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Endocrinology Vol. 138, No. 10 4401-4409
Copyright © 1997 by The Endocrine Society

ARTICLES

Transforming Growth Factor-ßs Inhibit Somatostatin Messenger Ribonucleic Acid Levels and Somatostatin Secretion in Hypothalamic Cells in Culture1

M. Quintela, R. M. SeñarÍs and C. Diéguez

Department of Physiology, Faculty of Medicine. University of Santiago de Compostela, Santiago de Compostela, Spain 15700

Address all correspondence and requests for reprints to: C. Diéguez, Departament of Physiology, Faculty of Medicine, University of Santiago de Compostela, 15700 Santiago de Compostela, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of hypothalamic cells in monolayer culture with transforming growth factor-ß1 (TGFß1) significantly reduced both basal and cAMP-induced somatostatin messenger RNA (mRNA) levels and somatostatin secretion. This inhibitory effect was dose- and time-dependent and not mediated by glial cells, as it was also observed in glial-free hypothalamic cell cultures treated with cytosine arabinonucleoside. TGFß2 and -ß3 mimicked the actions of TGFß1, which indicated that the three isoforms of the TGFß family expressed in the central nervous system displayed similar effects on the somatostatinergic neurons.

The blockade of synthesis of proteins with either cycloheximide or puromycin for 24 h prevented the inhibitory effect of TGFß1 on somatostatin mRNA. This implied that the reduction of this mRNA by TGFß1 required de novo protein synthesis.

We next studied whether TGFß1 acted at the transcriptional or posttranscriptional level by altering the stability of somatostatin mRNA. Examination of the rate of disappearance of somatostatin mRNA by Northern blot, after inhibition of mRNA transcription with either actinomycin D (AcD) or 5,6-dichloro-1ß-ribofuranosyl benzimidazole revealed that TGFß1 did reduce the stability of somatostatin mRNA. This effect was observed when we pretreated the cultures with TGFß1 4 h before the addition of AcD, but not when we administered TGFß1 simultaneously with AcD or 5,6-dichloro-1ß-ribofuranosyl benzimidazole.

Altogether these results demonstrated that the treatment of hypothalamic cells in culture with TGFß1, TGFß2, or TGFß3 resulted in a decrease in somatostatin mRNA levels and somatostatin secretion. TGFß1 reduced the steady state levels of somatostatin mRNA by inducing the synthesis of a protein (s), that appears to accelerate the degradation of the mRNA of somatostatin. Whether TGFß1 has additional effects on the transcription of the somatostatin gene will require further study.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SST) is a peptide originally isolated from the hypothalamus and shown to regulate the function of the anterior pituitary (1). Outside the hypothalamus SST is widely distributed throughout the central and peripheral nervous system, where it acts as a neurotransmitter/neuromodulator (1, 2).

In the hypothalamus SST expression and secretion are regulated by a large number of classical neurotransmitters and neuropeptides, hormones, metabolic fuels, and growth factors (1, 3, 4, 5, 6, 7, 8, 9). Furthermore, induction of SST immunoreactivity in some neuronal cell types is associated with in vivo neurodifferentiation (10). Among growth factors, there is an increasing awareness of the importance of the transforming growth factor-ß (TGFß) family in the development and function of the nervous system (11).

The TGFß family comprises five distinct, yet highly homologous, peptide isoforms (TGFß1–5) (12). TGFß1 and TGFß2 are 72% homologous in amino acid sequence and are interchangeable in most biological assays. TGFß3, -4, and -5 share 64–82% homology with TGFß1 and -2, and parallels in their function are presently being elucidated. TGFßs are pleiotropic peptides that can induce mitosis, differentiation, and inhibition of proliferation and function, depending upon the species, type, and maturity of the cell involved (12). It has been recently demonstrated that at least TGFß1–3 are expressed in the nervous system and exhibit a variety of neurotrophic actions on glial and neural cells (11). Thus, it has been shown that TGFßs can potentiate neuronal survival and substance P expression in neonatal dorsal root ganglia (13, 14), regulate LHRH gene expression and secretion in the hypothalamic cell line GT1 (15), inhibit proliferation of glial cells (16), and modulate the expression of several growth factor receptors (17). On the other hand, in the mature central nervous system, the expression of TGFß1 is increased by neural injury (18, 19), and exogenous TGFß1 provides neuroprotection from cerebral ischemia (20).

Taking into account that of all diencephalic areas the hypothalamus displayed the greatest density of TGFß-inmunostained cell bodies (21), we examined here the role of TGFß1, -ß2, and -ß3 on SST expression in hypothalamic cells in monolayer culture, assessing SST messenger RNA (mRNA) levels by Northern blot, and SST secretion by RIA. We studied the effect of TGFß1 on basal and cAMP-induced steady state SST mRNA levels and SST release. To examine whether the inhibitory effect of TGFß1 on SST mRNA content required de novo protein synthesis, we used two different inhibitors of protein synthesis: cycloheximide (Chx), and puromycin, and evaluated the action of TGFß1 on SST mRNA levels in the presence of these inhibitors.

Next, we studied the possible action of TGFß1 on the degradation rate of SST mRNA, analyzing the decay rate of SST mRNA when the RNA transcription was blocked with actinomycin D (AcD) or 5, 6-dichloro-ß-ribofuranosyl benzimidazole (DRB) in the presence or absence of TGFß1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fetal rat hypothalamic cell cultures
Primary monolayer cultures of hypothalamic cells were established as previously described (6). Briefly, fetal rat hypothalamic cells (embryonic day 18) were dissociated by a mechano-enzymatic method employing a protease treatment with 2 mg/ml Dispase (Boehringer Mannheim, GmBH Biochemicals, Mannheim, Germany) and 0.05 mg/ml deoxyribonuclease (DNAse) type I (Sigma Chemical Co., St. Louis, MO), with additional mechanical shearing. Plating of 3 x 106 hypothalamic cells per 60-mm2 tissue culture Petri dish (Nunc A/S, Roskilde, Denmark) was carried out in {alpha}-modified MEM (GIBCO, Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (GIBCO, Life Tecnologies), 2 mM L-glutamine (Flow Laboratories, Rockville, MD) and antibiotics at final concentrations of 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. The cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air, and the culture medium was replaced after 4 days. After 8 days in culture, cells were washed with Earle’s balanced salt solution (GIBCO) and then incubated in Krebs-Ringer bicarbonate containing 1 mg/ml BSA (Sigma) and bacitracin (Sigma) with test compounds or without them (control).

To obtain cultures basically free of nonneuronal cells, 1.5 µM cytosine arabinonucleoside (Ara-C) was added to the medium 24 h after plating and maintained in the medium during the entire culture period as shown by others (22, 23). Culture plates were previously coated overnight with a solution of 1 mg/ml polyethylenimine in 0.15 M sodium borate buffer, pH 8.3. The plates were washed three times with sterile PBS before addition of the medium. This treatment schedule resulted in suppression of nonneuronal cell proliferation (<5% glial cells present), as assessed by antiglial fibrillary acidic protein (GFAP) staining. The immunohistochemistry for GFAP was performed as previously described (24).

Determination of SST secretion
At the end of the experiment the medium was collected, immediately acidified to 1 N HCl, and extracted with a C-18 silica SepPak cartidge (Millipore, Bedford, MA), using standard procedures (elution of the sample from the SepPak with 80% acetonitrile in 0.1% trifluorocetic acid). The SepPak eluate was vacuum-dried and assayed for SST.

SST levels were measured by RIA as reported previously (6).

RNA extraction
Total RNA was extracted from the cells using the single-step guanidinum-thiocyanate-phenol-chlorofom procedure (25). Twenty micrograms of total RNA were electrophoresed on 1.5% agarose-formaldehyde gels and transferred to Nylon membranes (Hybond-N+, Amersham, Arlington Heights, IL) for Northern blot analysis. Membranes were then exposed to UV light for 5 min. The blots were hybridized with an 32P-labeled antisense SST riboprobe, transcribed in vitro by sp6 RNA polymerase from a 450-bp fragment of rat prepro-SST complementary DNA (cDNA) in pSP65 (6). Prehybridization was carried out at 42 C for 16 h in the prehybridization solution containing 50% deionized formamide, 10% dextran sulfate, 0.1% SDS, 10x Denhardt’s, 0.1% pyrophosphate, 1 M NaCl, and 100 µg/ml denatured salmon sperm DNA. For hybridization, 6 x 106 cpm of the labeled RNA probe were added in 20 ml hybridization buffer, and then the membranes were incubated at 65 C for 24 h.

After hybridization, the membranes were washed twice (5 min/each) in 2x SSC at 42 C, twice (30 min/each) in 2x SSC, 0.5% SDS at 65 C, and twice (30 min/each) in 0.2x SSC at 65 C.

Membranes were exposed to x-ray film (Hyperfilm, Amersham) with intensifying screens for 1–2 days at -80 C.

To ensure that equal amounts of RNA were loaded and transferred, membranes were washed and subsequently hybridized with a rat [{gamma}-32P]ATP-labeled 18S ribosomal RNA (rRNA) oligonucleotide (24 bp), as a control probe. Membranes were prehybridized for 16 h at 50 C in a solution containing 10x Denhardt’s, 0.1% SDS, 2x SSC, and 100 µg/ml denatured salmon sperm DNA. Hybridization was performed for 24 h at 50 C in the same buffer, after the addition of 4 x 106 cpm of the labeled probe. After hybridization, they were washed once (5 min) in 2x SSC at 65 C and three times (30 min/each) in 2x SSC at 40 C. Autoradiograms were developed after exposure to x-ray film for 4–6 h at -80 C using intensifying screens.

Hybridization signals were quantitated by densitometry, using a Hirschmann (Elscript 400, Hirschrann Gersetebau, Germany) scanning densitometer. Data were expressed in arbitrary densitometric units. The results of SST mRNA levels were expressed as the ratio of SST mRNA to 18S rRNA.

Statistical analysis
Data (mean ± SEM) were expressed as percentage change in relation to control values (control = 100). The results were evaluated using ANOVA and the least significant difference test (LSD test). P < 0.05 was taken as the criterion for significance; n = number of independent experiments.

The SST mRNA decay rates were obtained by regression analysis and evaluated using a Log Rank Statistic test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGFßs reduce SST mRNA levels and SST secretion in hypothalamic cells
The mean levels of SST release in our hypothalamic cultures were 2 ng/plate. Figures 1Go and 2Go show the time course and dose-dependent effect of TGFß1 on SST mRNA levels and SST secretion in monolayer hypothalamic cell cultures. SST mRNA content was already reduced after 4 h incubation with 5 ng/ml of TGFß1 (30% inhibition, P < 0.01) and continued suppressed at 12 and 24 h. On the other hand, in the presence of TGFß1 (5 ng/ml), SST secretion was not changed after 4 h, but was significantly reduced after 6 h (23% inhibition, P < 0.01), and progressively thereafter (9, 12 and 24 h). Figure 2Go also demonstrates the dose-response effect of TGFß1 for 24 h on SST secretion, with a maximum effect at 5 ng/ml (46% inhibition, P < 0.01).



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  		Figure 1. Prepro-SST mRNA levels assessed by Northern blot analysis. Hypothalamic cells in monolayer culture were treated with TGFß1 (1, 2, and 5 ng/ml) for 4 h (A), 12 h (B), and 24 h (C). In panel D, cells were treated for 24 h with vehicle (c), 5 ng/ml TGFß1, 10-4 M forskolin (FK), 10-4 M forskolin together with 5 ng/ml TGFß1 (FK+TGF ß1), 10-3 M (Bu)2cAMP (db-cAMP), and 10-3 M (Bu)2cAMP together with 5 ng/ml TGFß1 (db-cAMP+TGF ß1). Results were expressed as a ratio SST mRNA/18S rRNA. Data (mean ± SEM) are shown as percentage change in relation to control values (n = at least 3 independent experiments; **, P < 0.01).

 


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  		Figure 2. SST secretion determined by RIA in hypothalamic cells in monolayer culture treated with TGFß1 (1, 2, and 5 ng/ml) for 4, 6, 9, 12, and 24 h (A and B). In panel C, cells were treated for 24 h with TGFß1 (5 ng/ml), 10-4 M forskolin (FK), and 10-4 M forskolin together with 5 ng/ml TGF ß1 (FK+TGF ß1). Data (mean ± SEM) are expressed as percentage of control values (n = at least 3 independent experiments; **, P < 0.01).

 
To provide conclusive evidence that the effects of TGFß1 on SST reflect direct actions on neurons, we treated hypothalamic cultures with Ara-C (1.5 µM), which resulted in basically glial-free neuron cultures (22, 23), as determined by GFAP staining (Fig. 3Go). In Ara-C-treated cultures, mean levels of SST secretion in basal conditions were 25 pg/plate. Incubation with TGFß1 for 24 h (5 ng/ml) significantly reduced basal SST mRNA levels (38% inhibition, P < 0.01) and SST secretion (57% inhibition, P < 0.01) (Fig. 4Go).



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  		Figure 3. A, GFAP immunofluorescence histochemistry in mixed neuronal-glial cultures, 10 days after plating; B, negative control of GFAP immunofluorescence histochemistry in mixed neuronal-glial cultures, in the absence of the GFAP first antibody. C, GFAP immunofluorescence histochemistry in Ara-C-treated neuronal cultures, 10 days after plating; and D, negative control of GFAP immunofluorescence histochemistry in Ara-C-treated neuronal cultures, in the absence of the GFAP first antibody. The cells were photographed at 100x magnification with the same time of exponsure in a Zeiss fluorescence microscope (Carl Zeiss, Thornwood, NY).

 


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  		Figure 4. Prepro-SST mRNA levels (A) and SST release (B) in glial-free hypothalamic cultures (see Materials and Methods). Neurons were treated with TGFß1 (5 ng/ml) for 24 h. SST mRNA levels were expressed as a ratio SST mRNA/18S rRNA. Results (mean ± SEM) are shown as percentage change in relation to control values (n = at least 3 independent experiments; **, P < 0.01).

 
We next evaluated the role of TGFß2 and TGFß3 on SST mRNA and SST secretion in hypothalamic cell cultures. Cells were incubated for 24 h with 1, 2, and 5 ng/ml of TGFß1, -ß2, and -ß3, and SST mRNA content and SST secretion were measured (Figs. 5Go and 6Go, respectively). We found that the inhibitory effect of TGFß1 was mimicked by TGFß2 and -ß3.



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  		Figure 5. Prepro-SST mRNA content in hypothalamic cells in monolayer culture treated with TGFß1 (1, 2, and 5 ng/ml) (A), TGFß2 (1, 2, and 5 ng/ml) (B), and TGFß3 (1, 2, and 5 ng/ml) (C) for 24 h. Results were expressed as a ratio SST mRNA/18S rRNA. Data (mean ± SEM) were expressed in percentage of control values (n = at least 3 independent experiments; **, P < 0.01).

 


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  		Figure 6. SST release in hypothalamic cells in monolayer culture treated with TGFß1 (1, 2, and 5 ng/ml) (A), TGFß2 (1, 2, and 5 ng/ml) (B), and TGFß3 (1, 2, and 5 ng/ml) (C) for 24 h. Data (mean ± SEM) were expressed in percentage of control values (n = at least 3 independent experiments; **, P < 0.01).

 
TGFß1 reduces cAMP-induced SST mRNA levels and SST secretion
Treatment of hypothalamic cell cultures with 10-4 M forskolin or 10-3 M (Bu)2cAMP for 24 h increased SST mRNA levels by 2.5- and 3.5-fold, respectively, (P < 0.01, in both cases). This stimulatory effect was significantly reduced when cells were treated with forskolin or (Bu)2cAMP in the presence of TGFß1 (5 ng/ml) (Fig. 1Go and Fig. 7Go).



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  		Figure 7. Prepro-SST mRNA and 18S rRNA levels in hypothalamic cells in monolayer culture. Representative Northern blot. Cells were treated for 24 h with vehicle (C), TGF ß1 (5 ng/ml), 10-3 M (Bu)2cAMP (db-cAMP), and 10-3 M (Bu)2cAMP together with TGFß1 (db-cAMP+TGF ß1).

 
On the other hand, treatment of hypothalamic cell cultures with 10-4 M forskolin for 24 h increased SST release by 2-fold (P < 0.01). Moreover, incubation with 5 ng/ml TGFß1 prevented the stimulatory effect of forskolin on SST release (Fig. 2Go).

TGFß1 inhibition of SST mRNA levels is blocked by Chx or puromycin
To analyze whether the reduction of SST mRNA by TGFß1 required new protein synthesis, hypothalamic cell cultures were incubated for 4, 12, and 24 h with one of two different inhibitors of protein synthesis: cycloheximide (10 µg/ml) (26, 27), or puromycin (10 µg/ml) (27), in the presence or absence of TGF ß1 (5 ng/ml). These treatments did not affect neuronal survival as assessed by trypan blue staining (data not shown). Figure 8Go shows that the decrease in SST mRNA observed after treatment with TGFß1 was prevented when cells were incubated with Chx together with TGFß1. The same effect was observed when cells were treated with puromycin. This clearly indicates that the action of TGFß1 on SST mRNA content requires de novo protein synthesis.



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  		Figure 8. Prepro-SST mRNA content in hypothalamic cultures after treatment for 4, 12, and 24 h with vehicle (C), TGFß1 (5 ng/ml), 10 µg/ml Chx, and Chx together with TGFß1 (Chx+TGF ß1). Results are expressed as a ratio SST mRNA/18S rRNA. Data (mean ± SEM) are shown in percentage of control values (n = at least 3 independent experiments). **, P < 0.01.

 
TGFß1 increases the degradation rate of SST mRNA
To analyze whether SST mRNA degradation was involved in SST reduction induced by TGFß1, the decay of preexisting SST mRNA was determined by Northern blot analysis after blockade of new RNA synthesis with either AcD (5 µg/ml), or DRB (25 µg/ml) in the presence or absence of TGFß1 (5 ng/ml) for varying times (1, 2, 4, 6, 12, and 24 h). The effectiveness of the concentration of AcD or DRB used in this study in blocking new RNA synthesis has been assessed previously (28). Hybridization signals for SST mRNA were expressed in relation to the initial values (0 h). Plots corresponding to mean data of three independent experiments are shown in Fig. 9Go, demonstrating a similar decline in SST mRNA in the presence or absence of TGFß1.



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  		Figure 9. Rates of decay of prepro-SST mRNA in the presence or absence of TGFß1 (5 ng/ml) after inhibition of transcription with 5 µg/ml AcD (A) or 25 µg/ml DRB (B). RNA was isolated at the indicated times, and prepro-SST mRNA levels were determined by Northern blotting. Hybridization signals were corrected using a 18S rRNA oligonucleotide as a control probe. Data (mean ± SEM of three independent experiments) were expressed as percentage change in relation to the values at time 0. The decay curves were obtained by regression analysis.

 
On the other hand, the SST mRNA decay was also determined in cells that were treated with TGFß1 alone (5 ng/ml) and compared with that observed in cells treated with AcD or DRB together with TGFß1 (Fig. 10Go). Our data showed a significantly faster decay of SST mRNA in the cells treated with TGFß1 alone than in the presence of the transcriptional inhibitors (P < 0.05). These findings show that the effect of TGFß1 on SST mRNA was blocked by coincubation with transcriptional inhibitors and suggest that the TGFß1-mediated action on SST mRNA levels is dependent on the transcription of other factor(s).



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  		Figure 10. Decay curve of prepro-SST mRNA in hypothalamic cells in culture treated with TGFß1 (5 ng/ml) in the presence or absence of either 5 µg/ml AcD (A) or 25 µg/ml DRB (B). RNA was isolated and prepro-SST mRNA levels were analyzed as described in Fig. 9Go.

 
Furthermore, SST mRNA content was also examined in cells that were or were not pretreated with TGFß1 (5 ng/ml) for 4 h and then washed (time 0) and incubated for 12 h with AcD. Data were expressed in relation to the SST mRNA levels obtained at time 0. At this time the mean content of SST mRNA was significantly lower (P < 0.01) in TGFß1-treated than in untreated cell cultures. After 12 h with AcD, we found that SST mRNA content was significantly more reduced (by 44%, P < 0.01) in cells that were pretreated with TGFß1 than in cultures that received vehicle as pretreatment. This showed a faster degradation of this mRNA induced by TGFß1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data demonstrate for the first time a role of TGFß1, -ß2, and -ß3 on the regulation of SST mRNA levels and SST secretion in hypothalamic cells in culture. The administration of TGFß1 significantly reduced SST mRNA levels. This effect was dose-dependent and was shown evident after 4 h of treatment and thereafter. Furthermore, we found that TGFß1 also decreased SST secretion after treatments of 6 h and longer, but not after shorter incubations (4 h). This indicates that the primary effect of TGFß1 might be on SST biosynthesis, mediated by a rapid and significant inhibition of the steady state levels of the mRNA encoding SST, followed by a reduction in SST release.

The inhibitory effect of TGFß1 on SST mRNA and SST secretion was mimicked by the other TGFß isoforms expressed in the central nervous system (TGFß2 and TGFß3).

To provide more conclusive evidence that the inhibitory effects of TGFß1 on SST reflected direct actions on neurons, and not effects mediated by glial cells, we treated hypothalamic cultures with Ara-C. Our data showed that TGFß1 in these basically glial-free conditions (22, 23) also decreases basal SST mRNA levels and SST secretion.

As it is well known that SST gene transcription is regulated by cAMP through a cAMP-response element (29), we next studied the effect of TGFß1 on the forskolin and (Bu)2cAMP induced SST levels. Our data showed that incubation of the hypothalamic cells with TGFß1 for 24 h significantly reduced forskolin and (Bu)2cAMP-induced levels of SST mRNA content and SST release.

As with most other growth factors, the interaction between TGFß1 and its cell surface receptors (30) constitutes the first step of TGFß1-mediated actions. The cytoplasmic signal transduction mechanisms that follow TGFß1 binding to its receptor are not completely understood, but multiple nuclear factors have been implicated in transducing TGFß1 action to various TGFß-responsive genes. It has been demonstrated that TGFß1 may act either by inducing the synthesis of new factors (31) or by induction of posttranslational modifications of proteins or preexisting factors that bind to response elements (32). To establish whether the ability of TGFß1 to reduce hypothalamic SST mRNA levels depended on new protein synthesis, we examined the effect of TGFß1 on SST mRNA content in the presence of two different inhibitors of protein synthesis: cycloheximide, which traps mRNA on polysomes by inhibiting elongation of the nascent peptide; and puromycin, which removes mRNAs from polysomes (33). Our findings showed that the administration of either cycloheximide or puromycin for 4, 12, and 24 h prevented the inhibitory action of TGFß1 on SST mRNA levels, indicating that protein synthesis is necessary to obtain TGFß1’s full suppressive effects on SST mRNA.

The reduction of the steady state levels of SST mRNA could be due to an inhibition of transcription of the SST gene and/or to an acceleration of the degradation of the SST transcripts. To examine whether TGFß1 acted at the posttranscriptional level by altering the stability of SST mRNA, we determined by Northern blot analysis the decay rates of SST mRNA in hypothalamic cells in culture treated for 1, 2, 4, 6, 12, and 24 h with either of two inhibitors of new RNA synthesis: AcD, or DRB, in the presence or absence of TGFß1. We used here two transcriptional inhibitors with different modes of action: AcD, which intercalates into DNA and blocks almost all RNA synthesis, and DRB, an analog of adenosine, which selectively blocks the synthesis of heterogenous nuclear RNA (RNA polymerase II transcripts) (34, 35).

In this study, we found that the decay rate of SST mRNA, estimated after the administration of either AcD or DRB, was not significantly different in cultures treated with TGFß1 compared with untreated cultures. These results would suggest that TGFß1-induced reduction of SST mRNA content is not due to degradation of this mRNA. Nevertheless, as our data demonstrated that protein synthesis is necessary to obtain the TGFß1-inhibitory effect on SST mRNA, it could be possible that this newly synthesized protein(s) accelerated the degradation of SST mRNA, and that the synthesis of this protein was blocked by AcD or DRB. This notion was supported by our findings showing a faster rate of decay of SST mRNA in cells treated with TGF-ß1 alone compared with cells treated with a transcriptional inhibitor (DRB or AcD) together with TGF-ß1, indicating that in cells treated with TGF-ß1, SST mRNA can be stabilized by simultaneous administration of AcD or DRB. Finally, we found that when cells were pretreated with TGFß1 4 h before the administration of AcD, they presented a faster SST mRNA degradation than cells that were not pretreated with TGFß1.

All this together would indicate that TGFß1 induces the synthesis of a protein(s) that appears to accelerate the SST mRNA decay. As a result, the steady state levels of SST mRNA and SST secretion are also decreased. Whether TGFß1 has additional effects on SST gene transcription remains to be elucidated.

In conclusion, the present study clearly indicates that TGFßs exert a marked inhibitory effect on SST mRNA levels and SST secretion in fetal hypothalamic neurons in culture and, in addition, that TGFßs may play an important role on hypothalamic SST expression and release. Furthermore, our data suggest that in vitro assessment of neuropeptide gene expression by fetal neurons in monolayer culture can be a useful model in which to obtain further functional cues of the actions of TGFß isoforms at the hypothalamic level.


   Footnotes
 
1 This work was supported by grants from El Fondo de Investigaciones Sanitarias de la Seguridad Social (F.I.S.S.), Xunta de Galicia, and Fundación Ramón Areces. Back

Received February 10, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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