The regulation of macrophage cholesterol homoeostasis is of crucial importance in the pathogenesis of atherosclerosis, an underlying cause of heart attack and stroke. Several recent studies have revealed a critical role for the cytokine TGF-β (transforming growth factor-β), a key regulator of the immune and inflammatory responses, in atherogenesis. We discuss here the TGF-β signalling pathway and its role in this disease along with the outcome of our recent studies on the action of the cytokine on the expression of key genes implicated in the uptake or efflux of cholesterol by macrophages and the molecular mechanisms underlying such regulation.

The TGF-β (transforming growth factor-β) superfamily and the signal transduction pathway

The TGF-β superfamily comprises over 30 members that include TGF-β and a number of related factors (e.g. activins, nodals, bone morphogenetic proteins/growth and differentiation factors) [1]. The mature active forms of the family are typically dimers of 12–15 kDa subunits linked mainly by a single disulfide bond [1]. TGF-β refers to three isoforms: TGF-β1, TGF-β2 and TGF-β3. Although each of these is derived from distinct genes, they display greater than 70% sequence homology, have similar properties, at least in vitro, and are collectively assigned the generic term TGF-β [1].

The cytokine is synthesized as a homodimeric pro-protein (pro-TGF-β; molecular mass 75 kDa), which is cleaved in the Golgi apparatus [2,3]. The dimeric pro-peptide has a high affinity for the cleaved TGF-β, and the two are secreted as a small latent complex, which is unable to bind to the cytokine receptor. The pro-peptide is also able to bind by disulfide bonds to members of the latent TGF-β-binding proteins to form a trimolecular aggregate called the large latent complex [2,3]. The biological action of TGF-β requires its dissociation from such a complex, which has been shown to occur, at least in vitro, by several different proteases, heat treatment and changes in pH [2,3].

Figure 1 summarizes the TGF-β signal transduction pathway. The cytokine mediates its action by interacting with cell-surface transmembrane serine/threonine kinases [1,4,5]. The receptors for TGF-β are the constitutively active type II receptor [TGF-βRII (TGF-β receptor type II)] and the TGF-βRI, which may confer cell type specificity depending on its expression pattern [1,46]. The action of TGF-β is also modulated by its interaction with type III receptor, which does not have an intrinsic kinase activity [1,46]. Upon binding of TGF-β to the high-affinity TGF-βRII, a heteromeric complex is formed with TGF-βRI consisting of two of each receptor subtype. TGF-βRII then phosphorylates TGF-βRI, which produces a conformational change in the protein that allows it to phosphorylate Smads, a novel class of signal transducers and transcription factors [1,7,8]. The binding of TGF-βRI and Smads is often facilitated by membrane-associated proteins called Smad anchor for receptor activation [1,7,8].

Schematic representation of the TGF-β signalling pathway

Figure 1
Schematic representation of the TGF-β signalling pathway

See text for details. Co-Smads, common Smads; ERK, extracellular-signal-regulated kinase; I-Smads, inhibitory Smads; P, phosphorylation; p38, p38 mitogen-activated protein kinase; R-Smads, receptor-regulated Smads; TF, transcription factors.

Figure 1
Schematic representation of the TGF-β signalling pathway

See text for details. Co-Smads, common Smads; ERK, extracellular-signal-regulated kinase; I-Smads, inhibitory Smads; P, phosphorylation; p38, p38 mitogen-activated protein kinase; R-Smads, receptor-regulated Smads; TF, transcription factors.

There are three types of Smads: receptor-regulated (Smad-1, -2, -3, -5 and -8), common Smads (Smad-4) and inhibitory Smads (Smad-6 and -7) [7,8]. Smad-2 and/or -3 are phosphorylated by TGF-β and then interact with Smad4 to form a heteromeric complex. This complex migrates to the nucleus and regulates gene transcription by interacting with Smad-responsive elements in the regulatory regions of target genes [7,8]. However, Smads can also regulate gene expression without any direct DNA binding via protein–protein interactions with other transcription factors [7,8].

Although activation of Smads represents the major mechanism for TGF-β signalling, a number of other pathways are also activated by the cytokine (e.g. mitogen-activated protein kinases, phosphoinositide 3-kinase and protein kinase CK2) [9,10]. Some of these pathways act in conjunction with Smads, whereas others are independent [9,10].

TGF-β and macrophage cholesterol homoeostasis

The control of macrophage cholesterol homoeostasis is of critical importance in the pathogenesis of atherosclerosis, an underlying cause of heart attack and stroke [11,12]. Indeed, the transformation of macrophages into lipid-loaded foam cells is a critical early event in atherogenesis [1112]. Because this disease is considered as a form of chronic inflammation, cytokines will have a major impact on its initiation, progression and clinical complications. TGF-β, its receptors and Smads have all been found to be expressed at high levels in macrophages of atherosclerotic lesions [1315]. An anti-atherogenic role for TGF-β has been suggested by several lines of recent evidence. First, an inverse relationship has been found between the levels of circulating TGF-β and the development of atherosclerosis [16,17]. Secondly, regions in the aorta with a high probability of developing atherosclerosis are associated with low TGF-β expression [18]. Thirdly, a link between polymorphisms in the TGF-β gene along with its receptor and cardiovascular disease has been seen in some studies [19,20]. Fourthly, studies on animal models of atherosclerosis have shown that inhibition of TGF-β signalling, using neutralizing antibodies or by expression of a dominant-negative form of the receptor, accelerates the development of the disease [21,22]. Increased development of atherosclerosis is also seen when TGF-β signalling is specifically inhibited in T-cells [23,24].

TGF-β inhibits macrophage foam cell formation [25,26] and regulates the expression of a number of genes implicated in the control of cholesterol homoeostasis (Table 1). Simplistically, foam cell formation can be considered as a balance between the uptake and the efflux of cholesterol. We and others have found that TGF-β stimulates the expression of most of the genes implicated in cholesterol efflux and simultaneously inhibits the expression of those involved in the uptake of this sterol. The molecular mechanisms underlying such a regulation remain poorly understood. We have therefore investigated the mechanisms by which TGF-β regulates the expression of LPL (lipoprotein lipase) and ApoE (apolipoprotein E).

Table 1
TGF-β-regulated expression of key genes in macrophages implicated in the control of cholesterol homoeostasis

ABC transporter, ATP-binding-cassette transporter; LDL, low-density lipoprotein.

Gene Effect of TGF-β on its expression References 
ApoE Increase [27
ABC transporter-A1 Increase [25,26
ABC transporter-G1 Increase [25
Lectin-like oxidized LDL receptor-1 Increase [28,29
LPL Decrease [30
LDL receptor Decrease [25
Peroxisome-proliferator-activated receptor-γ Increase (early); decrease (late) [31
Scavenger receptor A Decrease [25,29
Scavenger receptor BI Decrease [32
Scavenger receptor CD36 Decrease [25,29,32
Scavenger receptor CD163 Decrease [33
Gene Effect of TGF-β on its expression References 
ApoE Increase [27
ABC transporter-A1 Increase [25,26
ABC transporter-G1 Increase [25
Lectin-like oxidized LDL receptor-1 Increase [28,29
LPL Decrease [30
LDL receptor Decrease [25
Peroxisome-proliferator-activated receptor-γ Increase (early); decrease (late) [31
Scavenger receptor A Decrease [25,29
Scavenger receptor BI Decrease [32
Scavenger receptor CD36 Decrease [25,29,32
Scavenger receptor CD163 Decrease [33

Molecular mechanisms underlying the TGF-β-mediated regulation of key genes implicated in the control of macrophage cholesterol homoeostasis

LPL catalyses the hydrolysis of the triacylglycerol component of circulating chylomicrons and very-low-density lipoproteins, thereby providing non-esterified fatty acids and 2-monoacylglycerol for tissue utilization. Several lines of evidence (reviewed by us in [3436]) have shown that the LPL expressed by macrophages is pro-atherogenic. Such an action involves both its catalytic activity and a non-catalytic bridging function that leads to the accumulation, and thereby uptake, of lipoproteins [3436]. We found that TGF-β inhibits LPL mRNA expression, protein levels and enzymatic activity in a range of macrophage sources from different species, including human monocyte-derived macrophages [30]. The action of TGF-β was mediated at the level of LPL gene transcription and not mRNA stability. We have previously delineated the molecular mechanisms underlying the transcriptional regulation of a number of genes [3740]. Analysis of the cis-acting regulatory sequences and the interacting DNA-binding proteins showed a crucial role for three conserved Sp1 (specificity protein-1) binding sites in the TGF-β-mediated inhibition of LPL gene transcription [30]. Mutations in these Sp1 sites attenuated the TGF-β response, whereas multimers of the sites could confer the response to a heterologous promoter [30]. Overall, the results showed that TGF-β inhibits LPL gene transcription by suppressing the transactivation potential of Sp1 without affecting its DNA-binding activity.

ApoE is a major component of several classes of plasma lipoproteins and plays a crucial role in lipid metabolism and transport by acting as a recognition signal for receptor-mediated uptake of lipoprotein particles (see [41] for a review]) ApoE promotes macrophage cholesterol efflux and mice deficient in this gene are severely hypercholesterolaemic and develop atherosclerosis even when fed with a low-fat diet [41]. Consistent with an anti-atherogenic role for ApoE, we have found that TGF-β induces the expression of ApoE mRNA and protein in monocytes and macrophages [27]. Using a series of pharmacological inhibitors against components of a number of signal transduction pathways, we have found that the TGF-β-induced ApoE expression was attenuated by inhibitors of JNK (c-Jun N-terminal kinase), p38 kinase and protein kinase CK2. TGF-β increased the activity and/or levels of activated, phosphorylated forms of these proteins and dominant-negative forms inhibited the cytokine-induced expression of ApoE in transfected cells [27]. The cytokine also increased the phosphorylation and expression of c-Jun, a downstream target for JNK action and a component of the AP-1 (activator protein-1) family of transcription factors, and dominant-negative c-Jun inhibited the induction of ApoE expression in response to the cytokine [27]. TGF-β induced the activity of AP-1 and the action of JNK, p38 kinase and CK2 converged on c-Jun/AP-1 activation [27].

In conclusion, TGF-β plays a key role in regulating macrophage cholesterol homoeostasis by regulating the expression of key genes implicated in the uptake and efflux of this sterol. TGF-β inhibits the expression of genes implicated in the accumulation of cholesterol, such as LPL, and stimulates the expression of those involved in its efflux (e.g. ApoE). We have identified key roles for Sp1 in the regulation of LPL and JNK, p38 kinase, CK2 and c-Jun/AP-1 in the case of ApoE. Further studies on the action of TGF-β in macrophages should lead to the development of novel therapeutic approaches for limiting the development of atherosclerosis.

Mechanisms of Gene Regulation: Research Colloquium at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by S. Graham (Glasgow, U.K.). Sponsored by Pfizer.

Abbreviations

     
  • AP-1

    activator protein-1

  •  
  • ApoE

    apolipoprotein E

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LPL

    lipoprotein lipase

  •  
  • Sp1

    specificity protein-1

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TGF-βRI/II/III

    TGF-β receptor type I/II/III

We thank the British Heart Foundation and Wellcome Trust for financial support.

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