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Endocrinology Vol. 142, No. 9 4055-4065
Copyright © 2001 by The Endocrine Society

ARTICLES

Evidence that TGFß May Directly Modulate POMC mRNA Expression in the Female Rat Arcuate Nucleus

Sebastien Bouret, Myriam Tran Van Chuoi-Mariot, Vincent Prevot, Dominique Croix, Toru Takumi, Sylvie Jegou, Hubert Vaudry, Jean-Claude Beauvillain and Valerie Mitchell

INSERM U422 (S.B., M.T.V.C.-M., V.P., D.C., J.-C.B., V.M.), IFR 22, Laboratoire de Neuroendocrinologie et Physiopathologie Neuronale, 59045 Lille, France; Department of Anatomy and Brain Science (T.T.), Kobe University School of Medicine, Kobe 650-0017, Japan; and INSERM U413 (S.J., H.V.), IFRMP 23, Université de Rouen, 76821 Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. Sebastien Bouret, INSERM U422, 1 Place de Verdun, 59045 Lille Cedex, France. E-mail: bouret{at}lille.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of the present study was to determine whether TGFß, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGFß1, and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10-10 M TGFß1 was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGFß receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGFß and the presence of type II receptor for TGFß, respectively, in POMC neurons. The results indicated that TGFß receptor I mRNA and TGFß receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGFß receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGFß1 downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (ß-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGFß-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGFß receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGFß receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGFß1 may directly modulate the activity of POMC neurons through the activation of TGFß receptors. Therefore, the present study provides additional evidence for the involvement of TGFß1 in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLIAL-TO-NEURON COMMUNICATION within the hypothalamus is currently a field of intense research interest (for review see 1). TGFß is one of the factors expressed in hypothalamic astrocytes that appears to be involved in the control of neuroendocrine secretions (2, 3, 4, 5, 6, 7). Growth factors belonging to the TGFß superfamily bind to, and signal through, a receptor complex comprising two membrane-type serine-threonine kinases, called type I and type II receptors (8). Activation of type I receptor by ligand bound type II receptor on the cell surface results in phosphorylation of the associated Smad2 and Smad3 molecules by type I receptor. Receptor-activated Smad molecules are released from the receptor complex, associate with the common-mediator Smad (Smad 4) in the cytosol, and are translocated to the nucleus, where they directly influence gene expression (for review see 9). Takumi et al. (10) have cloned a type I receptor for TGFß and activin, and we have previously shown that TGFß-RI mRNA is expressed in GnRH neurons and in various cells of hypothalamic nuclei implicated in the regulation of the GnRH neuroendocrine axis, particularly in the arcuate nucleus (5). These observations suggest that TGFß may act both directly on GnRH neurons and indirectly via neuronal systems of the arcuate nucleus.

POMC-derived peptides, especially the endogenous opioid peptide ß-endorphin, are clearly implicated in the regulation of the hypothalamo-pituitary-gonadal axis (for review, see 11). Opioid peptides inhibit basal LH release in ovariectomized (OVX) rats, and are involved in events that trigger the LH surge on proestrus and steroid-treated OVX rats (12, 13, 14). The main group of POMC-expressing perikarya is found in the arcuate nucleus of the hypothalamus (15), and these neurons send projections toward various brain regions, including the medial preoptic area, where GnRH neurons are located (16). The activity of POMC neurons is regulated by gonadal steroid hormones as well as neuropeptides and neurotransmitters (for review, see 17). The expression of TGFß-RI mRNA in the arcuate nucleus (5) suggests that TGFß might modulate POMC neuronal activity and, consequently, GnRH secretion.

The purpose of the present study was to examine whether TGFß1 is able to modulate directly arcuate POMC neurons. The primary objective was to determine the effect of TGFß1 on POMC mRNA levels in the mediobasal hypothalamus. We next investigated whether POMC neurons expressed type I and type II receptors for TGFß and whether these receptors are coexpressed in arcuate cells. We also examined whether POMC (ß-endorphin) neurons contain Smad2/3 downstream-signaling molecules. Since POMC neuronal activity is regulated by gonadal steroids (18), we also investigated the possible effects of gonadal steroids on the coexpression between TGFß-RI and POMC mRNAs in OVX, OVX E2-treated (OVX+E2), and OVX E2 plus progesterone-treated (OVX+E2P) female rats. The present report shows, for the first time, that: 1) TGFß1 decreases the relative POMC mRNA level; 2) TGFß receptors and Smad2/3 are expressed in POMC neurons; and 3) gonadal steroids modulate the number of POMC neurons expressing TGFß-RI mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals. Adult female Wistar rats (200–250 g) were purchased from CERJ (Saint-Berthevin, France) and housed under constant 14-h light, 10-h dark conditions, with lights on at 0500 h. They were given free access to tap water and rat chow. Ovariectomies were performed under ketamine (20 mg/kg) and xylazine (10 mg/kg) anesthesia. Rats were handled two times weekly until killed. Before death, blood was collected, and plasma was stored at -20 C for E2, progesterone, and LH RIAs. All experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC), regarding mammalian research.

Experimental design
Exp 1. We examined the effect of TGFß1 on relative POMC mRNA levels in the mediobasal hypothalamus. To this aim, mediobasal hypothalamic fragments from OVX animals (n = 5, per experimental condition) were incubated ex vivo and exposed either to TGFß1, TGFß1 with the soluble form of TGFß receptor II (sRII), or sRII alone. A single-label in situ hybridization was then performed from fragment sections to evaluate the relative cellular POMC mRNA content in the sections examined. For this analysis, the arcuate nucleus was divided into four equal rostrocaudal subdivisions.

Exp 2. We sought to learn whether POMC neurons expressed TGFß receptors. We performed on tissue sections, from OVX animals (n = 5), double-labeling in situ hybridization to determine whether POMC neurons expressed TGFß-RI mRNA; and immunohistochemical labeling, coupled to single in situ hybridization to determine whether POMC neurons contained TGFß-RII immunoreactivity. For these experiments, the arcuate nucleus was divided into four equal rostrocaudal subdivisions. We also determined the occurrence of TGFß-RI mRNA expression in TGFß-RII-immunopositive cells of the arcuate nucleus, by an immunohistochemical technique coupled to a single in situ hybridization. Double-labeling immunohistochemistry was finally performed to investigate the presence of Smad2/3 immunoreactivity in POMC (ß- endorphin) neurons.

Exp 3. We investigated whether gonadal steroids could modulate the expression of TGFß-RI in POMC neurons by comparing the number of POMC neurons expressing TGFß-RI mRNA among groups in OVX, OVX+E2, and OVX+E2P animals. For this study, the arcuate nucleus was divided into four rostrocaudal areas. Rats were OVX and allowed to recover for 2 wk. On d 0, at 1000 h, the animals were injected with E2 benzoate (E2; 30 µg/rat, sc). On d 2, at 1000 h, half of the E2-treated rats were injected with progesterone (2 mg/rat, sc) to induce the LH surge in the afternoon. The group of OVX+E2-treated rats (n = 5) was killed on d 2 at 1000 h. The group of OVX+E2P-treated rats (n = 5) was killed on d 2 at 1600 h.

Ex vivo experiment
Dissection procedure. Animals were killed by decapitation. After rapid removal of the brain, a block of neural tissue encompassing the mediobasal hypothalamus was microdissected under a binocular magnifying glass. The hypothalamic explant was delineated by the posterior border of the optic chiasm, the anterior border of the mamillary bodies, and (laterally and dorsally) by the fornix. The total dissection time was less than 3 min after decapitation.

Incubation system. After dissection, mediobasal hypothalamic fragments were washed twice in Krebs-Ringer bicarbonate/glucose buffer (KRB, pH 7.4) containing bacitracine (23 µM; Sigma-Aldrich Corp., Saint-Quentin Fallavier, France) in an atmosphere of 95% O2-5% CO2 and incubated in vitro at 35 C in KRB as previously described (19). Briefly, after a 60-min preincubation period, mediobasal hypothalamic fragments were incubated for 120 min with either 4 x 10-10 M TGFß1 (Sigma-Aldrich Corp.), 4 x 10-10 M TGFß1 with 6 x 10-8 M sRII, or 6 x 10-8 M sRII alone. The medium was changed every 30 min. Controls consisted of an incubation of the mediobasal hypothalamic fragments in KRB alone (without drugs) during the 120 min of the incubation period. At the end of each experiment, fragments were fixed by immersion in 4% paraformaldehyde/0.1 M phosphate buffer for 18 h at 4 C, washed for 6 h in 0.05 M Coons veronal buffer (pH 7.4) containing 20% sucrose, and then embedded in Tissue-Tek (Miles Laboratories, Naperville, CA), and frozen in liquid nitrogen. Frozen 14-µm coronal sections were collected, mounted onto gelatin-coated slides, and stored at -80 C until used. To evaluate the relative POMC mRNA level in the mediobasal hypothalamic fragments of each group, a single-label in situ hybridization was performed as described below.

Tissue preparation
To determine the expression of TGFß receptors in POMC neurons, animals were anesthetized with ketamine (20 mg/kg) and xylazine (10 mg/kg). They were perfused intracardially with 5–10 ml saline followed by 500 ml 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The brains were removed and immersed in the same fixative for 2 h. They were then washed overnight in 0.05 M Coon’s veronal buffer (pH 7.4) containing 20% sucrose, embedded in Tissue-Tek (Miles Laboratories), and frozen in liquid nitrogen. Frozen 14-µm coronal sections were collected from the level of -1.8 to -4.8 mm, relative to the Bregma, according to the atlas of Paxinos and Watson (20). The sections were mounted onto gelatin-coated slides and stored at -80 C until used.

Riboprobe preparation
35S-labeled TGFß-RI receptor complementary RNA probes. The plasmid vector pcDNA I containing a XbaI/HindIII fragment of 2300-bp of the full-length rat TGFß-RI (B1) receptor (10) was linearized by cutting at a single site with XbaI for the antisense probe or HindIII for the sense probe. In vitro transcription was performed using the T7 RNA polymerase for the antisense and SP6 RNA polymerase for the sense TGFß-RI probes and [35S]cytidine 5'-triphosphate (Amersham Pharmacia Biotech, Orsay, France).

35S-labeled POMC complementary RNA probes. The plasmid vector pCRTMII containing a HindIII/XhoI fragment of 409-bp of the rat POMC gene (position 221–629 bp of the POMC exon III) (generously provided by Dr. Drouin, Montréal, Canada) was linearized by cutting at a single site with HindIII for the antisense probe or XhoI for the sense probe. In vitro transcription was performed using the T7 RNA polymerase for the antisense and SP6 RNA polymerase for the sense POMC probes and [35S]cytidine 5'-triphosphate (Amersham Pharmacia Biotech).

Digoxigenin-labeled POMC complementary RNA probe. A digoxigenin- labeled cRNA probe for POMC mRNA was made from the same plasmid used for synthesis of the 35S-labeled probe. The probe was synthesized in vitro from 1 µg linearized DNA with 1x digoxigenin RNA labeling mixture (Boehringer-Roche Diagnostics, Meylan, France), RNA polymerase, and 1x transcription buffer. This mixture was incubated at 37 C for 2 h. Residual DNA was digested with deoxyribonuclease.

Histochemical labelings
In each of the in situ hybridization and immunohistochemical experiments, all experimental groups were processed in parallel.

Single-label in situ hybridization for POMC mRNA. Single label in situ hybridization was performed, following a protocol described previously in detail (21). Briefly, processed sections were hybridized with riboprobe-hybridization buffer mix, which contains the 35S-labeled POMC cRNA probe (25,000 cpm/µl). Overnight hybridization at 55 C was followed by ribonuclease (RNase) treatment, a series of stringent washes, including a high-stringency wash at 60 C. Hybridized slides were dehydrated in 70% and 100% ethanols in ammonium acetate and dipped in NTB2 emulsion (Integra Biosciences, Eaubone, France). After a 10-d exposure, slides were developed.

Dual-label in situ hybridization for POMC mRNA and TGFß-RI mRNA. Dual-labeling in situ hybridization was performed as previously described (22). Briefly, processed tissues were hybridized with the riboprobe-hybridization buffer mix, containing 35S-labeled TGFß-RI cRNA probe (30,000 cpm/µl) plus the digoxigenin-labeled POMC cRNA probe (1:200). Overnight hybridization at 55 C was followed by RNase treatment, a series of stringent washes, including a high-stringency wash at 60 C. Tissue slices were incubated with antidigoxigenin antibody conjugated to alkaline phosphatase (Boehringer-Roche Diagnostics), and incubated in chromogen solution (tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate) (Boehringer-Roche Diagnostics). The reaction was stopped after 3 h by rinsing twice for 15 min in Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). The slides were dehydrated in 70% ethanol containing ammonium acetate (300 mM) and 100% ethanol, and dipped in Ilford K5 emulsion (Saint-Priest, France). All sections were developed after a 20-d exposure.

Immunohistochemical (TGFß-RII) and in situ hybridization (TGFß-RI or POMC) labelings. Tissue sections were prepared as described above. Microwave-oven (Samsung, Roissy, France) heating of tissue sections was then performed. Slides were rinsed in citrate buffer (10 mM, pH 6.0) for 10 min and preincubated for 5 min at 800 W and for 2 x 4 min at 400 W in an appropriate box containing citrate buffer (10 mM, pH 6.0). After cooling, sections were rinsed in 0.1 M Tris-buffered saline (TBS) and incubated for 48 h at 4 C with a rabbit polyclonal TGFß-RII antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted at 1:250 with TBS containing 0.3% Triton. Antigen-antibody binding sites were visualized using biotinylated-linked goat antirabbit (1:200, for 90 min) (Caltag Laboratories, Inc. Burlingame, CA) and the standard peroxidase ABC amplification method (for 1 h30) (Vector Laboratories, Inc. Burlingame, CA). After the DAB reaction, sections were rinsed in TBS and then processed for a single in situ hybridization labeling, as described above, for the detection of POMC or TGFß-RI mRNA expression in TGFß-RII immunopositive cells.

Double immunohistochemistry for ß-endorphin and Smad2/3. After citrate/microwave-treatment as described above, the slides were incubated for 48 h at 4 C with a cocktail containing goat polyclonal anti-Smad2/3 (1:100) (Santa Cruz Biotechnology, Inc.) and rabbit polyclonal anti-ß-endorphin (1:2000) (generously provided by Dr. G. Tramu, University of Bordeaux, Talence, France) antibodies diluted in TBS containing 0.3% Triton. Immunoreactivities were detected by incubating the sections with fluorescein isothiocyanate-conjugated antirabbit IgG (1:50) (Amersham Pharmacia Biotech) for 90 min, then with biotinylated linked antigoat IgG (1:200) (Caltag Laboratories Inc.), followed by Texas-Red streptavidin (1:200) (Amersham Pharmacia Biotech) for 1 h. In a final step, all sections were stained for 5 min, at room temperature, with the nuclear stain 4',6-diamidino-2-phenylindole dilactate (DAPI), which was diluted 1:400 in TBS. The sections were then coverslipped with glycerol-PBS (3:1, vol/vol) containing 0.1% p-phenylenediamine, to avoid fading, and observed using a conventional fluorescent microscope (Leica Corp., Rueil Malmaison, France).

Quantitative analysis
For the analysis of double-labeling staining, the arcuate nucleus was divided into four areas (designated A, B, C, and D) of approximately equal length, in the rostral-caudal plane, as previously described (22). This partitioning is commonly used because POMC neurons are heterogeneous in terms of projections, or sensibility to different factors (16, 22, 23, 24, 25, 26). For each animal, four tissue sections per rostrocaudal area were analyzed. First, each POMC mRNA-expressing neuron was examined for the presence of TGFß-RI mRNA expression or TGFß-RII immunoreactivity and, conversely, each TGFß-RI mRNA or TGFß-RII immunopositive cell was examined for the presence of POMC mRNA. The number of single- or double-labeled cells were then counted and averaged in the four rostrocaudal areas of the arcuate nucleus. Second, the number of POMC neurons expressing TGFß-RI mRNA was counted, and averaged in the four rostrocaudal areas of the arcuate nucleus from OVX, OVX+E2, and OVX+E2P.

For the determination of the relative POMC mRNA levels in mediobasal hypothalamic fragments incubated ex vivo, the arcuate nucleus was also divided into four areas. For each fragment, five tissue sections per rostrocaudal area were analyzed. The grain density corresponding to relative POMC mRNA levels in mediobasal hypothalamic fragments was measured according to previously published protocols (21, 22). Briefly, sections were viewed using an axioskop microscope (Carl Zeiss, Gottingen, Germany). The density of silver grains corresponding to relative POMC mRNA levels was quantified in the neurons using the DensiRag program of Biocom (Les Ulis, France), under a x40 epiillumination darkfield objective.

The boundaries of the arcuate nucleus were determined by using the corresponding azure blue-stained sections. Neurons were identified as labeled with POMC probe if the number of silver grains over the perikaryon was at least three times higher than the background.

Statistical analysis
Differences among groups were assessed with a one-way ANOVA, followed by a post hoc Bonferroni’s t test. Differences among groups and areas were regarded as significant when P was less than 0.05.

Control experiments
Specificity controls of in situ hybridization included incubation of the sections with 35S- and digoxigenin-labeled sense probes, pretreatment with RNase, and coincubation with a 100-fold excess of unlabeled antisense probe. Controls of the immunohistochemical staining was verified by using the labeled secondary antiserum alone. The specificity of the anti-ß-endorphin has been described earlier (27). The specificity of the TGFß-RII or Smad2/3 antibodies has been previously checked (28, 29). The TGFß-RII antibody reacts with the C-terminal domain of the receptor. The Smad2/3 antibody reacts with the C-terminal domain of both Smad2 and Smad3. No labeling was observed on control sections.

Plasma LH, 17ß-E2, and progesterone determination
Plasma LH levels were measured by RIA using materials supplied by the NIDDK rat pituitary hormone distribution program (Baltimore, MD), and values were expressed as nanograms per milliliter, using the LH pituitary reference preparation RP-3. Assay sensitivity was 0.02 ng/tube, and the intra- and interassay coefficients of variation were 6% and 8.5%, respectively. Plasma E2 was measured using an RIA kit optimized for the direct quantitative determination of very low concentrations of 17ß-E2 in human serum and plasma (e.g. in children), purchased from SORIN Biomedica (Antony, France). Assay sensitivity was 0.2 pg/tube, and the intra- and interassay coefficients of variation were 5.6% and 7.3%, respectively. Progesterone levels were measured in plasma samples without extraction, using an RIA kit purchased from SORIN Biomedica. Assay sensitivity was 5 pg/tube, and the intra- and interassay coefficients of variation were 5.5% and 8.1%, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
We first determined the effect of TGFß1 on POMC mRNA levels in the arcuate nucleus. As shown in Figs. 1Go and 2Go, treatment of mediobasal hypothalamic fragments with TGFß1 provoked a decrease of the mean density of silver grains overlying labeled POMC neurons (P < 0.01). TGFß1 produced a 57.9 ± 1.4% and 41.7 ± 2.3% decrease of relative POMC mRNA levels, compared with controls, in areas A and B of the arcuate nucleus, respectively (Figs. 1Go, A–D; and 2A), and a 39.2 ± 2.1% and 26.4 ± 2.6% decrease in the areas C and D of the arcuate nucleus, respectively (Figs. 1Go, E–H; and 2B). Interestingly, the decrease in POMC mRNA levels induced by TGFß1 was significantly higher in the most rostral part of the arcuate nucleus (i.e. area A) than in areas B, C, and D (P < 0.01) (Fig. 2Go). Similarly, the decrease of POMC mRNA levels was significantly higher in areas B and C than in area D (P < 0.01) (Fig. 2Go). The inhibitory effect of TGFß1 on POMC mRNA levels was reversed by addition of sRII, which presents the ability to inhibit the effect of TGFß (Fig. 2Go). Incubation of the fragments with sRII alone induced a slight, but not significant, increase of POMC mRNA levels (Fig. 2Go).



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  		Figure 1. Darkfield (A, C, E, and G) and brightfield (B, D, F, and H) microphotographs showing POMC mRNA-expressing neurons in control (A, B, E, and F) and TGFß1-treated (C, D, G, and H) mediobasal hypothalamic fragments. A–D, POMC mRNA-expressing cells from the rostral part of the arcuate nucleus; E–H, POMC mRNA-expressing cells from the caudal part of the arcuate nucleus. Note that POMC mRNA expression is decreased concomitantly with the treatment with TGFß1. V, Third ventricle; scale bars (A, C, E, and G), 75 µm and (B, D, F, and H) 7.5 µm.

 


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  		Figure 2. Relative amounts of POMC mRNA in the four rostrocaudal (A–D) subdivisions of the arcuate nucleus, as determined by the density of grains per cell, from mediobasal hypothalamic fragments incubated with either 4 x 10-10 M TGFß1 alone, 4 x 10-10 M TGFß1 in the presence of 6 x 10-8 M of soluble receptor II (sRII), or 6 x 10-8 M sRII alone. Control consisted of incubating fragments with buffer alone. The values are mean ± SEM. Significant differences (P < 0.01) among the average values for the groups considered are noted as a vs. b (statistical analysis with the Bonferroni’s test).

 
Exp 2
We next investigated the occurrence of TGFß receptor mRNA or protein in POMC neurons. We used an in situ hybridization technique to examine the expression of type I receptor of TGFß and an immunohistochemical technique to examine the expression of type II receptor for TGFß. TGFß-RI mRNA-expressing cells (Fig. 3Go, B and E) and TGFß-RII immunoreactive cells (Fig. 3Go, C and F) were widely expressed throughout the arcuate nucleus, and their distribution overlapped with that of POMC mRNA-expressing neurons (Fig. 3Go, A and D).



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  		Figure 3. Brightfield (A, C, D, and F) and darkfield (B and E) microphotographs showing the expression of POMC mRNA-containing neurons (A and D), type I serine-threonine kinase receptor for TGFß mRNA-expressing cells (B and E), and type II serine-threonine kinase receptor for TGFß immunopositive cells (C and F) in the rostral (A–C) and caudal (D–F) subdivisions of the arcuate nucleus. Note that this labeling exhibits an overlapping distribution across the arcuate nucleus. V, Third ventricle; scale bar, 120 µm.

 
Analysis of double labeling revealed that TGFß-RI and TGFß-RII receptor types were found in POMC neurons (Fig. 4Go). TGFß-RI mRNA expression (Fig. 4Go, A and B) and TGFß-RII immunoreactivity (Fig. 4Go, C and D) were detected in numerous POMC neurons without preferential distribution in a locoregional aspect of the arcuate nucleus. The analysis according to the rostrocaudal aspect of the nucleus revealed that the highest proportion of POMC neurons expressing TGFß-RI mRNA was located in the rostral part of the arcuate nucleus (i.e. area A) (Fig. 5AGo; P < 0.01). Similarly, POMC neurons containing TGFß-RII immunoreactivity were preferentially expressed in the rostral part of the arcuate nucleus (Fig. 5BGo; P < 0.01).



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  		Figure 4. Brightfield photomicrographs of the arcuate nucleus showing cells (A and B) labeled with a digoxigenin-conjugated cRNA probe for POMC mRNA (dark staining) and 35S-labeled cRNA probe for type I receptor mRNA (black silver grains), or (C and D) labeled with a 35S-labeled cRNA probe for POMC mRNA (black silver grains) and immunoreactive for TGFß-RII (dark precipitate). Arrows point to double-labeled neurons; arrowheads point to POMC neurons that do not express TGFß receptor; open arrows point to TGFß receptor-containing cells that do not express POMC mRNA. Scale bar, 8 µm.

 


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  		Figure 5. Proportion of POMC neurons containing TGFß-RI mRNA (A) or TGFß-RII immunoreactivity (B) in the four rostrocaudal subdivisions of the arcuate nucleus. Area A is the most rostral, and D is the most caudal area of the nucleus. The values are the mean ± SEM. Significant differences (P < 0.01) among the areas are indicated by different superscripts (statistical analysis with the Bonferroni’s test).

 
Colocalization between TGFß-RI mRNA and TGFß-RII protein was observed in numerous cells of the arcuate nucleus (Fig. 6Go). However, some TGFß-RI mRNA-expressing cells did not contain TGFß-RII immunoreactivity and, reciprocally, some TGFß-RII- immunoreactive cells did not contain TGFß-RI mRNA (Fig. 6Go). In addition, all TGFß-RI mRNA- or TGFß-RII- expressing cells in the arcuate nucleus did not contain POMC mRNA (Table 1Go).



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  		Figure 6. (A) Representative photomicrograph of the arcuate nucleus showing coexpression of TGFß-RI mRNA (black silver grains) and TGFß-RII immunoreactivity (dark staining) under brightfield illumination. A higher magnification of the microphotograph is shown in B. Arrows point to dual-labeled cells; the open arrow points to TGFß-RI cell that does not express TGFß-RII; arrowheads point to TGFß-RII cells that do not express TGFß-RI. Scale bars, (A) 50 µm; (B) 5 µm.

 

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  		Table 1. Proportion of TGFß-RI or TGFß-RII cells expressing POMC mRNA in the four rostrocaudal subdivisions of the arcuate nucleus from OVX animals

 
Having demonstrated that TGFß-RI and TGFß-RII receptors were expressed in POMC neurons, we further tested whether Smad signaling occurred in arcuate POMC neurons. As shown in Fig. 7Go, we observed that Smad2/3 immunoreactivity was present in both the cytoplasm and nucleus of some ß-endorphin (POMC) neurons of the arcuate nucleus.



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  		Figure 7. Microphotomicrographs of a section through the arcuate nucleus showing double-immunofluorescent immunostaining with a ß- endorphin (ß-END) antiserum (green fluorescence) and Smad2/3 antiserum (red fluorescence). Each panel corresponds to the same frame photographed. A shows ß-endorphin immunoreactivity; B shows Smad2/3 immunoreactivity; C shows the merged images of ß-endorphin and Smad2/3 immunoreactivities. Arrows indicate example of double-stained neurons; arrowheads indicate cells expressing Smad2/3 but not ß-endorphin immunoreactivity. ß-endorphin-positive neurons exhibit Smad2/3 immunoreactivity in their cytoplasm (C). D, the nuclear DAPI stain (blue fluorescence) reveals that Smad2/3-immunoreactivity is also present in the nucleus of ß-endorphin neurons. Scale bar, 12 µm.

 
Exp 3
We also examined the effect of ovariectomy and hormonal replacement on the number of POMC neurons that expressed TGFß-RI mRNA. Before comparing the number of POMC neurons expressing TGFß-RI mRNA, it was necessary to determine whether the number of POMC mRNA-expressing neurons varied according to the hormonal status of the animal. As shown in Table 2Go, no significant differences in the mean number of POMC mRNA-expressing neurons were observed among OVX, OVX+E2, and OVX+E2P animals, whatever rostrocaudal subdivision of the arcuate nucleus was considered. TGFß-RI mRNA was observed in numerous POMC neurons, whatever the hormonal status of the animal; however, the number of POMC neurons that expressed TGFß-RI mRNA was affected by E2. In the rostral subdivisions of the nucleus, i.e. areas A and B, the number of POMC neurons that expressed TGFß-RI mRNA was significantly lower in OVX+E2 animals, compared with OVX animals (P < 0.01) (Fig. 8Go). In area A, administration of progesterone to OVX+E2 animals reversed the inhibitory effect of estrogens on the number of POMC neurons that expressed TGFß-RI mRNA (P < 0.01) (Fig. 8Go). Conversely, in areas C and D, no significant variations of the number of POMC neurons expressing TGFß-RI were observed among the experimental groups (P > 0.05) (Fig. 8Go).


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  		Table 2. Mean number of POMC mRNA-expressing neurons per hemisection in the four rostrocaudal subdivisions of the arcuate nucleus from OVX, OVX+E2, and OVX+E2P animals

 


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  		Figure 8. Mean number of POMC neurons expressing TGFß-RI mRNA per hemisection in OVX (black column), OVX+E2 (gray column), and OVX+E2P (dark column) female rats across the four rostrocaudal subdivisions (A–D) of the arcuate nucleus. The values are the mean ± SEM. Significant differences (P < 0.01) among the average values for the groups considered are noted as a vs. b (statistical analysis with the Bonferroni’s test).

 
Hormonal levels
Average plasma LH, E2, and progesterone levels are shown in Table 3Go. In OVX+E2P female rats, the mean plasma LH level, on d 2, at the time of death, exhibited the characteristic pattern of a typical LH surge. The levels of plasma E2 and progesterone in OVX+E2P rats were significantly higher, compared with OVX animals. The OVX+E2 animals showed significantly increased levels of E2.


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  		Table 3. Average ± SEM concentrations of plasma LH, E2, and progesterone in OVX, OVX+E2, and OVX+E2P rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study suggests that TGFß1 directly regulates the activity of POMC neurons located in the arcuate nucleus since: 1) exposure of mediobasal hypothalamic fragments to TGFß1 provoked a significant decrease in POMC mRNA expression; 2) type I and type II serine-threonine kinase receptors for TGFß were expressed in POMC neurons; and 3) Smad2/3 downstream signaling molecules are expressed in the cytoplasm and are translocated into the nucleus of some POMC (ß-endorphin) neurons. Our data also show that POMC neurons located in the rostral part of the arcuate nucleus are a preferential target for TGFß1 action. In addition, we demonstrate that the number of POMC neurons expressing type I TGFß receptor mRNA is decreased by estrogen treatment in rostral arcuate nucleus neurons, and that this effect is reversed by progesterone treatment in a subset of the most rostral POMC neurons.

Previous studies have indicated that TGFß1 is produced in the hypothalamus by astrocytes (2, 3, 6, 30). Growth factors secreted by glial cells, have been implicated in the regulation of neuroendocrine functions within the hypothalamus (see 1). The present study shows that, in the arcuate nucleus, TGFß1 modulates the activity of POMC neurons by decreasing POMC mRNA levels. To our knowledge, the present study is the first report suggesting the existence of glia-to-neuron communication processes involving TGFß1 within the arcuate nucleus. TGFß1 exerts its effects on target cells through binding to the heterodimeric complex of type I and type II receptors (8). The present study shows that both type I and type II receptors for TGFß are expressed throughout the arcuate nucleus. The expression of type I receptor has been previously described (5), but this is the first time that the expression of TGFß-RII is reported in the arcuate nucleus. Surprisingly, a previous study failed to detect the expression of mRNA encoding for a type II of TGFß receptor within the hypothalamus (31). This discrepancy may be attributable to the existence of various type II receptors for TGFß, or the mRNA level for this receptor might be below the level of cellular detection by in situ hybridization. Because TGFß-RI and TGFß-RII have been shown to mediate the action of TGFß (32, 33, 34, 35) and because these two receptors are colocalized in most of the arcuate cells, we conclude that TGFß signaling occurs in the arcuate nucleus. However, although the majority of TGFß-RI-expressing cells also express TGFß-RII, all TGFß-RI-expressing cells do not express TGFß-RII. This observation may be ascribed to the fact that TGFß-RI receptors heterodimerize with both TGFß type II and activin type II receptors. Thus, one can speculate that cells expressing TGFß-RI, but not TGFß-RII, express an activin type II receptor. In support of this hypothesis, expression of the activin receptor Act-RII has been previously reported in the hypothalamus (36).

Using double in situ hybridization and in situ hybridization combined with immunocytochemistry, we show that TGFß-RI and TGFß-RII receptors are expressed in a representative population of POMC neurons. The occurrence of a TGFß1 signaling within POMC neurons is further demonstrated by the fact that TGFß1 affects POMC gene expression, suggesting that TGFß-RI and TGFß-RII are functional in these neurons. These observations suggest that the TGFß1-induced down-regulation of POMC gene expression may result from the direct action of TGFß1 on POMC neurons. Noticeably, we observed that TGFß1 has a more potent effect on POMC mRNA levels in the rostral arcuate nucleus. This result might be related to our findings showing that the proportion of POMC neurons expressing TGFß receptors is much higher in the rostral part of the arcuate nucleus than in its caudal part. POMC neurons located in the rostral arcuate nucleus thus seem to be the preferential target for the action of TGFß. Recent advances in the identification of direct substrates for the TGFß type I receptor include the discovery that Smads may act as direct effectors for the TGFß type I receptor. The Smad family of proteins has been identified as mediators of the TGFß signal from the cytoplasm to the nucleus. Upon direct phosphorylation by type I TGFß receptor, Smad2 or Smad3 binds to its Smad4 partner to form a heterodimeric complex and translocates into the nucleus. Once in the nucleus, Smads can potentially regulate the transcription of target genes (9). The presence of Smad2/3 immunoreactivity in both the cytoplasm and nucleus of ß- endorphin neurons further demonstrates that the TGFß signaling pathway is present in POMC neurons. This observation, together with the expression of TGFß receptors in POMC neurons, suggests that TGFß1 modulates POMC gene expression, at least in part, directly.

The physiological significance of this TGFß-POMC interrelationship remains unknown. However, POMC neurons have been implicated in the regulation of physiological functions, such as food intake and reproduction. Our results demonstrate that gonadal steroids modulate TGFß signaling in POMC neurons: though E2 induces a decrease in the number of POMC neurons expressing TGFß-RI mRNA in the rostral parts of the arcuate nucleus, progesterone reverses this effect in a subset of the most rostral neurons. Interestingly, POMC neurons form a heterogeneous population, with respect to their projection toward the preoptic area (16), where GnRH perikarya are located. Using retrograde tracers, Cheung and Hammer (16) have shown that POMC neurons that project to the preoptic area originate mainly from the rostral arcuate nucleus. In addition to an effect on TGFß receptor expression, estrogens could affect the TGFß system by modulating TGFß1 expression in the hypothalamus. In this regard, recent studies have shown that estrogen induces an increase in TGFß1 release from hypothalamic astrocytes (6). These effects of estrogens seem to be direct, given that hypothalamic astrocytes are known to express estrogen receptors (6, 37, 38, 39). As well, mRNA levels of TGFß1 fluctuate in the hypothalamus during the various phases of the estrous cycle, the highest level of expression occurring during the day of proestrus (40). Taken as a whole, these observations suggest that gonadal steroids may regulate the expression of both TGFß1 and TGFß receptors. Specifically, the timing of the action of TGFß1 on POMC neurons located in the rostral part of the arcuate nucleus seems to be tightly regulated by estrogen and progesterone. We can thus hypothesize that, on the day of proestrus: 1) the high levels of estrogen would induce in parallel with an increase in TGFß1 concentrations in the arcuate nucleus, a decrease of the number of POMC neurons expressing TGFß–RI receptors preventing them from responding to TGFß1; and 2) in the afternoon of proestrous, the rise of progesterone would induce an up-regulation of TGFß-RI expression in the rostral POMC neurons, allowing the inhibitory influence of TGFß1 to occur on those neurons that may modulate GnRH neuron activity.

In conclusion, the present study provides compelling evidence for the involvement of TGFß in the regulation of the activity of arcuate cells. We show that TGFß1 modulates the activity of POMC neurons and that this effect is mediated by the activation of type I and type II serine-threonine kinase receptors for TGFß. Additionally, gonadal steroids seem to be important modulators of the TGFß-POMC communication, especially in rostral POMC neurons. These results suggest that TGFß potentially plays an important role in the regulation of neuronal activity within the arcuate nucleus and provide additional evidence for the existence of a glial-to-neuron communication within the hypothalamus that may play a key role in neuroendocrine regulation, especially in the control of reproductive function.


   Acknowledgments
 
The authors thank Mrs. G. Mortreux for her excellent technical assistance, the NIDDK for the rat LH RIA material, and the Service Commun d’Imagerie Cellulaire (University of Lille II) for the image analysis system.


   Footnotes
 
This work was supported by the Lille-Amiens-Rouen-Caen opioid peptide program and the University of Lille II.

Abbreviations: DAPI, 4',6-diamidino-2-phenylindole dilactate; KRB, Krebs-Ringer bicarbonate/glucose buffer; OVX, ovariectomized; OVX+E2P, OVX E2 plus progesterone-treated; RNase, ribonuclease; sRII, soluble form of TGFß receptor II; TBS, Tris-buffered saline.

Received January 10, 2001.

Accepted for publication May 7, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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