Novel Pituitary Actions of Epidermal Growth Factor: Receptor Specificity and Signal Transduction for UTS1, EGR1, and MMP13 Regulation by EGF

Epidermal growth factor (EGF) is a member of the EGF-like ligands family, which plays a vital role in cell proliferation, differentiation, and folliculogenesis through binding with EGF receptors, including ErbB1 (EGFR/HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). In mammals, many functional roles of EGF have been reported in the ovaries and breasts. However, little is known about the functions of EGF in the pituitary, especially in teleost. In this study, using grass carp pituitary cells as the model, we try to examine the direct pituitary actions of EGF in teleost. Firstly, transcriptomic analysis showed that 599 different expressed genes (DEGs) between the control and EGF-treatment group were mainly involved in cell proliferation, cell migration, signal transduction, and transcriptional regulation. Then, we further confirmed that EGF could significantly induce UTS1, EGR1, and MMP13 mRNA expression in a time-and dose-dependent manner. The stimulatory actions of EGF on UTS1 and EGR1 mRNA expression were mediated by the MEK1/2/ERK1/2 and PI3K/AKT/mTOR pathways coupled with both ErbB1 and ErbB2 in grass carp pituitary cells. The receptor specificity and signal transductions for the corresponding responses on MMP13 mRNA expression were also similar, except that the ErbB2 and PI3K/AKT/mTOR pathway were not involved. As we know, MMP13 could release EGF from HB-EGF. Interestingly, our data also showed that the MMPs inhibitor BB94 could suppress EGF-induced UTS1 and EGR1 mRNA expression. These results, taken together, suggest that the stimulatory actions of EGF on UTS1 and EGR1 mRNA expression could be enhanced by EGF-induced MMP13 expression in the pituitary.


Introduction
Epidermal growth factor (EGF) is a small protein of 6 kDa containing 53 amino acids, which comprises three disulfide bridges [1]. The biological effects of EGF are mediated mainly through four tyrosine kinase receptors, namely ErbB1 (HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) [2]. EGF is a potent mitogen growth factor, and so it is involved in the process of cell growth, differentiation, proliferation, metabolism, and tumorigenesis [3]. The EGF ligand and receptor could also play an important role in the renewal of stem cells in early embryonic development, skin, liver, and gut [4]. In the hypothalamus-pituitary-adrenal (HPA) axis, EGF could regulate adrenocorticotropic hormone (ACTH) release through the up-regulation of hypothalamic corticotropin releasing hormone (CRH) [5]. At the pituitary level, EGF could stimulate luteinizing hormone (LH) release [6] and gonadotrope mitosis [7] in rats, and even increase plasma follicle-stimulating hormone (FSH) and LH levels in vivo in ewes [8]. In addition, EGF could also induce prolactin (PRL) synthesis and reduce growth hormone (GH) synthesis in rat pituitary tumor cells [9].
In zebrafish, previous studies found that EGF could significantly enhance the final maturation of the oocytes [10]. Further studies found that EGF was predominantly expressed in the oocytes, whereas epidermal growth factor receptor (EGFR) was highly detected in the follicle cells, which suggested that EGF was a potential paracrine/juxtacrine factor from the oocytes to regulate the function of the follicle cells [11]. At the pituitary level, previous studies found that the EGFR could be detected in zebrafish pituitary cells, but EGF had no effect on the expression of FSHβ, LHβ, and GH [12]. Recently, our study found that EGF could significantly induce somatolactin α (SL α) and tachykinin receptor 3 (TACR3) secretion and synthesis in grass carp pituitary cells [13]. Besides, little is known about the direct pituitary actions of EGF in teleost.
To further examine the direct pituitary actions of EGF in teleost, the primary cultured grass carp pituitary cells were used as the model. Firstly, the global pituitary actions of EGF were examined by using the RNA-Seq technique. Secondly, we further investigated the receptor specificity and signal pathways for EGF-induced Urotensin1 (UTS1) and early growth response 1 (EGR1) mRNA expression in grass carp pituitary cells. Thirdly, we also examined the direct pituitary actions of EGF in matrix metallopeptidase 13 (MMP13) and tissue inhibitor of metalloproteinase 3 (TIMP3) gene expression. Finally, we further examined the functional role of MMP13 in EGF-induced UTS1 and EGR1 gene expression in pituitary cells.

Transcriptomic Analysis
To investigate the global regulation of EGF in fish pituitary, a high-throughput transcriptome was used to compare the transcript levels between the control and EGF-treatment groups. In total, 19,486 genes were identified in grass carp pituitary cells. Compared to the control group, 599 differential expression genes (DEGs) were detected after EGF (0.5 µM)-treatment, fragments per kilobase of exon per million fragments mapped (FPKM) > 5, p < 0.05, fold change (FC) > 1.5, including 195 up-regulated DEGs and 404 down-regulated genes. GO analysis showed that these DEGs were classified in three main ontologies, including cellular component, biological process, and molecular function ( Figure 1A). Within the cellular component category, the 'integral component of membrane', 'cytoplasm', 'nucleus', 'transcription factor complex', 'membrane', and 'plasma membrane' were the most enriched GO terms ( Figure 1A). In addition, the most abundant groups in molecular function were 'ATP binding', 'metal ion binding', 'zinc ion binding', 'GTP binding', and 'DNA binding transcription' ( Figure 1A). Finally, the GO enrichment analysis of biological process revealed that the top 46 up-regulated DEGs (Table 1) and top 48 down-regulated DEGs ( Table 2) were involved in cell migration, cell differentiation, signal transduction, metabolic process, phosphorylation, and transcriptional regulation ( Figure 2).
To further understand the direct pituitary functions of EGF, annotated pathways of DEGs were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results revealed that a total of 209 DEGs were enriched in the top 10 pathways. Among them, the up-regulated DEGs were mostly enriched in 'metabolic pathways' and 'pathways in cancer', and the down-regulated DEGs were mainly enriched in 'PI 3 K-Akt signaling pathway', 'metabolic pathway', and 'pathways in cancer' ( Figure 1B).     To further understand the direct pituitary functions of EGF, annotated pathways of DEGs were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results revealed that a total of 209 DEGs were enriched in the top 10 pathways. Among them, the upregulated DEGs were mostly enriched in 'metabolic pathways' and 'pathways in cancer', and the down-regulated DEGs were mainly enriched in 'PI3K-Akt signaling pathway', 'metabolic pathway',

EGF-Induced Critical DEGs in Grass Carp Pituitary Cells
Among the DEGs identified by RNA-Seq analysis, we focused on three up-regulated DEGs: MMP13, EGR1, and UTS1. To further confirm these DEGs, we incubated the grass carp pituitary cells with EGF to detect their mRNA expression by real-time PCR. The results showed that EGF could significantly induce pituitary UTS1 ( Figure   Effects of ErbB1 antagonist AG1478, ErbB2 antagonist AG879, and IGF receptor antagonist AG1024 on EGF-induced UTS1 mRNA expression, respectively. (F-H) The effects of EGF (0.5 μM) induced UTS1 mRNA transcription with the MEK inhibitor U0126 (10 μM), ERK1/2 inhibitor LY3214996 (10 μM), and p38MAPK inhibitor SB203580, respectively. (I-K) Co-treatment with the PI3K inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), and mTOR inhibitor Rapamycin (10 μM) on EGF (0.5 μm)-induced UTS1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05.  AKT inhibitor MK2206 (10 µM), and mTOR inhibitor Rapamycin (10 µM) on EGF (0.5 µm)-induced UTS1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. inhibitor Wortmannin (10 μM), AKT inhibitor MK2206 (10 μM), or mTOR inhibitor Rapamycin (10 μM) by EGF (0.5 μM)-induced EGR1 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. After drug treatment, total RNA was isolated and used for real-time PCR of UTS1 mRNA expression. The differences between groups were considered as highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. , and mTOR inhibitor Rapamycin (10 µM) induced MMP13 mRNA expression was examined, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. The differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05.

Receptor Specificity and Signal Transduction for EGF-Induced UTS1 and EGR1 Gene Expression
In this experiment, a pharmacological approach was recruited to clarify the receptor specificity for EGF-induced UTS1 and EGR1 gene expression. The results showed that the up-regulation of UTS1 and EGR1 mRNA expression was consistently observed in grass carp pituitary cells with EGF treatment for 24 h. These stimulatory effects on UTS1 mRNA expression could be totally abolished by co-treatment with the ErbB1 antagonist AG1478 ( Figure 3C; Supplementary Figure S2A) or ErbB2 antagonist AG879 ( Figure 3D; Supplementary Figure S2E), while the Insulin-like growth factor I receptor (IGF-IR) antagonist AG1024 was not effective in this regard ( Figure 3E). Similarly, the stimulatory effects of EGF on EGR1 mRNA expression could be also blocked by simultaneous incubation with ErbB1 antagonist AG1478 ( Figure 3C; Supplementary Figure S2B) or ErbB2 antagonist AG879 ( Figure 3D; Supplementary Figure S2F) but not the IGF-IR antagonist AG1024 ( Figure 3E).
To further elucidate the post-receptor signaling mechanism involved in the up-regulation of UTS1 and EGR1 mRNA expression by EGF, various pharmacological inhibitors targeting different signaling pathways were recruited. As a first step, cotreatment with the MEK1/2 inhibitor U0126, ERK1/2 inhibitor LY3214996, or p38MAPK inhibitor SB203580 could all block the stimulatory effects of EGF on UTS1 ( Figure 3F Figure S3D) mRNA expression could be suppressed/totally abolished by co-treatment with the PI 3 K inhibitor Wortmannin, AKT inhibitor MK2206, or mTOR inhibitor Rapamycin.

Receptor Specificity and Signal Transduction for EGF-Induced MMP13 mRNA Expression
To clarify the receptor specificity and signal transduction for EGF-induced MMP13 mRNA expression, a pharmacological approach was used. As shown in Figure 5, the stimulatory effects of EGF on MMP13 could be blocked by simultaneous incubation with ErbB1 antagonist AG1478 ( Figure 5C; Supplementary Figure S2C), but not ErbB2 antagonist AG879 ( Figure 5D; Supplementary Figure S2G) and the IGF-IR antagonist AG1024 ( Figure 5E). With the use of pharmacological blockers targeting different signaling pathways, the signal transduction mechanisms for the up-regulation of MMP13 mRNA expression were examined. The stimulatory effects of EGF on MMP13 mRNA expression were notably dispelled by simultaneous incubation with the MEK1/2 inhibitor U0126 ( Figure 5F; Supplementary Figure S3E), or ERK inhibitor LY3214996 ( Figure 5G), but not with the PI 3 K inhibitor Wortmannin ( Figure 5I; Supplementary Figure S3F), AKT inhibitor MK2206 ( Figure 5J), or mTOR inhibitor Rapamycin ( Figure 5K). To further confirm whether MEK/ERK cascades were involved in EGF-induced post-receptor signaling, the effects of EGF and EGFR inhibitor AG1478 treatment on ERK phosphorylation were tested in grass carp pituitary cells. As shown in Supplemental Figure S4, EGF could significantly induce the phosphorylation of ERK in grass carp pituitary cells. In addition, EGFR inhibitor AG1478 could significantly block EGF-induced ERK phosphorylation.

Functional Role of MMP13 in EGF-Induced UTS1 and EGR1
Previous studies have reported that TIMP3 is the endogenous inhibitor for MMP13. Interestingly, our present study found that EGF could inhibit the pituitary TIMP3 mRNA expression in a time-course ( Figure 6A; Supplementary Figure S1D) and dose-dependent manner ( Figure 6B). For the receptor specificity, the EGF-inhibited TIMP3 mRNA expression could be recovered by co-treatment with ErbB1 antagonist AG1478 ( Figure 6C), but not with the ErbB2 antagonist AG879 ( Figure 6D) or IGF-IR antagonist AG1024 ( Figure 6E). Furthermore, the EGF-induced UTS1 or EGR1 mRNA expression could be abolished by co-treatment with MMP inhibitor BB94 ( Figure 7A,B).
Previous studies have reported that TIMP3 is the endogenous inhibitor for MMP13. Interestingly, our present study found that EGF could inhibit the pituitary TIMP3 mRNA expression in a time-course ( Figure 6A; Supplementary Figure S1D) and dose-dependent manner ( Figure 6B). For the receptor specificity, the EGF-inhibited TIMP3 mRNA expression could be recovered by cotreatment with ErbB1 antagonist AG1478 ( Figure 6C), but not with the ErbB2 antagonist AG879 ( Figure 6D) or IGF-IR antagonist AG1024 ( Figure 6E). Furthermore, the EGF-induced UTS1 or EGR1 mRNA expression could be abolished by co-treatment with MMP inhibitor BB94 ( Figure 7A,B). , and IGF receptor antagonist AG1024 (10 μM) on TIMP3 mRNA expression for 24h, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. , and IGF receptor antagonist AG1024 (10 µM) on TIMP3 mRNA expression for 24 h, respectively. After drug treatment, total RNA was isolated for real-time PCR of MMP13 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. After drug treatment, total RNA was isolated for real-time PCR of UTS1 and EGR1 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p < 0.05 with different letters.

Discussion
Previous studies have reported that EGF could play an important role in mammalian pituitary [14]. However, little is known about the pituitary actions of EGF in lower vertebrate. Grass carp (Ctenopharyngodon idellus) is the most important aquaculture species in China, with a total production of 5.50 million tonnes in 2018 [15]. As we know, the pituitary is the crucial organ for the regulation of After drug treatment, total RNA was isolated for real-time PCR of UTS1 and EGR1 mRNA expression. In the data present (mean ± SEM), the differences between groups were considered as significant at p < 0.05 with different letters.

Discussion
Previous studies have reported that EGF could play an important role in mammalian pituitary [14]. However, little is known about the pituitary actions of EGF in lower vertebrate. Grass carp (Ctenopharyngodon idellus) is the most important aquaculture species in China, with a total production of 5.50 million tonnes in 2018 [15]. As we know, the pituitary is the crucial organ for the regulation of reproduction and growth in teleost, so it will be important to clear the pituitary actions of EGF in teleost. Using grass carp as a model, transcriptomic analysis showed that EGF could induce 195 genes and inhibit 404 genes. These DEGs were involved in cell migration, cell differentiation, signal transduction, metabolic process, phosphorylation, and transcriptional regulation. Similarly, in mammals, previous studies have also reported that EGF could regulate several pituitary functions, including cell proliferation, cell migration [7], and gland tumorigenesis [16].
CRH plays an important role in the HPA system in regulating stress physiology [17]. UTS1 was firstly isolated and purified from white sucker and common carp [18]. Further studies found that fish UTS1 had a closed structural and biological homology with ovine CRH and the frog skin peptide sauvagine [19]. In addition, the UTS1 could also induce ACTH release in mammalian and fish pituitary [20]. Previous studies have found that EGF could induce hypothalamic CRH release [5]. In the present study, we found that EGF could directly induce UTS1 mRNA expression in grass carp pituitary cells. These results suggest that EGF could also be involved in stress physiology mediated by UTS1 in grass carp pituitary. In addition, our present study also found that EGF could induce EGR1 mRNA expression in grass carp pituitary cells. EGR1 is a member of the immediate early gene family of transcription factors, which could regulate a wide variety of transcripts [21]. EGR1 could also be stimulated by many environmental signals, including growth factors [22]. A previous study reported that EGF-induced EGR1 expression could be involved in the down-regulation of matrix metallopeptidase 9 (MMP9) expression in lymphoma cells [23]. These results suggest that EGF could induce EGR1 expression to regulate several physiological functions in teleost pituitary.
MMP13, also called collagenase 3, is a member of the matrix metalloproteinase (MMPs) family, which is involved in embryonic development, reproduction, tissue remodeling, as well as disease processes [24][25][26]. In the present study, we found that EGF could induce pituitary MMP13 mRNA expression, but reduce TIMP3 mRNA expression in grass carp pituitary cells. TIMP3 is a member of tissue inhibitor of metalloproteinases (TIMP) gene family, which are the endogenous protein inhibitors of the MMPs family [27]. These results suggest that EGF could not only directly induce pituitary MMP13 mRNA expression, but could further enhance MMP13 expression via reducing its endogenous inhibitor TIMP3 expression in grass carp pituitary. In addition, previous studies reported that the activated MMPs could release EGF from heparin-bound EGF (HB-EGF) [28]. Interestingly, our present study found that BB94, which was the inhibitor of MMPs, could partially suppress EGF-induced EGR1 and UTS1 mRNA expression in grass carp pituitary cells. Based on these results, it is reasonable for us to speculate that EGF-induced MMP13 mRNA expression might be involved in the up-regulation of UTS1 and EGR1 mRNA expression by EGF in grass carp pituitary cells.
EGF receptors are transmembrane glycoprotein receptors containing an extracellular ligand-binding domain and an intracellular tyrosine kinase domain [29]. A previous study demonstrated that ErbB1 and ErbB2 have both been abundantly detected in normal pituitary corticotroph cells [30], but ErbB3 and ErbB4 were hardly detected in normal or tumoral corticotrophs [5]. Similarly, our previous study also found that both ErbB1 and ErbB2 were abundantly expressed in grass carp pituitary, but ErbB3 and ErbB4 were hardly detected in the pituitary [13]. In the present study, we found that EGF could induce UTS1 and EGR1 mRNA expression via the activation of both ErbB1 and ErbB2 in grass carp pituitary cells. Interestingly, EGF-regulated MMP13 and TIMP3 mRNA expression could only be mediated by ErbB1, but not ErbB2. Previous studies reported that ErbB1 could be activated by binding to growth factors of the EGF family [31]. However, ErbB2 has no ligand, it could bind with other activated ErbB receptors (ErbB1 or ErbB3) to form the highly active heterodimer [32,33]. Besides, the ErbB2 receptor could be activated by a ligand-independent mechanism, such as it could undergo the pH-dependent autophosphorylation [34]. These results suggest that EGF-induced pituitary UTS1 and EGR1 mRNA expression might be mediated by ErbB1/ErbB2 heterodimers, but EGF-regulated MMP13 and TIMP3 mRNA expression should be mediated by ErbB1 homodimers in teleost pituitary. For the post-receptor signaling pathway, our results found that EGF-induced UTS1 and EGR1 mRNA expression were coupled with the PI 3 K/AKT/mTOR and MEK 1/2 /ERK 1/2 pathways. However, EGF-induced MMP13 mRNA expression was only mediated by the MEK 1/2 /ERK 1/2 pathway, but not PI 3 K/AKT/mTOR pathway. Similarly, a previous study also reported that only the MEK 1/2 /ERK 1/2 pathway was involved in EGF-induced MMP13 mRNA expression in gastric cancer cells [35]. These results, taken together, suggest that MEK 1/2 /ERK 1/2 should be the critical signal transduction pathway in the up-regulation of MMP13 by EGF. Recently, MMP13 has emerged as a key target for the treatment of tumors [36]. These findings raise the possibility that MEK 1/2 and ERK 1/2 should be the critical signal transduction factors in EGF-induced tumors. In addition, it is confusing that the PI 3 K inhibitor Wortmannin could hugely induce MMP13 mRNA expression in grass carp pituitary cells. We speculated that some factors in the pituitary could inhibit MMP13 mRNA expression via the PI 3 K pathway, so the Wortmannin could induce pituitary MMP13 expression through blocking these inhibitory actions.
In summary, our present study tried to examine the global pituitary actions of EGF in grass carp pituitary cells. Based on the transcriptomic analysis, EGF could significantly regulate 599 DEGs, which were involved in cell migration, cell differentiation, signal transduction, metabolic process, and phosphorylation. Then, we focused on three critical EGF-induced DEGs, namely UTS1, EGR1, and MMP13. Firstly, we found that EGF could significantly induce UTS1 and EGR1 mRNA expression via the activation of both ErbB1 and ErbB2 in grass carp pituitary cells. However, EGF-regulated MMP13 and TIMP3 mRNA expression were only mediated by ErbB1. The stimulatory actions of UTS1 and EGR1 mRNA expression were mediated by the PI 3 K/AKT/mTOR and MEK 1/2 /ERK 1/2 pathways (Figure 8). The signaling mechanisms for MMP13 responses were also similar, except that PI 3 K/AKT/mTOR was not involved. As we know, MMP13 could release EGF from HB-EGF. In addition, our results found that the MMPs inhibitor BB94 could suppress EGF-induced EGR1 and UTS1 mRNA expression in grass carp pituitary cells. These results, taken together, suggest that EGF-induced MMP13 mRNA expression might be involved in the up-regulation of UTS1 and EGR1 mRNA expression by EGF in grass carp pituitary cells (Figure 8).

Animals and Chemicals
One-year-old grass carps (1+) (Ctenopharyngodon idellus) with a body weight (BW) of 1.0-1.5 kg were bought from local markets and kept in the aquaria at 20 ± 2 • C for seven days and without feeding for at least three days prior to use in the experiment. To prepare the pituitary cells, grass carps were anesthetized in well-aerated water containing 0.05% MS-222 (Sigma, St. Louis, MO, USA) according to the protocol approved by the committee for animal use at Huazhong Agricultural University (Ethical Approval No. HBAC20091138; Date: 15 November 2009). Human EGF was purchased from GenScript Corporation (Nanjing, China) and dissolved in double-distilled deionized water and stored as 0.1 mM stocks in small aliquots at −80 • C. The pharmacological agents for receptor specificity and signal pathways (listed in Supplemental Table S1) were prepared as 10 mM frozen stocks in small aliquots and diluted with pre-warmed culture medium to appropriate concentrations 15 min prior to drug treatment.

Figure 8.
Working modal of EGF-induced UTS1, EGR1, MMP13, and TIMP3 regulation in grass carp pituitary. EGF induced UTS1 and EGR1 mRNA expression were mediated by the PI3K/AKT/mTOR and MEK1/2/ERK1/2 pathways coupled with both ErbB1 and ErbB2. EGF-induced MMP13 mRNA expression was only through the MEK1/2/ERK1/2 pathway coupled with ErbB1 and inhibited TIMP3 mRNA expression via ErbB1. EGF-induced MMP13 might be involved in the up-regulation of UTS1 and EGR1 mRNA expression by EGF in grass carp pituitary cells. The solid arrows indicated that the actions were verified by our study, the dotted arrows indicated that the actions were verified basing on the references. And the dotted "T" represented the inhibited action basing on the references.

Animals and Chemicals
One-year-old grass carps (1+) (Ctenopharyngodon idellus) with a body weight (BW) of 1.0-1.5 kg were bought from local markets and kept in the aquaria at 20 ± 2 °C for seven days and without feeding for at least three days prior to use in the experiment. To prepare the pituitary cells, grass carps were anesthetized in well-aerated water containing 0.05% MS-222 (Sigma, St. Louis, MO, USA) according to the protocol approved by the committee for animal use at Huazhong Agricultural University (Ethical Approval No. HBAC20091138; Date: 15 November 2009). Human EGF was purchased from GenScript Corporation (Nanjing, China) and dissolved in double-distilled deionized water and stored as 0.1 mM stocks in small aliquots at -80 °C. The pharmacological agents for receptor specificity and signal pathways (listed in Supplemental Table S1) were prepared as 10 mM Figure 8. Working modal of EGF-induced UTS1, EGR1, MMP13, and TIMP3 regulation in grass carp pituitary. EGF induced UTS1 and EGR1 mRNA expression were mediated by the PI 3 K/AKT/mTOR and MEK1/2/ERK1/2 pathways coupled with both ErbB1 and ErbB2. EGF-induced MMP13 mRNA expression was only through the MEK1/2/ERK1/2 pathway coupled with ErbB1 and inhibited TIMP3 mRNA expression via ErbB1. EGF-induced MMP13 might be involved in the up-regulation of UTS1 and EGR1 mRNA expression by EGF in grass carp pituitary cells. The solid arrows indicated that the actions were verified by our study, the dotted arrows indicated that the actions were verified basing on the references. And the dotted "T" represented the inhibited action basing on the references.

Cell Culture, RNA Extraction and cDNA Library Construction
The grass carp pituitaries were rinsed three times with Hanks Balanced Salt Solution (HBSS; 400 mg KCl, 600 mg KH 2 PO4, 350 mg NaHCO 2 , 8 g NaCl, 48 mg Na 2 HPO 4 , and 1 g D-Glucose in 1 L ddH 2 O), and dispersed by trypsin/DNase II digestion method [37]. Then, grass carp pituitary cells were seeded in 24-well culture plates at a density of 2.5 × 10 6 cells/well/mL at 28 • C under 5% CO 2 for 15~18 h in plating medium. After that, the pituitary cells were incubated with EGF dissolved in testing medium for 24 h. Total RNA were harvested from the plate by adding 500 µL of Trizol reagent (Invitrogen, Carlsbad, CA, USA) to each well and shaking the plate for 10 min at 160~170 rpm on the shaker. The RNA was treated with DNase I to remove contaminating genomic DNA. The concentration and sample purity of total RNA were estimated using a Nanodrop 2000 spectrophotometer, and the quality of RNA was analyzed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Then, the RNA (RIN > 8.0) samples were sent to Majorbio Genome Center (Shanghai, China) for library preparation by TruSeq™ RNA sample prep Kit (Illumina, San Diego, CA, USA) and sequencing on HiSeq4000 (Illumina). A read depth of 600 million 150-bp single end reads was selected. An average of~90% of the reads mapped to the grass carp genome (http://bioinfo.ihb.ac.cn/gcgd). All raw-sequence read data were deposited in NCBI Sequence Read Archive (SRA)2 with accession number SRP148383.

Differential Expression Genes (DEGs) Analysis and Functional Enrichment
Clean data could be obtained by removing read operations containing adapters, poly-N, and low-quality reads from the raw data. High-quality clean reads were mapped to the grass carp genome using TopHat v2.0 (http://ccb.jhu.edu/software/tophat/index.shtml). In different samples, gene expression levels were estimated by the number of fragments per kilobase transcript (FPKM). The read counts were further normalized into FPKM values. The fold changes were calculated by using RSEM software v 1.2.7 [38] and the DEGs were analyzed by using the R Bioconductor package, edgeR which calculated assuming a negative binomial distribution for the transcript level. The p-value was used to set the threshold for the differential gene expression test. The threshold of the p-value in multiple tests was determined by the value for the false discovery rate (FDR) [39]. DEGs were screened with a cut-off conditions of fold change (FC) > 1.5, p < 0.05 and FDR < 0.001. Functional annotation of gene ontology (GO) terms was analyzed by using Blast2GO software (https://www.blast2go.com/) [40], and GO functional classification of unigenes were analyzed by using WEGO 2.0 software (http://wego.genomics.org.cn/) [41]. Functional enrichment analysis, including GO and KEGG, was performed using Goatools (or KOBAS) software (https://github.com/tanghaibao/GOatools) [42].

Real-Time Quantitative PCR Validation
Grass carp pituitary cells were seeded in 24-well culture plates at a density of 2.5 million/mL/well and incubated with test substances for the duration as indicated. After drug treatment, the total RNA was isolated from pituitary cells by Trizol reagent (Invitrogen) and reversely transcribed by HifairTM III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen Biotech, Shanghai, China). After RNA isolated and reversely transcribed, the ABI 7500 real-time PCR system was used to detect the mRNA transcription of MMP13, UTS1, EGR1, and TIMP3 with specific primers (see Supplementary Table S2 for primer sequences and PCR condition). In these experiments, plasmid DNA containing the gene coding sequence was subjected to gradient dilution as a standard for data calibration. In addition, parallel real-time PCR of β-actin was used as an internal control. The specific methods for dose-and time-dependent experiment, receptor specificity, signal transduction of EGF-induced UTS1, EGR1 and MMP13 mRNA expression could see Supplementary Methods in Supplemental Materials.

Data Transformation and Statistical Analysis
In this experiment, for real-time PCR of MMP13, UTS1, EGR1, and TIMP3 mRNA, standard curves with dynamic range of 10 5 and correlation coefficient > 0.95 were used for data calibration with ABI7500 software (Applied Biosystems, USA). MMP13, TIMP3, EGR1, and UTS1 mRNA data were normalized with β-actin transcript level, and then were transformed as a percentage of the mean value in the control group without drug treatment (as "%Ctrl"). In the present study, the eight replicates (expressed as Mean ± SEM) were pooled results from two individual experiments prior to statistical analysis; all data were tested for normality of distribution using the Shapiro-Wilk normality test. One-way ANOVA and two-way ANOVA were used to test the significant difference according to different experiments. The differences between groups were considered as significant at p < 0.05 ("*") or highly significant at p < 0.01 ("**"). The groups denoted by different letters represent a significant difference at p < 0.05. Acknowledgments: This article is dedicated to Anderson OL Wong (The University of Hong Kong) for his genuine interest in training young scientists in the field of comparative endocrinology.

Conflicts of Interest:
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.