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. 2017 Aug 24:8:211.
doi: 10.3389/fendo.2017.00211. eCollection 2017.

Grass Carp Follisatin: Molecular Cloning, Functional Characterization, Dopamine D1 Regulation at Pituitary Level, and Implication in Growth Hormone Regulation

Roger S K Fung  1 , Bai Jin  1 , Mulan He  1 , Karen W Y Yuen  1 , Anderson O L Wong  1
Affiliations

Grass Carp Follisatin: Molecular Cloning, Functional Characterization, Dopamine D1 Regulation at Pituitary Level, and Implication in Growth Hormone Regulation

Roger S K Fung et al. Front Endocrinol (Lausanne). .

Abstract

Activin is involved in pituitary hormone regulation and its pituitary actions can be nullified by local production of its binding protein follistatin. In our recent study with grass carp, local release of growth hormone (GH) was shown to induce activin expression at pituitary level, which in turn could exert an intrapituitary feedback to inhibit GH synthesis and secretion. To further examine the activin/follistatin system in the carp pituitary, grass carp follistatin was cloned and confirmed to be single-copy gene widely expressed at tissue level. At the pituitary level, follistatin signals could be located in carp somatotrophs, gonadotrophs, and lactotrophs. Functional expression also revealed that carp follistatin was effective in neutralizing activin's action in stimulating target promoter with activin-responsive elements. In grass carp pituitary cells, follistatin co-treatment was found to revert activin inhibition on GH mRNA expression. Meanwhile, follistatin mRNA levels could be up-regulated by local production of activin but the opposite was true for dopaminergic activation with dopamine (DA) or its agonist apomorphine. Since GH stimulation by DA via pituitary D1 receptor is well-documented in fish models, the receptor specificity for follistatin regulation by DA was also investigated. Using a pharmacological approach, the inhibitory effect of DA on follistatin gene expression was confirmed to be mediated by pituitary D1 but not D2 receptor. Furthermore, activation of D1 receptor by the D1-specific agonist SKF77434 was also effective in blocking follistatin mRNA expression induced by activin and GH treatment both in carp pituitary cells as well as in carp somatotrophs enriched by density gradient centrifugation. These results, as a whole, suggest that activin can interact with dopaminergic input from the hypothalamus to regulate follistatin expression in carp pituitary, which may contribute to GH regulation by activin/follistatin system via autocrine/paracrine mechanisms.

Keywords: activin; dopamine; follistatin; grass carp; growth hormone; pituitary.

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Figures

Figure 1
Figure 1
Sequence analysis, gene copy number, and tissue expression of grass carp follistatin. (A) Phylogenetic analysis of carp follistatin cDNA with corresponding sequences reported in other species using neighbor-joining method with MEGA 6.0. The numbers presented with the guide tree are the percentage of bootstrap values based on a 1,000 bootstraps. (B) 3D protein modeling of carp follistatin using SWISS-MODEL and DeepView with the crystal structure of human follistatin as the template. The regions in red and blue represent the helical motifs and β sheet structures, respectively, within the N-terminal and FSD1–3 domains. The four structural domains can be clustered into a “concave” structure, which is known to be essential for activin binding. (C) Gene copy number for carp follistatin deduced by genomic Southern. Southern blot was conducted with a DIG-labeled probe for follistatin in carp genomic DNA with prior digestion of restriction enzymes as indicated. (D) Tissue expression of follistatin in grass carp. Total RNA was isolated from selected tissues and brain areas as indicated and subjected to RT-PCR with primers specific for follistatin. The authenticity of PCR product was confirmed by PCR Southern with DIG-labeled probe for follistatin with parallel PCR for β actin as internal control. (E) Characterization of follistatin transcript in the carp pituitary. Total RNA was isolated from the carp pituitary, resolved in 1% agarose gel and subjected to Northern blot with DIG-labeled probe for follistatin. In this experiment, total RNA prepared form the gonad was used as a positive control and parallel blotting for β actin transcript was used as the internal control (M: size markers for RNA transcripts).
Figure 2
Figure 2
Functional characterization of carp follistatin in αT3 cells. (A) Validation of follistatin blocking of activin’s action in αT3 cells with pAR3-Lux transfection. In this experiment, αT3 cells were transfected with the pAR3-Lux reporter with 5′promoter carrying tandem repeats of activin-responsive elements and challenged for 24 h with human activin A (10 ng/ml) and B (10 ng/ml), respectively. In treatment group, activin A and B induction were conducted in the presence of human follistatin (100 ng/ml). (B) Grass carp follistatiin on activin-induced pAR3-Lux reporter activity expressed in αT3 cells. Conditioned medium obtained from CHO cells transfected with the expression vector for carp follistatin was used as the source of follistatin of carp origin. For functional testing of carp follistatin, αT3 cells transfected with pAR3-Lux were challenged for 24 h with activin A (10 ng/ml) and B (10 ng/ml) in the presence of the conditioned medium containing carp follistatin. Parallel incubation with conditioned medium harvested from CHO cells transfected with the blank vector pcDNA3.1 was used as the control treatment. In these experiments, cell lysate was prepared from αT3 cells after activin treatment (with/without follistatin) and used for luciferase activity measurement with a Dual-Glo® assay kit. Data presented are expressed as mean ± SEM (N = 6) and the groups denoted by different letters represent a significant difference at P < 0.05 (ANOVA followed by Newman–Keuls test).
Figure 3
Figure 3
Effects of activin A and B on follistatin, activin βA, and activin βB expression in grass carp pituitary cells. (A) Time course and (B) dose dependence of activin-induced follistatin mRNA expression. (C) Dose dependence of activin treatment on activin βA and βB mRNA expression. In time course experiment, pituitary cells were incubated with activin A (10 ng/ml) or activin B (10 ng/ml) for the duration as indicated up to 48 h. To test for dose dependence, the duration of drug treatment was fixed at 24 h with pituitary cells challenged with increasing levels of activin A or B, respectively. After activin treatment, total RNA was isolated and used for real-time PCR of follistatin, activin βA, and activin βB mRNA, respectively. Data presented are pooled from four separate experiments (N = 4) and the groups denoted by different letters represent a significant different at P < 0.05 (ANOVA followed by Newman–Keuls test).
Figure 4
Figure 4
Removal of endogenous activin by follistatin on follistatin, activin βA, and activin βB expression in grass carp pituitary cells. (A) Effects of follistatin treatment on basal and activin-induced follistatin mRNA expression. (B) Effects of follistatin treatment on activin βA and βB mRNA expression. (C) Effects of follistatin treatment on basal and activin-induced growth hormone (GH) mRNA expression. To examine the effect on basal expression of follistatin mRNA, pituitary cells were treated for 24 h with increasing doses of follistatin as indicated. To test the effects of follistatin on activin modulation of follistatin and GH gene expression, pituitary cells were challenged for 24 h with activin A (10 ng/ml) or B (10 ng/ml) with/without the co-treatment of follistatin (100 ng/ml). In these experiments, total RNA was isolated after drug treatment and subjected to real-time PCR of follistatin, activin βA, activin βB, and GH mRNA, respectively. Data presented are pooled from four separate experiments (N = 4) and the groups denoted by different letters represent a significant different at P < 0.05.
Figure 5
Figure 5
Dopaminergic regulation of follistatin and activin expression in carp pituitary cells. (A) Effects of dopamine (DA) and its non-selective agonist apomorphine (APO) on follistatin mRNA expression. Pituitary cells were treated for 24 h with increasing doses of APO (0.001–1 μM) or with a single dose of DA (1 μM). (B) Effects of DA and APO on activin βA and βB mRNA expression. Pituitary cells were treated for 24 h with DA (1 μM) or APO (1 μM). (C) Effects of DA D1 and D2 agonists on follistatin mRNA expression. Pituitary cells were treated for 24 h with increasing doses of the DA D1 agonist SKF77434 or D2 agonist Ly171555 as indicated. (D) DA D1 and D2 antagonists on the inhibitory effect of DA on follistatin mRNA expression. Pituitary cells were challenged with DA (1 μM) for 24 h in the presence or absence of the DA D1 antagonist SKF83566 (5 μM) or D2 antagonist sulpiride (5 μM). In these studies, total RNA was isolated after drug treatment and subjected to real-time PCR measurement for follistatin, activin βA, and activin βB mRNA, respectively. Data presented are pooled from four experiments (N = 4) and the groups denoted by different letters represent a significant different at P < 0.05.
Figure 6
Figure 6
Follistatin regulation by activin, growth hormone (GH), and human chorionic gonadotropin (hCG) and their interaction with dopaminergic D1 stimulation in grass carp pituitary cells. (A) Dopamine (DA) D1 activation on activin-induced follistatin mRNA expression. Pituitary cells were incubated for 24 h with activin A (10 ng/ml) or B (10 ng/ml) in the presence or absence of the D1 agonist SKF77434 (1 μM). (B) DA D1 activation on the differential effects of GH and hCG on follistatin mRNA expression. Pituitary cells were exposed to GH (30 ng/ml) or hCG (30 IU/ml) for 24 h with/without the co-treatment of the D1 agonist SKF77434 (1 μM). After drug treatment, total RNA was isolated and subjected to real-time PCR for follistatin mRNA measurement. Data presented are pooled from four experiments (N = 4) and the groups denoted by different letters represent a significant different at P < 0.05.
Figure 7
Figure 7
Effects of dopamine (DA) D1 activation on follistatin expression induced by activin and growth hormone (GH) in carp somatotrophs. (A) Follistatin expression in carp somatotrophs (GH cells), lactotrophs [prolactin (PRL) cells], and gonadotrophs [luteinizing hormone (LH) cells]. The three cell types (marked by green arrows) were identified by immunostaining using antisera for carp GH, PRL, and LH, respectively, and isolated on HS caps using laser capture microdissection technique. After that, RT-PCR was performed on the cells captured (~200 cells for individual cell types) using primers for follistatin. Mixed populations of pituitary cells were used as a positive control and RNA samples prepared with/without reverse transcription (±RT) were used to control for potential contamination with genomic DNA. The authenticity of PCR product was confirmed by PCR Southern using DIG-labeled probe for carp follistatin and parallel PCR for β actin mRNA was used as the internal control. (B) DA D1 activation on activin- and GH-induced follistatin mRNA expression in enriched somatotrophs. Enriched somatotrophs (86–92% pure) were prepared from mixed populations of carp pituitary cells by Percoll gradient centrifugation and the leftover cells (with only 3–4% somatotrophs, referred to as the “somatotroph-deficient” cells) were also harvested to serve as a parallel control. The two cell fractions were treated with activin A (10 ng/ml), activin B (10 ng/ml), and GH (30 ng/ml) for 24 h in the presence or absence of the D1 agonist SKF77434 (1 μM). After that, total RNA was isolated and used for real-time PCR for follistatin mRNA measurement. Data presented are pooled from four experiments (N = 4) and the groups denoted by different letters represent a significant different at P < 0.05.
Figure 8
Figure 8
Working model of dopaminergic D1 interaction with activin/follistatin system for growth hormone (GH) regulation in the carp pituitary. In the carp pituitary, local release of GH can induce activin A and B production, which can exert a negative feedback to inhibit GH secretion and GH gene expression. Meanwhile, pituitary expression of follistatin, especially in carp somatotrophs, is also up-regulated by activin A and B, which can trigger a feedback inhibition to suppress the stimulatory effects of activin on follistatin and GH gene expression. Dopaminergic input from the hypothalamus, which is well-documented to stimulate GH release via activation of pituitary dopamine (DA) D1 receptor, can inhibit both basal as well as activin- and GH-induced follistatin expression at pituitary level. This DA D1 action presumably can relief activin feedback on GH regulation from the inhibitory effect of follistatin, which is a major component for signal termination of activin/follistatin system at tissue level.
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