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Endocrinology Vol. 147, No. 12 5777-5785
Copyright © 2006 by The Endocrine Society

Thyroid Hormone Induction of Actin Polymerization in Somatotrophs of Hypothyroid Rats: Potential Repercussions in Growth Hormone Synthesis and Secretion

Francemilson Goulart da Silva, Gisele Giannocco, Marinilce Fagundes Santos and Maria Tereza Nunes

Departments of Physiology and Biophysics (F.G.d.S., G.G., M.T.N.) and Cell and Developmental Biology (M.F.S.), Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, SP, Brazil

Address all correspondence and requests for reprints to: Maria Tereza Nunes, Ph.D., Associated Professor, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, 05508-900, São Paulo, SP, Brazil. E-mail: mtnunes{at}icb.usp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone was shown to induce actin cytoskeleton polymerization in hypothyroid astrocytes and osteoblastic cells by a nongenomic mechanism. Polyadenylation of GH mRNA, a process that depends on cytoskeleton-associated proteins, was also shown to be regulated by thyroid hormone. Here we investigated by histochemistry and immunohistochemistry whether acute (100 µg per 100 g body weight, iv, for 30 min) or chronic (5 µg per 100 g body weight, ip, 5 d) administration of T3 to thyroidectomized (Tx) and sham-operated rats affects the somatotrophs F-actin cytoskeleton arrangement and its potential repercussion on GH synthesis and secretion. Thyroidectomy dramatically decreased the amount of somatotrophs F-actin content and induced the disassembly of the actin cytoskeleton. These effects were reversed by acute and chronic administration of T3. In addition, in Tx rat somatotrophs, GH labeling was detected mostly at the cell periphery. After 30 min of T3 administration, GH labeling decreased at periphery and increased in the perinuclear region, suggesting that GH secretion and synthesis were stimulated by T3. No differences were detected in the total actin protein content, although a decrease in the F- and increase in G-actin contents were detected in Tx rat pituitaries, a panorama that was reversed by acute T3 treatment, as shown by Western blotting analysis. The sham-operated animals’ somatotrophs were only mildly affected by acute T3 administration. The results indicate that the T3-induced rapid alterations on somatotroph actin cytoskeleton and GH cellular distribution resulted from actin filaments rearrangement, which characterizes a nongenomic action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (TH) effects on metabolism, growth, and development result from its interaction with thyroid hormone receptors present on specific regions of its target genes, by means of which it can inhibit or activate gene transcription (1, 2, 3).

However, some TH actions occur in a short period of time (minutes or even seconds) (4, 5, 6), are elicited by T4 as well as rT3 (7, 8), and occur even in the presence of drugs that block gene transcription, as actinomycin D (9, 10). These actions include alterations in Ca2+, Na+, and glucose transport through the plasma membrane, protein-kinases activity, translation efficiency, and half-life of specific mRNAs and mitochondrial respiration (11, 12, 13, 14, 15). These data and other accumulating evidences indicate that, besides its well-characterized genomic actions, TH also acts at the posttranscriptional level, the mechanisms of which are still not well understood (15, 16, 17, 18, 19).

One of the first evidences of TH nongenomic actions was reported by Leonard et al. (20) in astrocytes cultured in TH-depleted medium, a condition in which they present high type 2 deiodinase (D2) activity and a disassembly of the actin cytoskeleton. They observed that after 20 min of T4 administration, there was a decrease in D2 activity, which occurred concomitantly to an increase in cellular filamentous actin (F-actin). Moreover, the addition of cytochalasin B to the medium, a drug that blocks actin polymerization, prevented the T4-induced decrease of D2 activity, indicating that the effect of TH on D2 activity was associated with T4-induced actin polymerization (8, 20, 21). Additional evidence for actin polymerization by T3 was obtained in hypothyroid osteoblastic cells (22). Furthermore, T4 and rT3 exert direct positive control of the F-actin content in elongating neurites of cerebellar neurons (7). In conjunction, these data strongly indicate that the action of TH on cytoskeleton organization is through nongenomic pathways.

In parallel, Gereben et al. (23) demonstrated that TH decreases D2 content in HEK-293 and CHO cells through D2 ubiquitination, a process that leads to D2 degradation by proteasoma. Of note, ubiquitination involves addressing of ubiquitins to proteins that will be degraded and represents a posttranslational processing that also depends on structural modifications of cytoskeleton (24, 25, 26).

Previous studies carried out in our laboratory have also suggested a relationship between somatotrophs and the cytoskeleton that seems to be orchestrated by TH. We demonstrated that hypothyroid rats, in which plasma GH and gene expression levels are extremely low, exhibit a great increase in pituitary GH mRNA content when treated with subphysiological doses of T4 for 20–30 min (27). Even though it is known that TH can regulate the transcription of genes involved in posttranscriptional control of mRNA stability (28), this rapid response to the hormone suggests a nongenomic effect on the GH mRNA stabilization. Further studies have shown that the polyadenylation of the 3' end of the GH mRNA is increased in hypothyroid rats, a finding that has already been pointed out by Murphy et al. (29), and that the acute administration of T3 leads, in 30 min, to a further increase on it (30). It is important to emphasize that mRNA stability is commonly associated with the increase in mRNA polyadenylation and poly A tail protection from RNases, processes dependent on the poly A polymerase whose activity is regulated by cytoskeleton proteins (31) and poly A binding protein, which is a cytoskeleton-associated protein (32). Thus, disruption as well as rearrangement of the actin cytoskeleton could indirectly interfere with the mRNA stabilization process (33).

In light of this possibility, acute administration of TH might increase the GH mRNA content by increasing its stability through alterations on actin cytoskeleton organization in somatotrophs. In fact, TH was shown to induce posttranscriptional effects in the stability of several mRNAs, like tubulin and GH secretagogue receptor (34, 35).

Taking into account that actin cytoskeleton arrangement is involved in a variety of biological processes, like hormonal protein synthesis, secretion, cellular proliferation, cellular adhesion, gene transcription, and mRNA stability (7, 8, 21, 34, 36, 37, 38, 39, 40), we attempted to investigate whether T3, acutely or chronically administered to thyroidectomized (Tx) and control rats, affects F-actin cytoskeleton arrangement in somatotrophs and actin content in pituitary. We also address the potential repercussions of acute T3 administration on GH synthesis and secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
T3, methylmercaptoimidazole, Triton X-100, 4,6-diamino-2-phenylindole, and the antibody anti-ß actin were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum, BSA, protein molecular weight markers, and ß-mercaptoethanol were purchased from Life Technologies, Inc. (Grand Island, NY). Polyvinylidene difluoride filters (PVDF Hybond-P), enhanced chemiluminescent kit, and antimouse horseradish peroxidase antibody were purchased from Amersham Biosciences (Piscataway, NJ). Rhodamine-phalloidin was purchased from Molecular Probes (Eugene, OR), whereas the fluorescein isothiocyanate-conjugated goat antirabbit antibody was purchased from Jackson Immunolabs (West Grove, PA). The commercial kit RIA-gnost T3 was obtained from CIS Bio International (Gif-sur-Yvette, France). The optimum cutting temperature compound was obtained from Sakura Finetek U.S.A., Inc. (Torrance, CA). The rabbit antiserum against rat GH was purchased from Dr. A. F. Parlow (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA). All other reagents were purchased from Labsynth (São Paulo, SP Brazil).

Animals and treatments
Male Wistar rats weighing 200–250 g were obtained from our own breeding colony and maintained on rat chow and tap water ad libitum. They were housed in a room kept at constant temperature (23 ± 1 C) and on a 12 h-light, 12-h dark (lights on at 0700 h) schedule. The animals were made hypothyroid by surgical Tx after being deeply anesthetized with ketamine and xylazine and received 0.03% methylmercaptoimidazole plus 45 mM calcium chloride in drinking water during 20 d. The animals were divided into the following groups: 1) euthyroid sham-operated animals (SO); 2) euthyroid sham-operated rats treated with saturating doses of T3 administered iv 30 min before the animals were killed; 3) hypothyroid (Tx); 4) a T3 chronic group, in which the animals suffered thyroidectomy and after 20 d were treated ip with T3 during a 5-d period (Tx+ T3 5 d); and 5) a T3 acute group, in which the animals suffered thyroidectomy and after 20 d received saturating doses of T3 administered iv, 30 min before the animals were killed (Tx + T3 30'). The T3 dose used in chronic treatments was 5 µg per 100 g body weight (BW) (41, 42), whereas the dose in acute experiments was 100 µg T3 per 100 g BW, a pharmacological dose known to induce the saturation of thyroid hormone receptors (30, 43, 44, 45). At least four animals were used in each group.

Rats were weighed and killed by decapitation at the indicated intervals. The pituitaries were excised and used for the analysis of F-actin arrangement, GH distribution, and nonpolymerized actin (G-actin)-F-actin expression. The experimental protocol conformed with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation and was approved by the Institute of Biomedical Sciences/University of São Paulo-Ethical Committee for Animal Research.

Procedures
Fixation and cryoprotection.
Pituitaries were fixed in 2% paraformaldehyde solution in 0.1 M phosphate buffer (PB; pH 7.4), during 30 min at 4 C and cryoprotected with 30% sucrose in PB for at least 24 h at 4 C. After optimum cutting temperature embedding and freezing, 7-µm-thick sections were obtained and positioned in gelatin-coated slides.

Immunohistochemistry and F-actin staining.
Nonspecific binding sites were blocked with 10% normal goat serum diluted in PB/0.2% Triton X-100 for 1 h at room temperature (RT). Incubation with the primary rabbit antibody against rat GH (diluted 1:5 in PB/0.2% Triton X-100) was performed overnight at RT. After rinsing, sections were incubated with the secondary antibody (1:200) for 90 min at RT, followed by F-actin staining using rhodamine-phalloidin, according to the manufacturer’s instructions. Nuclear counterstaining was obtained with 4,6-diamino-2-phenylindole. Experiments were performed in duplicate, and for confocal analysis an LSM 510 microscope (Zeiss, Jena, Germany) was used. The confocal microscope allows an analysis of the labeling in depth, coupled to a semiquantitative analysis of labeling intensity and distribution. At least three to four stacks of representative photo micrographs were taken from each slide, and the picture showing the higher labeling intensity was used for the semiquantitative analysis in all groups. Two types of semiquantitative analysis were used: 1) a color distribution over the pictures, indicating labeling intensity, where blue less than green less than yellow less than red, and 2) a graph showing the distribution of the pixels in the picture according to their luminosity, ranging from values 0–250, also indicating labeling intensity (X axis) and the number of pixels (Y axis).

Protein measurement.
The pituitaries were homogenized in a Polytron in specific buffer [15 mM Tris-HCl (pH 7.4), 15 mM MgCl2, 300 mM NaCl, 1% Triton X-100] on ice. The homogenate was centrifuged at 12,000 rpm for 40 min at 4 C, the supernatant was removed, and the protein content was determined by the Lowry’s method (46). Forty micrograms of protein were lyophilized, dissolved in Laemmli’s sample buffer, and submitted to SDS-PAGE.

Actin fractionation.
The protocol was adapted from Posern et al. (47) and Haller et al. (48). The pituitaries were lysed in 0.5 ml lysis buffer [50 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 20 mM HEPES (pH 7.9)] on ice. Phalloidin (132 nM) was then added to the lysis buffer for 10 min. The homogenate was centrifuged at 100,000 x g for 30 min at 4 C, and the supernatant and pellet fractions were obtained. The pellet was resuspended in 0.1 ml of actin lysis buffer, sonicated, and the protein content of supernatant and pellet fractions determined by the Lowry’s method. Equal amounts of protein (40 µg) were prepared and submitted to SDS-PAGE.

Western blotting analysis
The Laemmli’s method was used (49), with a 5% acrylamide stacking gel and a 10% acrylamide resolving gel. Electrotransfer of proteins from the gel to nitrocellulose membranes (Hybond-C; Amersham Pharmacia Biotech, Little Chalfont, UK) was performed for 60 min at 100 V. Nonspecific protein binding to the nitrocellulose membrane was reduced by preincubating the membrane with blocking buffer (5% nonfat dry milk, 2.7 mM KCl, 137 mM NaCl, 8 mM NaHPO4, 1.4 mM KPO4, and 0.1% Tween 20) overnight at 4 C. Incubation with the monoclonal antibody anti-ß actin (1:5000 diluted in blocking buffer) was performed for 2 h at RT, whereas the incubation with the secondary antibody antimouse IgG conjugated to horseradish peroxidase (1:5000 in blocking buffer) lasted 1 h, also at RT. The reaction was visualized by chemiluminescence (enhanced chemiluminescent kit; Amersham, Aylesbury, UK). Blots were analyzed by scanning densitometry and quantified using the Image Master-1D-Pharmacia Biotech SW software (Pharmacia Biotech, Uppsala, Sweden).

Evaluation of the effectiveness of the treatments
The effectiveness of the treatments in the different experimental groups was evaluated by determining: the total serum T3 levels by RIA, using a standard curve prepared by adding different concentrations of T3 in iodothyronine-free rat serum, the ventricular dry weight, and the ratio between the ventricular and body weight.

Statistics
The data were expressed as mean ± SEM and subjected to ANOVA, followed by the Student-Newman-Keuls test. Differences were considered significant at P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effectiveness of thyroidectomy and T3 treatment was verified by determining total serum T3 concentration, ventricular dry weight, and ratio between ventricular weight and BW, all of which were decreased in Tx animals and increased after 5 d of T3 treatment (Tx + T3 5d), as expected. The Tx animals that received T3 acutely (Tx + T3 30') also presented increased T3 levels. These data are presented in Table 1Go and confirm the induction of hypo- and hyperthyroidism in our experimental groups.


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  		TABLE 1. Ventricular wet weight (VWW), ventricular dry weight (VDW), ventricular wet weight to body weight ratio (VWW/BW ratio), and serum T3levels of euthyroid (SO), hypothyroid (Tx), Tx rats acutely (Tx + T3 30':100 µg per 100 g) or chronically (Tx + T3 5d: 5 µg per 100 g/5 d) treated with T3

 
T3 effects on the anterior pituitary F-actin content
Figure 1Go shows the effect of T3 administration on the anterior pituitary F-actin content of rats subjected to the experimental conditions previously described. It contains five panels (A to E), which represent the pituitaries from the following: euthyroid (SO) (A) and Tx rats (B); Tx rats acutely (30 min) (C) or chronically (5 d) treated (D) with T3; and SO rats acutely (30 min) treated with T3 (E). The panels show: 1) the photo micrography of the pituitaries, in which the red color represents the polymerized actin (F-actin); 2) the semiquantitative analysis of their F-actin content, in which blue, green, yellow, and red colors indicate the intensity of the F-actin labeling in a crescent order; and 3) the graphic representation of the F-actin content, in which the curve deviation to the right indicates the presence of higher amount of F-actin.


Figure 1
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  		FIG. 1. F-actin distribution in anterior pituitary of euthyroid (A), hypothyroid (Tx) (B), Tx acutely (100 µg per 100 g BW) (C) or chronically (5 µg per 100 g BW, 5 d) (D) treated with T3 and euthyroid rats acutely treated with T3 (100 µg per 100 g BW) (E). The panels show on the first column the photo micrography of the pituitaries, in which the red color represents the polymerized actin (F-actin); on the second the semiquantitative analysis of their F-actin content, in which blue, green, yellow, and red colors indicate the intensity of the F-actin labeling in a crescent order; and on the third column the graphic representation of the F-actin content, in which the curve deviation to the right indicates the presence of higher amount of F-actin. The X axis contains an intensity scale indicating the luminosity of the pixels, whereas the Y axis shows the number of pixels. Except for the hypothyroid group (B), all groups reach maximum luminosity and show a similar distribution. The hypothyroid group, however, does not reach maximum luminosity and the graph shows a clear deviation to the left, indicating that the majority of the pixels in the figure have low luminosity and that the amount of F-actin is much lower. The actin content was determined by histochemical analysis, whereas the semiquantitative analysis of F-actin content was provided by a specific confocal software. Magnitude bars, 20 µm.

 
Figure 1BGo shows that the pituitary of the Tx animals presented a dramatic reduction of the F-actin content, compared with the euthyroid ones (Fig. 1AGo), which was promptly increased with acute administration of T3 (30 min; Fig. 1CGo). On the other hand, the F-actin content of the pituitaries from Tx animals chronically treated with T3 (Fig. 1DGo) as well as of those from the euthyroid rats acutely treated with T3 (Fig. 1EGo) were similar to the euthyroid group.

Semiquantitative analysis of F-actin content provided by a specific confocal software supported the above data: 1) a weak fluorescent signal (represented mostly by the blue color) and a curve deviation to the left, confirmed the disassembly of the actin in the pituitaries derived from Tx rats (Fig. 1BGo) and 2) a strong fluorescent signal (green/yellow/red) and a curve deviation to the right, in Fig. 1Go, C and D, indicated that T3 acutely (30 min) or chronically (5 d) administered to Tx rats induced pituitary actin polymerization. The fluorescent signal as well as the curve representation of F-actin content of pituitaries from euthyroid rats acutely treated (Fig. 1EGo) or not (Fig. 1AGo) with T3 did not differ considerably from each other.

TH effect on the F-actin and GH distribution in somatotrophs
Figure 2Go exhibits panels (Fig. 2Go, A–E) in which slices of pituitaries presenting somatotrophs are shown. On the left panel, the F-actin labeling is shown, in red; on the middle panel, the GH was labeled, by immunohistochemistry, and is presented in green; and on the right panel, both images were merged, demonstrating the F-actin distribution in somatotrophs. Figure 2BGo shows that F-actin content of Tx rats somatotrophs is decreased, when compared with the euthyroid rats (Fig. 2AGo). This panorama is not different from that observed with the totality of the Tx rats pituitary cells (Fig. 1BGo), which presented a disassembly of the actin cytoskeleton. The GH content of the somatotrophs of the Tx animals is also decreased, as expected, and the superposition of both images (F-actin and somatotrophs) corroborates these findings.


Figure 2
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  		FIG. 2. F-actin and GH distribution in somatotrophs of euthyroid (A), hypothyroid (Tx) (B), Tx acutely (100 µg per 100 g BW) (C) or chronically (5 µg per 100 g BW, 5 d) (D) treated with T3 and euthyroid rats acutely treated with T3 (100 µg per 100 g BW) (E). On the left the F-actin labeling is shown, in red; on the middle, the GH was labeled, by immunohistochemistry, and is presented in green color; and on the right both images were superimposed, demonstrating the F-actin distribution in somatotrophs. Magnitude bars, 20 µm.

 
Acute administration of T3 rapidly increased F-actin content in Tx rats somatotrophs concomitantly to a fast decrease in GH content (Fig. 2CGo). When Tx rats were chronically treated with T3, the F-actin content was not different from those acutely treated with T3, even though the GH content of the somatotrophs was greatly increased (Fig. 2DGo). Acute T3 administration to euthyroid rats did not induce relevant alterations on F-actin and GH distribution on somatotrophs (Fig. 2EGo).

We determined the subcellular distribution of F-actin and GH by confocal analysis of somatotrophs derived from euthyroid (SO) and Tx rats, before and after acute T3 treatment (Fig. 3Go). In this figure F-actin (Fig. 3Go, A, C, and E, at left) and GH (Fig. 3Go, B, D, and F, at right) distribution in somatotrophs of SO (Fig. 3Go, A and B), Tx (Fig. 3Go, C and D), and Tx rats acutely treated with T3 (Fig. 3Go, E and F) are shown. In SO somatotrophs F-actin was concentrated mainly in the cell cortex (Fig. 3AGo, arrowheads), whereas GH was observed in numerous secretory granules distributed throughout the cytoplasm (Fig. 3BGo). In hypothyroid (Tx) somatotrophs, however, the content of cortical F-actin appeared to be lower than in SO cells; actin was scattered throughout the cell, in a disorderly fashion (Fig. 3CGo), suggesting a less polymerized state. Secretory granules containing GH were mostly at the periphery of the cells and less numerous than in SO cells (Fig. 3DGo, arrows). This panorama changed completely when T3 was acutely administered to the Tx animals, suggesting that actin polymerization promoted by T3 increased the amount of F-actin in the somatotroph cortex (Fig. 3Go, E and F). In parallel to this cytoskeleton rearrangement, only a few GH granules were still present at the periphery of the cells (arrows), whereas a strong GH labeling clearly appeared at the perinuclear region of the somatotrophs (Fig. 3FGo, asterisk), in which organelles associated with the translational machinery, such as the endoplasmic reticulum and Golgi complex, predominate.


Figure 3
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  		FIG. 3. Confocal analysis of somatotrophs derived from control (A and B), hypothyroid (C and D), and hypothyroid rats acutely treated with T3 (100 µg per 100 g BW) (E and F) showing, on the left, the F-actin (in red) distribution and, on the right, the GH distribution in the same cells (in green). The cortical F-actin is pointed by arrowheads. Arrows point to secretory granules containing GH, and the asterisk shows the perinuclear GH labeling observed in hypothyroid rats acutely treated with T3. Magnitude bars, 5 µm.

 
Evaluation of the total and F- and G-actin content
Lastly, we performed Western blot analysis of pituitary actin content. In Fig. 4AGo, the total actin content of pituitaries excised from euthyroid (SO), Tx, and Tx animals subjected to acute (30 min) or chronic (5 d) T3 treatment is presented. No changes in the total actin content were observed between the experimental groups studied. However, the proportion between polymerized (F) and nonpolymerized (G) actin fractions of the Tx animals pituitaries was modified by the acute T3 treatment (Fig. 4Go, B and C). G-actin content was increased in pituitaries of Tx animals and decreased in Tx animals subjected to acute T3 treatment. The opposite was seen with F-actin (Fig. 4CGo). F-actin content decreased in Tx rat pituitaries and returned to SO values in Tx animals acutely treated with T3 (P < 0.05).


Figure 4
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  		FIG. 4. A, Total actin protein content of pituitaries excised from euthyroid (SO), hypothyroid (Tx), and hypothyroid animals subjected to acute (30 min) (100 µg per 100 g BW) (Tx + T3 30') or chronic (5 µg per 100 g BW, 5 d) (Tx + T3 5 d) T3 treatment. B and C, G- and F-actin, respectively, protein content of pituitaries excised from euthyroid (SO), hypothyroid (Tx), and hypothyroid animals subjected to acute (30 min) (100 µg per 100 g BW) T3 treatment (Tx + T3 30'). In A, proteins from total pituitaries extract (40 µg) were separated by SDS-PAGE and electrotransferred to a membrane. In B and C total pituitaries were homogenized in a buffer containing phalloidin. The supernatant, which contains the G-actin (B), and the pellet, which contains the F-actin (C), were obtained after ultracentrifugation, and 40 µg of the proteins contained in these fractions were separated by SDS-PAGE and electrotransferred to a membrane. Actin content was detected by Western blotting, using specific antibodies for G-actin. Blots from a representative experiment are shown. Data are shown as mean ± SEM for four independent experiments (n = 10 per group). *, P < 0.05 vs. Tx + T3 30' (G-actin); **, P < 0.05 vs. SO and Tx + T3 30' (F-actin) as determined by ANOVA and Student-Newman-Keuls post hoc test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have shown that pituitaries and, in particular, that somatotrophs of hypothyroid rats present a disassembly of the actin cytoskeleton, in which the G-actin prevails over the F-actin. This panorama is promptly reversed by a 30-min acute treatment with pharmacological doses of T3, as shown by the histochemical and Western blot analysis. This effect was restricted to the somatotrophs of hypothyroid rats because the same T3 treatment did not modify the actin cytoskeleton arrangement in euthyroid rats. This result supports our hypothesis that the hormone promotes the assembly of the actin cytoskeleton. In control animals the actin cytoskeleton is already organized, and this particular posttranslational effect of T3 cannot be seen.

The capability of thyroid hormone to induce rapid actin polymerization was first demonstrated by Siegrist-Kaiser et al. (8) in astrocytes cultured in a hypothyroid medium in which the addition of T4 and rT3, but not T3, was able to induce significant actin polymerization, indicating a novel extranuclear action of thyroid hormone. Our study corroborates these results, even though we have used T3 instead of T4, as well as the studies carried out by Banovac and Koren (22) in hypothyroid osteoblastic cells and Luegmayr et al. (50) in MC3T3-E1 osteoblastic cells. In fact, many studies have shown that not only T4 but also T3 in supraphysiological and, in a lesser extension, in physiological doses is able to induce nongenomic actions (9, 15, 16, 22, 51, 52).

The ability of TH in modulating the actin polymerization of somatotrophs, without interfering with the actin synthesis, points to a nongenomic action of T3 on the actin cytoskeleton arrangement and leads us to consider that processes like GH synthesis and secretion can also be altered by this mechanism when thyroid functional states are disturbed.

The lower expression of GH in hypothyroid rat somatotrophs was already expected because the transcriptional rate of GH is dramatically reduced in hypothyroid states (53), but an additional reduction after acute T3 treatment suggests that GH secretion has been elicited by T3.

In fact, preliminary studies performed in our laboratory have shown, by means of real-time PCR, that liver IGF-I mRNA expression, which is extremely reduced in hypothyroid rats, when compared with the control group, was drastically increased 30 and 60 min after T3 administration (data not shown). Considering that GH is the most important regulator of hepatic IGF-I synthesis and that thyroid hormones have relatively little or no direct effect on IGF-I synthesis, this result strengths our assumption that T3 increased GH secretion through the stimulation of actin polymerization (54, 55).

Actually, it is known that the exocytosis of secretory granules requires a highly organized cytoskeleton (56) as well as an increase in the intracellular calcium concentration; there are evidences that T3 activation of Na+/H+ exchanger, in L-6 myoblasts, occurs by a fast nongenomic mechanism, which requires the mobilization in intracellular calcium (16). Moreover, T4/T3 were shown to interact with the ß3-subunit of the {alpha}Vß3 integrin present in plasma membrane, which is supposed to link the extracellular matrix to actin cytoskeleton. Signaling pathways activated by integrin receptors usually regulate cytoskeletal arrangement through activation of rho GTPases (15, 57).

Furthermore, somatotrophs from hypothyroid rats chronically treated with T3 presented an enhanced GH content, without significant modification on the degree of actin polymerization, which denotes that in this case a genomic effect is taking place.

The detailed analysis of the F-actin and GH distribution in somatotrophs showed that, in the hypothyroid animals, the actin cytoskeleton is disassembled and GH secretory granules were less numerous and were present mostly at the cell periphery, suggesting that GH secretion is impaired. On the other hand, after the acute T3 administration, a strong GH labeling appeared at the perinuclear region of the somatotrophs, in which predominant organelles associated with the translational machinery, such as the endoplasmic reticulum and Golgi complex. This fact suggests that GH transcripts were promptly recruited and translated at the ribosome, clearly indicating that GH synthesis was also stimulated by T3.

Although the mechanisms underlying the mRNA targeting to the translational machinery are not completely clarified, cytoskeleton proteins are certainly involved in this process (37, 58). Indeed, the recruitment of some mRNAs by specific proteins (poly A binding protein is essential for this mechanism), the association of this complex to the cytoskeleton, and their subsequent addressing to specific areas, in which they are translated, have been reported in neuronal and nonneuronal cells (59). Furthermore, it is known that the mRNA translation at the polysome fraction depends on the mobility of specific proteins to the translational machinery, which is also under control of the actin cytoskeleton (40).

The possibility that T3 could interfere with the GH mRNA translation was addressed in a preliminary trial in our laboratory by means of the polysome profile analysis. The data obtained showed a decreased GH mRNA content in polysome fractions of hypothyroid rats pituitaries, when compared with the control rats, and a prompt increase on it, 30 min after T3 administration (60). These findings strongly indicate that the GH mRNA recruitment to the translational machinery is impaired in hypothyroid state and that T3 administration acutely reverts this picture.

Taking into account these considerations, we are compelled to believe that the rapid T3 induction of actin polymerization of somatotrophs occurs by nongenomic mechanisms and might potentially interfere with the GH synthesis and secretion.

The pathway used by T3 to promote actin polymerization is, so far, unknown. It is possible that T3 could activate specific kinases, such as rho kinase, which is known to promote the rearrangement of the G-actin already present in the cells, or others that could be linked to rho GTPase function. The possibility that integrins could mediate this effect, as mentioned elsewhere, is very attractive, considering that, besides the fact that they mediate the interaction between the extracellular matrix to the actin cytoskeleton, they can activate MAPK as well as rho GTPases (15, 55).

It is important to stress that T3 target genes, like those that code for the malic enzyme, tubulin, and GH, are known to be posttranscriptionally regulated by thyroid hormone as well (34, 61, 62, 63) and that most of these effects involve alterations in the stability of the message, which, as already pointed out, depends on the poly-A polymerase and poly-A binding protein, which are also associated with the actin cytoskeleton (31, 32).

Finally, the actin cytoskeleton disarrangement observed in pituitaries from hypothyroid rats is not restricted to the somatotrophs and could underlie some of the clinical aspects of hypothyroidism.


   Acknowledgments
 
The authors thank the technician Leonice Lourenço Poyares for the excellent technical assistance and Dr. Chao Yun Irene Yan for revising the manuscript.


   Footnotes
 
This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 02/07943-3) (to M.T.N.). F.G.d.S. and M.T.N. are recipients of Conselho Nacional de Pesquisa e Desenvolvimento fellowships.

Disclosure statement: the authors have nothing to disclose.

First Published Online September 21, 2006

Abbreviations: BW, Body weight; D2, type 2 deiodinase; F-actin, filamentous actin; G-actin, nonpolymerized actin; PB, phosphate buffer; RT, room temperature; SO, sham-operated animals; TH, thyroid hormone; Tx, thyroidectomized.

Received January 26, 2006.

Accepted for publication September 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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