Redox regulation of macrophage migration inhibitory factor expression in rat neurons

doi:10.1016/j.bbrc.2009.09.112

Biochemical and Biophysical Research Communications

Volume 390, Issue 1, 4 December 2009, Pages 171-175

https://doi.org/10.1016/j.bbrc.2009.09.112 Get rights and content

Abstract

Macrophage migration inhibitory factor (MIF) expression is induced by angiotensin II (Ang II) in normal rat neurons and serves a negative regulatory role by blunting the chronotropic actions of this peptide. The aim here was to determine whether hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that is a key intracellular mediator of the neuronal actions of Ang II, is a trigger for MIF production in neurons. Thus, we tested the effects of H₂O₂ on MIF expression in primary neuronal cultures from newborn normotensive (Wistar Kyoto [WKY] or Sprague–Dawley [SD]) rat brain, cells that respond to Ang II by increasing MIF levels. Treatment of WKY or SD rat neuronal cultures with a non-cytotoxic concentration of H₂O₂ elicited a significant, time-dependent increase in MIF mRNA and protein levels. Glucose oxidase, which produces H₂O₂ via oxidation of glucose in the cell-culture medium, elicited a similar increase in neuronal MIF mRNA levels. The stimulatory action of H₂O₂ was not apparent in neuronal cultures from spontaneously hypertensive rats (SHR), cells that fail to express increased MIF in response to Ang II. Finally, preincubation of SD rat cultures with either polyethylene glycol-catalase or actinomycin D abolished the H₂O₂-induced increase in MIF, suggesting that this ROS is acting intracellularly to increase transcription of the MIF gene. These results suggest the presence of a redox regulatory mechanism for induction of MIF in normotensive rat neurons.

Introduction

Macrophage migration inhibitory factor (MIF) is a small, highly conserved protein with nearly ubiquitous tissue distribution that was originally described as a cytokine in the 1960s. Recently, it has become apparent that MIF and its functions are far more complicated than originally thought. It is now known that MIF has many, sometimes seemingly disparate, biological and cellular functions. Physiologically, MIF plays modulatory roles in the immune, endocrine, and nervous systems [1]. Pathologically, MIF contributes to a wide range of inflammatory disorders and plays a pivotal role in tumor biology [2], [3].

The functions of MIF can be divided into two basic categories. Much of the traditional literature focuses on the pro-inflammatory, cytokine-like function of MIF. However, in recent years, it has been discovered that MIF has two enzymatic activities, one of which is a thiol-protein oxidoreductase (TPOR) activity that lies between amino acid residues 57 and 60. Accordingly, it has recently been suggested that MIF be re-classified as not only a cytokine, but also a thioredoxin (Trx) family member due to the fact that it contains this TPOR motif, an identifying characteristic of Trx proteins [4]. Members of the Trx family are essential to maintaining redox balance in the intracellular environment by scavenging of reactive oxygen species (ROS), and may also influence the activity of other proteins by reducing critical cysteines [5]. Whether MIF acts as a pro-inflammatory cytokine or an antioxidant protein seems to depend on the concentration of MIF in the tissue, with relatively low concentrations acting as a pro-inflammatory mediator and higher concentrations serving an antioxidant function [6].

MIF is relevant to the field of blood pressure regulation due to its ability to serve as a negative regulator of the central actions of angiotensin II (Ang II). In vitro, Ang II up-regulates MIF in neurons cultured from normotensive rat hypothalamus and brainstem [7], and increased intracellular levels of MIF protein exert a negative regulatory action over the neuronal chronotropic effects of Ang II via its type 1 receptor (AT1R) [8]. In vivo, CNS injections of Ang II in normotensive rats increase MIF expression in the paraventricular nucleus (PVN) of the hypothalamus, an area that has a key role in regulating sympathetic outflow and hypothalamus/pituitary axis activity. Increased MIF within PVN sympathetic regulatory neurons blunts the AT1R-mediated increases in discharge of these cells elicited by Ang II and the increases in blood pressure produced by CNS-injected Ang II [9]. Thus, it is of major interest to understand the intracellular mechanisms that control MIF expression in normal rat neurons. It is established that Ang II, acting via the AT1R, induces ROS production in neurons by activating NADPH oxidase [10]. This leads to superoxide production, which is metabolized to hydrogen peroxide (H₂O₂) by superoxide dismutases [11]. Many studies have established that H₂O₂ is a product of Ang II signaling in many cell types, including neurons [12]. Furthermore, it has been demonstrated that H₂O₂ can cause induction of MIF expression in peripheral tissues [13]. Therefore in the present study the aim was to determine whether H₂O₂, a key intracellular mediator of the neuronal actions of Ang II, elicits MIF production in neurons from normotensive rats.

Section snippets

Methods

Animals. Newborn SD or WKY rats or SHR, derived from our breeding colony, were used for the production of neuronal cultures. Adult breeder rats were purchased from Charles River Farms (Wilmington, MA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee.

Cell cultures. Neuronal cultures (∼90% neuronal, 10% glial) were prepared from the forebrain of newborn rats as described previously [7]. Cultures were grown in Dulbecco’s modified

Hydrogen peroxide stimulates an increase in MIF levels in neurons cultured from normotensive rats

In the first set of experiments we determined whether H₂O₂ can induce expression of MIF mRNA in primary neuronal cultures from normotensive rats. Incubation of primary neuronal cultures prepared from newborn SD or WKY rats with H₂O₂ (30 μM; 1, 2 or 3 h.) resulted in time-dependent increases in MIF mRNA levels in cells from both strains (Fig. 1A and B). Importantly, this concentration of H₂O₂ had no cytotoxic action on the cultured cells. For example, in the cultures treated with 30 μM H₂O₂ the

Discussion

This study represents the first demonstration that ROS can regulate the expression of MIF in CNS neurons. Furthermore, we have shown that this effect of H₂O₂ involves intracellular events that are specific to neurons, and the data suggest that the increase in MIF involves de novo transcription. Finally, the observation that H₂O₂ fails to elicit an increase in MIF in neurons cultured from SHR, in contrast to their normotensive controls, provides support for the contention that the MIF gene

Acknowledgment

This work was supported by NIH Grant 1R01HL-076803.

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Redox regulation of macrophage migration inhibitory factor expression in rat neurons

Abstract

Introduction

Section snippets

Methods

Hydrogen peroxide stimulates an increase in MIF levels in neurons cultured from normotensive rats

Discussion

Acknowledgment

Immunity

J. Biol. Chem.

FEBS Lett.

Toxicology

Neuroendocrine properties of macrophage migration inhibitory factor (MIF)

Immunol. Cell Biol.

Macrophage migration inhibitory factor: gene polymorphisms and susceptibility to inflammatory diseases

Clin. Infect. Dis.

Link between macrophage migration inhibitory factor and cellular redox regulation

Antioxid. Redox Signal.

Thioredoxin and related molecules – from biology to health and disease

Antioxid. Redox Signal.

Signal transduction. A most interesting factor

Nature

Novel role of macrophage migration inhibitory factor in angiotensin II regulation of neuromodulation in rat brain