Des-acyl ghrelin inhibits the capacity of macrophages to stimulate the expression of aromatase in breast adipose stromal cells
Introduction
Aromatase is the rate-limiting enzyme in estrogen biosynthesis, responsible for catalyzing the conversion of androgens into estrogens. Prior to menopause, estrogens are mainly produced by the ovaries and fluctuate with every menstrual cycle [1]. They affect reproductive function, mammary gland development, as well as the normal functioning of a number of tissues and organ systems, including maintaining bone and joint health, cognitive function and cardio-metabolic health [1]. After menopause, the ovaries no longer produce measurable levels of estrogens and normal functioning of these tissues is ensured via the local expression of aromatase, with estrogens acting locally in an autocrine and paracrine manner [1]. Aromatase expression at extra-gonadal sites is responsive to a number of stimuli. Of all sites to produce estrogens after menopause, the adipose tissue is by far the most important in terms of tissue volume. In the adipose tissue, aromatase is elevated with obesity, and this is thought to occur, at least in part, due to chronic low grade inflammation that is associated with obesity [2]. Inflammatory mediators, including prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF) stimulate aromatase expression in adipose stromal cells (ASCs), the main cell type responsible for aromatase expression in the adipose tissue [3], [4].
Obesity is also associated with inflammation in breast white adipose tissue and an increase in breast aromatase expression [5]. The local production of estrogens in the breast fat is believed lead to a hormonal milieu conducive to cancer development and growth [6]. In obesity, white adipose tissue inflammation occurs as a consequence of the recruitment of immune cells, e.g. macrophages, to dead or dying adipocytes. Macrophages can be classified into two major populations: the classically activated M1 macrophage, characterized by CD11c surface expression and responsible for the production of pro-inflammatory factors including IL-6, TNFα, and IL-1β, and the alternatively activated M2 macrophage, that expresses CD206 and CD301 and secretes anti-inflammatory cytokines [7]. In obesity, the number of M1 macrophages increases in the adipose tissue and correlates with the degree of tissue inflammation [7].
Ghrelin is a 28-amino acid peptide hormone, predominantly produced from the stomach, which has well-characterized roles in the stimulation of appetite and growth hormone release [8]. It is known to act via the growth hormone secretagogue receptor (GHSR1a) to mediate effects in the hypothalamus [8]. Recently, ghrelin has been shown to play a crucial role in glucose and energy homeostasis and bone metabolism, and to inhibit the production of pro-inflammatory cytokines IL-1β and TNFα in lipopolysaccharide-stimulated RAW264.7 macrophages, effects that were antagonized in the presence of a GHSR1a-specific antagonist [9]. Conversely, signaling involving GHSR1a has been shown to stimulate M1 macrophage polarization and adipose tissue inflammation during aging [10]. The physiological levels of ghrelin and its unacylated form, des-acyl ghrelin, are in the range 0.1–0.5 nM [11]. Des-acyl ghrelin circulates more abundantly than ghrelin and does not bind to GHSR1a at physiological concentrations. Despite this, it has been shown to exert a number of biological effects, including regulating adipocyte differentiation and function, reducing adipose tissue inflammation, inhibiting skeletal muscle atrophy, promoting glucose uptake, and stimulating osteoblast proliferation [11]. Effects of des-acyl ghrelin to prevent the dysregulation of glucose metabolism in a diet-induced mouse model of obesity are also associated with a switch in macrophage polarization in epididymal white adipose tissue [12].
Recently, we have demonstrated that des-acyl ghrelin inhibits aromatase expression in isolated ASCs [13]. In the current study, we aimed to determine whether des-acyl ghrelin also had direct effects on macrophage polarization and impacted their capacity to stimulate aromatase expression in isolated ASCs.
Section snippets
Isolation of ASCs, ATMs and tissue culture
Primary human breast adipose stromal cells (ASCs) were isolated from breast reduction surgery and cultured as previously described [14]. Immune cells, including adipose tissue macrophages (ATMs), were isolated after ASC preparation was allowed to adhere for 1 h and characterized based on CD14 and CD16 expression (data not shown). ATMs were plated at a density of 100,000 cells/mL, 10 mL per dish, and allowed to adhere overnight. Mouse macrophages RAW264.7 cells were purchased from American Type
Des-acyl ghrelin inhibits Ptgs2 and Tnf expression in macrophages and suggests a shift towards M2 polarization
The effect of 10pM and 100pM des-acyl ghrelin on Cd11c and Cd206 expression in RAW264.7 cells was examined by qRT-PCR. Treatment with 100pM des-acyl ghrelin significantly stimulated the expression of the M2-specific cell surface marker Cd206 and inhibited the expression of M1-specific cell surface marker Cd11c (Fig. 1). Effects of des-acyl ghrelin on TNF responses were also examined by qRT-PCR and ELISA from RAW264.7 cells and conditioned media, respectively. Des-acyl ghrelin caused a
Discussion
In the current study, we demonstrate that des-acyl ghrelin directs macrophage polarization towards an anti-inflammatory M2 phenotype, decreases Tnf and Ptgs2 expression, and reduces the macrophage-dependent stimulation of aromatase expression in breast adipose stromal cells.
Our findings demonstrating that des-acyl ghrelin inhibits the expression of M1-specific genes, including Cd11c and Tnf is consistent with other groups’ findings demonstrating an anti-inflammatory effect of ghrelin and
Conclusion
Taken together, our findings suggest that in addition to direct effects on aromatase expression in breast ASCs, des-acyl ghrelin also has the capacity to inhibit macrophage-derived factors that contribute to the increased expression of aromatase in obesity (Fig. 4). Our studies have been performed in the context of adipose tissue inflammation; however, it is possible that similar effects may be observed in tumor-associated macrophages, also believed to be key drivers of aromatase in breast
Acknowledgements
This work was supported by a grant from the National Breast Cancer Foundation (NC-14-011), and by the Victorian Government Operational Infrastructure Support Program. CCA is supported by an Endocrine Society of Australia Research Higher Degree Scholarship. KAB was supported by an NHMRC (Australia) Career Development Award GNT1007714, the Endocrine Society of Australia Ken Wynne Memorial Postdoctoral Research Award, and by the Mavis Robertson Fellowship from the National Breast Cancer Foundation
References (22)
Sources of estrogen and their importance
J. Steroid Biochem. Mol. Biol.
(2003)- et al.
Aromatase overexpression in dysfunctional adipose tissue links obesity to postmenopausal breast cancer
J. Steroid Biochem. Mol. Biol.
(2015) - et al.
Exogenous ghrelin modulates release of pro-inflammatory and anti-inflammatory cytokines in LPS-stimulated macrophages through distinct signaling pathways
Surgery
(2008) - et al.
Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice
Cytokine
(2011) Impact of obesity on mammary gland inflammation and local estrogen production
J. Mammary Gland Biol. Neoplasia
(2014)- et al.
Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene
Endocrinology
(1996) - et al.
Tumor necrosis factor-alpha stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter 1.4
Mol. Endocrinol. (Baltimore Md)
(1996) - et al.
Inflammation and increased aromatase expression occur in the breast tissue of obese women with breast cancer
Cancer Prev. Res. (Phila)
(2011) - et al.
The macrophage switch in obesity development
Front. Immunol.
(2015) - et al.
Ghrelin: much more than a hunger hormone
Curr. Opin. Clin. Nutr. Metab. Care
(2013)