Molecular characterization of two suppressor of cytokine signaling 1 genes (<i>SOCS1a</i> and <i>SOCS1b</i>) in chickens

doi:10.15302/J-FASE-2015044

Front. Agr. Sci. Eng.

2015, Vol. 2

Issue (1) : 73-83 https://doi.org/10.15302/J-FASE-2015044

RESEARCH ARTICLE

Molecular characterization of two suppressor of cytokine signaling 1 genes (SOCS1a and SOCS1b) in chickens

Xue XU,Jiannan ZHANG,Juan LI,Yajun WANG(

)

Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China

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Abstract

Suppressor of cytokine signaling 1 (SOCS1) protein can inhibit the signal transduction triggered by some cytokines or hormones and thus are important in many physiological/pathological processes, including innate and adaptive immunity, inflammation, and development in mammals. However, there is sparse information about their structure, tissue expression, in birds, where their biological functions remain unknown. In this study, we cloned and characterized two SOCS1 genes (named cSOCS1a and cSOCS1b) from chickens. SOCS1a is predicted to encode a 207-amino acid protein, which shares high amino acid sequence identity (64%–67%) with human and mouse SOCS1. Besides SOCS1a, a novel SOCS1b gene was also identified in chickens and other non-mammalian vertebrates including Xenopus tropicalis. Chicken SOCS1b is predicted to encode a 212-amino acid protein, which shares only 30%–32% amino acid sequence identity with human SOCS1 and cSOCS1a. RT-PCR assay revealed that both cSOCS1a and cSOCS1b are widely expressed in all chicken tissues. Using a luciferase reporter assay system, we further demonstrated that transient expression of cSOCS1a and cSOCS1b can significantly inhibit chicken growth hormone (GH)- or prolactin (PRL)-induced luciferase activities of Hep G2 cells expressing cGH receptor (or cPRL receptor), indicating that SOCS1a and SOCS1b proteins can negatively regulate GH/PRL signaling. Taken together, these data suggest that both cSOCS1a and cSOCS1b may function as negative regulators of cytokine/hormone actions, such as modulation of GH/PRL actions in chickens.

Keywords chicken SOCS1a SOCS1b growth hormone prolactin

Corresponding Author(s): Yajun WANG

Just Accepted Date: 16 March 2015 Online First Date: 24 April 2015 Issue Date: 22 May 2015

Cite this article:

Xue XU,Jiannan ZHANG,Juan LI, et al. Molecular characterization of two suppressor of cytokine signaling 1 genes (SOCS1a and SOCS1b) in chickens[J]. Front. Agr. Sci. Eng. , 2015, 2(1): 73-83.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2015044
https://academic.hep.com.cn/fase/EN/Y2015/V2/I1/73

Tab.1 Primers used in this study^a

Fig.1 Amino acid sequence alignment of SOCS1 proteins from chicken and other species. (a) Alignment of chicken SOCS1a (Ch-SOCS1a, XM_414929) with SOCS1a (or SOCS1) from duck (Du-SOCS1a: XP_005027549), human (Hu-SOCS1), mouse (Mo-SOCS1), pig (Pi-SOCS1), alligator (Al-SOCS1a), Xenopus tropicalis (Xe-SOCS1a), coelacanth (Co-SOCS1a) and zebrafish (zf-SOCS1a1, NM_001003467; zf-SOCS1a2, JN800507); (b) alignment of chicken SOCS1b (Ch-SOCS1b, HQ917699) with that of zebra finch (Fi-SOCS1b), falcon (Fa-SOCS1b), pigeon (Po-SOCS1b), penguin (Pe-SOCS1b), alligator (Al-SOCS1b), Xenopus tropicalis (Xe-SOCS1b), coelacanth (Co-SOCS1b), or with that of chicken SOCS1a (Ch-SOCS1a) or human SOCS1 (Hu-SOCS1). Dots indicate the amino acid residues identical to chicken SOCS1a and dashes represent gaps in the alignment. The conversed SH2 domain, and SOCS box are shaded; the kinase inhibitory domain (KIR) is shaded and shown in bold; the Extended SH2 domain (ESS) is bold and underlined; the BC box is shaded and boxed. The amino acid sequences of SOCS1a or SOCS1b genes from other species were either retrieved from GenBank or predicted according to genomic sequences.

Fig.2 Gene structure (a) and tissue expression (b) of chicken SOCS1a and SOCS1b. (a) Exon (E) organization of cSOCS1a and cSOCS1b genes. The coding regions of cSOCS1a and cSOCS1b are intronless and shaded. cSOCS1a contains one non-coding exon (Exon 1, 25 bp) upstream of the translation start site (ATG), while cSOCS1b contains two non-coding exons (Exon 1, 67 bp; Exon 2, 121 bp) upstream of ATG codon. The numbers in the boxes indicate the size of non-coding or coding regions (shaded), and the number in italics indicates the size of the intron; (b) RT-PCR detection of cSOCS1a and cSOCS1b mRNA expression in adult chicken tissues, including the brain (Br), heart (He), small intestine (In), kidneys (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), testes (Te), pituitary (Pi), and spleen (Sp). No PCR band was detected in the negative controls (-). Numbers in parenthesis indicate the PCR cycles used.

Fig.3 Synteny analyses showing the existence of SOCS1a (a) and SOCS1b (b) in non-mammalian vertebrates. (a) SOCS1a gene is located in a syntenic region conserved between chickens, humans, Xenopus tropicalis and zebrafish. The two copies of SOCS1a (SOCS1a1, NM_001003467; SOCS1a2, JN800507) genes in zebrafish are likely to be paralogs generated by the teleost-specific whole genome duplication (WGD) event; (b) SOCS1b gene is located in a syntenic region conserved between chickens and Xenopus tropicalis. SOCS1b gene is likely lost in zebrafish and human genomes. Dashed lines denote the genes of interest (SOCS1a or SOCS1b), while dotted lines indicate the syntenic genes identified in these species. Genes were labeled according to their gene symbols in the human genome. Gray bars appeared in (b) indicate the SOCS1b gene likely lost in these species. SOCS1a gene identified in chickens, Xenopus tropicalis, and zebrafish is orthogous to mammalian (human) SOCS1 gene. Chr, chromosome; WGD, teleost-specific whole genome duplication.

Fig.4 Inhibition of GH/PRL signaling by cSOCS1a (a, c) and cSOCS1b (b, d). (a, b) transient expression of cSOCS1a or cSOCS1b inhibits cGH-induced (200 ng·mL^–1, 18 h) luciferase activities of Hep G2 cells expressing chicken GH receptor, monitored by a 5 × STAT luciferase reporter system; (c, d) transient expression of (b,d) transient expression of cSOCS1a or cSOCS1b inhibits cPRL-induced (200 ng·mL^–1, 18 h) luciferase activities of Hep G2 cells expressing chicken PRL receptor, monitored by a 5 × STAT luciferase reporter system; The luciferase activity of Hep G2 cells in each treatment group was expressed as relative increase compared to the control group (without hormone treatment). Each data point represents mean±SEM of three replicates. **, P<0.001 vs control (without hormone treatment); #, P<0.01; ##, P<0.001 vs hormone treatment group (without SOCS1a/SOCS1b added).

Fig.5 Phylogenetic tree (constructed by Neighboring-Joining method) showing the evolutionary relationship of SOCS1a, SOCS1b, SOCS3, SOCS2 and CIS genes from non-mammalian and/or mammalian vertebrates. Numbers near each branch point indicate the bootstrap values. The amino acid sequences of all SOCS or CIS genes were either retrieved from GenBank or predicted according to genomic sequences.

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