Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions

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<ul><li><p>RESEARCH ARTICLE</p><p>Insulin-like growth factor binding protein-3 (IGFBP-3):Novel ligands mediate unexpected functions</p><p>Robert C. Baxter</p><p>Published online: 23 May 2013# The International CCN Society 2013</p><p>Abstract In addition to its important role in the regulationof somatic growth by acting as the major circulating trans-port protein for the insulin-like growth factors (IGFs), IGFbinding protein-3 (IGFBP-3) has a variety of intracellularligands that point to its function within major signalingpathways. The discovery of its interaction with the retinoidX receptor has led to the elucidation of roles in regulatingthe function of several nuclear hormone receptors includingretinoic acid receptor-, Nur77 and vitamin D receptor. Itsinteraction with the nuclear hormone receptor peroxisomeproliferator-activated receptor- is believed to be involvedin regulating adipocyte differentiation, which is also modu-lated by IGFBP-3 through an interaction with TGF/Smadsignaling. IGFBP-3 can induce apoptosis alone or in con-junction with other agents, and in different systems canactivate caspases 8 and 9. At least two unrelated proteins(LRP1 and TMEM219) have been designated as receptorsfor IGFBP-3, the latter with a demonstrated role in inducingcaspase-8-dependent apoptosis. In contrast, IGFBP-3 alsohas demonstrated roles in survival-related functions, includ-ing the repair of DNA double-strand breaks through inter-action with the epidermal growth factor receptor and DNA-dependent protein kinase, and the induction of autophagythrough interaction with GRP78. The ability of IGFBP-3 tomodulate the balance between pro-apoptotic and pro-survival sphingolipids by regulating sphingosine kinase 1and sphingomyelinases may be integral to its role at thecrossroads between cell death and survival in response to avariety of stimuli. The pleiotropic nature of IGFBP-3 activ-ity supports the idea that IGFBP-3 itself, or pathways withwhich it interacts, should be investigated as targets of ther-apy for a variety of diseases.</p><p>Keywords IGFBP-3 . apoptosis . DNA damage repair .</p><p>authophagy . sphingosine kinase . GRP78</p><p>Introduction</p><p>The insulin-like growth factors, IGF-I and IGF-II (encodedin humans by the genes IGF1 and IGF2) are ubiquitousgrowth factors that influence cell proliferation, differentiation,survival and migration. The IGFs, which are structurally andfunctionally related to insulin (Clemmons 2012), signalthrough the type 1 IGF receptor (IGF1R), a heterotetramerwith tyrosine kinase activity, as well as the related insulinreceptor (notably insulin receptor isoform A which mediatesIGF-II signalling) and hybrids of the two receptor types(Martin and Baxter 2011; Siddle 2012). IGF-like signalingpathways have been strongly conserved through evolutionand have been shown to modulate longevity in C. elegans(Lapierre and Hansen 2012).</p><p>In contrast to insulin, IGF-I and IGF-II are not known tobe stored intracellularly, but are secreted by their cells oforigin. In the extracellular environment and the circulation,they are predominantly bound by IGF binding proteins(IGFBPs), a family of six highly conserved proteins (Firthand Baxter 2002) encoded by the genes IGFBP1 toIGFBP6. These proteins are characterized by high affinityfor both IGFs, with association constants above 109 L/mol(Baxter 2000; Forbes, et al. 2012). Although the IGFBPnomenclature has been applied to other proteins with weakstructural homology to the IGFBPs, in particular mac25(sometimes called IGFBP-7) and members of the CCNfamily, there is now wide consensus that only the six proteinswith high affinity for the IGFs should be known as IGFBPs.</p><p>The conserved IGFBP structure can be divided into threedomains of approximately equal size: cysteine-rich amino-and carboxy-terminal domains, which are highly conserved,joined by an unconserved central or linker domain. Both</p><p>R. C. Baxter (*)Kolling Institute of Medical Research, University of Sydney,Royal North Shore Hospital, Level 8, Kolling Building,St Leonards, NSW 2065, Australiae-mail:</p><p>J. Cell Commun. Signal. (2013) 7:179189DOI 10.1007/s12079-013-0203-9</p></li><li><p>mutagenesis studies and structural determination by NMRand X-ray crystallography have revealed that high-affinityIGF binding involves residues in both the amino- andcarboxy-terminal domains (Baxter 2000; Forbes et al.2012). Two of the six IGFBPs (IGFBP-3 and IGFBP-5)form complexes in the circulation that contain either IGF-Ior IGF-II, and a third protein, the acid-labile subunit or ALS(encoded by the IGFALS gene). The IGFBP binding site forALS consists of a highly basic motif in the carboxy-terminaldomain (Firth, et al. 2001; Firth, et al. 1998). Basic residuesin this domain also form a bipartite nuclear localizationsignal (NLS) (Schedlich, et al. 2000). IGFBP-6, which alsohas a basic carboxy-terminal motif, is unable to bind ALS(Twigg, et al. 1998), but appears to have a functional NLS(Iosef, et al. 2008).</p><p>IGFBPs were first characterized for their IGF transportfunction in the circulation, where they are known to act as areservoir of IGFs, mainly bound to IGFBP-3 in ternarycomplexes with ALS. Circulating IGFBPs regulate the bio-availability of the IGFs by controlling their egress from thecirculation to the tissues (Baxter 1993; Payet, et al. 2004;Rajaram, et al. 1997). Over the past decade, it has becomeclear that IGFBPs have many ligands apart from the IGFsand ALS, and have significant functions in the pericellularand intracellular spaces in addition to the circulation. Thisreview, concentrating on IGFBP-3, will discuss recent dis-coveries of other (i.e. non-IGF) ligands, and the biologicalfunctions in which they have been implicated. These in-teractions may explain, at least in part, the dichotomousgrowth-inhibitory and -stimulatory effects that have beenattributed to IGFBP-3.</p><p>Nuclear hormone receptors</p><p>In 2000, Liu et al. reported the unexpected finding, by yeasttwo-hybrid screen, that IGFBP-3 interacts with the retinoid Xreceptor, RXR (Liu, et al. 2000). RXR is a member of theclass II nuclear hormone receptors, and typically acts either asa homodimer or, more commonly, as a heterodimer with othermembers of this group including the retinoic acid receptors(RARs), the vitamin D receptor VDR, the thyroid hormonereceptor TR, liver X receptors (LXRs) and peroxisomeproliferator-activated receptors (PPARs) (Dawson and Xia2012). This important study revealed IGFBP-3 as a potentialtranscriptional regulator, activating effects mediated throughthe RXR response element but inhibiting signaling throughthe RAR response element (RARE, activated by ligand bind-ing to RXR-RAR heterodimers) (Liu et al. 2000).</p><p>Breast cancer cells with a basal-type molecular subtype havehigh IGFBP-3 expression, and growth inhibition of the basalcell lines Hs578TandMDA-MB-231 by the RAR ligand, all-trans-retinoic acid (atRA), was found to be modulated by</p><p>endogenous IGFBP-3, such that immunoneutralizing IGFBP-3sensitized both cell lines to atRA inhibition (Schedlich, et al.2004). Further investigation showed that IGFBP-3 inhibitedRARE transactivation by binding to RAR and blockingRXR-RAR heterodimerization, with no effect on atRA bind-ing. Thus, in this context, IGFBP-3 is potentially growth-stimulatory to these basal-type cell lines, by blocking theirgrowth inhibition by RAR ligands. Similarly, IGFBP-3 has beenfound to block the RAR-dependent differentiation of myeloidleukemia cells, while it enhanced differentiation induced by anRXR-selective ligand (Ikezoe, et al. 2004).</p><p>Contrasting with its effect in breast cancer cells, in22RV1 human prostate cancer cells, IGFBP-3 was reportedto induce apoptosis by a mechanism involving anotherRXR dimerization partner, Nur77 (Lee, et al. 2005). ThisIGFBP-3 effect involved the rapid mitochondrial transloca-tion of RXR-Nur77 dimers, resulting in cytochtome crelease, and required direct cytoplasmic interaction betweenIGFBP-3 and Nur77 (Lee, et al. 2007), but the precise de-tails of the mechanism are still unclear. IGFBP-3 also bindsdirectly to VDR, as does the related binding protein IGFBP-5(Schedlich, et al. 2007b), and IGFBP-3 is inhibitory to VDRtranscriptional activity (Ikezoe et al. 2004).</p><p>The metabolic regulator PPAR has an important roleduring adipogenic differentiation. Since PPAR signals as aheterodimer with RXR, it was proposed that IGFBP-3might interfere with this process (Chan, et al. 2009), partic-ularly since IGFBP-3 knockout mice are described as hav-ing increased adiposity (Yakar, et al. 2009). However, theadipogenic differentiation of bone marrow derived mesen-chymal stem cells was not enhanced by IGFBP-3 knockout(Fritton, et al. 2010). Using the 3T3-L1 preadipocyte modelof adipogenesis, both exogenous and overexpressed IGFBP-3 were found to be inhibitory to adipogenic differentiation(Chan et al. 2009). This inhibition was associated with thedirect interaction of IGFBP-3 and PPAR, demonstrated bycoimmunoprecipitation from 3T3-L1 cell lysates, and theinhibition of PPAR-RXR dimerization and ligand-induced transcriptional activity. Recently we have observedthe same effect of IGFBP-3 in human breast cancer cell linesalthough, paradoxically, IGFBP-3 did not reverse thegrowth-inhibitory effect of PPAR ligands in these cells,but acted to enhance the growth inhibition (Pon et al.,unpublished data).</p><p>A few studies have investigated structural determinantsinvolved in nuclear receptor-IGFBP-3 interactions, usingmutants of both RXR and IGFBP-3, including the basicdomain IGFBP-3 mutant originally characterized for itsdeficient ALS binding and nuclear translocation (Firth etal. 1998; Schedlich, et al. 1998). Using GST-pulldown as-says, IGFBP-3 was found to interact with the DNA-bindingdomain (C domain) of RXR. No binding was observed tothe aminoterminal (A/B) domain, the hinge (D) domain,</p><p>180 R.C. Baxter</p></li><li><p>or the carboxyterminal ligand-binding (E/F) domain(Schedlich, et al. 2007a). Examining binding determinantson IGFBP-3, both aminoterminal residues (T58, R60) andbasic carboxyterminal residues (220222, 228232) werefound to be important for RXR binding (Schedlich et al.2007a). Similar residues are involved in IGFBP-3 interactionwith PPAR (Chan et al. 2009). Non-RXR binding mutantswere unable to block atRA signaling, demonstrating the es-sential role of the IGFBP-3-RXR interaction in the inhibitionof retinoic acid action. The 228232 mutant also failed toinhibit PPAR-dependent adipogenesis (Chan et al. 2009)and interestingly, appeared to exert a dominant negative effectin preventing breast cancer cell growth inhibition by thePPAR ligand rosiglitazone, in contrast to wild-typeIGFBP-3 which enhanced the rosiglitazone effect (Ponet al., unpublished data).</p><p>Thus it appears that, similar to the interaction betweenIGFBP-3 and the IGFs, its interaction with RXR involvesboth aminoterminal and carboxyterminal residues. However,in contrast to IGF binding (Yan, et al. 2004), RXR bindingwas not inhibited by mutating the key IGF-binding determi-nants, L77, L80 and L81 (Schedlich et al. 2007a). Theseresidues are part of an LXXLL motif, sometimes termed theNR box and known to be involved in coactivator binding ofnuclear receptors (Savkur and Burris 2004). The lack ofinvolvement of these residues of IGFBP-3 in nuclear hormonereceptor binding suggests that, despite its regulatory role,IGFBP-3 should not be categorized as a nuclear receptorcoactivator, a conclusion also supported by its demonstratedinteraction with the DNA-binding domain of RXR.</p><p>LRP1 and TGF signaling</p><p>The ways in which extracellular IGFBP-3 regulates intracel-lular events apart from simply preventing IGFs from bindingand activating IGF1R have remained elusive over manyyears. IGFBP-3 association with the surface of cells has beenrecognized for over two decades (Martin, et al. 1992) but theoriginal characterization of its binding sites as functional re-ceptors (Oh, et al. 1993) was not fully substantiated. A largecell-surface protein termed the type V transforming growthfactor- receptor (TRV) was later shown to bind IGFBP-3,but again its putative role in IGFBP-3 signaling was not wellestablished (Leal, et al. 1997). This protein was subsequentlydetermined to be identical to the low density lipoproteinreceptor-related protein LRP1, also known as the 2-macroglobulin receptor, and its mediation of IGFBP-3growth-inhibitory signaling was said to involve the dephos-phorylation of insulin receptor substrate-2 (IRS-2) (Huang, etal. 2004). Another, unrelated report also described an inhibi-tory role for IGFBP-3 mediated through protein dephosphor-ylation (Ricort and Binoux 2002).</p><p>LRP1 is an endocytosing receptor and its impairment hasbeen shown to result in extracellular accumulation ofIGFBP-3, supporting a role for this receptor in IGFBP-3internalization (Lee, et al. 2006). Other studies found thatIGFBP-3 endocytosis requires a caveolin-binding structuralmotif and involves its binding to transferrin and internaliza-tion through the transferrin receptor (Lee, et al. 2004; Singh,et al. 2004). Interactions with 1 integrin and caveolin-1have also been reported in other studies (Perks, et al. 2011).Since LRP1 can bind caveolin-1 and associate with caveolae(Zhang, et al. 2004), these mechanisms are not necessarilymutually exclusive. However, a dynamin 2-dependentendocytic pathway for IGFBP-3 has also been demonstratedin osteosarcoma cells (Micutkova, et al. 2012), but thekinetics of transferrin and IGFBP-3 uptake were found tobe different, suggesting that uptake through the transferrinreceptor is unlikely in these cells. How the various proposedcellular uptake mechanisms might inter-relate and mediatethe regulation of cell signaling by IGFBP-3 remains to beelucidated.</p><p>IGFBP-3 inhibitory signaling through LRP1/TRV wasoriginally described as occurring without phosphorylation ofthe canonical TGF signaling intermediates, the Smad pro-teins (Leal, et al. 1999), but other studies showed that IGFBP-3 could activate Smad2 and Smad3 phosphorylation andrequired the type I and type 2 TGF receptors (TRI andTRII). In T47D breast cancer cells, that lack TRII, resto-ration of this receptor by transfection sensitized the cells toSmad2 and Smad3 phosphorylation, and inhibition of cellproliferation, by IGFBP-3 (Fanayan, et al. 2000). TRI wasalso phosphorylated in response to IGFBP-3, suggesting thatIGFBP-3 required an intact TGF signaling pathway forgrowth inhibition of this cell line (Fanayan, et al. 2002).Subsequently Smad activation by IGFBP-3 has been demon-strated in a variety of other cell lines. In intestinal smoothmuscle cells, IGFBP-3 was shown to stimulate TRI andSmad2 phosphorylation, resulting in the inhibition of prolif-eration (Kuemmerle, et al. 2004). Similarly in humanplacental explants, IGFBP-3 inhibition of cytotrophoblastproliferation was shown to involve Smad2 activation andhave a requirement for the TRI/TRII system (Forbes,et al. 2010).</p><p>Recently we also demonstrated that IGFBP-3 stimulatesSmad2 activation in 3T3-L1 preadipocytes, possibly...</p></li></ul>


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