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 c CRH2 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 TSH β 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.

New Insights Into the Evolution of Corticotropin-Releasing Hormone Family With a Special Focus on Teleosts

“…In contrast we identified a third gene, corresponding to crh2 according to our phylogenetic analysis, in representative species of other amphibian groups, Gymnophiona (Microceacilla unicolor), and Caudata (axolotl, Ambystoma mexicanum) (Figure 1). The three genes (crh1, crh2 and ucn1) were present in representative species of the various groups of sauropsids including birds, as recently reported (48), as well as in protherian and metatherian mammals, in agreement with previous studies (21) (Figure 1; Table S1).…”

“…The loss of crh2 gene would thus be specific to the anuran lineage. In sauropsids, our study further assessed the conservation of the three crh genes, crh1, crh2 and ucn1 in various lineages, including birds, as recently reported (48). In mammals, we found a crh2 gene only in monotremes and marsupials, confirming its loss in the eutherian lineage (15,21).…”

Expression patterns and evolution of urocortin and corticotropin‐releasing hormone genes in a cichlid fish

“…CRF, UCN1, and UCN3 expression in several tissues has also been reported in the amphibian Xenopus laevis (Boorse et al, 2005;Stenzel-Poore et al, 1992). Similarly, CRH1, CRH2, and urocortin expression was described in some birds, including chicken (Bu et al, 2019;Grommen et al, 2017;Vandenborne et al, 2005). However, a detailed description of expression patterns for all the fish urocortin genes within neural and peripheral tissues of a single species, which would be important for understanding their diverse functions, is not yet available.…”

KLF7 Promotes Preadipocyte Proliferation via Activation of the Akt Signaling Pathway by Cis-regulating CDKN3

“…DF-1, an immortalized chicken fibroblast cell line, can be easily transiently transfected with exogenous DNA. DF-1 is often used for transcriptional regulation analysis in birds and is an ideal cell model for luciferase reporter gene assays [28][29][30][31]. ICP1 and DF-1 cells were respectively cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 and high-glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Biological Industries, Germany) and 1% penicillin-streptomycin solution (Beyotime, Shanghai, China), at 37 °C in the presence of 5% CO2.…”