Expression and regulation of beta-defensin 11 in the oviduct in response to estrogen and in ovarian tumors of chickens

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Abstract

Avian beta-defensins (AvBDs), also known as gallinacins, are small cationic peptides having three cysteine disulfide bonds between their cysteine residues. They play essential roles in the innate immune system as well as stimulate proliferation of epithelial cells and fibroblasts. Although we found the avian homolog of human beta-defensin 11 to be highly expressed in chicks treated with the diethylstilbestrol (DES, a synthetic estrogen agonist), little is known about the hormonal and transcriptional regulation of AvBD-11 in the chicken oviduct and its expression in cancerous ovaries of chickens. Results of this study of young chicks revealed that DES induced AvBD-11 mRNA and protein in the oviduct, specifically luminal and glandular epithelial cells. In addition, microRNA-1615 was discovered to influence AvBD-11 expression via its 3′-UTR which suggests post-transcriptional regulation of AvBD-11 expression in chickens. Furthermore, we compared the expression patterns of the AvBD-11 gene in normal and cancerous ovaries from laying hens which are models for human epithelial ovarian cancer. Our results demonstrated that AvBD-11 is most abundant in the glandular epithelium of endometrioid-type ovarian tumors, but not normal ovaries of laying hens. Collectively, these results suggest that AvBD-11 is an estrogen-induced gene during oviduct development and that it may be used as a biomarker for diagnosis of ovarian cancer and for monitoring effects of therapeutics on progression of ovarian carcinogenesis.

Highlights

► We examine hormonal regulation of AvBD-11 in the chicken oviduct. ► We determine AvBD-11 gene expression in cancerous ovaries of laying hens. ► AvBD-11 is a novel estrogen-induced gene in chickens during oviduct development. ► AvBD-11 is abundant in the glandular epithelium of laying hens with ovarian tumors.

Introduction

The laying hen is one of the best animal models for studies of oviduct growth and development and ovarian tumorigenesis. Indeed many researchers use the chicken oviduct due to its sensitive response to sex steroids and rapid growth and differentiation (Dougherty and Sanders, 2005). Estrogen, as a sex hormone affecting the oviduct, induces epithelial cells within the chick oviduct to mature into tubular gland cells thorough cell proliferation and cytodifferentiation and to induce expression of egg-white protein genes such as ovalbumin (Socher and Omalley, 1973, Palmiter and Wrenn, 1971). In addition, laying hens are the most relevant animal model to identify biomarkers for epithelial ovarian cancer. It is believed that incessant laying of eggs (ovulation) in hens increases the possibility of genetic mutations and damage in the ovarian surface epithelium that leads to epithelial ovarian cancer as occurs in women (Murdoch et al., 2005). Furthermore, several biomarkers such as ovarian carcinoma antigen 125 (CA125; also known as mucin 16, MUC16), epidermal growth factor receptor (EGFR), and V-ERB-B2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB-2) for human ovarian cancer (Jackson et al., 2007, Rodriguez-Burford et al., 2001, Anderson et al., 2010, Johnson, 2009) are highly expressed in similar patterns to those for chicken ovarian tumors (Hakim et al., 2009).

Defensins are small host defense peptides that play a pivotal role in innate immunity (Klotman and Chang, 2006). They consist of conserved cysteine-rich cationic domains and beta-pleated sheet structures connected by three intra-disulfide bonds (Ganz, 2003). In mammals, they are divided into three subgroups, alpha-, beta- and theta-defensins, depending on the arrangement of disulfide bonds among cysteine residues (Klotman and Chang, 2006, Ganz, 2003). Avian defensins, known as gallinacins (Harwig et al., 1994), were originally purified from chicken leukocytes and classified as members of the beta-defensin subfamily involved in antimicrobial activity against microorganisms such as Gram-positive/-negative bacteria and fungi (Xiao et al., 2004, van Dijk et al., 2008). At present, 14 classes of avian beta-defensin (AvBD) genes have been identified in the chicken (Abdel Mageed et al., 2009) of which 11 have been detected in the chicken oviduct and five of those AvBD mRNAs increase in response to lipopolysaccharides (Mageed et al., 2008). Of interest, we found the avian homolog of human beta-defensin 11 transcript to be highly expressed in chicks treated with the DES (Song et al., 2011). However, there is little known about the hormonal regulation of AvBD-11 in the chicken oviduct or its expression in cancerous ovaries of laying hens. Thus we focused on this gene in the present study.

The objectives of this study were to: (1) determine if estrogen regulates expression of AvBD-11 during oviduct development in chicks; (2) determine whether expression of AvBD-11 is regulated by post-transcriptional actions of specific microRNAs and (3) compare expression of AvBD-11 in normal and cancerous ovaries from laying hens. Results of the present study indicate that AvBD-11 is an estrogen-stimulated gene during development of the chicken oviduct and that it may be an initial biomarker gene for epithelial ovarian carcinogenesis in laying hens that may be used in research into the etiology of epithelial ovarian cancer in laying hens and perhaps in women.

Section snippets

Experimental animals and animal care

The experimental use of chickens for this study was approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-070823-5). All White Leghorn (WL) chickens were exposed to a light regimen of 15 h light and 9 h dark, ad libitum access to feed and water, and standard management practices for laying hens.

Tissue samples

Study one, Following euthanasia of mature WL hens, tissue samples were collected from brain, heart, liver, kidney, small intestine, gizzard, ovary, oviduct and testis of

Multiple sequence alignment, pairwise comparisons, and phylogenetic analysis

The AvBD-11 gene spans 2,619 bp on chicken chromosome 3 and consists of four exons (Xiao et al., 2004). AvBD-11 mRNA has 652 bp encoding a protein with 104 amino acid residues (Supplementary Fig. 1A). The amino acid sequence of chicken AvBD-11 was compared to those of six other species. The pair-wise comparisons of AvBD-11 orthologs revealed that chicken AvBD-11 protein is well conserved relative to other avian AvBD-11 proteins, but not with mammalian beta defensin 11 proteins (38.0–93.3%, Table 1

Acknowledgments

This research was funded by the World Class University (WCU) program (R31-10056) and by Basic Science Research Program (2010-0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology, and also by a Grant from the Next-Generation BioGreen 21 Program (No. PJ008142), Rural Development Administration, Republic of Korea.

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