Effects of interleukin-6 −174C/G and metallothionein 1A +647A/C single-nucleotide polymorphisms on zinc-regulated gene expression in ageing

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Abstract

Decreased zinc ion availability in ageing is associated with altered immune response. One of the main regulators of zinc availability is metallothionein. Metallothionein induction is under the control of interleukin-6, a pro-inflammatory cytokine whose production is associated with poor ageing. The production of interleukin-6 is controlled, in part, by variability in the −174 nucleotide position. Under conditions of chronic inflammation, such as in ageing, zinc release by metallothionein is limited and may reduce zinc availability. Understanding the precise nature of the interactions between interleukin-6 and metallothioneins will aid in identifying individuals who are at risk of zinc deficiency. In the current study, we used gene arrays to investigate the effects of in vitro zinc supplementation on gene expression in elderly donors with described interleukin-6 and metallothionein 1a polymorphisms. Ingenuity Pathway Analysis™ identified several zinc-responsive genetic networks uniquely regulated only in elderly individuals with the pro-inflammatory interleukin-6 polymorphism. These include zinc-dependent decreased transcription of pro-inflammatory cytokines and alterations in metabolic regulatory pathways. The genomic effects of zinc increased in significance in the presence of the metallothionein 1a +647 C/A transition, suggesting that the interleukin-6 and metallothionein 1a genes act in a concerted manner to control zinc-regulated gene expression.

Introduction

In human ageing, zinc bioavailability is diminished resulting in impaired immune response and anti-oxidant activity (Mocchegiani et al., 1998). Metallothioneins (MTs) play an important role in regulating zinc homeostasis due to their high affinity for zinc. An important function of MTs is in releasing zinc in response to oxidative/nitrosative stress in order to up-regulate the zinc-dependent antioxidant enzymes (Mocchegiani et al., 2006a, Mocchegiani et al., 2006b). This response is inadequate in states of chronic inflammation and in ageing individuals (Mocchegiani et al., 2003).

A recent report has demonstrated that polymorphisms in the MT coding region are involved in regulating zinc release by MT and are associated with longevity (Cipriano et al., 2006). Cipriano et al. have shown that carriers of the C-allele at position +647 of the MT1a gene show enhanced zinc release, low metallothionein levels, and reduced IL-6 plasma concentrations. In addition, metallothionein gene polymorphisms have been associated with cardiovascular disease, hyperglycaemia and mild zinc deficiency in diabetic patients (Giacconi et al., 2005). Metallothionein induction and expression are under the control of IL-6 (Jacob et al., 1999). These data suggest that in the presence of heightened IL-6 production, the MT1a +647 C/A transition may impact negatively on inflammation through up-regulation of IL-6 pro-inflammatory cytokine production, thereby creating vicious pro-inflammatory feed-back cycle. Since increased IL-6 production has been linked to disease and is a poor indicator of survival (Fagiolo et al., 1993, Ferrucci et al., 1999), these studies have profound implications for understanding the link between genes, health and longevity.

It is critically important to determine the precise nature of the relationship between metallothionein status and IL-6 production. Heightened levels of metallothionein, IL-6 and low zinc ion availability are observed in diseases characterised by chronic inflammation and in syndromes of accelerated ageing (Mocchegiani et al., 2002). In addition, high levels of IL-6 production is common in ageing and is detrimental for successful ageing (Fagiolo et al., 1993, Ferrucci et al., 1999). Genetic variation at the −174 region within the IL-6 promoter affects cytokine production. The presence of the C-allele in the −174 position of the IL-6 promoter decreases the level of IL-6 production. Loss of the C-allele may be associated with risk of disease in ageing and may predict poor ageing outcome (Bonafe et al., 2001, Olivieri et al., 2002). In fact −174 C/G transitions have been associated with susceptibility to age-related diseases and may predict longevity (Bonafe et al., 2001, Franceschi et al., 2005, Olivieri et al., 2002). C-allele-negative elderly individuals displayed impaired innate immune response, zinc deficiency, enhanced metallothionein expression, and low zinc ion availability compared to C-allele carriers (Giacconi et al., 2004). Taken together, these data suggest that the IL-6 C/G polymorphism may interact with MT to impact on zinc homeostasis.

In ageing, it is possible that enhanced IL-6 production may impact on metallothionein levels, thereby altering zinc availability. In turn, metallothionein homeostasis and regulation of zinc turnover may affect IL-6 production, causing a cycle by which increasing IL-6 expression further alters zinc homeostasis and depletes available zinc. Another relevant aspect to be taken into account is that in the presence of alterations in mechanisms regulating zinc homeostasis, zinc itself might also have side effects on oxidative stress by promoting the production of reactive oxygen species (Frazzini et al., 2006). Therefore, genetic variability at the IL6 −174 C/G locus may be useful for both screening of elderly subjects who are at risk for zinc deficiency and impaired immune response and also in identifying subjects that are more or less able to regulate zinc homeostasis (Mocchegiani et al., 2006a, Mocchegiani et al., 2006b).

Section snippets

Human subjects and sample collection

Sixteen elderly subjects (age range 69–79 years) were selected for study participation. Elderly subjects were recruited in accordance to immunogerontological ‘SENIEUR’ protocol (Ligthart, 2001). 15 ml whole blood was collected and peripheral blood mononuclear cells (PBMC) were recovered. Cell cultures were prepared in zinc-free RPMI medium containing 10% of heat-inactivated FCS, 4 mM l-glutamine, 100 U/ml penicillin and streptomycin (Invitrogen, San Giuliano Milanese, MI, Italy). Cells were

Results

In order to examine the effect of zinc supplementation on gene expression profiling in elderly individuals, RNA samples were obtained from PBMC isolated from elderly donors selected on the basis of IL-6 −174 and MT1a +647 genotype and treated in vitro with normal media or media supplemented with zinc for a period of 24 h. Donor information is shown in Table 1. Gene expression profiling of cells cultured in the presence of zinc was compared to cells cultured in the absence of exogenous zinc. Gene

Discussion

In the current study, we investigated whether variability in IL-6 −174 and MT1a +647 loci influenced zinc-regulated gene expression in ageing. The hypothesis was taken that an anti-inflammatory molecule such as zinc might impact positively on IL-6 production in the elderly. Since MT1a polymorphisms are associated with increased IL-6 production and could potentially impact on zinc homeostasis, we further segmented the elderly population recruited for investigation based on MT1a polymorphism. It

Acknowledgements

We thank Ann Scarborough for technical assistance. This study was supported by European Union Framework 6 programme project ZINCAGE (FOOD-CT-2003-506850).

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