‘As time goes by’: angiotensin II-mediated transactivation of the EGF receptor comes of age

Gunter Wolf

Department of Internal Medicine III, University of Jena, Germany

Correspondence and offprint requests to: Gunter Wolf, MD, Department of Internal Medicine III, University Hospital Jena, Erlanger Allee 101, D-07740 Jena, Germany. Email: gunter.wolf{at}med.uni-jena.de



   The renin–angiotensin system (RAS) and progression of renal disease
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Clinical studies have provided ample evidence that inhibition of the RAS with ACE inhibitors and/or AT1-receptor blockers is an effective strategy in slowing the progression of chronic renal disease [1–3]. Although it was initially thought that inhibition of the RAS is renally protective through haemodynamic mechanisms (for example by reduction of hyperfiltration), it is now clear that angiotensin II (ANG II) is a multifactorial cytokine exhibiting growth stimulatory, proinflammatory and profibrotic effects [4–6]. These more pleiotrope effects of ANG II on the kidney were initially greeted with some skepticism [5]. At a time when the specific receptors for ANG II have only just been cloned and the ANG II-receptor blocker, saralasin, was only recently replaced with more specific agents, these reservations were not surprising [6].

ANG II binds to various receptors [6]. The AT1-subtype is involved in many of the deleterious effects of the peptide. Signalling through the AT1-receptor (a receptor with seven membrane-spanning domains) involves G-proteins (Gq) leading to activation of phospholipase C and the subsequent generation of diacylglycerol and inositol trisphosphate, which in turn stimulate protein kinase C and increase intracellular calcium [3]. In addition, activation of various protein kinases such as extracellular signal kinases 1,2 (Erk 1,2), the phosphatidylinositol 3-kinase (PI3K)-dependent kinases Akt, and the mTOR/S6 kinase pathway are necessary for ANG II-mediated growth responses including cardial and renal hypertrophy [7–9]. Although activation of some of these kinases, for example Erk 1,2, could be explained by protein kinase C, stimulation of other signal kinases is difficult to understand because the AT1-receptor lacks intrinsic tyrosine kinase activity.



   Relationship between ANG II and the EGF receptor
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Earlier findings suggested a close relationship between ANG II and epidermal-growth factor (EGF)-mediated cellular effects. Norman et al. [10] studied the potential interaction between ANG II and EGF in proximal tubular cells already in 1987. They found that ANG II itself was not mitogenic for the cells, but it amplified the proliferative effect of EGF and the dose-response curve of EGF-stimulated mitogenesis was shifted to the left in the presence of ANG II. However, ANG II had no effect on the binding of EGF to its putative receptor and also did not influence receptor down-regulation. Norman et al. [10] concluded that ‘ANG II potentiates EGF-induced mitogenesis at one or more postreceptor steps’. In 1990, we described in a cultured murine proximal tubular cell line that ANG II-pretreatment further enhanced EGF-induced cell division by ~40% [6]. Although there were similarities between EGF and ANG II in the induction of immediate early genes, EGF stimulated mitogenesis whereas ANG II induced hypertrophy in proximal tubular cells [6,11]. These early findings provide evidence of a potential interaction between ANG II and EGF-mediated signalling.

In 1996 Axel Ullrich's group discovered a pathway showing how G-protein coupled receptors such as the AT1-receptor could cause phosphorylation and activation of the EGF receptor [12]. ANG II was initially not studied, but the investigators found that endothelin 1, lysophosphatic acid and thrombin all activate G-coupled receptors without a kinase domain, phosphorylate tyrosine residues on the EGF receptor and thereby activate other downstream kinases [12]. These findings were rapidly extended to ANG II. For example, in cardiac fibroblast ANG II stimulates tyrosine phosphorylation of the EGF receptor, a process named transactivation [13]. Further studies revealed that stimulation of G-protein coupled receptors leads to EGF-receptor phosphorylation through activation of metalloproteinases (MMP). After ligand binding to the G-protein coupled receptors, MMP is activated inducing cleavage of pro-heparin-binding EGF (HB-EGF), which liberates a soluble HB-EGF that binds to and activates the EGF receptor [14,15].



   A novel mechanism of ANG II-mediated transactivation of the EGF receptor
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The study by Lautrette et al. [16] provided an important new twist to this fascinating story. The investigators studied transgenic mice overexpressing a dominant negative form of the EGF receptor [16]. These mice, which could not activate the EGF receptor and downstream kinases, were protected from renal lesions (e.g. glomerulosclerosis, mononuclear cell infiltration, tubular fibrosis and atrophy) induced by 2 months of ANG II-infusion compared with normal non-transgenic mice that bear a functioning EGF receptor. ANG II-infused EGF receptor mutant mice exhibited significantly less proteinuria compared to ANG II-treated wild-type mice, but tail-cuff measured arterial blood pressure was apparently identical in both groups. These results indicate that the damaging effects of ANG II-infusion are mediated by EGF receptor transactivation rather than by systemic hypertension. Lautrette et al. [16] next studied potential mechanisms by which ANG II transactivates the EGF receptor during renal injury. Incubation of a rat liver cell line with ANG II induced secretion of transforming growth factor-{alpha} (TGF-{alpha}), a potential ligand of the EGF receptor. TGF-{alpha} is released from a larger integral membrane precursor called pro-TGF-{alpha}. An important enzyme in cleaving TGF-{alpha} from its transmembrane precursor is TACE (tumor necrosis factor {alpha} converting enzyme), first identified in the processing of membrane-bound precursors of tumor necrosis factor {alpha} [17]. A pharmacological EGF receptor antagonist as well as specific TACE inhibitor prevented ANG II-mediated kinase activation in rat hepatocytes. Lautrette et al. [16] then tested whether similar mechanisms are operative in ANG II-induced renal injury in vivo. First, ANG II infusion stimulated a marked increase in tubular (ascending limb of Henle's loop and distal tubule) TGF-{alpha} protein expression without concomitant mRNA increase, suggesting that more membrane-bound pro-TGF-{alpha} is converted to active TGF-{alpha}. In parallel, immunostaining for TACE but not mRNA expression increased in ANG II-infused mice at the same tubular localization as TGF-{alpha}. It appears that ANG II-infusion leads to a TACE translocalization from the perinuclear compartment to the cell surface. Treatment of ANG II-infused animals with a TACE inhibitor blunted ANG II-induced TGF-{alpha} accumulation, EGF-receptor phosphorylation and renal damage without influencing blood pressure. Similar effects were also observed in TGF-{alpha} ‘knockout’ mice in which ANG II failed to transactivate the EGF receptor. Since ANG II infusion is a rather unphysiological model of kidney injury, the authors finally studied a renal ablation model in mice (75% reduction of renal mass). Immunohistological and western blot analyses revealed that both TGF-{alpha} and TACE, but not EGF protein levels, increased 2 months after ablation at a time when renal injury developed. Treatment with an AT1-receptor antagonist prevented these changes, indicating that renal ablation has led to intrarenal RAS activation as previously described [for review see 3]. In summary, these elegant experiments suggest the following signal transduction pathway (Figure 1). ANG II binds to G-protein coupled AT1-receptors leading to a redistribution of TACE from the cytoplasm to the cell surface. How this works is currently unclear. Cell surface-associated TACE has an increase in half-life time and cleaves adjacent membrane-associated pro-TGF-{alpha} to release active TGF-{alpha}. TGF-{alpha}, in turn, binds to the EGF receptor in an autocrine or paracrine manner and activates the receptor-associated kinase cascade leading to phosphorylation of mTOR/S6 kinase, PI3K/Akt and Erk 1,2 (Figure 1). This quite complicated signal transduction pathway goes from the outside (ANG II) to the inside (TACE), again to the outside (TNF-{alpha}) and finally returns to the inside of the cell (EGF-receptor phosphorylation). Nature is complicated.



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Fig. 1. Schematic overview of the novel mechanism of ANG II-mediated transactivation of the EGF receptor as described by Lautrette et al. [16]. ANG II binds extracellularly to the G-protein coupled AT1-receptor that lacks an intracellular kinase domain. This activation leads through currently unclear mechanisms to a translocalization of the metalloprotease TACE from the perinuclear/cytoplasmatic space to the cell surface. Perhaps ANG II also stimulates expression of TACE. The cell surface localized TACE has a longer half-life time and can now act on the membrane-associated pro-TNF-{alpha} releasing soluble TNF-{alpha}. This TNF-{alpha}, in turn, binds to the EGF receptor leading to autophosphorylation of the C-terminal end, and recruitment of cytoplasmatic mediators that bind to phosphotyrosine residues of the receptor through SH2- or PTB-domains. The result is activation of various kinase pathways including mTOR/S6 kinase, phosphatidylinositol 3-kinase (PI3K)-dependent kinase Akt and Erk 1,2. Interestingly, it has been previously shown that ANG II-mediated injury is associated with activation of these kinases.

 


   Anything wrong with this landmark study?
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I have some doubts in believing that this is the only mechanism by which ANG II mediates renal damage. Previous studies have shown that ANG II or its degradation products (e.g. angiotensin IV) bind to receptors other than AT1 and could mediate proinflammatory and profibrotic effects through these receptors [18,19]. It is currently unclear whether ANG II-induced TACE translocalization and expression is only mediated by AT1-receptors. In addition, it remains to be established whether the TACE inhibitor used may also inhibit other enzymatic systems. For example, assuming that the TACE inhibitor interferes with other matrix-degrading proteins, disruption of the basement membrane, the first process in initiation epithelial–mesenchymal transition that is important in interstitial fibrosis [20], would be prevented. In addition, EGF receptor transactivation has been linked to the release of HB-EGF and activation of another enzyme metalloprotease (ADAM12) in ANG II-stimulated cardiac hypertrophy [21,22], The potential role of HB-EGF in the current renal injury model requires further study because TACE may also release HB-EGF. Finally, inducing chronic renal injury and making physiological measurements (e.g. blood pressure measurements) is a challenging task in mice [23]. Measurements of blood pressure with the tail-cuff method could be difficult [23]. In contrast to findings by Lautrette et al. [16] who observed a similar degree of ANG II-induced hypertension in wild-type mice compared to TGF-{alpha} ‘knockout’ animals, application of antisense oligonucleotides against the EGF receptor significantly reduced hypertension in ANG II-infused rats indicating that EGF-receptor transactivation is also involved in ANG II-mediated blood pressure regulation [24]. Since there is increasing evidence that growth factors such as transforming growth factor-ß or EGF may be involved in regulation of renal haemodynamics [25,26], it would be interesting to know whether treatment of ANG II-infused mice with the TACE inhibitor influences renal haemodynamics.



   What do the novel findings mean for the practicing nephrologist?
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One could argue that the mechanism of inhibition of the RAS that slows progression of renal diseases does not matter as long as ACE inhibitor and AT1-receptor antagonists do their job. However, this treatment strategy is far from being perfect and patients are still reaching end-stage renal failure [27]. Experimental models have provided convincing evidence that high doses of ACE inhibitors or AT1-receptor antagonists could induce regression of previously deduced fixed structural renal changes such as fibrosis [28,29]. The study by Lautrette et al. [16] identified novel potential targets to treat chronic renal disease. It would be worthwhile to study pharmacological inhibition of TACE to decrease release of ANG II-mediated TNF-{alpha} and transactivation of the EGF receptor in patients with chronic renal diseases. Presumably, such treatment could provide therapeutic benefit beyond dual blockade of the RAS with ACE inhibitors and sartanes. Such innovative strategies are urgently needed to deal with the future deluge of patients with chronic kidney disease.

Sam, the piano player in Michael Curtiz's 1942 classic ‘Casablanca’, sings in ‘As Time Goes By’: ‘And no matter what the progress, or what may yet be proved, the simple facts of life are such they cannot be removed’. Although Sam originally addressed the more romantic facts of life, he was wrong in terms of renal disease progression. The simple pathophysiological fact of progression could be efficiently halted by RAS blockade and experimental evidence suggests that the simple facts can even be removed. A better understanding of the many pathophysiological effects of ANG II including the novel observations made by Lautrette et al. [16] convincingly contribute to such a progress.

Conflict of interest statement. None declared.



   References
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