Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells

Abstract

Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1β-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-κB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-κB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

Introduction

Human adiponectin is a 247-amino acid and 30-kDa protein hormone and cytokine encoded by the gene apM1 located on chromosome 3, locus 3q27 [1], [2], [3] and secreted mainly by adipocytes [4]. Adiponectin is also called 30-kDa adipocyte complement-related protein, Acrp30, adipoQ, APM-1 and gelatine-binding-protein-29 and is the most abundant cytokine produced by adipocytes [5]. Full-length adiponectin (fAd) contains a N-terminal signal sequence, a collagenous domain and a C-terminal C1q-like globular domain. A 16.5 kDa truncated peptide, containing only the globular portion (gAd) can be generated by proteolytic cleavage [3], [6]. Both fAd and gAd circulate in serum at high concentrations and both forms are biologically active. Different actions and signal transduction activation have been reported for the isoforms, although the full significance of these actions remain to be determined [7], [8], [9], [10].

Adipose tissue is now known to have many important immunologic and endocrine functions [11], [12] and obesity is an important risk factor for many human diseases including colon cancer and diabetes mellitus [13]. Studies have suggested that a biological explanation for this link can be provided by adipokines secreted by adipose tissue [14]. For example, leptin, the best characterised adipokine, has regulatory roles in glucose metabolism, insulin action, body weight and energy homeostasis [15], but has also been shown to stimulate growth of both normal and neoplastic colonic epithelia and promote invasiveness of colon cancer cells [14], [16].

Whereas leptin levels are usually proportional to body fat mass, adiponectin levels are reduced in obesity, diabetes mellitus and insulin resistance [2], [3], [12], [17]. Adiponectin has anti-diabetic actions: administration to mice lowered glucose levels and reduced insulin resistance [17] and improved insulin sensitivity was also demonstrated in transgenic mice with increased circulating adiponectin levels [18]. These studies have all suggested that decreased adiponectin secretion by adipose tissues in obesity may be an important contributor to insulin resistance and the metabolic syndrome.

Adiponectin also protected leptin-deficient obese mice and apo-E-deficient mice against atherosclerosis and initial studies suggested that adiponectin may have anti-inflammatory properties that contribute to the anti-atherosclerotic effects [3], [19], [20], [21]. More specifically adiponectin has been reported to reduce TNF-α and IL-6 secretion by lipopolysaccharide (LPS)-activated human and porcine macrophages [19], [22], [23], reduce interferon-γ secretion by human macrophages [24], inhibit human macrophage phagocytosis [19], reduce LPS-stimulated IL-6 mRNA expression by porcine adipocytes and LPS-stimulated IL-6 release by 3T3-L1 adipocytes [25]. Adiponectin also inhibited the inflammatory activity of aortic endothelial cells [20], [21] and increased the secretion of anti-inflammatory IL-10 and IL-1 receptor antagonist by monocytes and dendritic cells [24] and inhibited the proliferation of macrophage progenitors [19].

Two recently cloned receptors, AdipoR1 (ubiquitous but heavily expressed in skeletal muscle) and AdipoR2 (mainly expressed in the liver), have been shown to mediate the energy-metabolic effects of both gAd and fAd [5], [17]. These metabolic effects of adiponectin have led to the suggestion that adiponectin or analogues could be useful therapies for diabetes mellitus, obesity and related conditions such as non-alcoholic fatty liver [26].

Like adipocytes, colonic epithelial cells are known to be immunologically active [27], [28]. Colonic epithelial cells and the more commonly studied colon cancer cells lines secrete an array of pro-inflammatory mediators in a similarly regulated manner. This response is probably important in the defence against enteric pathogens as well in the perpetuation of inflammation seen in human inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis. The secreted mediators (called cytokines here for the sake of brevity) include those predominantly involved in non-specific immunity such as granulocyte-macrophage colony stimulating factor (GM-CSF) and a variety of chemokines, such as IL-8 and ENA-78 (neutrophil chemoattractants), monocyte chemotactic protein-1 (MCP-1) (a predominantly monocyte chemoattactant) and T cell chemoattractants such as RANTES [27], [29], [30]. In addition to roles in inflammation, these mediators may be involved in carcinogenesis and scarring. IL-8 enhances the migratory capacity of a wide variety of cells including malignant cells and it has been reported to play a role in angiogenesis [31]. IL-8 protein and mRNA are upregulated in colorectal cancers with high degree microsatellite instability [32] and may be crucial to the metastatic capacity of colon cancer [31]. GM-CSF stimulates the proliferation of gastrointestinal cancer cells [33] and MCP-1 has recently been implicated in the fibrosis typical of Crohn's disease [34].

Given that obesity is an important risk factor for colon cancer [14] and that leptin, another adipokine, has important effects in stimulating immune system as well as enhancing proliferation in normal and neoplastic colonic epithelium [14], [16], it is possible that adiponectin may also have important biologic effects on the colon. To our knowledge there are no previous studies examining the effects of adiponectin on colonic (or any gastrointestinal) cells, although recent data have shown that mesenteric adipose tissue also secretes adiponectin and that secretion is enhanced in fat tissue adjacent to inflamed bowel, suggesting that adiponectin may be a paracrine mediator of colonic epithelial function [35], [36]. As adiponectin is typically regulated in an inverse manner to leptin and many of the effects of adiponectin oppose those of leptin, we initially hypothesized that adiponectin may have beneficial anti-inflammatory and anti-proliferative effects. Consequently, we have studied the effects of adiponectin on proliferation and cytokine secretion by colonic epithelial cells with particular interest in the mechanisms of any possible effects.

We have used the HT-29 cell line for characterisation of these effects. This cell line has been widely used to assess colonic proliferative responses [14], [37], [38] and it secretes a regulated complex array of cytokines similar to primary colonic epithelial cells [27]. Of these many mediators we have initially concentrated on GM-CSF as a stimulant of non-specific immunity, IL-8 as a neutrophil chemoattractant and MCP-1 as a monocyte chemoattractant because of the involvement of these mediators in inflammatory bowel diseases [39].