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Endocrinology Vol. 144, No. 2 732-740
Copyright © 2003 by The Endocrine Society

ARTICLE

Rat Anterior Pituitary Folliculostellate Cells Are Targets of Interleukin-1ß and a Major Source of Intrapituitary Follistatin

Louise M. Bilezikjian, Angela M. O. Leal, Amy L. Blount, Anne Z. Corrigan, Andrew V. Turnbull and Wylie W. Vale

Clayton Foundation Laboratories for Peptide Biology (L.M.B., A.L.B., A.Z.C., W.W.V.), The Salk Institute, La Jolla, California 92037; Hospital das Clínicas da FMRP-USP (A.M.O.L.), Ribeirão Preto-Sao Paolo, 14049-900, Brazil; and AstraZeneca (A.V.T.), Mereside, Alderley Park, Macclesfield SK10 4TG, Cheshire, United Kingdom

Address all correspondence and requests for reprints to: Louise M. Bilezikjian, Ph.D., The Salk Institute, Clayton Foundation Laboratories for Peptide Biology, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: bilezikjian{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [125I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express {alpha}, ßA, and ßB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1ß (0.005–5 nM) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1–27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1ß, glucocorticoids, and other agents on the response of pituitary cells to activins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULOSTELLATE CELLS OF the anterior pituitary comprise a group of S100-antigen-positive nonendocrine cells whose function remains poorly defined. They are postulated to play an important role as a source of paracrine factors that act locally to modulate pituitary responses to hypothalamic and peripheral signals (1, 2). Electrophysiological and morphological studies suggest that folliculostellate cells form a functional network of interconnecting cells that facilitate synchronization and long-range communication among different parts of the pituitary (3). These cells also have been viewed as a pool of progenitor cells capable of differentiating into specialized endocrine cells under certain conditions (4).

Activins, along with their functional antagonists, the inhibins and follistatins, are an important group of autocrine/paracrine factors involved in the local modulation of the anterior pituitary (5, 6, 7, 8, 9, 10, 11). Activins and inhibins are members of the TGFß family of growth and differentiation factors, and as is characteristic of this family of proteins, they are expressed in most tissues and exert a broad spectrum of local or endocrine effects (5, 12). Activin A, B, and AB; homo/heterodimers of inhibin/activin ßA- and ßB-subunits; and inhibin A and B, heterodimers of the same ßA- or ßB- subunits with the structurally related inhibin/activin {alpha}-subunit are documented to exert pituitary effects and be expressed in this tissue (5, 13). Activin effects are mediated by the widely distributed activin-specific type II (ActRII and ActRIIB) and type I (ActRIB or ALK4) receptors (12, 14). By contrast, the antagonistic function of inhibin appears to be restricted to a subset of activin-responsive cell types that express inhibin-specific binding proteins (15, 16, 17, 18). Most anterior pituitary cell types are responsive to activins (7, 13), but gonadotropes seem to be the only known targets of inhibin within this tissue (5, 13, 16, 19).

Follistatins are widely distributed activin-binding glycoproteins that serve an important function by their ability to biologically inactivate the activins (6, 20, 21). The observation that follistatin-deficient mice suffer many abnormalities and die within a few hours of birth is presumed to reflect the absence of an important mechanism that modulates the actions of activins and possibly other TGFß family members also known to bind to follistatin (22). Two forms of follistatins differing in length at the C terminus, FS315 and FS288, are encoded by two alternatively spliced mRNA products of a single gene (23). The relative distribution of the two follistatins is not well characterized, but FS315 is likely to be the circulating form because truncated forms have a higher affinity for cell surface heparin sulfate proteoglycans and the mRNA encoding FS315 is more abundant than the shorter version (24, 25, 26). Consistently, follistatins present in human serum display characteristics of the longer FS315 form, whereas those in human follicular fluid are likely to be the FS288 splice variant probably involved in autocrine/paracrine functions (27). Follistatins and inhibin/activin subunits often exhibit an overlapping pattern of expression. Pituitary follistatin expression is strongly induced by activins (8, 28, 29). Follistatin mRNA is present in most cell types of the anterior pituitary, including folliculostellate cells. Various preparations of anterior pituitary cultures or enriched pituitary folliculostellate cells obtained from a number of different species have been reported to express the mRNA or secrete the protein (24, 30, 31, 32, 33).

In addition to follistatins, preparations of rat, mouse, bovine, or primate primary pituitary folliculostellate cells or cell lines derived from them have been shown to also produce IL-6 (34, 35, 36, 37), lipocortin 1 (38), vascular endothelial growth factor (VEGF) (30, 39), leukemia inhibitory factor (40), TGFß (41, 42), and basic fibroblast growth factor (43, 44). Moreover, these cells have been reported to be responsive to a number of agents with known pituitary effects, including TGFß, activin, pituitary adenylate cyclase-activating polypeptide (PACAP), calcitonin, IL-1ß, IL-6, glucocorticoids, and lipopolysaccharide (LPS) (37, 39, 42, 44, 45, 46, 47). It has been postulated that these agents exert effects on the endocrine cells of the anterior pituitary, in part, by modulating the production of paracrine factors from folliculostellate cells (1, 2). Along these lines, it has been proposed that the reciprocal relationship between follistatin and FSHß mRNA levels seen in nonhuman primate anterior pituitary cell preparations reflects the importance of folliculostellate cells as a local source of follistatin (48).

Studies with rat anterior pituitary preparations have also suggested that follistatin derived from folliculostellate cells exerts a paracrine influence on the regulation of gonadotropes by activin (33). Similarly, the influence of PACAP on gonadotropes may reflect its ability to modulate follistatin production from folliculostellate cells (46). Finally, one of the proposed mechanisms by which IL-1ß modulates the hypothalamo-pituitary-adrenal or hypothalamo-pituitary-gonadal axis is by stimulating IL-6 and follistatin production from folliculostellate cells and thereby modifying ACTH or FSH secretion from the pituitary (49, 50). To gain further insight into their role, a previously described strategy was used to obtain enriched preparations of rat anterior pituitary folliculostellate cells (33). These cells were subjected to dilution cloning and independent lines were selected for their ability to proliferate, express S100, and secrete measurable follistatin. One of these lines, FS/D1h, was characterized further and shown to secrete follistatin and IL-6 in response to IL-1ß and be responsive to activin A, dexamethasone, PACAP, and LPS, confirming that a subset of S100-positive folliculostellate cells are targets of IL-1ß and a source of pituitary follistatin and IL-6. These studies with the FS/D1h cell line also provide further support for a role of rat anterior pituitary folliculostellate cells in mediating the suppressive effects of IL-1ß on activin-mediated FSH secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Enrichment and selection of FS/D1h folliculostellate cells
Folliculostellate cells were enriched by a previously published method (33). Anterior pituitary cells from male Sprague Dawley rats (180–200 g) were digested with collagenase and the dispersed cells [rat anterior pituitary cells (RAP)] were cultured in ßPJ medium (51) supplemented with 15% fetal bovine serum (FBS) and 6 mM Gln (5 x 106 cells per 10-cm dish). The medium was changed every 2–3 d, and 2 wk later a population of flat adherent cells that covered the dish were visible. These adherent cells were trypsinized and replated at a dilution of 1:6 to 1:8 and allowed to grow to confluence. This process was repeated several times, and the cells that survived after the fourth passage were distributed into several 24-well plates at a low density. Cells that produced a factor with FSH inhibitory activity were removed by trypsinization, pooled, and subjected to dilution cloning by culturing them in two 48-well plates at a calculated density of less than 1cell/well. After approximately 2 wk, 8 of 96 wells contained visible colonies of proliferating cells, and medium conditioned by cells in one of these wells contained measurable follistatin levels. The cells recovered from this well were subjected to another round of dilution cloning, and one of the wells ultimately yielded the cell line designated FS/D1h.

Follistatin and IL-6 measurements
Follistatin secretion was measured by an indirect RIA using a rabbit anti-rhFS288 serum (no. 5542) that binds either free follistatin or the follistatin:activin complex. Briefly, serial dilutions of conditioned medium or recombinant human (rh) FS288 were incubated for 18–20 h at 4 C with anti-rhFS288 (1:5,000) in the presence of approximately 100,000 cpm [125I]activin A (~3 x 106 cpm/pmol), 0.1% BSA, Aprotinin (1:500), and 0.01% Triton X-100 in a final volume of 0.5 ml. Activin A was labeled as described previously (52). The follistatin:[125I]activin A complexes bound to anti-FS288 were immunoprecipitated by first incubating the samples with sheep antirabbit IgG (1:100) and normal rabbit serum (1:1000) for 30 min at 4 C and then centrifuging them in the presence of 5% polyethylene glycol in a final volume of 1 ml. Nonspecific interactions were determined by substituting normal rabbit serum for anti-FS288. After discarding the supernatants, radioactivity remaining in the pellets was determined on a {gamma}-counter, and specifically immunoprecipitated [125I]activin A values were determined by subtracting nonspecific counts. Regression analysis was used to analyze the data with Prism 3.0a for MacIntosh (GraphPad Software, Inc., San Diego, CA). All samples and standard curves were measured in triplicate. The minimum detectable concentration of the assay was in the range of 0.05–0.1 ng/ml rhFS288, and the assay was linear over approximately two log units.

The secretion of IL-6 bioactivity was determined using a 7TD1 cell survival assay, essentially as described (53). Briefly, 7TD1 cells were cultured in RPMI 1640 containing 5% FBS, 50 µM 2-ß-mercaptoethanol, antibiotics, and 2 ng/ml rhIL-6. Samples or standards were serially diluted (1:2) in assay medium and added to cells cultured in 96-well plates and 72 h later, the plates were centrifuged (300 x g for 5 min) to remove the medium. Cell proliferation was measured colorimetrically using the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and quantification at 570 nm test and 640 nm reference wavelengths using a microplate reader (MR700, Dynatech Corp. Laboratories, Chantilly, VA). Concentrations of unknown samples were determined by measuring the displacement of their dilution curves from the known standard (the detection limit of the assay was 0.08–0.25 international interim units/ml). All samples were assayed in triplicate (on separate plates), and IL-6 values are presented as international interim units, based on comparisons with the standard.

S100 immunocytochemistry
The FS/D1h cells were cultured in eight-chambered Falcon CultureSlides (Becton Dickinson and Co. Labware, Bedford, MA) at a density of 50,000 cells/chamber. Three days later, they were washed with PBS, fixed with 4% paraformaldehyde for 15 min on ice, rewashed, and permeabilized with 0.2% Triton X-100 for 10 min and washed again with PBS. Nonspecific sites were blocked by incubation for 30 min at ambient temperature with PBS containing 2% normal donkey serum, 0.1% BSA, 0.1% Tween 20 before adding affinity-purified rabbit anti-S100 IgG (1:250) (Sigma, St. Louis, MO) in the same buffer for 1 h. After several washes, Texas Red dye-conjugated affinity-purified donkey antirabbit IgG (1:300, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added and allowed to incubate for 1 h at ambient temperature before washing, applying ProLong Antifade reagent (Molecular Probes, Inc., Eugene, OR) and cover slipping. Specificity was determined by either excluding the primary antibody or substituting it with normal rabbit IgG. Immunostaining for glial fibrillary acidic protein (GFAP) was evaluated using guinea-pig antihuman GFAP (1:250) (Advanced ImmunoChemical Inc., Long Beach, CA) and Texas Red dye-conjugated affinity-purified donkey anti-guinea pig IgG (1:400; Jackson ImmunoResearch Laboratories, Inc.). Fluorescent signals were captured using an eclipse E600 microscope (Nikon, Melville, NY) fitted with a mercury epifluorescence mercury light source and a CoolSNAP monochromatic digital camera and analyzed using the Image-Pro Plus (version 4.5) image analysis software (Media Cybernetics, Inc., Silver Spring, MD).

Ribonuclease (RNase) protection
Total RNA was extracted using the RNeasy kit (QIAGEN, Hilden, Germany). Steady-state inhibin/activin {alpha}, ßA, ßB, follistatin, and Smad7 mRNA levels were measured by RNase protection, using rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control as previously described (28, 54). For the analysis of primary follistatin transcripts, a template containing 203 bp of exon 3 of the rat follistatin gene and 260 bp of the adjacent third intron (in pBluescript IISK, Stratagene, La Jolla, CA) was digested with HindIII and used to measure unprocessed transcript and mature mRNA simultaneously. The antisense riboprobe was synthesized using T3 RNA polymerase and levels were measured using total RNA. The ability of this riboprobe to detect primary transcript was validated by analyzing nuclear and cytoplasmic RNA samples individually and by kinetic studies to establish an optimal time point for these measurements. Quantitative analysis was performed using the PhosphorImager system and the ImageQuant 4.0 software package (Molecular Dynamics, Inc., Sunnyvale, CA). Reported data reflect transcript levels normalized to the internal GAPDH control. Treatments were replicated at least three times, and the results are reported as means ± SEM.

Cell proliferation
Cells were cultured in 96-well plates at a density of 103 cells/well in 0.1 ml complete medium and equilibrated overnight. Test substances were added into triplicate wells, and cell proliferation was assessed after 4 d using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide proliferation assay kit (Cell Titer 96, Promega Corp., Madison, WI).

FSH measurements and statistical analysis
For the measurement of FSH secretion, RAP cells were plated in 48-well plates (Costar, Cambridge, MA) (0.15 x 106 cells/well) and allowed to recover 3 d in ßPJ medium (51) supplemented with 2% FBS. The cells were washed, and test substances or conditioned medium was added to the wells and secretion was measured in a final volume of 0.5 ml. FSH was quantified using RIA reagents generously provided by Dr. Parlow through the National Pituitary and Hormone Distribution Program at NIDDK. All treatments were performed in triplicate, and data from at least three determinations were subjected to either the t test or ANOVA followed by Dunnett’s multiple comparison test, as indicated in the figure legends.

Reagents
The rh-activin-A was provided by Genentech, Inc. (San Francisco, CA); rhFS288 was obtained through the National Hormone and Pituitary Program of NIDDK; rhIL-1ß was a gift from Dr. Liu of Neurocrine Biosciences Inc. (La Jolla, CA); rhIL-6 was purchased from Endogen, Inc. (Woburn, MA), and LPS was from Sigma (Escherichia coli serotype 026:B6m code L3755, lot 20H4025).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection and establishment of a rat anterior pituitary folliculostellate cell line, FS/D1h
An indirect RIA was used for measuring follistatin secretion from rat anterior pituitary cells. As shown in two examples of a typical standard curve, the minimum concentration of free FS288 detectable by this indirect immunoassay was in the range of 0.05–0.1 ng/ml, and the assay was linear over approximately two log units (Fig. 1Go). Concentrations of activin A greater than 0.01 nM lowered the sensitivity of the assay because of competition with radiolabeled activin A, indicating that the assay provides an estimate of only free follistatin (Fig. 1AGo). This assay was used to measure relative changes in follistatin levels by comparing the linear portion of serially diluted media to the standard curve, as shown by a typical example of the analysis of a set of control and treated samples (Fig. 1BGo).



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  		Figure 1. Typical FS288 standard curves obtained from an indirect RIA in which follistatin concentrations were quantified based on follistatin:[125I]activin A complexes immunoprecipitated with anti-FS288 (no. 5542). Approximately 100,000 cpm [125I]activin A were incubated with the indicated concentrations of FS288, with and without cold activin A (A), or with serially diluted medium conditioned by FS/D1h cells treated as indicated (B). The samples were immunoprecipitated with anti-FS288 sheep antirabbit IgG and processed as described in Materials and Methods. The values reflect the mean ± SEM of triplicate determinations from typical experiments.

 
After culturing rat anterior pituitary cells for 2–3 wk in high FBS (15%), medium conditioned by the surviving cells contained no measurable GH, FSH, LH, or ACTH (data not shown). However, the medium was observed to contain an activity that suppressed basal FSH secretion and attenuated the effect of submaximal concentrations of activin A (Fig. 2AGo). Subjecting this population of cells to multiple dilution cloning steps, as described in Materials and Methods, yielded several independent lines that await further analysis. One of these lines, herein referred to as FS/D1h cells, was determined to express considerably more follistatin mRNA, compared with the other lines or to the mixed population of rat anterior pituitary cells (data not shown), and secrete substantial amounts of follistatin (Fig. 2BGo). Conditioned medium from the FS/D1h line when applied to cultured rat anterior pituitary cells also suppressed basal FSH secretion, consistent with the expected action of follistatin on gonadotropes (Fig. 2CGo).



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  		Figure 2. The suppression of FSH secretion by medium conditioned by enriched rat anterior pituitary folliculostellate cells (A) and the evaluation of medium conditioned by several selected folliculostellate lines for follistatin secretion (B) and effects on FSH secretion (C). A, Control or conditioned medium (0.3 ml) from enriched folliculostellate cells in 10-cm dishes was added to rat anterior pituitary cells cultured in 48-well plates (Costar) at a final volume of 0.5 ml/well, with and without various concentrations of activin A. B, Follistatin levels in medium conditioned for 7 d with the initial pool of cells or several selected folliculostellate lines were quantified by an indirect RIA described in the text. C, 0.2 ml control or conditioned medium by the same lines shown in panel B were tested for effects on FSH secretion as described for panel A. The effect of FS288 on FSH secretion is shown at the far right. The data are means ± SEM of triplicate determinations of representative experiments. The data within each panel were subjected to ANOVA and Dunnett’s multiple comparison test. For A, statistical comparisons are relative to control medium for each x-axis value. For B and C, comparisons are relative to FSH or follistatin (FS) secretion from the pool: **, P < 0.001; #, P < 0.01; *, P < 0.05.

 
Immunocytochemical evaluation revealed that all FS/D1h cells expressed the S100 antigen (Fig. 3Go). This cell-associated staining was no longer visible when anti-S100 was omitted or substituted with normal rabbit IgG, verifying the specificity of the S100 immunoreactivity (Fig. 3BGo). The FS/D1h cells were also positive for GFAP immunostaining, another marker of the anterior pituitary cell population (1) (data not shown). The FS/D1h cells had a doubling time of approximately 48 h, formed a monolayer, and essentially stopped growing when they reached confluence. Light microscopy indicated the presence of large vacuolar structures in the cytoplasm (data not shown). The FS/D1h cells established cell-cell contact via long cytoplasmic processes, and during the logarithmic growth phase, they had a more stellate appearance but became more rounded at higher densities (data not shown). Because folliculostellate cells have been proposed to be the source of pituitary IL-6, the production of this cytokine from FS/D1h cells was evaluated and levels were compared with those of RAP cells. The results indicated that FS/D1h cells secrete considerably more IL-6, compared with RAP cells (Fig. 4AGo). Moreover, the cells responded to IL-1ß by an increase in the production of not only IL-6 but also follistatin (Fig. 4BGo). These observations indicate that FS/D1h cells possess at least some of the characteristics usually associated with pituitary folliculostellate cells. The cells were amenable to propagation through multiple passages (up to 35–40 passages tested) without loss of phenotypic characteristics such as follistatin and IL-6 production, morphological appearance, and growth pattern; S100 immunoreactivity was not evaluated beyond passage no. 19.



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  		Figure 3. Immunocytochemical analysis for S100 protein expression in FS/D1h cells (passage no. 5). Texas Red fluorescence was captured at x40 magnification. B, The signal obtained when the primary S100 antiserum was omitted is shown.

 


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  		Figure 4. The secretion of IL-6 and follistatin from FS/D1h cells (passage no. 10) and cultured RAP cells in response to 0.5 nM IL-1ß. The data are means ± SEM of triplicate determinations of a representative experiment. The t test was used to compare IL-6 or follistatin (FS) secretion from the indicated cell types incubated with or without IL-1ß. ND, Not detectable. **, P < 0.001; #, P < 0.01.

 
Regulation of free follistatin production from FS/D1h cells
The effects of IL-1ß, glucocorticoids, and PACAP(1–27) on follistatin production were evaluated because folliculostellate cells have been proposed to be responsive to these agents (37, 39, 42, 46, 55). The FS/D1h cell line responded to IL-1ß by a concentration-dependent increase in the secretion of free follistatin (Fig. 5AGo). This effect was lost on heat inactivation of IL-1ß (data not shown). Follistatin secretion from the FS/D1h cell line was also stimulated by dexamethasone in a concentration-dependent manner (Fig. 5AGo). A 50-nM concentration of testosterone had no effect (data not shown). Although PACAP has been shown to be a potent stimulus for the secretion of IL-6 from another established pituitary folliculostellate line, TtT/GF (39, 46), treatment of FS/D1h cells with 250 nM PACAP(1–27) caused only a small but statistically significant increase in measurable free follistatin in the medium (Fig. 5AGo). Of additional agents tested, LPS increased medium-free follistatin levels, whereas forskolin and 12-O-tetradecanoylphorbol-13-acetate had no effect (Fig. 5Go, B and C).



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  		Figure 5. The effects of increasing concentrations of IL-1ß and dexamethasone (DEX) or 250 nM PACAP(1–27), 5 ng/ml LPS, 10 µM forskolin, and 20 nM 12-O-tetradecanoylphorbol-13-acetate on follistatin (FS) levels in medium conditioned by FS/D1h cells (passage no. 9) during a 48-h incubation period. The data are means ± SEM of triplicate determinations of representative experiments. Data from each panel were subjected to ANOVA followed by Dunnett’s multiple comparison test to compare follistatin secretion in the presence of the indicated treatments relative to untreated control (open bars). ND, Not detectable. **, P < 0.001; #, P < 0.01.

 
Regulation of mRNA levels
Experiments in which RNase protection analysis was employed revealed that FS/D1h cells express the mRNA for follistatin and all three inhibin/activin subunits, {alpha}, ßA, and ßB, known to be present in the anterior pituitary (Fig. 6Go). Consistent with the stimulatory effects of IL-1ß and dexamethasone on follistatin secretion, both agents increased follistatin mRNA levels of FS/D1h cells (Fig. 7AGo). By contrast, IL-1ß caused a small but statistically significant decrease in ßB mRNA levels, whereas dexamethasone slightly increased those of ßA (Fig. 7Go, B and C). The effect of IL-1ß on ßA mRNA levels was not statistically significant. Neither of these treatments altered the levels of inhibin/activin {alpha} mRNA (data not shown).



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  		Figure 6. An image of an RNase protection gel showing the expression of follistatin (FS) and inhibin/activin subunits {alpha}, ßA, and ßB in FS/D1h cells (passage no. 6). Alternating lanes include the internal GAPDH control. Total RNA (1–2 µg for follistatin and approximately 10 µg for {alpha}, ßA, and ßB) from FS/D1h cells was hybridized with the indicated probes and resolved on a 5% polyacrylamide/8 M urea gel.

 


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  		Figure 7. The effects of 24-h treatment with 5 nM IL-1ß or 25 nM dexamethasone on follistatin and inhibin/activin ßA and ßB mRNA levels of FS/D1h cells. Each band was normalized to the internal GAPDH control, and values are presented as percentages of untreated cells. The results were subjected to ANOVA followed by Dunnett’s multiple comparison test to compare mRNA levels shown in each panel in the presence of the indicated treatments relative to the corresponding untreated control (open bars). The data are means ± SEM of five independent experiments, each performed in triplicate. ***, P < 0.0001; **, P < 0.001; *, P < 0.05.

 
The response of FS/D1h cells to activin A was also evaluated. Cross-linking experiments with [125I]activin A and Western analysis of cell lysates indicated that FS/D1h cells express type II (ActRII and ActRIIB) and type I (ALK4 or ActRIB) activin receptors (data not shown). Activin A caused a 3-fold induction of the well-characterized activin/TGFß-responsive reporter, 3TP-Lux (56), transiently transfected into FS/D1h cells (data not shown), indicating that the cells are responsive to activins. However, unlike RAP cells (8, 28, 29), activin A failed to increase follistatin mRNA levels of FS/D1h cells (Fig. 8AGo). On the other hand, similar to RAP cells (54), activin A rapidly induced Smad7 mRNA levels in this cell line (Fig. 8BGo). To further evaluate this unexpected observation, the effect of activin A on the transcriptional activation of the follistatin promoter was evaluated and compared with IL-1ß action. In RNase protection experiments using a probe that spanned an intron/exon junction of the follistatin gene, activin A again failed to stimulate follistatin expression, whereas IL-1ß increased both primary and mature transcript levels (Fig. 9Go). Activins and other TGFß superfamily members are well established to exert antiproliferative effects on responsive cells (12). Treatment with activin A suppressed the growth of FS/D1h cells to the same extent as IL-1ß (Fig. 10Go). These results indicated that, despite the negative results with follistatin, FS/D1h cells probably express most if not all the pathway components necessary for signaling in response to activin A.



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  		Figure 8. The effects of treatment with 3 nM activin A on 24 h follistatin (FS) or 1 h Smad7 mRNA levels of FS/D1h cells (passage no. 11). Each band was normalized to GAPDH and values are presented as percentages of untreated cells. The data are means ± SEM of triplicate determinations of a representative experiment. The Student’s t test was used for statistical comparison of follistatin or Smad7 mRNA levels with and without Activin A: *, P < 0.05.

 


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  		Figure 9. Transcriptional effects of 3 nM activin A or 5 nM IL-1ß on the follistatin gene. Confluent FS/D1h cells (passage no. 7) in 10-cm dishes were treated for 2 h and total RNA was extracted and used for hybridization with a riboprobe that spanned the junction of the third intron and exon then resolved on a 5% polyacrylamide/8 M urea gel. The intensity of the bands protected with either the entire follistatin riboprobe (primary = 260 nt) or corresponding to only the exon portion of the riboprobe (mature mRNA = 203 nt) were normalized to the internal GAPDH control and are presented relative to untreated control values. The data are means ± SEM of triplicate determinations of a representative experiment. They were subjected to ANOVA followed by Dunnett’s multiple comparison test to compare the effect of the indicated treatments on transcript levels relative to the corresponding controls (open bars). **, P < 0.001.

 


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  		Figure 10. The concentration-dependent effects of IL-1ß and activin A on FS/D1h cell proliferation at the end of 96 h. The data were normalized to untreated controls and are means ± SEM of normalized data from four individual experiments each performed in triplicate. They were subjected to ANOVA followed by Dunnett’s multiple comparison test to determine the concentration-dependent effects of IL-1ß relative to control (open bar). **, P < 0.001; #, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the present study was to evaluate the potential importance of the folliculostellate cells of the rat anterior pituitary as targets of agents known to regulate the local production of follistatin. Thus, a clonal line, FS/D1h, representative of folliculostellate cells, was selected from cultured rat anterior pituitary cells. Experiments confirmed that these FS/D1h cells express S100, a known marker of the folliculostellate cell population, and these cells secrete measurable amounts of follistatin. The secretion of follistatin from FS/D1h cells was stimulated by the addition of IL-1ß, dexamethasone, PACAP(1–27), and LPS, all of which are thought to modulate pituitary cell function in part by actions on folliculostellate cells. These results suggest that a subpopulation of folliculostellate cells from which FS/D1h cells were derived may be involved in mediating follistatin-dependent paracrine control on the anterior pituitary.

A number of morphologic features distinguish the endocrine cells from the folliculostellate cell population of the anterior pituitary. These nonendocrine star-shaped cells are devoid of secretory granules but are characterized as having lysosomal organelles, phagocytotic vesicles, and S100 immunoreactivity (1, 2). The selected FS/D1h cell line exhibited all of these characteristics. Folliculostellate cells have also been proposed to be a major source of pituitary IL-6 and follistatin, both of which were produced by the FS/D1h cell line. The expression of other paracrine factors that have also been associated with mouse, rat, or primate folliculostellate cells, including lipocortin 1 (38), VEGF (30, 39), leukemia inhibitory factor (40), TGFß (41, 42), and bovine fibroblast growth factor (43, 44) was not evaluated.

Follistatin mRNA is expressed by most anterior pituitary cell types, but it has not been established whether they also express the protein (32). On the other hand, a number of studies using various preparations of primary bovine, rat, and nonhuman primate folliculostellate cells have confirmed that they express follistatin mRNA and secrete the protein (30, 31, 33, 46). Indeed, follistatin produced by this cell type is likely to participate in the paracrine modulation of gonadotropes and pituitary responses that are dependent on endogenous and/or exogenous activins (33, 48, 57). Folliculostellate cell lines have been established and used for evaluating the functional importance of this population of anterior pituitary cells. One of these, the mouse TtT/GF line, has been a useful model for studying the regulated production of factors such as IL-6 and VEGF in response to PACAP, LPS, dexamethasone, TGF{alpha}, and calcitonin (35, 37, 39, 45, 46, 58). Unfortunately, the TtT/GF cells have not been amenable to the evaluation of follistatin production because an RNase protection assay specific for mouse follistatin detected only a hint of a protected band, and the protein, if present, was not detectable by our immunoassay (Bilezikjian, L. M., personal observations). Human pituitary tumor-derived folliculostellate cell lines have been reported to express follistatin mRNA but not the protein (47).

In an attempt to establish a folliculostellate cell line useful for studies of follistatin production, a previously described strategy was used (33). This method was reported to enrich cells that contained S100 immunoreactivity and secreted a paracrine factor that interfered with the action of activin A on FSH cells and whose effect could be immunoneutralized with a follistatin antiserum, therefore likely to be follistatin. In the current study, the clonal selection of cells enriched by a similar strategy yielded the FS/D1h cell line with high levels of endogenously expressed follistatin mRNA and protein. The RNase protection assays predict that the expressed protein is likely to be the longer FS315 splice variant and not the shorter FS288 form. Although cell lines other than FS/D1h were also obtained by this selection strategy, most that were tested did not produce levels of follistatin protein detectable by the assay described in the present study.

The fact that FS/D1h cells also express inhibin/activin {alpha}, ßA, and ßB subunit mRNAs indicates they might produce inhibins and/or activins. All three inhibin/activin subunits are expressed in the anterior pituitary and, although gonadotropes are presumed to be the major sites of {alpha} and ßB expression, other pituitary cell types may express them at low levels (59). Medium conditioned with RAP cells has been reported to contain immunoreactive activin A and B, and immunoneutralizing antisera directed to the inhibin/activin {alpha} subunit have provided indirect evidence that inhibins are also present (8, 9). The extent to which folliculostellate cells contribute to the local production of activins and/or inhibins is not known. The observation that follistatin mRNA expression is significantly greater (~25-fold) in the FS/D1h cells relative to RAP cells but the expression of inhibin/activin subunit mRNAs is enriched to a much lesser extent (data not shown) suggests that folliculostellate cells play a more important role as a pituitary source of follistatins.

Several functional assays confirmed that FS/D1h cells are responsive to activin and possess all the necessary activin signaling components. The addition of exogenous activin A induced Smad7 mRNA expression; activated an activin/TGFß-responsive luciferase reporter, 3TP-Lux; and suppressed the growth rate of FS/D1h cells. However, these effects required slightly higher concentrations of activin A, compared with RAP cells (13) probably because of binding and inactivation by endogenously produced follistatin. Surprisingly, activin A failed to modulate inhibin/activin subunit mRNA expression or produce the strong induction of follistatin expression seen in RAP cells (28). This did not reflect a generalized problem because IL-1ß clearly increased primary transcript levels suggestive of a transcriptional effect at the promoter level. Exogenous activin may have been ineffective to induce follistatin expression because the concentrations of activin produced by FS/D1h cells may have been sufficient to maximally induce the follistatin promoter by an autocrine mechanism. Alternatively, pituitary folliculostellate or FS/D1h cells may lack cellular or nuclear factors required for activin effects on follistatin expression.

The FS/D1h cells were also determined to be responsive to IL-1ß. The cytokine stimulated IL-6 secretion from FS/D1h cells and exerted a growth-inhibitory effect on them. In these cells, IL-1ß also increased free follistatin secretion and follistatin mRNA expression but concomitantly lowered steady-state inhibin/activin ßA and ßB subunit mRNA levels, although the change in ßA mRNA did not quite reach statistical significance. These results indicate that, in contrast to reported effects of systemic LPS administration (60), IL-1ß effects on follistatin production in FS/D1h cells is likely not to be secondary to a rise in activin A. The exact pituitary targets of IL-1ß are not fully defined, but paracrine factors originating from folliculostellate cells have been proposed to mediate some pituitary effects of this cytokine (49, 61). Follistatin may be one of these paracrine mediators of IL-1ß. It is known that IL-1ß modulates reproductive function and stress responses by exerting effects at all levels of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes (49, 61, 62, 63). Experiments with cultured rat anterior pituitary cells previously demonstrated that IL-1ß attenuates FSH secretion in response to activin A (50). This study also demonstrated that the treatment of cultured rat anterior pituitary cells with IL-1ß or the administration of LPS, in vivo, modulates pituitary inhibin/activin subunit and follistatin mRNA levels (50). Based on these observations, it was hypothesized that cytokine-induced changes in the local concentration of follistatin may be a mechanism by which IL-1ß or LPS modulate FSH secretion from gonadotropes (50). The current study supports this model and provides direct evidence for the ability of IL-1ß to regulate follistatin production from folliculostellate cells.

Dexamethasone effects on FS/D1h cells were also tested to evaluate the possibility that activin and/or follistatin production from folliculostellate cells mediate some pituitary actions of glucocorticoids. The majority of rat folliculostellate cells have been reported to express glucocorticoid receptors (55). Consistently, dexamethasone stimulated follistatin mRNA and protein levels and increased inhibin/activin ßA but not ßB mRNA accumulation of FS/D1h cells. These observations suggest that, unlike IL-1ß, dexamethasone effects on follistatin production may have been secondary to an increase in activin A. In primary cultures of anterior pituitary cells from female rats, glucocorticoids have been shown to positively regulate FSH production by an effect postulated to involve changes in the local activin/follistatin circuitry (64). Although corticosterone effects on follistatin and inhibin/activin ßB mRNA levels were not noted in the latter study, observations from FS/D1h cells suggest that folliculostellate cells may participate in mediating these reported effects of glucocorticoids on FSH production.

In summary, the present study describes the establishment and characterization of an immortalized cell line, FS/D1h, derived from the folliculostellate population of the rat anterior pituitary cells. The cell line was selected based on the presence of S100 immunoreactivity and measurable follistatin secretion as well as the proliferative and survival potential of the cells in culture through multiple passages, without loss of differentiated functions. The results of this study indicate that the FS/D1h cell line provides a model that should be useful for the evaluation of the role of folliculostellate cells as a source of paracrine factors that locally modulate anterior pituitary function.


   Acknowledgments
 
Cynthia J. Donaldson, Joan Vaughan, and Yaira Haas are acknowledged for providing expert technical support.


   Footnotes
 
This work was supported in part by NIH Grant HD-13527 and the Foundation for Medical Research. W.W.V. is a senior investigator for the Foundation for Medical Research.

Abbreviations: FBS, Fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; LPS, lipopolysaccharide; PACAP, pituitary adenylate cyclase-activating polypeptide; RAP, rat anterior pituitary cell; rh, recombinant human; RNase, ribonuclease; VEGF, vascular endothelial growth factor.

Received July 10, 2002.

Accepted for publication October 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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