TGF-β1 upregulates CX3CR1 expression and inhibits fractalkine-stimulated signaling in rat microglia

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Abstract

Following peripheral nerve transection, CX3CR1 and TGF-β1 are increased in a time-dependent manner within the injured facial motor nucleus. To explore the relationship between TGF-β1 and CX3CR1 in the CNS, the effects of TGF-β1 on CX3CR1 mRNA, protein and fractalkine-dependent stimulation of signal transduction cascades in primary cultures of rat microglia were examined. TGF-β1 increased steady state levels of CX3CR1 mRNA, 125I-fractalkine binding sites and blunted fractalkine-stimulated ERK1/2 phosphorylation. The half-life of CX3CR1 mRNA was unaltered by TGF-β1 and two potential Smad binding elements (SBEs) were identified in the rat CX3CR1 promoter. TGF-β1 may shift fractalkine-dependent signaling away from activation of ERK1/2 towards other pathways and/or may provide a mechanism for microglia to more strongly adhere to neurons.

Introduction

It is now well established that fractalkine (CX3CL1) and its receptor, CX3CR1, are constitutively expressed in the CNS although the expression of these genes are regulated in a variety of CNS diseases and pathological insults. Fractalkine, the unique member of CX3C chemokine family, is primarily expressed on neurons while the majority of CX3CR1 is associated with microglial cells Harrison et al., 1998, Nishiyori et al., 1998. This pattern of expression suggests a crosstalk between neurons and microglial cells through this ligand–receptor pair. Additional roles for fractalkine in the CNS could involve recruitment of CX3CR1-expressing cells into the CNS subsequent to inflammatory insults to the brain or spinal cord. At a cellular level, recent studies have established that fractalkine can induce, via activation of CX3CR1, a cascade of signal transduction events in microglial cells, including stimulation of intracellular Ca2+ mobilization Harrison et al., 1998, Boddeke et al., 1999, as well as ERK1/2 and Akt/PKB phosphorylation (Maciejewski-Lenoir et al., 1999). Fractalkine also has been shown to inhibit LPS-induced TNF-α release from microglia, implicating an anti-inflammatory function of this chemokine (Zujovic et al., 2000). Collectively, these data suggest that fractalkine modulates microglial migration, activation and survival. However, studies aimed at establishing roles for fractalkine and CX3CR1 in CNS pathology are still deplete, with most data derived from studies of either cell culture systems or expression analysis of fractalkine and CX3CR1 in normal or pathologic tissue. Results from the characterization of the expression of fractalkine and CX3CR1 in the CNS suggest that changes in fractalkine expression are directed toward attracting or activating CX3CR1-expressing cells within sites of injury. However, CNS phenotypes associated with either fractalkine or CX3CR1 gene disrupted mice have not provided evidence for these or any such roles Jung et al., 2000, Cook et al., 2001. A recent report suggests that fractalkine may participate in post ischemic brain injury; a slight reduction in infarct size subsequent to transient focal cerebral ischemia was apparent in fractalkine gene depleted mice (Soriano et al., 2002).

Microglial cells serve as resident immune cells in the brain. In response to certain stimuli, they become activated to perform the function of macrophages including antigen presentation, free radical and NO secretion, synthesis of a variety of cytokines, chemotaxis and phagocytosis. In a variety of CNS diseases such as multiple sclerosis Li et al., 1993, Prineas and Wright, 1978, and its animal model EAE (Bauer et al., 1994), Alzheimer's disease (Meda et al., 1995) and AIDS dementia (Price et al., 1988), microglial cells become activated and release substances toxic to neurons and oligodendrocytes. In these various scenarios, the expression levels of chemokines and their receptors have been shown to change, implying some involvement in the disease process (Asensio and Campbell, 1999). A few chemokine receptors such as CCR5, CXCR4 and CX3CR1 have been localized to microglial cells and changes in their expression may be critical in regulating chemokine-dependent functions in the CNS (Bacon and Harrison, 2000).

Cytokines such as IFN-γ and TGF-β, together with LPS and other factors, play major roles in regulating the expression of chemokine receptors. TGF-β consists of three closely related isoforms (TGF-β1, -β2 and -β3), displaying broad functional diversity including inhibition and stimulation of cell proliferation, immune suppression, neuroprotection and modulation of cytokine production. TGF-β is nearly absent in normal brain while increased expression by astrocytes and microglia is evident in the injured or diseased CNS O'Brien et al., 1994, Kiefer et al., 1995, Pratt and McPherson, 1997. TGF-β may function as a potent suppressor of microglial cell activation and play an important role in neurodegenerative diseases and CNS trauma Suzumura et al., 1993, Semple-Rowland et al., 1995, Lodge and Sriram, 1996. It has been shown to modulate chemokine and chemokine receptor expression both in peripheral systems and within the CNS. Previous data showed that TGF-β1 upregulated CXCR3 and CXCR4 expression on human NK cells (Inngjerdingen et al., 2001) and CCR1 expression on rat astrocytes (Han et al., 2000). TGF-β exerts its functions through a group of signaling proteins called Smads. TGF-β activates cytoplasmic, receptor-regulated Smads (Smad2, Smad3), which in turn associate with common mediator Smads (Smad4). These complexes translocate into the nucleus, bind DNA, and activate gene transcription (Massague and Wotton, 2000). Co-factors and co-activators such as AP1 and SP1 are often required with Smads to bind to DNA and promote transcriptional activation (Wrana, 2000).

Following facial motor nerve axotomy in the adult rat, microglial cells are activated and recruited rapidly to the injured motor neuron pool. In this scenario, microglial cells do not become phagocytic and the injured facial motor nerves regenerate. Following peripheral facial motor nerve injury, both microglial TGF-β1 and CX3CR1 are upregulated in a comparable time-dependent manner within the facial nucleus suggesting that these two cytokine systems interact Kiefer et al., 1993, Harrison et al., 1998. To explore the relationship between TGF-β1 and fractalkine in the CNS, we examined the effects of TGF-β1 on CX3CR1 mRNA, protein and fractalkine-dependent stimulation of signal transduction cascades in primary cultures of rat microglia. In this report, we show that TGF-β1 can increase CX3CR1 expression on rat microglial cells both at the mRNA and protein levels without changing its affinity for fractalkine. Although CX3CR1 is increased on microglial cells, stimulation of ERK1/2 by fractalkine is diminished by TGF-β1. Furthermore, the mechanism by which TGF-β1 increases CX3CR1 in microglia is likely due to enhanced transcription of the CX3CR1 gene.

Section snippets

Primary microglial cell cultures

Cerebra were dissected from newborn Sprague–Dawley rats, stripped of meninges and mechanically minced in solution D (137 mM NaCl, 5.4 mM KCl, 0.2 mM NaH2PO4, 0.2 mM KH2PO4, 1 g/l glucose, 0.25 μg/ml fungizone, 106 U/l penicillin/streptomycin, pH 7.4). The tissues were digested with 0.25% trypsin at 37 °C on a bidirectional rotator for 30 min. An equal volume of DMEM (Life Technologies, Rockyville, MD) containing 10% FBS (Life Technologies) was added to stop the trypsin and the mixture was

TGF-β1 enhances microglial CX3CR1 mRNA in a time- and concentration-dependent manner

Previous studies have shown that mRNA levels of both TGF-β1 and CX3CR1 were upregulated, in a comparable time-dependent manner, within the rat facial motor nucleus after peripheral nerve axotomy Kiefer et al., 1993, Harrison et al., 1998. To investigate the relationship between TGF-β1 and expression of CX3CR1, primary cultured rat microglial cells were treated with various concentrations of TGF-β1 (0.001–10 ng/ml) for 16 h and total RNA was extracted. Northern blot analysis of stimulated

Discussion

The principal goal of this study was to determine the effect of TGF-β1 on CX3CR1 expression and function in primary rat microglial cells. The major findings of this work are threefold. Firstly, TGF-β1 upregulated CX3CR1 expression, in terms of both steady state mRNA and cell surface protein levels. Secondly, despite the significant TGF-β1-dependent increase in CX3CR1, the function of the receptor was inhibited in the TGF-β1-pretreated cells, as fractalkine was unable to stimulate any known

Acknowledgements

This work was supported by a PHS grant NIH NS34901.

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