Insulin-Like Growth Factor Receptor Type I (IGF1R) Signaling and Inflammation
The IGF1R, a Protein Tyrosine Kinase Receptor, is expressed in many tissues and can be activated by its physiological ligands IGF-I and IGF-II, which are available systemically and from local sources. Activation of the IGF1R promotes cell growth and survival in a cell-autonomous manner. Because of a wide distribution of IGF1R and good bioavailability of its ligands, under pathological conditions this cytoprotective intracellular signaling pathway may be activated in cells that also receive stimulation by pathology-specific factors. Cross-regulation between IGF1R signaling and pathways activated by inflammation has recently been in the focus of attention in several laboratories because pro-inflammatory factors are a common cause of cellular pathology in many different conditions (O’Connor et al. 2008).
Resident parenchymal cells activated by pro-inflammatory cytokines in tissues affected by inflammation are characterized by several features, including increased activity of the stress-related Mitogen-Activated Kinases (MAPK) and transcription factors of nuclear factor-κB ( NF-κB) family, stress of the endoplasmic reticulum (ER), and generation of reactive oxygen species (ROS). Several components in inflammation-activated pathway may be cross-regulated by IGF1R-activated signaling, and there are signaling molecules in the IGF1R pathway that are subject to cross-regulation by pro-inflammatory factors. Here IGF1R signaling has been reviewed and biochemical events that may facilitate cross-regulation of this pathway and those activated by inflammation have been elucidated. The generic signaling networks presented here are based on molecular interaction shown in non-transformed nonimmune cells. There is a considerable variation between cells of different lineages and differentiation states in usage of these networks.
IGF1R Signaling Cascade
IGF1R Signaling Complex
Ligand binding initiates formation of a protein complex consisting of kinases and adaptor proteins around the IGF1R. An important component of this complex is Insulin Receptor Substrate (IRS), a scaffolding protein containing N-terminal Pleckstrin Homology (PH) domain, the Src Homology 2 (SH2) domain, and several tyrosine and serine residues that can be substrates of protein kinases. Isoforms 1–4 of IRS have been implicated in IGF1R signaling, with IRS-1 and -2 being the most widely distributed. The SH2 domain of IRS-1 binds to tyrosine phosphorylated Asn-Pro-X-Tyr motifs in the juxtamembrane region of the IGF1R. Another signal positioning IRS near the activated IGF1R is the interaction between PH domain of IRS and 3′-phosphorylated phosphoinositides formed in the vicinity of the activated IGF1R as described below.
To various degrees, all four groups of conventional MAPK are involved in signal transmission from the activated IGF1R. These include Erk1/2, c-jun NH2-terminal protein kinase (JNK) 1/2/3, p38α/β/γ/δ, and Erk5. MAPK are activated by dual specificity MAPK kinases (MKK or MAP2K) phosphorylating Thr and Tyr residues within a conserved Thr-X-Tyr motif in the MAPK activation loop. Upstream of MAP2K are their activators MAP2K kinases (MKKK or MAP3K) (Cargnello and Roux 2012). Signal wiring within the MAPK cascade is dependent on substrate specificity of second and third tire MAPK and participation of scaffolding proteins. Increased signaling activity of MAPK is associated with their nuclear translocation.
Erk1 and Erk2, the first MAPK to be cloned and characterized in early 1990, are most frequently associated with mitogenic effects of the activated IGF1R and other receptor tyrosine kinases in different cell lineages (Cargnello and Roux 2012). The Erk1/2 module of the MAPK cascade includes the MAP2K MEK1 and MEK2, as well as the MAP3K Raf1. The serine/threonine kinase Raf1 is activated by the small guanosine triphosphate (GTP)-binding protein Ras. Ras is inactive when bound to guanosine diphosphate (GDP) and becomes activated when associated GDP is replaced by GTP. This exchange is promoted by the guanine nucleotide exchange factor (GEF) son of sevenless, which is activated by recruitment to the IGF1R signaling complex via interaction with Grb2 (Fig. 4) and a mechanism that requires Shp2. In addition, IGF1R-agonist-induced Raf1 activation may occur independently of IGF1R autocatalytic activity, and Raf1-independent Erk1/2 activation in response to IGF1R ligation has been shown.
The stress MAPK p38 and JNK, which are usually associated with apoptosis and cell differentiation, as well as Erk5, are also involved in IGF1R signaling, but less frequently than Erk1/2. IGF1R-induced JNK activation may be dependent on receptor-interacting protein, a death domain-containing serine/threonine kinase with a well-documented function of linking JNK activation to the Tumor Necrosis Factor Receptor (TNFR) type 1.
Ubiquitously Expressed Adaptors
The activation status of the IGF1R signaling cascade is a subject to modulation by several ubiquitously expressed scaffolding or adaptor proteins. IRS family proteins belong to the group classified by some authors as docking proteins (Brummer et al. 2010) on the basis of having a N-terminal PH domain and SH2 domain, as well as multiple tyrosine residues. In addition to IRS, docking proteins participating in IGF1R signaling include members of two families, Grb-associated binder (Gab) and downstream of tyrosine kinases (Dok). Gab1, one of the best studied docking proteins of Gab family, binds to several components of the IGF1R signaling complex, including Shp2, p85, and Grb2, and its deficiency is associated with reduced proliferation in response to IGF-I.
Another adaptor, Grb10, binds to a diverse range of partners through SH2 and PH domains, including the IGF1R, Raf1, MEK, Akt, the regulatory subunit of PI3K and Gab1, and has the capacity to positively and negatively regulate IGF1R signaling. Grb10 may promote Ras-independent activation of Raf1 in response to IGF1R activation. Grb10 also provides a link between the IGF1R and Nedd4, an E3 ubiquitin ligase, which is required for optimal IGF1R signaling (Fig. 4).
Among molecules interacting with Grb10 is the ubiquitously expressed adaptor 14-3-3 which has affinity to phosphorylated serine and threonine residues. Binding partners of 14-3-3 isoforms are found in different intracellular signaling pathways. Regulatory effects of 14-3-3 isoforms are mediated by restricting subcellular localization of client proteins and shielding them from interaction with other molecules (Darling et al. 2005). Several signaling molecules in IGF1R- and inflammation-activated pathways, including IGF1R, IRS-1, PI3K, ASK1-interacting protein 1 (AIP1), as well as the third tire MAPK Raf, apoptosis signal-regulating kinase 1 (ASK1), and MEKK3, may generate binding sites for this adaptor by phosphorylation of serine and threonine residues. Therefore, serine/threonine phosphorylation of a signaling molecule in a given pathway by a kinase activated in another signaling pathway may be a 14-3-3-dependent mechanism of cross-talk between these pathways.
Grb10 and 14-3-3 may be involved in one of the IGF1R-dependent anti-apoptotic pathways. Association of 14-3-3 with IGF1R C-terminal serines, phosphorylated during receptor activation by a mechanism that might involve autophosphorylation, may induce translocation of the MAP3K Raf1 to the mitochondrion where it inhibits the pro-apoptotic Bcl-2 Family protein BAD. The effect of Raf1 is independent of Erk1/2 activation and may require interaction of Raf1 with Grb10 and Nedd4 (Fig. 4). This may provide cytoprotection against mitochondrial cell death, which is often induced by pro-inflammatory conditions in resident cells.
Cellular Responses to IGF1R Activation and Intracellular Signaling Pathways Involved
Another IGF1R-dependent effector is mammalian target of rapamycin ( mTOR), which positively regulates protein translation by activating p70 Ribosomal S6 Kinase (Rsk), a kinase activating the 40S ribosomal protein. Akt and other kinases in the IGF1R signaling cascade, including Pdk-1, PKCζ, and Erk1/2, can activate mTOR and Rsk, thereby promoting protein synthesis (Kuemmerle 2003). Finally, IGF1R signaling activates upstream binding factor 1, which regulates cell size by transcriptionally activating RNA polymerase I, a limiting factor in ribosome biosynthesis (Fig. 5).
By promoting cell growth IGF1R signaling indirectly promotes cell proliferation. This pathway also has a direct mitogenic effect on selected lineages, for example, myoblasts, by modulating activity of proteins regulating cell cycle progression, such as Cyclins, their kinases and c- Myc, effects that are often dependent on Erk1/2. Furthermore, Akt and the aforementioned 14–3-3/Raf1-dependent mechanism mediate anti-apoptotic effects of activated IGF1R by targeting BAD. In addition, IGF-I and -II are differentiation factors for several cell lineages, including osteoblasts, chondroblasts, and myoblasts, because in these cells they induce expression of lineage-specific genes. Finally, IGF1R signaling may lead to cell activation and increased functional competence in several cell types, for example, steroidogenesis in ovarian cells or expression of cytokines in various immune and non-immune cells (Fig. 5).
Cross-Regulation Between IGF1R and Inflammation-Activated Intracellular Signaling Pathways
Perhaps unsurprisingly, cytoprotective effects of the IGF1R pathway are in part mediated by inhibition of ASK1 (Fig. 6). ASK1 may be inhibited by recruitment to the IGF1R signaling complex via a mechanism that requires Raf1. In addition, two modifications of ASK1 molecule that lead to its inactivation can be induced by the IGF1R signaling pathway. They are dephosphorylation of Thr838 by protein phosphatase 5 (Fig. 6), which is activated by mTOR, and Akt-induced phosphorylation on Ser83.
On the other hand, ASK1 may inhibit IGF1R signaling. This may involve AIP1. Under inflammatory conditions, AIP1 is activated in response to cytokines and ER stress. AIP1 has the ability to inhibit Ras because it has Ras-GTPase activity. It also contains domains that bind ASK1 and Akt, bringing them together. This sequesters Akt in cytosol, preventing its translocation to the plasma membrane and interaction with PIP3. The Akt-inhibiting function of AIP1 is increased by phosphorylation at Ser604 induced by ASK1 (Xie et al. 2009). Therefore, AIP1 inhibits both Erk1/2 activation and the PI3K/Akt pathway. These properties implicate AIP1 in the inhibition of IGF1R signaling during inflammation (Fig. 6).
Stress of the ER
Proteins are synthesized and folded in ER. Overloading of ER with nascent protein molecules or decrease in its functional capacity may lead to an adaptive cellular response, known as unfolded protein response (UPR), which attempts to restore ER functions. The major adaptive effect of UPR is inhibition of translation, resulting from inactivation of eukaryotic initiation factor 2 by double-stranded RNA-activated protein kinase ( PKR) and PKR-like ER kinase. These kinases are activated together with the other sensors of ER stress, which include activating transcription factor 6 and inositol-requiring enzyme-1 (Ire1). Activated Ire1α subunit removes an internal fragment from X box-binding protein 1 (XBP1) transcript, generating thereby a stable transcriptionally active splice variant of XBP1 (Zhang and Kaufman 2008). UPR leads to the induction of gene expression programs that restore protein folding capacity of ER. Therefore, adaptive role of UPR is mediated by relieving ER overload, increasing its protein folding capacity and sparing cellular resources.
UPR is a common feature of cellular pathology in inflammation. Moreover, since IGF1R activation has a potent anabolic effect, it leads to increased loading of ER with nascent proteins, also predisposing cells to ER stress. This, however, does not compromise IGF1R-dependent cytoprotection because IGF1R signaling can promote cell adaptation to ER stress, regardless of its causes, by inducing expression of chaperones, such as Grp78 (Pfaffenbach and Lee 2012), and via several other mechanisms.
NF-κB Signaling Pathway
NF-κB family of transcription factors transduce signals from several receptors activated by pro-inflammatory factors, including the receptors of TNFα and IL-1β, as well as TLR, receptors recognizing pathogen molecular patterns (Wajant and Scheurich 2012). The five members of this family of transcription factors form homo- and heterodimers maintained in transcriptionally inactive state by interaction with an inhibitor of κB (IκB). NF-κB dimers are activated when IκB is inhibited by phosphorylation mediated by IκB kinase complex (IKK). The sequence of events leading to nuclear translocation of NF-κB dimers after they have been released from IκB-mediated inhibition is known as the classical pathway of NF-κB activation. This pathway is characterized by predominant formation of heterodimers consisting of p50 and p65 proteins of NF-κB family (Wajant and Scheurich 2012). Activation of the classical NF-κB pathway in resident nonimmune cells at sites of inflammation may induce production of pro-inflammatory cytokines and improve cell survival, which in many instances results from inhibition of JNK-induced apoptosis.
NF-κB does not appear to be a major signaling pathway mediating cellular responses to IGF1R activation, but nuclear translocation of p65 and NF-κB-mediated transcriptional control have been implicated in a diverse range of biological effects elicited by IGF1R signaling, including stimulation of endothelial cell migration, chondroblast proliferation, lung fibroblast differentiation, cytoprotection of keratinocytes from UV, neuroprotection, and MHC I promoter activation. IGF1R-induced NF-κB activation is dependent on MAPK and Akt, but, unlike NF-κB activation in response to pro-inflammatory factors, may not require IκB phosphorylation.
NF-κB signaling activated by pro-inflammatory factors may be inhibited or modified by co-activation of the IGF1R, as shown in untransformed cells of different lineages, including keratinocytes, astrocytes, and vascular smooth muscle cells. Co-activation of the IGF1R may change the composition of NF-κB dimers formed in response to pro-inflammatory factors. This may inhibit nuclear translocation of NF-κB or change the array of genes activated by this pro-inflammatory pathway. These effects of IGF1R signaling might be in part mediated by phosphorylation of NF-κB subunits.
ROS are generated during oxygen metabolism, predominantly in mitochondria by electron transfer from nicotine amid dinucleotide phosphate (NADPH) to O2 by NADPH oxidases (Nox). Pro-inflammatory cytokines, as well as insulin and IGF-I, have the capacity to increase cellular oxidative potential by inducing increased Nox activity (Meng et al. 2008). Nox are regulated by changes in the phosphorylation status of their regulatory subunits. The association of IGF1R activation with increased ROS production may also be mediated by Rac1, a member of Rho Family GTPases (Meng et al. 2008).
IRS as a Target of Inflammation-Activated Pathways
IRS-1 is a substrate of several serine/threonine kinases found in the signaling pathways activated by the IGF1R, as well as those activated during inflammation, such as Erk1/2, JNK, IKK, mTOR, Rsk, PKR, Protein Kinase C δ, and PKCζ. Serine phosphorylation of IRS inhibits its function as a signaling molecule in the IGF1R complex by inducing dephosphorylation of IRS tyrosines, which results in IRS dissociation from the IGF1R and plasma membrane. Targeting IRS by serine kinases activated in response to IGF-I may mediate negative feedback, whereas IRS inhibition by inflammation-activated kinases may reduce cellular sensitivity to cytoprotective effects of IGF-I under inflammatory conditions. IRS inhibition, together with some other mechanisms, implicates low-level chronic inflammation in the development of systemic insulin resistance (Hotamisligil 2010). Dephosphorylation of tyrosines by protein phosphatases is also inhibitory for IRS-1.
Few serine phosphorylation sites are found in IRS-2, making this isoform less amenable to regulation by differential phosphorylation. IRS-2, however, may be transcriptionally inhibited during ER stress.
The IGF1R Signaling Pathway and Inflammatory Diseases
Inflammatory mechanisms are involved in tissue degeneration in many diseases. These include a group of chronic tissue-specific autoimmune conditions, which are characterized by inflammation-dependent tissue loss followed by functional deficit, with examples including demyelination in multiple sclerosis, cartilage destruction in rheumatoid arthritis, and inflammation-mediated degeneration of pancreatic β-cells in type I diabetes. Both IGF1R-mediated cytoprotection and inhibition of IGF1R signaling by pro-inflammatory factors have been shown in these conditions (Glass 2005; Ye et al. 2007). It remains to be elucidated to what extent cytoprotective mechanisms mediated by endogenous IGF-I and -II modify the course of these inflammatory diseases.
Pro-inflammatory factors, including cytokines, ER stress, and ROS, contribute to the pathogenesis of type 2 diabetes and the metabolic syndrome by suppression of intracellular signal transmission from insulin receptor and IGF1R. Interplay between IGF1R signaling and inflammation may also play an important and complex part in pathogenesis of atherosclerosis. IGF1R signaling is required for maintenance of vascular smooth muscle cells in differentiated status, which is an atherosclerosis-inhibiting effect, but dedifferentiated vascular smooth muscle cells proliferate and migrate in response to IGF1R activation, thereby promoting atherosclerotic lesion formation. Furthermore, tissue hyperplasia in several inflammatory conditions, such as Crohn’s disease and allergic airway inflammation, may be dependent on IGF1R-mediated signaling.
Testing experimentally all possible interactions mediating cross-regulation of the IGF1R signaling cascade by inflammation-activated signaling and vice versa may be inefficient because of the complexity of these intracellular signaling networks. Computational modeling is, therefore, an important approach for understanding network dynamics and underlying mechanisms. In one of the pioneering studies in this area, a discrete modeling approach in combination with experimental data analysis has been applied in order to analyze the involvement of PKR in cross-regulation of the insulin receptor signaling network (Wu et al. 2009).
Cells of different lineages express the IGF1R beginning from early developmental stages into adulthood. This receptor mediates effects of the growth factors IGF-I and -II. Cellular responses to IGF1R activation include increased cell growth, inhibition of apoptosis, cell division, differentiation and acquisition of new phenotypic features required for functional competence. Depending on the cell type these responses are activated in various combinations. Effects of IGF1R ligation are mostly mediated by activation of MAPK family proteins and PI3 kinase. Phosphatidylinositol 3,4,5 triphosphate generated by PI3K from plasma membrane lipids activates Akt and several other pleckstrin homology domain containing proteins, which regulate cell growth and survival by targeting several effectors, including GSK3, FOXO transcription factors, and mTOR. As IGF1R is so widely distributed, it is important to understand how this cytoprotective signaling cascade interacts with signaling pathways activated by pathological processes leading to tissue degeneration, in particular, inflammation-activated pathways. IGF1R activation may counteract cytotoxic effects of pro-inflammatory factors by inhibiting some of the components in their signaling pathways, including the stress MAPK JNK and p38, NF-κB transcription factors, pro-apoptotic Bcl-2 family members, and mediators of endoplasmic reticulum stress. On the other hand, inflammation-activated signaling pathways may inhibit IGF1R signaling, for example, by targeting IRS and Akt. Reactive oxygen species, which are generated during cell activation by IGF1R ligands and pro-inflammatory factors, may also mediate cross-regulation between the IGF1R and inflammation-activated signaling pathways. This cross-talk, rather than an isolated response to pro-inflammatory factors, may drive a number of pathological processes, including tissue-specific autoimmune diseases, atherosclerosis, and the metabolic syndrome.