Guian Huang, Chen He, Fengyan Meng, Juan Li, Jiannan Zhang, and Yajun Wang

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1 THYROID-TRH-TSH Glucagon-Like Peptide (GCGL) Is a Novel Potential TSH-Releasing Factor (TRF) in Chickens: I) Evidence for Its Potent and Specific Action on Stimulating TSH mrna Expression and Secretion in the Pituitary Guian Huang, Chen He, Fengyan Meng, Juan Li, Jiannan Zhang, and Yajun Wang Key Laboratory of Bioresources and Ecoenvironment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu , People s Republic of China Our recent study proposed that the novel glucagon-like peptide (GCGL), encoded by a glucagonlike gene identified in chickens and other lower vertebrates, is likely a hypophysiotropic factor in nonmammalian vertebrates. To test this hypothesis, in this study, we investigated the GCGL action on chicken pituitaries. The results showed that: 1) GCGL, but not TRH, potently and specifically stimulates TSH secretion in intact pituitaries incubated in vitro or in cultured pituitary cells monitored by Western blotting or a cell-based luciferase reporter assay; 2) GCGL (0.1nM 10nM) dose dependently induces the mrna expression of TSH but not 5 other hormone genes in cultured pituitary cells examined by quantitative real-time RT-PCR, an action likely mediated by intracellular adenylate cyclase/camp/protein kinase A and phospholipase C/inositol 1,4,5-trisphosphate/Ca 2 signaling pathways coupled to GCGL receptor (GCGLR); 3) GCGLR mrna is mainly localized in pituitary cephalic lobe demonstrated by in situ hybridization, where TSH-cells reside, further supporting a direct action of GCGL on thyrotrophs. The potent and specific action of GCGL on pituitary TSH expression and secretion, togetherwiththepartialaccordanceshownamongthetemporalexpressionprofilesofgcgl in the hypothalamus and GCGLR and TSH in the pituitary, provides the first collective evidence that hypothalamic GCGL is most likely to be a novel TSH-releasing factor functioning in chickens. The discovery of this novel potential TSH-releasing factor (GCGL) in a nonmammalian vertebrate species, ie, chickens, would facilitate our comprehensive understanding of the hypothalamic control of pituitary-thyroid axis across vertebrates. (Endocrinology 155: , 2014) ISSN Print ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received April 22, Accepted July 22, First Published Online July 30, 2014 In vertebrates, anterior pituitary, as one of the most important endocrine organs, contains multiple types of hormone-producing cells, such as somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs. These cells can synthesize and secrete GH, prolactin (PRL), TSH, ACTH, and gonadotropins (FSH and LH), respectively, which consequently regulate many vital physiological processes, such as growth, development, metabolism, stress, behavior, and reproduction (1). It is generally believed that the secretion of each pituitary hormone is tightly controlled by its specific hypothalamic factor(s), for instance, GH secretion by somatotrophs is under the stimulatory control of hypothalamic GHRH in mammals and chickens (2 5). Pituitary glycoprotein hormones, including TSH, FSH, and LH, are composed of 2 subunits ( - and -subunits). The -subunit is common among the 3, and the -subunits are unique to each hormone, eg, TSH -subunit is unique to TSH, hence conferring hormone specificity. It is well documented that TSH produced by thyrotrophs plays critical roles in stimulating thyroid hormone (T 4 and T 3 )secretion and, thus, in turn controls many physiological pro- Abbreviations: AC, adenylate cyclase; 2-APB, 2-Aminoethoxydiphenyl borate; cgcgl, chicken GCGL; CRH-R1, CRH type I receptor; E, embryonic day; ED, effective dose; GCG, glucagon; GCGL, glucagon-like peptide; GCGLR, GCGL receptor; IHC, immunohistochemical; IP3, inositol 1,4,5-trisphosphate; ISH, in situ hybridization; PKA, protein kinase A; PLC, phospholipase C; PRL, prolactin; qrt-pcr, quantitative real-time RT-PCR; TRF, TSH-releasing factor endo.endojournals.org Endocrinology, November 2014, 155(11): doi: /en

2 doi: /en endo.endojournals.org 4569 cesses in vertebrates, including metabolism, growth, and development. In mammals, pituitary TSH secretion is, in principle, controlled by a hypothalamic TRH (6), although TRH displays a secondary role in the regulation of PRL and GH secretion under certain physiological/pathological conditions (7, 8). In nonmammalian vertebrates, a hypothalamic releasing factor specific for TSH secretion, however, remains elusive. Despite the full conservation of TRH structure across vertebrates, TRH action on the pituitary in nonmammalian vertebrates seems to be nonidentical to that in mammals (9, 10). In chicken embryos or growing chicks, injection of TRH stimulates TSH secretion and elevates plasma T 4 levels (9, 11 13). However, this thyrotropic activity seems to be diminished in adult chickens (14, 15). In contrast, TRH potently induces pituitary GH secretion both in vitro and in vivo, and thus, it is more likely to act as a GH-releasing factor in chickens (3, 14 20). In amphibians, TRH stimulates pituitary TSH release moderately in adult frogs but not in tadpoles, and it also induces PRL and GH secretion effectively (10, 21 23). Interestingly, CRH, a well-known ACTH-releasing factor in mammals (24, 25), has been demonstrated to act as a TSH-releasing factor (TRF) in chickens, amphibians, and teleosts (16, 21, 26 30), in addition to its classic action on pituitary ACTH secretion (9, 16, 29, 31 35). In view of the fact that TRH and CRH can stimulate the secretion of multiple pituitary hormones in chickens and other lower vertebrates, it raises an intriguing, but fundamental, question, whether a hypothalamic releasing factor specific for TSH secretion exists and functions in nonmammalian vertebrates, such as in chickens. Recently, we identified a novel ligand-receptor pair, namely glucagon-like peptide (GCGL)-GCGL receptor (GCGLR), in chickens and other lower vertebrates (36). And our finding has been confirmed by a later study, in which GCGL-GCGLR was designated as GCRP-GCRPR (37). GCGL is a 29-amino acid peptide encoded by the novel glucagon-like gene (GCGL, EU718628) and shares high amino acid identity with chicken and mammalian glucagon (GCG) and low identity to other structurally related peptides, including GHRH (38), thus being classified as a new member of the GCG/GHRH/secretin superfamily (39). GCGLR (EU718627) is a receptor specific to GCGL and shares 53% identity with GCG receptor (40) and is viewed as a new member of GPCR B1 subfamily (41, 42). Interestingly, GCGL mrna expression is mainly restricted to several brain regions with the highest expression noted in the chicken hypothalamus, whereas GCGLR is abundantly expressed in the pituitary (36). The expression of GCGL-GCGLR in the hypothalamus-pituitary axis led us to hypothesize that GCGL may function as a novel hypophysiotropic factor to regulate pituitary functions in chickens (36). As an initial step to substantiate this hypothesis, the present study aimed to 1) investigate the GCGL action on pituitary hormone secretion; 2) examine whether GCGL stimulates pituitary hormone gene expression; and 3) reveal the temporal expression patterns of GCGL-GCGLR in the developing hypothalamus-pituitary axis. Our study, for the first time, demonstrated that GCGL can stimulate TSH secretion and expression specifically and potently in vitro, suggesting that GCGL may function as a novel TRF in chickens. Considering the existence of GCGL-GCGLR in chickens and other nonmammalian vertebrates (36), this study, together with future studies on exploring the functions of GCGL as a potential TRF in other nonmammalian vertebrate groups, would greatly advance our comprehensive understanding of neuroendocrine control of pituitary-thyroid axis across vertebrates. Materials and Methods Chemicals, hormones, antibodies, and primers All chemicals were obtained from Sigma-Aldrich, and restriction enzymes were obtained from TaKaRa unless stated otherwise. TRH was purchased from Sigma-Aldrich. Chicken GCGL (cgcgl) was synthesized using solid-phase Fmoc chemistry (GL Biochem) (36). cgcgl was prepared as 1mM stock solution dissolved in 0.05N NaOH and then diluted to the desired concentrations with serum-free medium before use. The pharmacological agents, including H89, MDL-12330A, U73122, 2-Aminoethoxydiphenyl borate (2-APB), thapsigargin, calmidazolium, and PD98059, were purchased from Calbiochem (Merck KGaA). Monoclonal antibodies for -actin and ERK1/2 were purchased from Cell Signaling Technology, Inc, and monoclonal anti-flag M2-peroxidase (HRP) antibody was from Sigma. The polyclonal antibodies against recombinant full-length chicken GH and PRL were prepared in our laboratory (43, 44), whereas the polyclonal antibody against chicken TSH partial sequence (DSNGKKLLLKSALSQN) was produced in rabbits according to a protocol previously reported (28). All primers used in this study were synthesized by Invitrogen and listed in Supplemental Table 1. RT-PCR and quantitative real-time RT-PCR (qrt-pcr) assay Adult chickens, 1-week-old chicks, 2-week-old chicks, and chicken embryos at embryonic day (E)8, E12, E16, and E20 (Lohmann Sandy strain) of both sexes were purchased from local commercial company and killed. All animal experiments were performed according to the guidelines provided by the Animal Ethics Committee of Sichuan University. Anterior pituitaries and hypothalami were collected either for cell culture or for total RNA extraction. Total RNA was extracted from chicken pituitaries (or hypothalami) or from cultured pituitary cells using RNAzol (Molecular Research Center) and reversely transcribed using moloney murine leukemia virus reverse transcriptase (Ta- KaRa). RT samples were then used for RT-PCR assay of

3 4570 Huang et al GCGL Is a Novel TRF in Chickens Endocrinology, November 2014, 155(11): GCGLR mrna expression (36) or for quantification of the mrna levels of target genes listed in the Supplemental Table 1. To measure the mrna levels of cgcgl, cgcglr, cgh, cprl, ctsh, cacth (also called POMC), clh, and cfsh genes, qrt-pcr assay was conducted on the CFX96 Real-time PCR Detection System (Bio-Rad), as described in our previous study (45). In situ hybridization (ISH) To probe the spatial distribution of cgcglr mrna in chicken pituitaries, ISH was performed. Briefly, adult chicken pituitaries were fixed in 4% paraformaldehyde, embedded in paraffin wax, and sectioned into 5- m-thick slices that were subsequently dewaxed with xylene and rehydrated. These pituitary sections were then digested with 10- g/ml proteinase K (Roche Diagnostics) for 20 minutes at 37 C, rinsed with PBS, and refixed by 4% paraformaldehyde. After 1-hour prehybridization at 60 C, Digoxigenin-labeled antisense/sense RNA probes (800 ng/ml) prepared by in vitro transcription (Supplemental Figure 1) were added to the hybridization buffer (20mM Tris-HCl [ph 8.0], 2.5mM EDTA, 1 Denhardt s solution, 7.5% dextran sulfate, 60% formamide, and 20- g/ml yeast trna), and the pituitary sections were incubated at 60 C for 16 hours. Posthybridization washing was conducted in formamide/ saline sodium citrate solution at 60 C. After the removal of unbound RNA probes by Ribonuclease A (100 g/ml) treatment, the hybridization signal was detected using anti-digoxigenin antibody conjugated to alkaline phosphatase and visualized by color development in a solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/ BCIP) (Roche). Immunohistochemical (IHC) staining Anterior pituitaries collected from adult chickens were fixed in 4% paraformaldehyde and embedded in paraffin wax for IHC staining as described in our recent study (44). Briefly, IHC staining was performed in chicken pituitary sections of 5 m in thickness using a Streptavidin-Biotin-Peroxidase Complex (SABC) kit (Boster Biological Technology Ltd) according to the manufacturer s instructions. Polyclonal antibody for ctsh (1:300) was used to probe the spatial distribution of ctsh protein in the pituitary. Sections incubated with rabbit preimmune serum, instead of anti-ctsh, were used as negative controls. Evaluation of cgcgl action on pituitary hormone secretion using intact pituitaries incubated in vitro The intact anterior pituitaries of nearly identical size collected from 1-week-old chicks or adult chickens were washed with PBS (ph 7.4) 5 times to remove the blood cells and then placed on a 48-well plate (NUNC) (6 chick pituitaries per well or 4 adult pituitaries per well) supplemented with 400- L serum-free Medium 199. After 1-hour 37 C incubation, the medium was replaced by 100- L serum-free Medium 199 containing different doses (0.1nM 10nM) of cgcgl (or TRH) peptide and incubated for 4 hours. Then, hormones (cgh, cprl, and ctsh) released into the incubation medium by whole pituitaries were examined by Western blot analysis using polyclonal antibodies for cgh (1:1000), cprl (1:800), and ctsh (1:500), as previously described (44). Parallel blotting of -actin (1:2000), cgh, cprl, and ctsh proteins in pituitary tissue lysate was conducted to serve as internal controls. Evaluation of cgcgl effect on pituitary hormone secretion and gene expression in cultured pituitary cells Anterior pituitaries collected from 1-week-old chicks were cut into pieces and digested by 0.25% trypsin at 37 C for 20 minutes. The dispersed pituitary cells were cultured at a density of cells per well in Medium 199 supplemented with 15% fetal bovine serum in a Corning CellBIND 48-well plate (Corning) at 37 C with 5% CO 2. After 24 hours of culture, the culture medium was replaced with serum-free M199 Medium (300 L per well), and the cells were treated with different doses (0.1nM 10nM) of cgcgl for 2 or 4 hours. Then, the cgh/cprl/ctsh levels in the culture medium were examined by Western blot analyses as described in our recent study (44) (or by a more sensitive cell-based luciferase reporter assay [for ctsh only]; for detailed information, please refer to the Supplemental Figure 2), whereas the intracellular cgh/cprl/ctsh (or -actin) levels were also examined in the pituitary cell lysates by Western blot analysis and used as internal controls (44). To examine whether cgcgl (or TRH) can induce pituitary hormone gene expression, the cultured pituitary cells were treated by different doses of peptide (0.1nM 10nM) for 24 or 48 hours. The total RNA was then extracted from pituitary cells and used for qrt-pcr assay of gene expression. Data analysis Quantification of bands from Western blottings was performed using the ImageJ program (ImageJ software; National Institutes of Health), and the relative hormone (ctsh/cgh) levels in culture medium were first calculated as the ratio to that of intracellular -actin and then expressed as the fold increase compared with respective control (without treatment) (44). The relative mrna level of each gene was first calculated as the ratio to that of -actin and then expressed as the percentage compared with respective control, or to that of 1-week-old chick pituitary (or hypothalamus). The data were analyzed by the Student s t test (for 2 groups), or by one-way ANOVA followed by the Dunnett test (for comparing treatment groups with control) or the Newman-Keuls test (for comparing all pairs of groups) with the use of GraphPad Prism 5 (GraphPad Software). To validate our results, all experiments were repeated at least 2 or 3 times. Results cgcglr mrna is mainly localized in cephalic lobe of chicken anterior pituitary Using ISH, we first examined the spatial distribution of cgcglr mrna in adult chicken anterior pituitary. As shown in Figure 1, strong cgcglr signals were mainly restricted to the cephalic lobe but not the caudal lobe. Using quantitative RT-PCR assay, we further confirmed the dominant expression of cgcglr mrna in the cephailc lobe, although a very low mrna level of cgcglr was noted in the caudal lobe. The uneven distribution of

4 doi: /en endo.endojournals.org wk-old chicks) incubated in vitro was treated with cgcgl for 4 hours, and the hormones (cgh, cprl, and ctsh) secreted into the incubation medium were detected by Western blot analyses (note, the specificity of antictsh antibody used here was evaluated via multiple approaches, including IHC and Western blot analysis shown in Figures 1 and 2, whereas the specificity of antibodies against cgh and cprl used here was described in elsewhere) (44). As shown in Figure 2, cgcgl treatment dose dependently (0.1nM 10nM) stimulates the secretion of ctsh with the minimal effective dose (ED) observed at 0.1nM but exerts no effect on cgh and cprl secretion. Moreover, we noted that in adult chicken pituitaries, cgcgl treatment (1nM, 4 h) still strongly induces the secretion of ctsh, but not cgh and cprl (Figure 2), indicating that cgcgl action is specific and not age dependent. It is reported that TRH can stimulate pituitary ctsh in chickens (16). Therefore, we also conducted parallel experiments to examine the effects of TRH on ctsh secretion of 1-week-old chick pituitaries by Western blot analysis. As shown in Figure 3, TRH (0.1nM 10nM, 4 h) stimulates both ctsh and cgh secretion dose dependently, suggesting that its effects are less specific than cgcgl. Figure 1. A, Quantitative real-time PCR assay of cgcglr mrna expression in the caudal lobe (Ca) and cephalic lobe (Ce) of adult chicken anterior pituitaries. ***, P.001 vs Ca. Each data point represents mean SEM of 4 individuals (n 4). B, Localization of cgcglr mrna in adult chicken anterior pituitaries by ISH. Strong GCGLR signals (dark purple color) are mainly restricted to cephalic lobe (Ce) but not caudal lobe (Ca), whereas no hybridization signal is observed in negative controls using sense RNA probe (data not shown). C, ctsh immunoreactivity (stained with anti-ctsh, brown color) is mainly localized in the cephalic lobe (Ce) (right part) of adult chicken anterior pituitary as previously reported (28, 49), whereas no immunoreactivity is observed in negative controls (data not shown). Note, the strongest signals of cgcglr (B) and ctsh (C) are mainly localized in the ventral region of cephalic lobe (Ce) (marked by arrows), suggesting that cgcgl may stimulate ctsh secretion and expression specifically via activation of cgcglr, as demonstrated in the next experiments. cgcglr mrna in the pituitary suggests that cgcgl may exert specific control over the secretion of (a) pituitary hormone(s). cgcgl, but not TRH, stimulates ctsh secretion in intact chicken pituitaries both potently and specifically To examine whether cgcgl can stimulate pituitary hormone secretion, the whole anterior pituitaries (from cgcgl specifically stimulates ctsh secretion in cultured chicken pituitary cells To further confirm the effect of cgcgl on pituitary ctsh secretion, we also examined whether cgcgl treatment (0.1nM 10nM, 2 h) could stimulate ctsh secretion in cultured chick pituitary cells. Due to the low ctsh level present in the medium and the limited sensitivity of Western blot analysis, a more sensitive cell-based luciferase reporter assay system was established to measure the relative ctsh levels in the culture medium, in which CHO cells cotransfected with ctsh receptor expression plasmid and a pgl3-cre-luciferase construct were shown to be capable of monitoring TSH levels sensitively (Supplemental Figure 2). As shown in Figure 4, cgcgl treatment can dose dependently induce ctsh secretion by the cultured pituitary cells, monitored by this cell-based luciferase reporter assay. In contrast, cprl and cgh secretion in the cultured pituitary cells are not affected by cgcgl treatment as revealed by Western blot analysis (Figure 4). These findings, together with the cgcglr mrna expression in cultured pituitary cells detected by RT-PCR (Figure 4), support that cgcgl stimulates ctsh secretion via activation of cgcglr.

5 4572 Huang et al GCGL Is a Novel TRF in Chickens Endocrinology, November 2014, 155(11): Figure 2. A and B, Validation of the specificity of the polyclonal antibody against chicken TSH subunit (anti-ctsh ) by Western blot analyses. A, An approximately 19-kDa band of expected size was detected in the lysate of HEK293 cells transfected with the expression plasmid encoding a ctsh -FLAG fusion protein (ctsh ), but not in the lysate of HEK293 cells transfected with the empty pcdna3.1 ( ) vector (PC), by Western blotting using either anti-ctsh or anti-flag antibody (for information of ctsh -FLAG plasmid and transfection, please refer to the Supplemental Figure 3). B, Anti-cTSH antibody detected the ctsh -subunit ( 18-kDa band) in the lysate of cephalic lobe (Ce), but not the caudal lobe (Ca), of adult chicken anterior pituitary, as previously reported (28). C, Western blot analyses showed that cgcgl treatment (0.1nM 10nM, 4 h) increased TSH levels (stsh ), but not GH (sgh) and PRL (sprl) levels, in the incubation medium of 1-week-old chicken pituitaries incubated in vitro. D, Western blot analyses showed that GCGL treatment (1nM, 4 h) increased TSH levels (stsh ), but not GH (sgh) and PRL (sprl) levels, in the incubation medium of adult pituitaries incubated in vitro. C and D, The TSH (itsh ), GH (igh), PRL (iprl), and -actin (actin) levels in the pituitary tissue lysate were also examined in parallel, and no obvious change in intracellular hormone concentrations was observed after GCGL treatment. The relative TSH levels (stsh band) in the incubation medium were quantified by densitometry, normalized by that of -actin band in pituitary tissue lysate, and then expressed as fold increase of respective control (CTL, without GCGL treatment). Each data point represents mean SEM of 4 replicates (n 4). *, P.05; **, P.01; ***, P.001 vs respective control; representative Western blottings for each hormone (TSH/GH/PRL) are shown in C and D. Note, arrows indicate the 2 major PRL bands of approximately 27 kda (glycosylated form) and approximately 24 kda detected in all experiments, whereas only a single major GH band of approximately 23 kda was detected (44). Both secreted (prefix s) and intracellular (prefix i) GH/PRL/TSH were detected and presented in Figures 2 4. cgcgl specifically induces ctsh mrna expression in cultured chicken pituitary cells To examine whether cgcgl could regulate pituitary hormone gene expression, the cultured pituitary cells from 1-week-old chicks were treated with different doses (0.1nM 10nM) of cgcgl for 24 or 48 hours, and the mrna expression of 6 hormone genes, including ctsh, cgh, cprl, cacth, cfsh, and clh, was examined by qrt-pcr. AsshowninFigure5,24-hourcGCGL treatment only stimulates ctsh mrna expression dose dependently with the minimal ED noted at 0.1nM. Likewise, 48-hour cgcgl treatment also dose dependently induces the mrna expression of ctsh but not any of the other 5 hormone genes examined. In contrast to that of cgcgl, the stimulatory effect of TRH (0.1nM 10nM, 24 or 48 h) on ctsh mrna expression is much less effective in cultured pituitary cells. Only a slight, but significant, increase in ctsh mrna expression was noted at 10nM TRH treatment for 24 hours. However, TRH treatment (0.1nM 10nM) for 24 or 48 hours can consistently induce cgh mrna expression dose dependently, with the minimal ED observed at 1nM (Figure 5). cgcgl action on ctsh expression is likely mediated by adenylate cyclase (AC)/cAMP/ protein kinase A (PKA) and phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/Ca 2 signaling pathways To investigate the postreceptor signaling pathways involved in mediating cgcgl action on ctsh mrna expression, the cultured pituitary cells were treated with various pharmacological drugs targeting AC/cAMP/PKA and PLC/IP3/Ca 2 signaling pathways, both of which

6 doi: /en endo.endojournals.org 4573 including IP3 and Ca 2, may also be involved in mediating cgcgl action on ctsh mrna expression. Our preliminary data showed that inhibition of MEK by PD98059 (10 M) did not block the stimulatory effect of GCGL on ctsh mrna expression (data not shown), implying that MEK/ERK signaling cascade may not be involved in mediating this action. However, cgcgl treatment (5nM, 10 min) consistently enhances ERK2 phosphorylation in cultured pituitary cells, as demonstrated by Western blot analysis (Figure 6). Figure 3. A, Western blot analyses showed that TRH treatment (0.1nM 10nM, 4 h) increased TSH levels (stsh ) in the medium, in which chick pituitaries were incubated. B, Western blot analyses showed that TRH treatment (0.1nM 10nM, 4 h) increased GH levels (sgh) in the incubation medium of intact chick pituitaries. A and B, TSH (itsh) and GH (igh) levels in the pituitary tissue lysate were also examined in parallel, and no obvious change was observed after TRH treatment. The relative TSH levels (stsh ) (A) or GH levels (sgh) (B) in the incubation medium of intact chick pituitaries were quantified by densitometry, normalized by that of -actin protein in pituitary tissue lysate, and then expressed as fold increase of respective control (CTL, without peptide treatment). Each data point represents mean SEM of 4 replicates (n 4). *, P.05; **, P.01; ***, P.001 vs respective control. Representative Western blottings are shown above each graph. have been reported to be functionally coupled to most GPCR B1 subfamily members (36, 42). As shown in Figure 6, 8-bromo-cAMP (a camp analog, 0.25mM 1.5mM) and forskolin (an AC activator, 1 M 10 M) treatment mimic cgcgl action and induce ctsh mrna expression dose dependently, whereas administration of either an AC inhibitor, MDL-12330A (100 M), or a PKA inhibitor, H89 (10 M), not only decreases the basal ctsh mrna levels significantly but also completely abolishes the stimulatory effect of cgcgl (5nM, 24 h). These findings suggest that the AC/cAMP/ PKA signaling pathway is likely involved in mediating the stimulatory action of cgcgl on ctsh mrna expression. Interestingly, the stimulatory effect of cgcgl is also abolished by pharmacological drugs targeting PLC/IP3/ Ca 2 signaling pathways, including U73122 (a PLC inhibitor, 20 M), 2-APB (an IP3 receptor antagonist that can block IP3-triggered calcium mobilization from endoplasmic reticulum, 100 M), thapsigargin (an inhibitor of sarco[endo]plasmic reticulum Ca 2 -ATPase that can deplete intracellular calcium stores, 100nM), and calmidazolium (a calmodulin antagonist, 1 M). These findings imply that PLC and its downstream signaling molecules, Temporal expression profiles of cgcgl-gcglr in the developing hypothalamic-pituitary axis Using qrt-pcr, the cgcgl and cgcglr mrna expression was also examined in the hypothalamus and pituitary, respectively, from embryonic to adult stages. As shown in Figure 7, in the hypothalami, GCGL mrna level remains minimal at E8 and E12, increases dramatically from E16 to 1-week posthatch stage, and is maintained at a high level at posthatch 2-week and adult stages. Similarly, the cgcglr mrna level in developing pituitaries is extremely low at E8 and E12 but increases abruptly at later developmental stages from E16 to 1-week posthatch stage. Interestingly, cgcglr mrna level at 2-week stage shows an approximately 2.5-fold reduction when compared with that at 1-week stage. However, a high cgcglr mrna level is noted at adult stage, despite the large variation among individuals. Interestingly, the temporal expression profile of ctsh was found to be in partial accordance with those of cgcglr in the pituitary and cgcgl in the hypothalamus. Pituitary ctsh mrna level is extremely low at E8, increases from E12 to E16, but decreases temporarily at E20, and then is maintained after it reaches the high level at 1 week (Figure 7). Discussion In the present study, GCGL is demonstrated to stimulate chicken pituitary TSH secretion and expression both specifically and potently in vitro, and this action is likely mediated by GCGLR, which is mainly localized in cephalic lobe of anterior pituitary, strongly suggesting that GCGL is a novel potential hypophysiotropic factor specific for pituitary TSH release and expression. To our knowledge, our study represents the first to identify a potential specific TRF in nonmammalian vertebrates and provides a new perspective to decipher the hypothalamic control of pituitary-thyroid axis across vertebrates.

7 4574 Huang et al GCGL Is a Novel TRF in Chickens Endocrinology, November 2014, 155(11): Figure 4. A, RT-PCR detection of cgcglr mrna expression in cultured pituitary cells from 1- week-old chicks., RT with reverse transcript;, RT without reverse transcriptase. B, cgcgl treatment stimulates ctsh secretion in cultured pituitary cells. Pituitary cells were treated with various doses of cgcgl (2 h), and the culture media (10 L) from each treatment group were collected, and their ctsh activity was assayed using the cell-based luciferase reporter assay established (for details, please refer to Supplemental Figure 2). 0.1CM, 1CM, and 10CM represent the culture media collected from cultured pituitary cells treated by 0.1nM, 1nM, and 10nM cgcgl, respectively, whereas 0CM denotes the culture medium from pituitary cells without cgcgl treatment. Expectedly, 10- L culture media collected from pituitary cells treated by cgcgl can significantly increase luciferase activity of ctsh receptor (ctshr)-expressing CHO cells (but not CHO cells transfected with empty pcdna vector), indicating that cgcgl can induce ctsh secretion from cultured pituitary cells, and the amount of ctsh secreted to medium seems to be positively correlated with the dosage of GCGL used. Each data point represents mean SEM of 4 replicates (n 4). **, P.01 vs control (0CM). C and D, Western blot analyses showed that GH (sgh) and PRL (sprl) levels in the culture medium of 1-week-old chicken pituitary cells were not affected by cgcgl treatment (0.1nM 10nM, 4 h). The GH (igh), PRL (iprl), and -actin (actin) levels in cell lysates were also examined in parallel, and no change in intracellular hormone concentrations was observed after GCGL treatment (similarly, the intracellular TSH levels also showed no obvious change after GCGL treatment, data not shown). Note, arrows indicate the 2 major cprl bands of approximately 27 and approximately 24 kda detected in the culture medium or in the cell lysate of pituitary cells. cgcgl, but not TRH, is a novel potential TSHspecific releasing factor in chickens GCGL-GCGLR is a novel ligand-receptor pair identified in chickens and other nonmammalian vertebrates in our recent study (36). The highest mrna level of GCGL restricted to the hypothalamus, coupled with the abundant mrna expression of GCGLR noted in the pituitary and several brain regions, including hypothalamus, led us to hypothesize that 1) GCGL may act as an anorexic factor, similar to its structurally related peptide (eg, glucagon-like peptide 1) played in the hypothalamus of chickens (46, 47) and 2) GCGL may function as a novel hypophysiotropic factor to control pituitary functions (36). The first notion has been substantiated by a recent report, in which intracerebroventricular injection of GCGL inhibits feeding behavior of 8-day-old male chicks (48), whereas the action of GCGL on pituitary remains an open question. In this study, using ISH and qrt- PCR, cgcglr mrna is revealed to be mainly restricted to the pituitary cephalic lobe, within which lactotrophs, corticotrophs, and thyrotrophs reside (1). Moreover, we noted that strong cgcglr signals seem to partially overlap with ctsh -immunoreactivity, which is intensively distributed in the ventral region of pituitary cephalic lobe as previously reported (Figure 1) (28, 49), implying that cgcgl may control ctsh secretion. This idea is directly substantiated by the follow-up experimental results that cgcgl rapidly and potently stimulates ctsh secretion of whole pituitaries collected either from 1-week-old chicks or from adult chickens. In sharp contrast, cgcgl stimulates neither cprl secretion, nor cgh release, under the same condition, further elucidating its specific action on thyrotrophs. Using a cell-based luciferase reporter assay, cgcgl is demonstrated to stimulate the secretion of bioactive ctsh from cultured pituitary cells, and the minimal ED was noted at 0.1nM (Figure 4), further supporting the notion that GCGL is likely a physiologically relevant TRF identified in chickens. Owing to the lack of specific antibodies for chicken ACTH, FSH, and LH in our laboratory, Western blotting was not performed to test whether cgcgl can stimulate the secretion of these pituitary hormones. However, using the CHO cells cotransfected with receptor expression plasmid encoding cfsh receptor (or clh receptor, or cacth receptor) and a pgl3-cre-luciferase construct to monitor the secretion of these hormones (a cell-based approach similar to that used for monitoring ctsh secretion shown in Figure 4 and Supplemental Figure 2), our preliminary studies showed that cgcgl (0.1nM 10nM;

8 doi: /en endo.endojournals.org 4575 Figure 5. Effects of cgcgl and TRH on pituitary hormone gene expression in cultured chicken pituitary cells examined by qrt-pcr. Effects of 24-hour (A) and 48-hour (C) cgcgl treatments (0.1nM 10nM) on the mrna expression of TSH, PRL, GH, LH, FSH, and ACTH (POMC) genes in cultured pituitary cells. Effects of 24-hour (B) and 48-hour (D) TRH treatments (0.1nM 10nM) on the mrna expression of TSH and GH genes in cultured pituitary cells. Each data point represents means SEM of 4 replicates (n 4). *, P.05, vs respective control (without peptide treatment); **, P.01 vs respective control. 1, 2, or 4 h) failed to stimulate their secretion from cultured pituitary cells (data not shown). In contrast to cgcgl, TRH, a major TRF in mammals (6), stimulates both pituitary ctsh and cgh secretion effectively in chickens (Figure 3). Our finding is consistent with the previous reports in chickens (9, 14). Interestingly, it is reported that TRH can stimulate ctsh secretion and consequently elevate the plasma T 4 levels in chicken embryos, or in growing chickens, but this action seems to be diminished in adult chickens (14, 15). Unlike TRH, cgcgl effectively induces ctsh secretion of pituitary from both 1-week-old chicks and adult chickens (Figure 2). These findings suggest that GCGL, but not TRH, may play a more specific stimulatory action on the pituitarythyroid axis at all stages of chickens. There are lines of evidence supporting that CRH may function as a TRF in chickens (16), frogs, and teleosts (10, 21, 23, 29, 30, 32). In chickens, CRH not only induces pituitary ACTH secretion (31) but also stimulates pituitary TSH secretion potently (16). The dual roles of CRH in chicken pituitary are likely mediated by CRH type I receptor (CRH-R1) and CRH type II receptor (CRH-R2) expressed in corticotrophs and thyrotrophs, respectively (50). In adult frogs, CRH can stimulate TSH secretion and elevate plasma T 4 levels, accompanied by a concurrent release of pituitary ACTH (23, 30, 33). Moreover, injection of ovine CRH into amphibian larvae is also reported to increase pituitary TSH secretion, elevate plasma thyroid hormone levels, and accelerate tadpole metamorphosis (16, 32, 51 53), a developmental process which involves the morphological and physiological changes orchestrated by thyroid hormone (30, 32, 53, 54). The involvement of both CRH and TRH in controlling thyroidal axis in chickens and frogs may suggest a much more primitive, but complex, hypothalamic control than that in mammals, in which CRH and TRH can control adrenal and thyroidal axes, respectively (Figure 8). Conceivably, the discovery of cgcgl as a novel potential TRF adds a further level of complexity to decipher the hypothalamic control of thyroidal axis in chickens (Figure 8). In view of the existence of GCGL-GCGLR in reptiles, amphibians, and teleosts (36), it led us to speculate that as in chickens, GCGL may act as a TRF in other, if not all, classes of nonmammalian vertebrates. Future studies on GCGL actions in other nonmammalian vertebrates, such as its possible role(s) in amphibian pituitary TSH secretion and metamorphosis, will undoubtedly advance our understanding of the hypothalamic control of thyroidal axis across vertebrates. cgcgl specifically stimulates pituitary ctsh gene expression In this study, cgcgl is shown to stimulate the mrna expression of TSH gene but not the other 5 pituitary hormone genes. This finding further supports the specific action of cgcgl on thyrotrophs. Unlike cgcgl, TRH is more effective in inducing cgh mrna expression than inducing ctsh mrna expression. The weak stimulatory effect of TRH on ctsh mrna expression, demonstrated in the present study and previous studies (55, 56), hints that TRH may not be a potent stimulatory factor for ctsh mrna expression. In contrast, the specific and potent stimulatory effect of cgcgl on both TSH secre-

9 4576 Huang et al GCGL Is a Novel TRF in Chickens Endocrinology, November 2014, 155(11): Figure 6. Effects of the different pharmacological drugs on basal or cgcgl-induced ctsh mrna expression levels in cultured chick pituitary cells examined by qrt-pcr. A, Effect of 8- bromo-camp (8-Br-cAMP) (0.25mM 1.5 mm, 24 h) on basal ctsh mrna expression. B, Effect of forskolin (1 M 10 M, 24 h) on basal ctsh mrna expression. C, Effects of MDL-12330A (MDL) (100 M) and H89 (10 M) on basal and cgcgl (5nM, 24 h)-induced ctsh mrna expression. D, Effects of U73122 (20 M), 2-APB (100 M), thapsgargin (TG) (100nM), and calmidazolium (Cad) (1 M) on basal and cgcgl (5nM, 24 h)-induced ctsh mrna expression. C and D, Each letter C in these graphs represents the control group without cgcgl treatment, whereas each letter T represents cells treated by 5nM cgcgl (24 h). Each drug was added 1 hour before cgcgl treatment. NS indicates no statistical difference between the 2 groups. Note, MDL-12330A, H89, 2-APB, and calmidazolium treatment could down-regulate the basal mrna levels of ctsh significantly. Each data point represent means SEM of 4 replicates (n 4). **, P.01; ***, P.001 vs control (without peptide treatment). E, cgcgl treatment (5nM, 10 min) was shown to enhance ERK2 phosphorylation (perk2) levels in cultured chick pituitary cells by Western blot analysis (the -actin and ERK2 contents in cell lysates were also examined and used as internal controls). Note, only a single band for perk2 or for total ERK2 ( 42 kda) was detected in cell lysate, because ERK1 gene is absent in the chicken genome (65). The representative sets of independent experimental duplicates are shown here. tion and expression strongly suggests that cgcgl is most likely to be a specific and physiologically relevant TRF identified in chickens, although future studies on cgcgl distribution within the hypothalamic nuclei and median eminence, and its concentrations in the hypophyseal portal blood, are needed to extend and substantiate this hypothesis. GCGL action on ctsh expression is likely mediated by AC/cAMP/PKA and PLC/Ca 2 signaling pathways coupled to cgcglr In this study, inhibition of AC/cAMP/PKA signaling pathway by pharmacological drugs is demonstrated to totally abolish the stimulatory effect of cgcgl on ctsh mrna expression, whereas activation of this signaling pathway by forskolin and 8-bromocAMP mimics the GCGL action on TSH expression. These findings further support that the AC/cAMP/ PKA signaling pathway is coupled to cgcglr, as previously reported (36), and is capable of mediating cgcgl action on ctsh expression (Figure 8). Interestingly, the stimulatory effect of cgcgl on ctsh mrna expression is also blocked by pharmacological drugs targeting the components of PLC/IP3/Ca 2 signaling pathway. This finding implies that GCGLR activation may activate the Gq-PLC signaling pathway, leading to IP3-triggered calcium mobilization from endoplasmic reticulum, which consequently activates CaM-dependent cascade and regulates ctsh expression. It is well documented that PLC activation can also increase intracellular diacylglycerol levels and in turn activate the protein kinase C signaling pathway (Figure 8). However, the involvement of protein kinase C activation in the regulation of ctsh mrna expression needs further investigation. The proposed functional coupling of cgcglr to both AC/cAMP/PKA and Gq-PLC signaling pathways is in fact not surprising (Figure 8), because GCG receptor, which was duplicated from an ancestral receptor gene common to cgcglr (36, 37, 57), has been reported to be functionally coupled to both AC/cAMP/PKA and Gq-PLC signaling pathways (42, 58). Clearly, future in-depth studies on the signaling pathways coupled to cgcglr and their involvement in ctsh expression and secretion in pituitary would be required to substantiate our hypothesis (Figure 8). Although calcium mobilization from intracellular calcium stores triggered by cgcgl was suggested to be involved in the regulation of ctsh mrna expression, and perhaps also critical for ctsh secretion, the question remains whether cgcglr activation can stimulate calcium influx through membrane-anchored calcium channel and thus control TSH expression and/or secretion like other

10 doi: /en endo.endojournals.org 4577 Figure 7. Developmental expression of cgcgl mrna in the hypothalami (A) and cgcglr mrna (B) and ctsh mrna (C) in the pituitaries of chicken embryos (E8, E12, E16, and E20), 1-week (1w)-old and 2-week (2w)-old chicks, and adult chickens (Ad). A C, mrna levels of all genes were examined by qrt-pcr. The relative mrna level of each gene was first calculated as the ratio to that of -actin and then expressed as the percentage compared with that of 1-weekold (1w) chick pituitary (or hypothalamus). Each data point represent as means SEM of 6 12 individuals (n 6 12). Values significantly different (P.05) between stages are indicated by different letters. GPCR family B members, eg, GHRH receptor (42, 59) (Figure 8). Temporal expression profiles of GCGL-GCGLR in the hypothalamus-pituitary axis, implications for their potential roles in thyrotrophs In this study, cgcgl-gcglr genes are found to be expressed in the hypothalamus-pituitary axis at all stages examined, and their temporal expression profiles are partially accordant with that of TSH gene in the pituitary, as revealed by the present study or previous studies (55, 60). Although the mrna levels of cgcgl in the hypothalami and cgcglr in the pituitaries are extremely low at E8 and E12, their mrna levels at respective sites increase abruptly and synchronously on E16 till the end of incubation (E20) (Figure 7). Interestingly, the temporal expression profile of cgcglr in embryonic pituitaries also contrasts to those of receptors for TRH (TRH type I receptor) and CRH (CRH-R2) expressed in the thyrotrophs, which have been reported to decrease from E14 to E20 (50, 60, 61). These findings suggest that during late embryogenesis, pituitary thyrotrophs may be much more sensitive to the elevated cgcgl levels, rather than to TRH and CRH, and hence, cgcgl may be actively involved in inducing TSH synthesis and secretion, which results an increase in plasma T 4 and T 3 levels essential for hatching and transition to posthatch endothermic life during late embryogenesis (55, 60, 62). Moreover, the synchronized elevation in both GCGL and GCGLR mrna levels during late embryogenesis raises another possibility that hypothalamic GCGL may be involved in inducing thyrotroph expansion (49, 63), a trophic effect also possessed by other classic hypophysiotropic factors, such as GHRHinduced somatotroph expansion in mammals (64). At 1-week posthatch stage, further increases in mrna levels of both genes are noted in the hypothalamus-pituitary axis. This finding, together with the potent action of GCGL on TSH secretion of whole pituitaries collected from 1-week chicks, implies that GCGL-GCGLR axis may play an important role in stimulating and maintaining TSH and thyroidal hormone levels during this period (62). Interestingly, at the 2-week posthatch stage, only a high mrna level of GCGL is noted in chicken hypothalamus, whereas the GCGLR mrna level in the pituitary decreases abruptly, possibly due to the negative feedback regulation by the high levels of plasma thyroid hormone (62). At adult stage, the high mrna levels of both GCGL and GCGLR are still observed in the hypothalamus and pituitary, respectively. This finding, together with the potent TSH-releasing effect of cgcgl demonstrated in adult chicken pituitaries, suggests that cgcgl may still act as a potent TRF at adult stage, when TRH and CRH become less effective in stimulating TSH secretion and elevating plasma T 4 levels (14, 15, 26). The temporal expression profiles of GCGL-GCGLR in chicken hypothalamus-pituitary axis tend to support an active role of cgcgl in the regulation of thyrotroph functions and possibly thyrotroph expansion as well. However, the study on TSH-releasing effect of cgcgl in vivo is lacking. Hence, future studies on cgcgl effect on ctsh secretion and plasma thyroid hormone levels in chickens at different stages, from embryonic to adult stages, would be required to substantiate this hypothesis. These studies, together with our pioneering study on the specific action of cgcgl on thyrotrophs, will undoubtedly open up a new portal into exploring the TSH-releasing effect of GCGL-GCGLR in other nonmammalian vertebrates, given that this ligand-receptor pair does exist in other nonmammalian vertebrate groups (36, 37, 57). In summary, cgcgl is proved to stimulate pituitary TSH secretion and expression in chickens both specifically and potently, and this specific action is mediated by GCGLR expressed in the cephalic lobe of anterior pituitary. Moreover, the temporal expression profiles of GCGL-GCGLR in the hypothalamus-pituitary axis are revealed to be in partial accordance with pituitary TSH expression, implying their active involvement in controlling pituitary-thyroid axis at various stages of chickens. Evidence presented here strongly suggests that GCGL is a

11 4578 Huang et al GCGL Is a Novel TRF in Chickens Endocrinology, November 2014, 155(11): Figure 8. A, Proposed model for GCGL actions on chicken pituitary thyrotrophs. In this model, GCGL stimulates pituitary ctsh secretion and expression both specifically and potently, and this stimulatory action is mediated by GCGLR expressed in thyrotrophs, which is likely to be functionally coupled to Gs-AC/cAMP/PKA and Gq-PLC/IP3/Ca 2 signaling pathways. Interestingly, GCGL treatment also activates ERK2 signaling pathway in pituitary cells. However, it remains unclear whether ERK2 activation is involved in controlling ctsh mrna expression and other processes, such as thyrotroph expansion. Calcium mobilization triggered by IP3 is likely involved in controlling ctsh gene expression (and secretion), yet the question whether cgcgl can trigger a calcium influx through calcium channels and subsequent induction of ctsh expression (and secretion) needs further clarification. B, Hypothetical model for the hypothalamic control of pituitary TSH secretion in chickens. In this model, pituitary TSH secretion is stimulated by the 3 hypothalamic TRFs, GCGL, CRH, and TRH (9). Among the 3 TRFs identified in chickens, only GCGL may act as a TRF specific for TSH secretion and expression, whereas TRH and CRH can also effectively stimulate pituitary GH and ACTH secretion, respectively (9, 14, 16). The question remains whether GCGL can act as a TRF in other nonmammalian vertebrates. C, The hypothalamic control of pituitary TSH secretion in mammals is distinct from that in chickens. Pituitary TSH secretion is primarily stimulated by TRH in mammals, whereas hypothalamic CRH is responsible for ACTH secretion. Interestingly, GCGL, a potential TRF in chickens demonstrated in the present study, was highly likely lost in mammals (36). novel potential TRF identified in chickens. Expectedly, future extensive studies on GCGL-GCGLR regulation of pituitary-thyroid axis in chickens, as well as in other nonmammalian vertebrates, would greatly advance our understanding of hypothalamic control of thyroidal axis across vertebrates. Acknowledgments We thank Professor Li Qintong (College of Life Sciences, Sichuan University, China) and Professor Wang Deshou (School of Life Sciences, Southwest University, China) for their suggestion and help to our study. Partial content of this work was presented in the form of conference abstract in the 17th International Congress of Comparative Endocrinology (ICCE2013), July 15 19, 2013, Barcelona, Spain. Address all correspondence and requests for reprints to: Professor Yajun Wang, Key Laboratory of Bioresources and Ecoenvironment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu , People s Republic of China. cdwyjhk@gmail.com. This work was supported by National Natural Science Foundation of China Grants and and the National High Technology Research and Development Program of China Grant 2013AA Disclosure Summary: The authors have nothing to disclose. References 1. Scanes CG. Introduction to endocrinology: pituitary gland. In: Sturkie s Avian Physiology. 5th ed. 2000; Harvey S, Scanes CG. Comparative stimulation of growth hormone secretion in anaesthetized chickens by human pancreatic growth

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