Identification of a Novel Functional Corticotropin-Releasing Hormone (CRH2) in Chickens and Its Roles in Stimulating Pituitary TSHβ Expression and ACTH Secretion

Abstract

Corticotropin-releasing hormone (CRH), together with its structurally and functionally related neuropeptides, constitute the CRH family and play critical roles in multiple physiological processes. Recently, a novel member of this family, namely CRH2, was identified in vertebrates, however, its functionality and physiological roles remain an open question. In this study, using chicken (c-) as the animal model, we characterized the expression and functionality of CRH2 and investigated its roles in anterior pituitary. Our results showed that (1) cCRH2 cDNA is predicted to encode a 40-aa mature peptide, which shares a higher amino acid sequence identity to cCRH (63%) than to other CRH family peptides (23-38%); (2) Using pGL3-CRE-luciferase reporter system, we demonstrated that cCRH2 is ~15 fold more potent in activating cCRH receptor 2 (CRHR2) than cCRHR1 when expressed in CHO cells, indicating that cCRH2 is bioactive and its action is mainly mediated by CRHR2; (3) Quantitative real-time PCR revealed that cCRH2 is widely expressed in chicken tissues including the hypothalamus and anterior pituitary, and its transcription is likely controlled by promoters near exon 1, which display strong promoter activity in cultured DF-1 and HEK293 cells; (4) In cultured chick pituitary cells, cCRH2 potently stimulates TSHβ expression and shows a lower potency in inducing ACTH secretion, indicating that pituitary/hypothalamic CRH2 can regulate pituitary functions. Collectively, our data provides the first piece of evidence to suggest that CRH2 play roles similar, but non-identical, to those of CRH, such as its differential actions on pituitary, and this helps to elucidate the roles of CRH2 in vertebrates.

Keywords: ACTH and TSH; CRH receptor; CRH2; chicken; pituitary.

Figure 1

(A) Nucleotide and predicted amino acid sequences of cCRH2. The putative signal peptide was underlined and in italic. The predicted mature peptide was shaded and in bold, and the putative polyadenylation signal (ATTAAA) was shaded. (B) Exon (E)-intron organization of cCRH2. The coding region of cCRH2 (324 bp) was intron-less and colored in gray. Numbers in the boxes indicate the size of non-coding or coding regions (shaded), and number in italic indicates the size of intron 1. The asterisk (*) indicates the stop codon.

Figure 2

Amino acid alignment of chicken CRH2 precursor (cCRH2: KU887752) with that of zebra finches (zbCRH2), flycatchers (flCRH2), turtles (tuCRH2), lizards (lzCRH2), coelacanth (coCRH2), spotted gars (sgCRH2), elephant sharks (shCRH2), or with chicken CRH (cCRH: NP_001116503.1), UCN1 (cUCN1: XP_015140488.2), UCN2 (cUCN2: APU52336.1), and UCN3 precursor (cUCN3: AGC65587.1). The mature peptide regions were marked and dashes denote gaps in the alignment. Identical amino acid residues between cCRH2 and other precursors are shaded in black, and similar ones in gray.

Figure 3

Synteny analysis showing the chromosomal location of CRH2 in vertebrates. CRH2 is located in a syntenic region conserved between chickens, anole lizards, coelacanth, spotted gars, sharks and platypus. Based on existing genome assemblies, CRH2 is absent in humans and zebrafish genomes and likely lost in Xenopus tropicalis. Dotted lines indicate the syntenic genes; dashed lines denote genes of interest (CRH2); the number in bracket (M) indicate the location of CRH2 gene in corresponding chromosome or scaffold.

Figure 4

Effects of cCRH2 (A) or cCRH (B) in activating chicken CRHR1 and CRHR2 expressed in CHO cells, as monitored by the pGL3-CRE-luciferase reporter system. cCRH2 and cCRH treatment did not stimulate luciferase activity of control CHO cells co-transfected with the empty pcDNA3.1(+) vector and a pGL3-CRE-luciferase reporter construct. Each data point represents mean ±SEM of three replicates (N = 3).

Figure 5

Tissue distribution of CRH2 in adult chickens. (A) qPCR detection of CRH2 expression in various chicken tissues, including the whole brain (Br), spinal cord (Sc), anterior pituitary (Pi), heart (He), duodenum (Du), kidney (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), testes (Te), spleen (Sp), pancreas (Pa), fat (Fa), and skin (Sk). (B) qPCR assay of cCRH2 expression in adult chicken brain regions including the telencephalon (Tc), midbrain (Mb), cerebellum (Cb), hindbrain (Hb), and hypothalamus (Hp). The mRNA levels of CRH2 were normalized to that of β-actin and expressed as the fold difference compared with that of the whole brain (Br) or telencephalon (Tc). Each data point represents the mean SEM of 4 adult chickens (N = 4).

Figure 6

(A) Detection of promoter activities of the 5′-flanking region of cCRH2 in cultured DF-1 and HEK293T cells. Various stretches of the 5′-flanking regions were cloned into pGL3-Basic vector for the generation of multiple promoter-luciferase constructs. These promoter-luciferase constructs were then co-transfected into DF-1 or HEK293T cells along with pRL-TK vector and their promoter activities were determined by the Dual-luciferase reporter assay. Each value represents the mean ± SEM of four replicates (N = 4). **P < 0.01 vs pGL3-Basic. Dotted line marked by an X indicates the deleted DNA fragment. (B) Nucleotide sequence of CRH2 promoter region (−989/+170; Accession no. MK550695). Transcriptional start site (TSS, nucleotide “G”) determined by 5′-RACE is boxed and designated as “+1,” and the first nucleotide upstream TSS is designated as “−1.” Exon 1 (35 bp) is shaded and in bold. Tanslation start codon (ATG) is located on exon 2. The TATA box and binding sites for transcriptional factors (e.g., CREB, Sp1, AP1) were predicted using online software TFBind (http://tfbind.hgc.jp/). CREB: cAMP response element binding protein; AP1, activation protein 1; Sp1, specificity protein 1. Whether these sties are functional remains to be clarified.

Figure 7

(A) Amino acid alignment of cCRH2 with cCRH, cUCN1, cUCN2, and UCN3. Identical amino acid residues were shaded in black, and similar ones were labeled in gray. (B,C)Western blot showing dose-dependent effects of cCRH2 (B) and cCRH (C) on ACTH secretion in cultured chick pituitary cells. Pituitary cells were incubated with designated dose of cCRH2 (0.1–100 nM) or cCRH (0.1–10 nM) for 6 h, then the culture medium and cell lysate were used for Western blot detection of ACTH and β-actin, respectively. The sACTH band at ~5 kDa (secretory ACTH level detected in culture medium) were semi-quantified by densitometric analysis. Their relative levels were normalized by that of β-actin in pituitary cell lysate, and then expressed as fold increase compared to control (0 nM). Each data point represents mean SEM of 3 replicates (N = 3). *P < 0.05, **P < 0.01, vs. control (0 nM). Representative set of Western blot is shown at the bottom of each graph; (D,E) qPCR assay of cCRH2 (1–100 nM) effect on pituitary POMC (D) and TSHβ (E) expression. Each data point represents mean SEM of 4 replicates (N = 4).**P < 0.01 vs. control (0 nM).

Figure 8

Proposed model for CRH2 action on chicken anterior pituitary. Like CRH, CRH2 (produced in the pituitary or hypothalamus) can potently stimulate TSH expression via the activation of CRHR2 expressed in thyrotrophs (marked by solid lines), suggesting that cCRH2 may play an important role in the hypothalamus-pituitary-thyroid axis (HPT axis). Unlike CRH, CRH2 has a low potency (≥10 nM) in stimulating ACTH secretion (marked by dotted lines) possibly via activating CRHR1 expressed in corticotrophs. In this model, CRH2 is expressed in the hypothalamus-pituitary axis, however, it remains to be clarified which type(s) of pituitary cell secrete CRH2, or whether hypothalamic CRH2 can directly control TSH/ACTH expression and/or secretion (marked by question marks).