Pancreatic islet development is impaired in mice lacking EGFRs (epidermal growth factor receptors). Even partial tissue-specific attenuation of EGFR signalling in the islets leads to markedly reduced β-cell proliferation and development of diabetes during the first weeks after birth. Out of the many EGFR ligands, betacellulin has been specifically associated with positive effects on β-cell growth, through both increased proliferation and neogenesis. EGFR action is also necessary for the β-cell mitogenic activity of the gut hormone GLP-1 (glucagon-like peptide 1). Finally, in vitro models demonstrate a central role for EGFR in transdifferentiation of pancreatic acinar and ductal cells into endocrine islet cells. EGFR thus plays an essential role in β-cell mass regulation, but its mechanisms of action remain poorly understood.

Introduction

EGF (epidermal growth factor) was discovered 45 years ago, and, in the years following, many structural homologues (EGF family of growth factors) and specific receptors (ErbB receptor family) were found. EGFR (EGF receptor) signalling was first implicated in developmental biology, recently in oncology as a target for modern chemotherapy and lately also in pancreatic β-cell biology. This review gives an overview of the current knowledge of EGFR signalling in β-cell mass regulation.

ErbB tyrosine kinase receptors and their ligands

EGFR belongs to the ErbB receptor family, which consist of four transmembrane tyrosine kinase receptors, namely EGFR (ErbB1/HER1), ErbB2 (neu/HER2), ErbB3 (HER3) and ErbB4 (HER4). EGFR is the prototype of a type I receptor tyrosine kinase and is highly conserved among different species (reviewed in [1]). All ErbB receptors are structurally similar and contain an extracellular part, which is responsible for the ligand binding, a single membrane-spanning domain and a cytoplasmic part that contains the tyrosine kinase domain and multiple autophosphorylation domains. When activated by binding of a ligand, ErbB receptors form homo- or hetero-dimers, which is followed by phosphorylation of their tyrosine residues and secondary messenger recruitment. It is noteworthy that no ligand is currently known to bind to ErbB2. Furthermore, the cytoplasmic domain of ErbB3 lacks tyrosine kinase activity. Homodimers of ErbB2 or ErbB3 cannot mediate the growth factor signal under normal conditions, but they readily form heterodimers with EGFR and ErbB4.

There are 11 known ligands for ErbBs in mammals that can be divided into three groups depending on the binding specificity. The first group consists of EGF, TGF (transforming growth factor)-α and AR (amphiregulin), which bind specifically to EGFR. The second group contains BTC (betacellulin), HB-EGF (heparin-binding EGF), epiregulin and epigen, which bind to both EGFR and ErbB4. The last group contains NRGs (neuregulins) 1–4, which can be classed into two groups: NRG-1 and -2, which bind to both ErbB3 and ErbB4, and NRG-3 and -4, which bind exclusively to ErbB4.

EGF family in the pancreas

Our understanding of the biological role of the ErbB receptors and their ligands has been advanced through the study of a number of transgenic mouse models. A summary focusing on their pancreatic phenotype is presented in Table 1.

Table 1
Overview of the genetically modified erbB receptor and their ligands in transgenic mouse models
Target gene Pancreatic phenotype Reference(s) 
Overexpression of ligands   
 Elastase/AR Proliferation of small intralobular ducts and centroacinar cells [53
 β-Actin/BTC Reduction in pancreatic weight [28
 Insulin/EGF Disorganized, increased islet size, some neogenesis in the ducts [54
 Pdx1/HB-EGF Fibrosis, epithelial metaplasia, abnormal islets, diabetes [23
 Metallothionein/TGF-α Fibrosis, ductal metaplasia [22
Down-regulation of erbB signalling   
 BTC−/− None [55
 EGF/AR/TGF-α−/− None [56
 TGF-α−/− None [57,58
 EGFR−/− Streak-like islets, poor branching [8,16
 Pdx1/EGFR-DN Reduced β-cell mass, diabetes [7
 ErbB2−/− Embryonically lethal at E10.5, before pancreatic development [11,12
 ErbB3−/− Embryonically lethal at E13.5, abnormal pancreas [11,14,15
 ErbB4−/− Embryonically lethal at E10.5, before pancreatic development [17
Target gene Pancreatic phenotype Reference(s) 
Overexpression of ligands   
 Elastase/AR Proliferation of small intralobular ducts and centroacinar cells [53
 β-Actin/BTC Reduction in pancreatic weight [28
 Insulin/EGF Disorganized, increased islet size, some neogenesis in the ducts [54
 Pdx1/HB-EGF Fibrosis, epithelial metaplasia, abnormal islets, diabetes [23
 Metallothionein/TGF-α Fibrosis, ductal metaplasia [22
Down-regulation of erbB signalling   
 BTC−/− None [55
 EGF/AR/TGF-α−/− None [56
 TGF-α−/− None [57,58
 EGFR−/− Streak-like islets, poor branching [8,16
 Pdx1/EGFR-DN Reduced β-cell mass, diabetes [7
 ErbB2−/− Embryonically lethal at E10.5, before pancreatic development [11,12
 ErbB3−/− Embryonically lethal at E13.5, abnormal pancreas [11,14,15
 ErbB4−/− Embryonically lethal at E10.5, before pancreatic development [17

Receptors

Three independent studies describing EGFR-knockout mouse models were published in 1995 [24]. The phenotypes ranged from pre-implantation lethality to postnatal lethality. The EGFR−/− animals that survived until birth had generalized epithelial immaturity and abnormalities in organs developing through branching morphogenesis.

During development, EGFR and many of its ligands are broadly expressed in the gastrointestinal tract, including the pancreas [5]. In the adult pancreas, the receptor is dominantly expressed in the islets of Langerhans and ductal epithelial cells [6]. In our studies, phosphorylated EGFR was nearly exclusively detected in the islets after systemic injection of EGF in mice [7].

A more detailed analysis of the pancreatic phenotype of the EGFR−/− mice revealed that the size of the organ was reduced as a result of impaired branching of the ductal tree. Furthermore, morphogenesis of the islets was disturbed, characterized by endocrine cells residing adhered to the ducts. This was apparently due to a reduced matrix metalloproteinase activity, resulting in defective delamination of the developing islets. There also appeared to be a specific defect in β-cell differentiation, since the formation of insulin-expressing cells was reduced by nearly 50% in cultures of embryonic pancreas, whereas the other islet cell types developed normally [8].

EGFR−/− mice die soon after birth, making it impossible to study the role of EGFR in the postnatal pancreas. In order to overcome this problem, we generated mice expressing dominant-negative kinase-deficient EGFR under the control of the Pdx1 promoter (EGFR-DN mice). It is important to note that this model does not represent a complete abolishment of EGFR activity; only a 40% reduction in pancreatic EGFR, ERK (extracellular-signal-regulated kinase) and Akt phosphorylation was detected in mice injected with EGF [7]. Still, the animals became frankly diabetic within 2 weeks of birth. An apparent explanation of this was a reduced β-cell mass. Interestingly, this defect was essentially postnatal and appeared to result from a markedly decreased β-cell proliferation during the critical period of rapid postnatal β-cell expansion, demonstrating that an intact EGFR signalling pathway is crucial for this process. Furthermore, heterozygous EGFR-DN mice, which are non-diabetic and have only a marginal defect in their β-cell EGFR signalling, are not able to increase their β-cell mass in response to an increased insulin demand introduced by a high-fat diet (E. Hakonen, J. Ustinov, P. Miettinen and T. Otonkoski, unpublished work).

ErbB2 is expressed in the developing mouse pancreatic ducts [9] as well as in the proliferating human adult ductal cells [10], suggesting that the receptor has a role in pancreatic development. However, since the ErbB2−/− mice die before pancreatic organogenesis at E (embryonic day) 10.5 because of arrested cardiac and neural development, the exact physiological role of ErbB2 in the pancreas is not known [11,12]. ErbB2 is overexpressed in pancreatic carcinomas [13] and, as in breast cancer, is an indicator of tumour progression and poor prognosis.

ErbB3 is expressed in the developing mouse pancreatic ducts [9] and mesenchyme surrounding the pancreas [11]. Also, ErbB3 can be found in the proliferating pancreatic ductal cells [10]. ErbB3−/− mice die at E13.5. They exhibit a thinned mesenchyme surrounding the branched pancreatic epithelium, suggesting a primary role for ErbB3 in the pancreatic mesenchyme [11,14,15].

ErbB4 protein has been detected in the pancreas as early as E12.5 [16], and it is expressed in the fetal mouse pancreatic ducts [9] and also weakly in the proliferating human adult pancreatic duct cells [10]. Analogous to ErbB2, ErbB4−/− mice die at E10.5 owing to cardiac and neural defects [12,17], and their pancreatic phenotype cannot be studied.

Ligands

All of the ErbB ligands that bind to EGFR have six cysteine residues and share a conserved EGF motif, which is of importance for their tertiary structure and receptor binding. The ErbB ligands are processed through membrane-bound precursors from which the ligand is proteolytically cleaved to form a soluble molecule. The free ligand is then released to the extracellular space, where it interacts with its receptors or becomes bound to an extracellular store for later release.

EGF has been shown to stimulate the formation of duct-like structures in E12.5 mouse pancreatic rudiments cultured in collagen gels, but, in that setting, had no influence on the development of the endocrine part [18]. EGF is a powerful growth factor for embryonic pancreatic epithelium, demonstrating a clear-cut balance between EGF-induced proliferation and MAPK (mitogen-activated protein kinase) inhibitor-activated endocrine differentiation [19]. In accordance with this concept, systemic administration of EGF in pigs resulted in induction of ductal proliferation with only few insulin-positive cells [20]. However, EGF−/− mice have no pancreatic phenotype, showing that there is extensive functional redundancy among the different EGFR ligands (Table 1).

TGF-α is expressed in duct cells, acini and islets of human fetal and adult pancreas [5,16], where it has been suggested to regulate ductal cell proliferation and differentiation [21]. Systemic overexpression of TGF-α led to a major pancreatic phenotype characterized by interstitial fibrosis and transdifferentation of acinar to ductal cells [22]. Similar features were detected after pancreatic overexpression of HB-EGF, another EGFR ligand [23]. Co-overexpression of TGF-α and gastrin in the pancreas allowed further differentiation of the ductular cells into insulin-expressing cells [21].

BTC was first found from a mouse tumoral β-cell line [24]. It is strongly expressed in the liver, kidney and small intestine. In the pancreas, its primary expression sites appear to be α-cells and ducts. It is also expressed in glucagonomas and insulinomas [25]. BTC is known to induce proliferation of insulinoma cells [26] and to induce endocrine differentiation of the exocrine cell line AR42J [27]. Out of a number of EGF family growth factors, BTC was the only one that potently induced the differentiation of β-cells in embryonic mouse pancreatic explants [16]. This effect was entirely dependent on EGFR because it was abolished in EGFR−/− pancreas and not affected by blocking of the ErbB4 receptor. Ubiquitous overexpression of BTC did not result in any major pancreatic phenotype, except for a slightly decreased overall pancreas mass, suggesting a negative effect on the exocrine compartment [28]. A novel secreted splice isoform of BTC, BTC-δ4, has been found that lacks the transmembrane domain and a part of the EGF motif [29]. It stimulates β-cell differentiation both in vitro and in vivo [29]. This effect is apparently mediated by an as yet unknown receptor, because BTC-δ4 binds neither EGFR nor ErbB4 [29]. It is obvious that the mechanisms of BTC action in the β-cell are still incompletely understood.

In addition, the ErbB4 ligand NRG4 is expressed in the pancreas as early as E13 [16,30]. In our experimental model of E12.5 mouse pancreatic explants, we found that NRG-4 specifically stimulates the differentiation of somatostatin-producing δ-cells, while at the same time suppressing α-cell development [16]. These observations indicate that signalling through the ErbB receptors has a special role in the lineage determination of developing pancreatic β-cells.

Signalling networks used by EGFR in the β-cell

EGFR activation affects cell functions on multiple levels, depending on the signalling pathways that are activated. This depends on the differential autophosphorylation of the five tyrosine residues in the intracellular domain of the receptor, which in turn leads to recruitment of different phosphotyrosine-binding proteins and second messengers. The Ras- and Shc-activated MAPK pathway and the PI3K (phosphoinositide 3-kinase)-activated Akt pathway are the most important downstream signalling networks of most ErbBs, particularly EGFR [31].

EGFR has also been identified as a necessary link in GLP-1 (glucagon-like peptide-1)-(7–36)-induced β-cell proliferation (Figure 1). GLP-1 acts through its G-protein-coupled receptor, expressed on the cell surface of pancreatic β-cells and ducts [32,33]. GLP-1 receptor activation has been shown to lead to Src-mediated release of anchored BTC or other EGF-like ligands from the cell membrane, which then bind to the EGFR, thus activating the PI3K pathway and leading to β-cell proliferation and increased insulin secretion [34,35]. Recently, forkhead transcription factor FoxO1 (forkhead box O1) has also been implicated in this pathway [36]. FoxO1 is active when it is in an unphosphorylated state in the nucleus (reviewed in [37]). GLP-1 blocked its function through phosphorylation-dependent nuclear exclusion and this could be inhibited using an EGFR inhibitor. The exact molecular mechanisms linking GLP-1, EGFR and FoxO1 are not yet known.

Role of EGFR signalling in β-cell proliferation: a theoretical model

Figure 1
Role of EGFR signalling in β-cell proliferation: a theoretical model

Binding of GLP-1 to its G-protein-coupled receptor leads to activation of a metalloproteinase and an increase in the amount of soluble EGF from its membrane-bound precursor. After ligand binding, EGFR homodimerizes and activates the MAPK [MEK (MAPK/ERK kinase)] and PI3K pathways. The former results in up-regulation of cyclin D1 and cell-cycle progression, whereas the latter is suggested to lead to phosphorylation of FoxO1 and thus its inactivation and exclusion from the nucleus. Shuttling of FoxO1 to the cytoplasm enables Pdx-1 (pancreatic and duodenal homeobox 1) transcription and conceivably also up-regulation of cyclin D2 and progression of the cell cycle. See [37] for a review.

Figure 1
Role of EGFR signalling in β-cell proliferation: a theoretical model

Binding of GLP-1 to its G-protein-coupled receptor leads to activation of a metalloproteinase and an increase in the amount of soluble EGF from its membrane-bound precursor. After ligand binding, EGFR homodimerizes and activates the MAPK [MEK (MAPK/ERK kinase)] and PI3K pathways. The former results in up-regulation of cyclin D1 and cell-cycle progression, whereas the latter is suggested to lead to phosphorylation of FoxO1 and thus its inactivation and exclusion from the nucleus. Shuttling of FoxO1 to the cytoplasm enables Pdx-1 (pancreatic and duodenal homeobox 1) transcription and conceivably also up-regulation of cyclin D2 and progression of the cell cycle. See [37] for a review.

β-Cell expansion: proliferation, neogenesis and transdifferentiation

In rodents, β-cell mass is maintained throughout adult life mainly through replication of pre-existing β-cells, although a low-grade neogenesis through stem cell differentiation or from ductal cells cannot be excluded [38]. β-Cells can increase their proliferation rate at times of stress, such as obesity or pregnancy. The best known factors mediating this growth stimulus include glucose, insulin, lactogenic hormones, such as prolactin, and gut hormones, such as GLP-1. However, in situations of pancreatic regeneration, other mechanisms are likely to be even more important. There is plenty of evidence for beneficial effects of EGFR ligands in rodent models of β-cell regeneration. In vitro studies have also demonstrated transdifferentation of acinar into endocrine cells mediated through EGFR-dependent processes. To what extent this is relevant for the human pancreas remains an open question.

Many studies have shown that expression of EGF ligands is increased during experimental β-cell regeneration. Gastrin and TGF-α are highly expressed during islet cell neogenesis in experimental duct-ligation models in the rat [39], and TGF-α, BTC and HB-EGF in partially duct-ligated mouse [40]. Mice overexpressing interferon γ in the pancreas have high expression of EGF, TGF-α and EGFR in the acini undergoing metaplastic transdifferentiation into duct-like structures [41]. There is also direct evidence of EGF ligands enhancing restoration of a normal β-cell mass. A special synergistic effect of EGF and gastrin has been observed using various approaches. Mice overexpressing both gastrin and TGF-α in the pancreas have an increased islet mass [21]. Furthermore, gastrin and EGF have been shown to induce β-cell neogenesis after alloxan treatment and to restore normoglycaemia [42]. In this model, the regenerating β-cells were suggested to originate from ductal cells based on detection of insulin/cytokeratin double-positive cells [42]. NOD mice treated with EGF and gastrin increased their β-cell mass, again seemingly by neogenesis from the ducts, and reversed hyperglycaemia [43]. There is also evidence for a stimulatory effect of EGF and gastrin on islet neogenesis from ducts in human pancreatic cultures [44].

BTC has long been studied as a potential therapeutic factor for β-cell regeneration. BTC has been shown to promote both β-cell neogenesis from ducts and improve glycaemic control after selective β-cell destruction with alloxan [45] or after 90% pancreatectomy [46]. When combined with activin A, a member of the TGF-β superfamily, it also stimulated proliferation of β-cells and neogenesis from ductal precursor cells in rats treated with streptozotocin [47]. Furthermore, gene therapy approaches have successfully restored insulin secretion in streptozotocin-induced diabetic mice or rats by NeuroD+BTC or Pdx1+BTC respectively [48,49].

Transdifferentiation of acinar cells into functional islet cells has recently been documented both in the rat [50] and the mouse [51]. In both of these cases, EGFR was found to be essential. In cultures of rat acinar cells, addition of EGF and LIF (leukaemia inhibitory factor) induced rapid β-cell neogenesis, and transplantation of the newly formed β-cells restored normoglycaemia in alloxan-treated mice [50]. An in vitro genetic lineage-tracing strategy in mouse acinar cultures showed that the neogenic β-cells were derived from exocrine cells through an EGFR-stimulated process [51]. Lineage tracing was also used to demonstrate that EGFR signalling can lead to true transdifferentiation of acinar cells into ductal epithelial cells [52].

Concluding remarks

As shown in this brief review, various lines of evidence point out that ErbB-, and particularly EGFR-, mediated signalling is of crucial importance for β-cell mass expansion. A more complete understanding of these mechanisms is important in order to develop therapeutic approaches against β-cell failure.

Pancreatic β-Cell: Birth, Life and Death: A joint Biochemical Society, Juvenile Diabetes Research Foundation (JDRF) and EU ‘SaveBeta’ Consortium Focused Meeting held at King's College London School of Medicine, London, U.K., 3–4 December 2007. Organized and Edited by Stephanie Amiel (King's College London, U.K.), David Dunger (University of Cambridge, U.K.), Decio Eizirik (Université Libre de Bruxelles, Belgium), Peter Jones (King's College London, U.K.), Jo Lilleystone (Juvenile Diabetes Research Foundation, U.K.), Guy Rutter (Imperial College London, U.K.), James Shaw (Newcastle University Medical School, U.K.) and David Tosh (Bath, U.K.).

Abbreviations

     
  • AR

    amphiregulin

  •  
  • BTC

    betacellulin

  •  
  • E

    embryonic day

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFR

    EGF receptor

  •  
  • EGFR-DN

    dominant-negative kinase-deficient EGFR under the control of the Pdx1 promoter

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FoxO1

    forkhead box O1

  •  
  • GLP-1

    glucagon-like peptide 1

  •  
  • HB-EGF

    heparin-binding EGF

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • NRG

    neuregulin

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • TGF

    transforming growth factor

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