Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion

<div><p>Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.</p></div

Signal transduction for GH-induced activin βA and βB gene expression in carp pituitary cells.

<p>Pituitary cells were treated for 24 hr with porcine GH (300 ng/ml) in the presence or absence of (A) the JAK₂ inhibitor AG490 (400 μM) or STAT₅ blocker IQDMA (50 μM), (B) MEK_1/2 inhibitor U0126 (10 μM) or ERK_1/2 inactivator FR180204 (2 μM), and (C) PI3K inhibitor LY294002 (10 μM) or Akt blocker HIMOC (20 μM). After treatment, total RNA was isolated and subjected to real-time PCR of activin βA (right panels) and βB mRNA (left panels), respectively. Data presented are pooled results from four experiments and individual groups denoted by different letters represent a significant different at P < 0.05 (ANOVA followed by Newman-Keuls test).</p

Sequence analysis, gene copy number and pituitary expression of grass carp activin βA and βB.

<p>(A) Phylogenetic analysis of carp activin βA and βB cDNA sequences with corresponding sequences reported in other species using neighbour-joining method with MEGA 6.0. The numbers presented with individual nodes of the guide tree are the percentage of bootstrap values based on a 1000 bootstraps. (B) 3D protein modelling of grass carp activin βA and βB using SWISS-MODEL and DeepView with the crystal structures of human activin βA and βB as the template, respectively. The regions in red represent the α2 and α3 helical domains while the two pairs of antiparallel β strands forming the main core of activin β subunit are marked in blue. (C) Gene copy number for activin βA and βB deduced by genomic Southern. Southern blot was conducted with DIG-labelled probes for activin βA and βB, respectively, in grass carp genomic DNA with prior digestion of the restriction enzymes as indicated. (D) Characterization of activin βA and βB transcripts expressed in the carp pituitary. Total RNA was isolated from the carp pituitary, resolved in 1% agarose gel and subjected to Northern blot with DIG-labelled probes for activin βA and βB. Parallel blotting for β actin mRNA was used as the internal control. (MK: Size markers for RNA transcripts).</p

Regulation of activin and follistatin expression by local release of GH in carp pituitary cells.

<p>(A) GH treatment on activin βA, activin βB and follistatin mRNA expression at pituitary level. Pituitary cells were challenged for 24 hr with increasing concentrations of porcine GH as indicated. (B) Removal of endogenous GH by immunoneutralization on activin βA, activin βB and follistatin mRNA expression in carp pituitary cells. Pituitary cells were incubated for 24 hr with increasing levels of antiserum raised against carp GH and parallel treatment with normal rabbit serum (NRS, 1:2500) was used as a negative control. In these experiments, total RNA was isolated after drug treatment and used for real-time PCR measurement of activin βA, activin βB and follistatin mRNA, respectively. Data presented are pooled results from four separate experiments and individual groups denoted by different letters represent a significant different at P < 0.05 (ANOVA followed by Newman-Keuls test).</p

Tissue distribution of activin βA and βB expression in grass carp.

<p>(A) RT-PCR of activin βA and βB expression in selected tissues and brain areas. Total RNA was isolated from the tissues/brain areas as indicated and subjected to RT-PCR with primers specific for carp activin βA (Act βA) and βB (Act βB), respectively. The authenticity of PCR products was confirmed by PCR Southern with DIG-labelled probes for respective gene targets with parallel PCR for β actin as internal control. (B) LCM capture of immuno-identified lactotrophs (PRL cells), gonadotorphs (LH cells) and somatotrophs (GH cells) from grass carp pituitary cells. The three cell types (marked by black arrows) were identified by immunostaining using antisera for PRL, LH and GH, respectively. After pulsed with infrared laser, the target cells with signals for respective hormones were captured on LCM HS cap and subjected to RT-PCR with primers for different targets. (C) Expression of activin βA and βB as well as their receptors ActRIB and ActRIIB in immuno-identified pituitary cells. Pure populations of PRL, LH and GH cells captured on LCM HS cap (×25 or ×250 cells/cap) were used for RT-PCR with primers for carp activin βA and βB, respectively (upper panel). For detection of ActRIB and ActRIIB, RNA samples prepared from the respective cell types (×250 cells/cap) with reverse transcription (+RT) were used for RT-PCR using primers for carp ActRIB and ActRIIB, respectively (lower panel). Parallel PCR in RNA samples without reverse transcription (-RT) was used as negative control and PCR with the RT sample prepared from mixed populations of pituitary cells (Pit cells) was used as positive control. In these experiments, RT-PCR for β actin was routinely performed to serve as the internal control.</p

Regulation of activin and follistatin expression by local release of LH in carp pituitary cells.

<p>(A) LH and (B) FSH treatment on activin βA, activin βB and follistatin mRNA expression at pituitary level. Pituitary cells were treated for 24 hr with increasing levels of equine LH and FSH as indicated. (C) Removal of endogenous LH by immunoneutralization on activin βA, activin βB and follistatin mRNA expression in carp pituitary cells. Pituitary cells were incubated for 24 hr with increasing levels of antiserum raised against carp LH and parallel treatment with NRS (1:5000) was used as a negative control. In these experiments, total RNA was isolated after drug treatment and used for real-time PCR measurement of activin βA, activin βB and follistatin mRNA, respectively. Data presented are pooled results from four experiments and individual groups denoted by different letters represent a significant different at P < 0.05 (ANOVA followed by Newman-Keuls test).</p

Functional testing of grass carp activin A and B in GH3 cells.

<p>(A) Validation of actvin responsiveness in GH3 cells with pAR3-Lux transfection. GH3 cells were transfected with the activin-responsive pAR3-Lux reporter and challenged for 24 hr with human activin A (10 ng/ml) and B (10 ng/ml), respectively. To confirm that the effects observed were specific for activin, parallel experiments were conducted with activin A and B induction in the presence of human follistatin (300 ng/ml). (B) Effects of carp activin A and B on pAR3-Lux reporter activity expressed in GH3 cells. Conditioned media obtained from CHO cells transfected with the expression vectors for carp activin βA (gc.Act βA) and βB (gc.Act βAB) were used as the source of carp activin A and B, respectively. For functional testing of carp activins, GH3 cells with pAR3-Lux transfection were treated for 24 hr with the conditioned media containing activin A and B, respectively, with parallel treatment of conditioned medium harvested from CHO cells transfected with the blank vector pcDNA3.1(+) as the control. To confirm that the effects on pAR3-Lux reporter activity were indeed mediated by activin, parallel studies with the respective conditioned media were also repeated with co-treatment of follistatin (300 ng/ml). In these experiments, cell lysate was prepared from GH3 cells after drug treatment and used for luciferase (LUC) activity measurement using a Dual-Glo luciferase assay. Data presented are expressed as Mean ± SEM (N = 4) and the groups denoted by different letters represent a significant difference at P < 0.05 (ANOVA followed by Newman-Keuls test).</p

Inhibitory effects of activin and SMAD2/3 on GH promoter activity expressed in GH3 cells.

<p>(A) Activin A and B treatment on GH promoter activity expressed in GH3 cells. After pGH(-986).LUC transfection, GH3 cells were challenged for 24 hr with increasing doses of activin A and B as indicated. (B) 5’ Deletion analysis of GH promoter to delineate the activin-responsive region. GH3 cells were transfected with LUC reporters carrying decreasing lengths of GH promoter as indicated and treated for 24 hr with activin A (30 ng/ml) and B (30 ng/ml), respectively. (C) SMAD2 and SMAD3 expression on GH promoter activity expressed in GH3 cells. GH3 cells were transfected with pGH(-986).LUC in the presence of increasing doses of the expression vectors for SMAD2 and SMAD3, respectively. (D) Expression of dominant negative (DN) mutants of SMAD2 and SMAD3 on activin inhibition of GH promoter activity expressed in GH3 cells. GH3 cells were transfected with pGH(-986).LUC in the presence of the expression vector for SMAD2 DN mutant (Smad2 DN) or SMAD3 DN mutant (Smad3 DN). Parallel transfection with pcDNA3.1, the blank vector for DN mutants, was used as the control treatment. After transfection, the cells were challenged for 24 hr with activin A (30 ng/ml) and B (30 ng/ml), respectively. In these studies, cell lysate was prepared after drug treatment/SMAD expression and used for measurement of luciferase (LUC) activity with a Dual-Glo luciferase assay. Data presented for LUC activity are pooled results from four separate experiments and individual groups denoted by different letters (ANOVA followed by Newman-Keuls test) or marked with asterisks (Student’s t test comparing to the respective control) represent a significant difference at P < 0.05.</p

GH regulation by activin and follistatin in grass carp pituitary cells.

<p>Effects of increasing doses of human activin A and B on (A) GH secretion and GH cell content, and (B) GH mRNA expression in carp pituitary cells. In parallel experiments, the effects of removing endogenous activin with increasing levels of human follistation on (C) GH secretion and GH cell content, and (D) GH mRNA expression were also examined. In these studies, the duration of drug treatment was fixed at 48 hr. After treatment, culture medium (for GH release) and cell lysate (for GH cell content) were harvested and subjected to Western blot with GH antiserum with parallel blotting of β actin expression as the internal control. In separate experiments, total RNA was also isolated for real-time PCR of GH mature mRNA. GH mRNA data presented (Mean ± SEM) are pooled results 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).</p

Inhibitory actions of activin on GH- and hCG-induced GH gene expression in carp pituitary cells.

<p>(A) Activin A and B treatment on GH-induced GH mRNA expression. Pituitary cells were treated for 48 hr with porcine GH (100 ng/ml) in the presence of human activin A (30 ng/ml) and B (30 ng/ml), respectively. (B) Activin A and B treatment on hCG-induced GH mRNA expression. Pituitary cells were exposed to hCG (40 IU/ml) for 48 hr with/without the co-treatment of human activin A (30 ng/ml) and B (30 ng/ml), respectively. After drug treatment, total RNA was isolated and subjected to real-time PCR for GH mRNA measurement. Data presented are pooled results 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).</p