent inhibitor of lactotroph cell growth (15), whereas TGF-ß3 is known to stimulate the proliferation of normal lactotrophs either directly or via TGF-ß3-induced bFGF (11, 12). Estradiol is known to suppress TGF-ß1 production and to stimulate TGF-ß3 expression (7, 15). In this process, Smad3 could play an important role, because it has recently been shown that Smad3 is critically in the cross-talk between estrogen receptor signaling and the TGF-ßsignal transduction pathway (33). The estrogen-induced alteration in the TGF-ß1/ß3 ratio could induce a shift in the TGF-ß isoform-mediated growth balance toward lactotroph cell growth. It is thought that this could be part of the mechanism by which estrogens induce lactotroph hyperplasia during pregnancy and lactation. Moreover, alterations in the TGF-ß isoform ratio could play a role in estradiol-induced prolactinoma formation in Fischer 344 rats (15). Development of prolactinomas, and probably also pituitary hyperplasia, is accompanied by neovascularization through angiogenesis (34, 35). Stimulation of the most potent angiogenic factor, VEGF, by TGF-ß isoforms suggests that estrogen-induced alterations of TGF-ß isoforms may not only increase proliferation of lactotrophs, but may stimulate in parallel VEGF and, thus, neovascularization. However, because both TGF-ß1 and -ß3 stimulate VEGF production by FS cells, it would be necessary that the stimulatory effect of E2 on TGF-ß3 is stronger than its inhibitory effect on TGF-ß1, to gain a net increase in intrapituitary TGF-ßisoform secretion and subsequent VEGF production. Estradiol-induced activation of FS cells and the over expression of VEGF during the early stages in prolactinoma formation in Fischer rats had been reported (36, 37, 38, 39, 40). However, whether an E2-induced net increase of TGF-ß isoform production is responsible for these effects needs to be studied.

In summary, we have shown that TGF-ß isoforms not only regulate the function and growth of lactotrophs within the pituitary, but are also auto- or paracrine-acting stimulators of VEGF production by FS cells. Thus estrogen-induced alterations in TGF-ß isoform production may affect VEGF-stimulated vascular permeability within the normal pituitary. Moreover, as discussed above, complex intrapituitary interactions may exist among TGF-ß isoforms, bFGF, and VEGF, which play a role in angiogenesis during estrogen-induced pituitary hyperplasia or prolactinoma formation.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft: Re 851/1-1.

Abbreviations: bFGF, Basic fibroblast growth factor; E2, estradiol; FS, folliculostellate; PRL, prolactin; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Received March 8, 2002.

Accepted for publication June 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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