The complexity of the IGF1 gene splicing, posttranslational modification and bioactivity.

The insulinlike growth factor-I (IGF-I), also called somatomedin C, is a cellular and secreted growth factor which is criti- cal for normal body growth, develop- ment and maintenance, and has impor- tant roles in multiple biological systems (1–3). A variety of cellular responses are induced by IGF-I, including cell prolifer- ation, differentiation, migration and sur- vival (4–8). These cellular responses have implicated IGF-I in several conditions such as the pathophysiology of several cancers (9–11), or the mitogenic and myogenic processes during muscle de- velopment, regeneration or hypertrophy, since, unlike other growth factors, IGF-I acts as both a mitogen and a differentia- tion factor (12,13). IGF-I is produced by many tissues, in- dicating that a significant component of IGF-I action is due to its autocrine and paracrine mode of function, although it also acts as a classical circulating hor- mone. In the endocrine mode of action, IGF-I acts as a mediator (somatomedin C) of the growth-promoting effects of pi- tuitary growth hormone (GH, soma- totropin), which induces the synthesis and release of IGF-I by the liver (2,14,15). Circulating IGF-I is mainly derived from the liver, but also from skeletal muscle (3,16–18), and is mostly bound to high affinity IGF-binding proteins, which pro- tect it from proteolytic degradation and modulate its bioavailability to the IGF-I receptors (2,19). Different IGF-I mRNA transcripts are produced as a result of the alternative splicing of the IGF1 gene, encoding for several IGF-I precursor proteins. These IGF-I protein isoforms differ by the structure of their extension peptides, or E-peptides, on the carboxy-terminal end and by the length of their amino- terminal signal peptides. However, they share the same mature peptide, which is the common part of all the IGF-I precur- sors (20–23). IGF-I mediates its actions through the binding and activation of several receptors and the IGF-I domain The Complexity of the IGF1 Gene Splicing, Posttranslational Modification and Bioactivity Anastassios Philippou,1 Maria Maridaki,2 Spiros Pneumaticos,3 and Michael Koutsilieris1 1Department of Experimental Physiology, Medical School, 2Department of Sports Medicine and Biology of Physical Activity, Faculty of Physical Education and Sport Science, and 3Third Department of Orthopaedic Surgery, Medical School, National and Kapodistrian University of Athens, Athens, Greece The insulinlike growth factor-I (IGF-I) is an important factor which regulates a variety of cellular responses in multiple biological systems. The IGF1 gene comprises a highly conserved sequence and contains six exons, which give rise to heterogeneous mRNA transcripts by a combination of multiple transcription initiation sites and alternative splicing. These multiple transcripts code for dif- ferent precursor IGF-I polypeptides, namely the IGF-IEa, IGF-IEb and IGF-IEc isoforms in humans, which also undergo posttransla- tional modifications, such as proteolytic processing and glycosylation. IGF-I actions are mediated through its binding to sever al cell-membrane receptors and the IGF-I domain responsible for the receptor binding is the bioactive mature IGF-I peptide, which is derived after the posttranslational cleavage of the pro-IGF-I isoforms and the removal of their carboxy-terminal E-peptides (that is, the Ea, Eb and Ec). Interestingly, differential biological activities have been reported for the different IGF-I isoforms, or for their E-peptides, implying that IGF-I peptides other than the IGF-I ligand also possess bioactivity and, thus, both common and unique or complementary pathways exist for the IGF-I isoforms to promote biological effects. The multiple peptides derived from IGF-I and the differential expression of its various transcripts in different conditions and pathologies appear to be compatible with the dis- tinct cellular responses observed to the different IGF-I peptides and with the concept of a complex and possibly isoform-specific IGF-I bioactivity. This concept is discussed in the present review, in the context of the broad range of modifications that this growth factor undergoes which might regulate its mechanism(s) of action. Online address: http://www.molmed.org doi: 10.2119/molmed.2014.00011 Address correspondence to Anastassios Philippou, Department of Experimental Physiol- ogy, Medical School, National and Kapodistrian University of Athens, 75 Micras Asias, Goudi-Athens, 115 27, Greece. Phone: + 30210 7462690; Fax: + 30210 7462690; E-mail: [email protected] Submitted January 18, 2014; Accepted for publication March 11, 2014; Epub (www.molmed.org) ahead of print March 12, 2014. REVIEW ARTICLE MOL MED 20:202-214, 2014 | PHILIPPOU ET AL. | 203 which is responsible for the receptor binding is the biologically active mature peptide. It is derived after the posttrans- lational cleavage of the pro-IGF-I (iso)forms and the removal of the E-peptides (23–26). Interestingly, it has been proposed that the E-peptides also possess bioactivity that is distinct from that of mature IGF-I (20,27). Thus, during the last decade, many in vitro and in vivo studies have investigated the aspect of the differential IGF-I iso- forms or their E-peptides actions in vari- ous conditions and pathologies (28–38). This concept was further supported by recent findings which revealed differen- tial, E-peptide– or IGF-I isoform–specific signaling (31,33,34,38–40). In the present review, focus has been on the propounded concept of the differ- ential roles and bioactivity of the IGF-I isoforms or peptides, in the context of the complexity that characterizes the al- ternative splicing, posttranscriptional regulation and posttranslational modifi- cations of this growth factor and which might modulate its mechanism(s) of action. HUMAN IGF1 GENE STRUCTURE AND ALTERNATIVE SPLICING The IGF1 gene spans a region of over 80 kb of genomic DNA located on the long arm of chromosome 12 in humans, it is a highly conserved sequence in mammals and primates (23), and con- tains six exons, which give rise to hetero- geneous mRNA transcripts by a combi- nation of multiple transcription initiation sites (that is, alternative leader se- quences), alternative splicing and differ- ent polyadenylation signals (41,42). These multiple IGF-I transcripts code dif- ferent precursor polypeptides, which also undergo posttranslational modifica- tions (26,43,44), (Figures 1A–C). More specifically, the different leader sequences result in two different classes of IGF-I mRNA variants: class 1 tran- scripts have their initiation sites on exon 1 (promoter 1), whereas class 2 tran- scripts use exon 2 as leader exon (pro- moter 2), and class 1 (exon 1 to exon 3) or class 2 (exon 2 to the exon 3) mRNA transcripts are produced by differential splicing of exons 1 and 2 to the common exon 3. Alternative splicing of exon 5 also results in different mRNA variants containing exon 5, generally defined as class B (IGF-IEb), or containing exon 6 (and excluding exon 5) defined as class A (IGF-IEa) (26,45,46), (see Figures 1A, B). A third variant, the IGF-IEc, which corre- sponds to IGF-IEb in rodents, also is gen- erated by alternative splicing in the human IGF1 gene and contains both exon 5 and 6 (22), (see Figure 1C). Simi- lar to the human IGF1 gene, multiple forms of pro-IGF-I mRNA have been de- scribed in other species in which IGF-I genomic sequences have been deter- mined, such as the designated pro-IGF-I Ea-1, Ea-2, Ea-3 and Ea-4 in teleosts (23,47–50). All possible combinations between promoter usage and terminal exon (5 or 6) can occur in different IGF-I transcripts (45,51,52). It has been proposed that the use of promoter 1 could be associated with the synthesis of paracrine IGF-I and may influence interactions with insulin- like growth factor binding proteins (IGF- BPs), or promote the formation of the truncated IGF-I peptide (46), (see below: IGF-I Processing, Secretion and Glycosy- lation). Transcripts initiating at promoter 1 are widely expressed in many tissues, whereas transcripts initiating at pro- moter 2 are expressed mainly in the liver (circulating forms) and kidney (53) and are thought to be more GH-dependent (23,54–58), or equally GH-responsive (59,60). However, the two promoters are probably not mutually exclusive, and GH can also stimulate the expression of tissue-specific (local) transcripts, al- though the existent evidence is still equivocal (48,61–65). IGF-IEa transcript derives from the splicing pattern exon 1 or 2–3–4–6 of the IGF1 gene, which represents the main pro-IGF-I mRNA produced in liver (sys- temic IGF-IEa) but also in other tissues with similar exon sequence (22,66), (see Figure 1A). IGF-IEb transcript is a splice variant of exon 1 or 2–3–4–5. Its expres- sion was firstly detected in the human liver (67), while it was also found to be expressed in lung carcinoma cells (20), in skeletal muscle (31,62,68) and more re- cently in various tissues and cells such as prostate, endometrium and lens epithe- lial cells (34,35,65,69), (see Figure 1B). Whether there is equivalent to human IGF-IEb splice pattern in nonhuman pri- mates is not known (23,43,70). IGF-IEc mRNA transcript is an exon 1 or 2–3–4–5–6 splice variant (see Figure 1C), which was initially identified also in human liver, where, however, it is ex- pressed approximately at 10% relative to the main IGF-IEa transcript (22). Struc- turally, its cDNA differs from the IGF-IEa variant by the presence of the first 49 base pairs from exon 5 (52 bp in rodents), It results from a splice acceptor site in the intron preceding exon 6 and, due to a reading frame shift, it gives rise to a dif- ferent carboxy-terminal peptide sequence and a premature stop codon in exon 6. This transcript was named mechano- growth factor (MGF) since it was found to be upregulated in response to muscle stretch and/or damage (71); for review see (72). However, its expression also has been identified in various tissues such as endometrium (35), normal and cancerous prostatic cells (34), as well as in osteoblast-like osteosarcoma cells (73). The biological significance of IGF-I splice variants is currently unknown and the physiological and molecular mecha- nisms that regulate their expression are unclear; however, the presence of distinct transcripts is indicative of diverse re- sponses of cells to different stimuli (74) and they probably reflect the complexity of IGF-I actions mediated via its various isoforms (75,76). Recent studies in humans have shown that the IGF-I splice variants are differen- tially transcribed in response to varying conditions and pathologies, such as exer- cise-induced muscle damage (31,68), en- dometriosis (35), and prostate (34), or cervical cancer (69), as well as in some human cell lines after hormonal treat- ment (65,73), while their differential tran- scription is possibly a function of age 204 | PHILIPPOU ET AL. | MOL MED 20:202-214, 2014 IGF-I SPLICING, MODIFICATION AND BIOACTIVITY (64,72). The differential expression of the IGF-I splice variants observed in various pathologies is of particular interest, as it could indicate distinct regulatory mecha- nisms and biological roles of the different IGF-I isoforms; however, their particular functions remain as yet unclear. The different IGF-I mRNA transcripts encode the corresponding precursor pro- teins IGF-IEa, IGF-IEb and IGF-IEc (23,44). The 5′ end, by alternative splic- ing of exons 1, 2 and 3, encodes for the signal peptide of the IGF-I prohormone. Four different transcription start sites are present in exon 1 and their positions rel- ative to the translational initiation codons (that is, Met-48 located in exon 1, and Met-25 and Met-22 located in exon 3) can give rise to three distinct IGF-I signal peptides from class 1 mRNAs (exon 1 to exon 3), (23,43). However, translation of mRNAs initiated at the four transcription start sites mentioned above is expected to produce signal sequences of 48 (tran- scription start sites 1, 2 and 3) and 25 (transcription start site 4) amino acids, since between two translation start sites contained in an mRNA, (for example, Met-25 and Met-22, located downstream of the transcription start site 4), the pref- erence is given to the upstream site (42,44,55,66), (see Figures 1A–C). It was suggested that when translation initiates at Met-25, the nucleotide sequence corre- sponding to the first 21 amino acids (en- coded by exon 1) of the sequence of the Figure 1. Gene structure, alternative splicing, amino acid sequence and posttranslational modifications of human IGF-I. Posttranslational cleavage of pro-IGF-I polypeptides can occur at a unique pentabasic motif and at sites containing the general PC consensus se- quence (panels 1A–C; potential processing sites are indicated by arrows; see text for details). Only the Ea-peptide of human IGF-I con- tains an N-linked glycosylation site. Mature IGF-I peptide residues that interact with IGFBPs are shown in orange color and underlined whereas residues that interact with IGF-IR are in purple color and marked with an asterisk (*) (panel 1A). Continued on next page REVIEW ARTICLE MOL MED 20:202-214, 2014 | PHILIPPOU ET AL. | 205 48 amino acids (aa) signal peptide may have a specific function as part of the promoter 1, or may play a role in post- transcriptional regulation of IGF-I mRNA by GH (23,55,58). From class 2 (exon 2 to exon 3) tran- scripts, three transcription start sites and their upstream position relative to the translational initiation codon Met-32 (lo- cated in exon 2) give rise to an IGF-I pre- cursor polypeptide with 32 aa long signal sequence (23,42,44), (see Figures 1A–C). The mature IGF-I peptide is coded by exons 3 (25 aa) and 4 (45 aa). The first 16 amino acids of the amino-terminal por- tion of the IGF-I E-peptide are coded by exon 4. Exons 5 and 6 encode, by alter- native splicing, distinct portions of the E-peptide with alternative carboxy- terminal sequences that contain also dis- tinct termination codons (22,44). Three different E-peptides have been identified in humans, encoded by three mRNA variants produced by alternative splic- ing of the 3 ′ end of the pre-IGF-I mRNA. Exon 4 to exon 6 mRNA splicing en- codes the Ea-peptide, which contains 35 aa. The first 16 aa, which are common in all the E-peptides, are encoded by the exon 4 and the remaining 19 are en- coded by exon 6 (44,66). Splice variant of exon 4 to exon 5 yields the Eb-peptide which, apart from the 16 common aa en- coded by the exon 4, contains 61 addi- tional aa encoded by exon 5, resulting in the 77 aa long Eb-peptide (44,67). The third mRNA splice variant, which con- tains exon 4, only 49 bp from exon 5, and then exon 6, produces the Ec- peptide with a predicted length of 40 aa, that is, 16 aa from the exon 4, 16 aa from the exon 5 and 8 aa from the exon 6 (22,44), (see Figures 1A–C). It is noted, that the last 8 aa of Ec-peptide are en- coded by exon 6, however they differ from the corresponding Ea-peptide se- quence, because of a frameshift at the splice point. Ec-peptide is thought to Figure 1. Continued. Continued on next page 206 | PHILIPPOU ET AL. | MOL MED 20:202-214, 2014 IGF-I SPLICING, MODIFICATION AND BIOACTIVITY occur by use of a cryptic IGF 633 donor splice site, which is located 49 bp down- stream from the 5 ′ end of the exon 5. When this cryptic IGF 633 donor splice site is not used, the alternative splicing of exon 4–5 occurs, that is, the Eb pep- tide (22,43). The predicted molecular mass and the residues of the three differ- ent isoforms of the human IGF-I precur- sor polypeptide as well as of their vari- ous forms and partial peptides (based on the amino acid sequence derived from the various IGF-I mRNA transcripts) are summarized in Table 1. In general, the complexity introduced by the transcriptional and splicing vari- ants, posttranscriptional regulation and posttranslational modifications of the IGF1 gene (77), giving rise to various IGF-I isoforms, probably indicate their different biological roles under various conditions or pathologies and following different stimuli (78). The development of epitope-specific antibodies for distin- guishing the different IGF-I E-peptides (20,38,79–82) can contribute to a more definitive analysis of IGF-I isoforms ex- pression in various tissues and physio- logical or pathophysiological conditions. IGF-I PROCESSING, SECRETION AND GLYCOSYLATION Post translational processing of IGF-I precursor protein may be a regulatory mechanism of the IGF-I activity, as indi- cated by the unique processing features of IGF-I precursor polypeptides that have been described (25,78). Posttransla- tional endoproteolysis of those polypep- tides produces the signal, the mature and the E-peptides (E domains). It is thought that the signal peptide at the start of a precursor is removed after facilitating the passage of the polypeptide into the en- doplasmic reticulum and the secretory pathway, with possibly no further bio- logical significance (23,83). The se- quences of the signal peptides and the E-peptides are less strongly conserved compared with mature IGF-I peptide, though to a variable extent (23). Figure 1. Continued. REVIEW ARTICLE MOL MED 20:202-214, 2014 | PHILIPPOU ET AL. | 207 The mature peptide comprises four domains, that is, the B amino-terminal domain, C and A domain and D carboxy- terminal domain, of IGF-I polypeptides (25,84). In addition, two other protein products have been identified in the human brain; the tripeptide glycyl- prolyl-glutamate (GPE) corresponding to the NH 2-terminal of the B domain of ma- ture IGF-I and a truncated IGF-I form (–3N:IGF-I) that lacks the first three amino acids of the amino terminal end of mature peptide, probably due to alter- nate signal peptides or the combined ac- tion of some peptidases (78,85,86). Re- moval of the NH 2-terminal tripeptide could be a mechanism for increasing the biological potency and availability of IGF-I, since the truncated –3N:IGF-I has less affinity for IGF-binding proteins than mature IGF-I, thus, increasing its bioactivity (78), (see Figure 1A) (see below: IGF-I Receptors and Binding Proteins). The mature IGF-I is a 70 aa long single-chain peptide and a highly con- served sequence among primate species (23,24). Cleavage of pro-IGF-I removes the carboxyl-terminal E domain and can occur at the highly conserved, unique pentabasic motif K 65-X-X-K68-X-X-R71-X- X-R74-X-X-R77. More specifically, the Arg71-Ser72 bond is cleaved followed by the removal of the Arg residue by the ac- tion a carboxypeptidase (25), (see Fig- ures 1A–C). In general, proproteins can be processed at this specific motif, usu- ally residing at the end of their pro re- gions, by proprotein convertases (PCs) such as furin (25,87,88). Furin belongs to the subtilisin-related PCs (SPCs), a major family of endoproteolytic processing en- zymes of the secretory pathway in mam- mals (89,90). Seven mammalian PCs have been identified, namely PC1, PC2, furin, PC4, PC5, paired basic amino acid cleav- ing enzyme 4 (PACE4) and PC7, and a

of prediction of the general PC- specific or furin-specific cleavage sites has been proposed (88). PCs process pre- cursors at sites usually containing the specific consensus sequence [R/K]-Xn- [R/K], where X indicates any amino acid residue, and n, the number of spacer amino acid residues, which is 0, 2, 4 or 6 (91), (see Figures 1A–C). However, furin appears to have a more stringent speci- ficity and preferentially recognizes sites that contain the sequence motif R-X- [R/K]-R (87), while R-X-X-R is its mini- mal cleavage sequence (88). Thus, apart from the cleavage site Arg 71-Ser72 for ma- ture IGF-I, another furin-mediated pro- cessing at Arg 77 has been observed in overexpression studies, which produces an extended, 76 aa long mature IGF-I (25,78), (see Figures 1A–C). PC-mediated processing of pro-IGF-I to mature peptide has been shown to occur intracellularly (92), as expected for intracellular convertases such as furin, which are located in the secretory path- way (92,93). Nevertheless, evidence has been provided that the E domains are not cleaved intracellularly (94), and the secretion of unprocessed pro-IGF-IEa iso- form, both glycosylated and nonglycosy- lated, has been reported (25,30,95–97), while there are potential proprotein con- vertases that could process pro-IGF-I ex- tracellularly (97,98). Conversion of pro-IGF-I to mature peptide cleaves off the E domains of the IGF-I precursors and one peptide is pro- duced from the human Ea domain after posttranslational processing of pro-IGF- IEa isoform (23,80), (see Figure 1A). However, the Eb domain contains po- tential processing sites (see Figure 1B), and at least two putative peptide prod- ucts of the human Eb peptide have been identified: the EB 1 peptide (residues 103–124 aa) with a C-terminal amide and the EB 2 peptide amide (residues 129–142) (20). The Ec domain also con- tains potential processing sites, from which one corresponds to the EB 1 pep- tide cleavage site and cleaves off the last eight residues of the Ec domain encoded by the exon 6 (26,82), (see Figures 1B, C). However, so far only the proform of IGF-IEc isoform and not the Ec peptide or other cleavage products have been detected (31,33–35,73,82). The Ea-peptide of human IGF-I con- tains an N-linked glycosylation site at Asn 92 based on the consensus sequence Asn-X-Ser/Thr, where X represents any encoded amino acid except proline (99,100), (see Figure 1A). However, it has been found that the human IGF-IEa pre- cursor with a signal sequence of 48 aa (Met-48) is not glycosylated, implying that the greater the number of the amino acids contained in the signal peptide the lesser the extent of a glycosylation process (101). The human Eb- and Ec-peptides Table 1. Predicted molecular mass and residues of the three different isoforms (i.e., IGF-IEa, IGF-IEb, IGF-IEc), as well as of their various forms and partial peptides, of the human IGF-I precursor polypeptide. Molecular Number of NCBI reference Class IGF-I form mass (kDa) residues (aa) sequence 1 Signal peptide 5.35 or 2.73 48 or 25 CAA24998/AAA52543 2 Signal peptide 3.51 32 NP_001104754 1or 2 Mature IGF-I 7.65 70 CAA01954 1 Pre-pro-IGF-I Ea 17.03 or 14.41 153 or 130 CAA24998/AAA52543 2 Pre-pro-IGF-I Ea 15.18 137 NP_001104754 1or 2 Pro-IGF-I Ea 11.69 105 NP_001104754 1or 2 Ea-peptide 4.05 35 AAA52543/NP_001104754 1 Pre-pro-IGF-I Eb 21.84 or 19.22 195 or 172 NP_001104755 2 Pre-pro-IGF-I Eb 19.99 179 1or 2 Pro-IGF-I Eb 16.51 147 NP_001104755 1or 2 Eb-peptide 8.87 77 NP_001104755 1 Pre-pro-IGF-I Ec 17.76 or 15.14 158 or 135 NP_001104753/EAW97695 2 Pre-pro-IGF-I Ec 15.91 142 1or 2 Pro-IGF-I Ec 12.43 110 NP_001104753/EAW97695 1or 2 Ec-peptide 4.79 40 NP_001104753/EAW97695 208 | PHILIPPOU ET AL. | MOL MED 20:202-214, 2014 IGF-I SPLICING, MODIFICATION AND BIOACTIVITY lack this cotranslational modification due to the reading frame shift resulted from the inclusion of the exon 5 in these two IGF-I isoforms (see Figures 1B, C). N-linked glycosylation involves the trans- fer of a lipid-linked tetradecasaccharide (GlcNAc 2-Man9-Glc3) to an asparagine side chain (100,102), (see Figure 1A). It oc- curs in endoplasmic reticulum and the subsequent diversification of the conju- gates occurs both in the endoplasmic reticulum and Golgi apparatus (100,102). Recently, it has been found that the gly- cosylation status of pro-IGF-IEa does not affect its processing (97), while the secre- tion of a peptide similar or identical to Ea from a human B-lymphocyte cell line, in a partially glycosylated form has been reported (80). Although the deglycosyla- tion process is expected to occur intracel- lularly, whether the deglycosylation of pro-IGF-IEa or the Ea-peptide also could be clipped extracellularly is not known. Considering the unique role of glycosy- lation in the protein biosynthesis process (100), it is possible that the Ea-peptide glycosylation might play a role in interac- tions with chaperones in the endoplasmic reticulum (78), or in regulation of the bioavailability of the different species of this IGF-I isoform (that is, pro-IGF-I Ea, mature IGF-I or Ea-peptide) (78,97,103). Thus, the existence of an N-linked glyco- sylation site in the Ea-peptide, which is absent in the Ec- and Eb-peptide, might reflect a differential and specific biologi- cal action of the IGF-IEa isoform medi- ated by this posttranslational modifica- tion of its Ea-peptide (25,43,97). Recently, it has been shown that the species of IGF-I produced by the IGF-IEa isoform has a differential ability to activate IGF- IR. Glycosylated pro-IGF-IEa is less effi- cient at receptor activation than pro-IGF-I and mature IGF-I (97), resembling the de- creased receptor- binding affinity of pegy- lated IGF-I forms (104,105) and implying that glycosylated pro-IGF-IEa may serve as a reservoir for IGF-I that can be stored until needed (97). The strong conserva- tion observed in the sequence of the Ea domain also suggests a specific biological function for the Ea-peptide (23). Nevertheless, whether the E-peptides are more stable and/or bioactive within their pro-IGF-I forms, or they are processed to act directly on their targets remains to be elucidated (see further dis- cussion in next sections). Moreover, it would be essential to verify whether the IGF-I isoforms could be released in the circulation as different proforms or E-peptides (3,79), or the final peptide that enters the circulation after extracellular endoproteolysis of the IGF-I prohormone is only the mature peptide (103,106). IGF-I RECEPTORS AND BINDING PROTEINS IGF-I actions are mediated through its binding to several receptors, such as IGF-IR (or type I IGF receptor) and IGF-IIR (or type II IGF receptor), insulin receptor (IR), and some atypical receptors such as the hybrid IR/IGF-IR (107–109). More specifically, the mature IGF-I peptide, which is responsible for binding to the receptors, binds IGF-IR with the highest affinity, IGF-IIR with low affinity and is also able to interact with IR. The IGF-IR exhibits a high degree of homol- ogy to IR (110) and, given the significant structural similarity between IGF-I and insulin, these ligands can cross-activate both receptors, while the IGF-IR signal- ing pathways share multiple intracellular mediators with the insulin signaling cas- cade (19,52). The IGF-IR/IR hybrid re- ceptor is thought to function predomi- nantly as an IGF-I receptor, since its binding affinity for insulin is lower than that for IGF-I, however the functional importance of IGF-IR/IR hybrid receptor remains poorly understood (74,109,111). It is widely recognized that most of the observed IGF-I biological effects on cell growth, differentiation, invasion and sur- vival depend on the binding and activa- tion of IGF-IR, which is a ligand-activated receptor tyrosine kinase (4,112). Specifi- cally, functional epitope mapping of IGF-I has revealed that the IGF-IR interacts with the residues 21, 23, 24, 44 as well as the ty- rosines 31 and 60, which are located in the C and A domains (113), (see Figure 1A). IGF-IR is a transmembrane protein con- sisting of two extracellular α-subunits, which contain the cysteine-rich ligand binding site, and two transmembrane β-subunits that have a cluster of three ty- rosine residues, which undergo phospho- rylation and activation upon IGF-I bind- ing (2,110,114). A structural rearrangement in the transmembrane β subunits of the receptor is caused by binding of IGF-I to IGF-IR, resulting in transautophosphory- lation of the cytoplasmic tyrosine kinase domain of the receptor, as one kinase do- main phosphorylates the other, and thus destabilizing the autoinhibitory conforma- tion within the kinase domain (115,116). This conformational change permits unre- stricted access to the binding sites for pro- tein substrates (117), thus recruiting spe- cific cytoplasmic molecules, such as insulin receptor substrate (IRS) proteins, and activating specific intracellular path- ways including Ras/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/ Akt (118). Biological actions of IGF-I are modu- lated by a family of at least six IGFBPs (52,75,119–21), which interact mainly with the residues 1–3 and 49–51 of ma- ture IGF-I (113), (see Figure 1A). In gen- eral, IGFBPs transport IGF-I and in- crease its half-life in the circulation; most of the circulating IGF-I is protected from proteolytic degradation by form- ing a ternary complex with IGFBP-3 and the glycoprotein acid-labile subunit (ALS) (74,122). IGFBPs also would be expected to modulate and control, both in the circulation and in the extracellu- lar environment, the extent of IGF-de- pendent cellular effects via regulation of free IGF-I concentration and its local bioavailability in the tissue, since IGF- BPs provide tissue specificity for the local action of IGF-I (74,119,123–126). In addition, IGFBPs compete with IGF-IR and normally have higher binding affin- ity to IGF-I than IGF-IR does. Therefore, binding of IGFBPs to IGF-I prevents the ligand from interacting with the recep- tor and, thus, suppresses IGF-I actions (74,124,125). REVIEW ARTICLE MOL MED 20:202-214, 2014 | PHILIPPOU ET AL. | 209 However, some IGFBPs can exhibit IGF-I potentiating effects and their IGF-I-inhibitory or stimulatory activities are dictated by factors such as the tissue- specific distribution of particular IGFBPs and the ratio between free (active) IGF-I and IGFBP-IGF-I bound (4,11). Moreover, it has been shown that certain IGFBPs have IGF-independent activities, imply- ing that they can modulate cell survival and apoptosis, or inhibit tumor growth in the absence of the ligand (11,127). In addition, proteolytic fragments of IGFBP-3 were reported to possess mito- genic activity in the peritoneal fluid of women with endometriosis (128). There is also a group of cysteine-rich proteins, known as IGFBP-related pro- teins (IGFBP-rPs), that share important structural similarities with the IGFBPs but they have low binding affinity to IGFs. It has been proposed that these proteins and the IGFBPs constitute an IGFBP superfamily (129,130), however the functions of the IGFBP-rPs regarding the IGFs actions are as yet unclear (131). IGF-I PEPTIDES ACTIONS AND SIGNALING Although, by the general consensus, IGF-I is thought to exert its biological actions predominantly through mature peptide, differential biological activities have been reported for the different IGF-I isoforms (propeptides), or for their E-peptides, exogenously adminis- trated or overexpressed in various in vivo (28,29,36,132–134) and in vitro mod- els (20,27,31–33,135,136), implying that there are peptides other than the IGF-I ligand that also possess bioactivity and, thus, both common and unique or com- plementary pathways exist for the IGF-I isoforms to promote biological effects (31,36). A differential expression profile of the IGF-I isoforms have been shown in vari- ous conditions or pathologies in humans, such as skeletal muscle damage (31,68), endometriosis (35) or prostate (34), cervi- cal (69) and colorectal cancer (137). The diversity and the patterns of differential expression have been proposed to reflect potential biological activities associated with the E domain peptides (27,52). Di- vergent actions and signaling of the dif- ferent pro-IGF-I forms or mature IGF-I lacking any E-peptide have been re- ported after viral-mediated expression of the IGF-I isoforms (IGF-IA and IGF-IB) in mouse skeletal muscle (36,132). Specifi- cally, it has been shown that overexpres- sion of mature IGF-I in skeletal muscle did not promote muscle hypertrophy in young mice, suggesting that the pro-IGF- I forms are required for this effect and that E-peptides, either as a part of pro- IGF-I or independent of mature IGF-I, may be necessary for IGF-I-mediated muscle hypertrophy (36). Moreover, by using the MKR transgenic mouse model where functional IGF-IRs are lacking in muscle fibers (as a dominant negative IGF-IR is expressed specifically in skele- tal muscle), it was shown that, regardless of which isoform is overexpressed, IGF-I receptors on muscle fibers are required for IGF-I-mediated hypertrophy (36). In- terestingly, however, after viral-mediated delivery of murine IGF-I isoforms into skeletal muscle, even though both iso- forms caused increased phosphorylation of the IGF-IR, increased expression of IGF-IB (murine IGF-IEb) drove both main pathways downstream of IGF-IR, that is, the PI3K/Akt pathway and the MAPK pathway, whereas IGF-IA (murine IGF-IEa) overexpression resulted in increased Akt phosphorylation only (132). These findings imply distinguish- ing IGF-I isoform–specific actions, re- gardless of any potential receptor(s) acti- vated (see also discussion in following sections). In addition, constitutively overexpression of the IGF-IEa isoform specifically in cardiac muscle was shown to protect the heart from oxidative stress via Sirtuin 1 (SirT1)⁄c-Jun N-terminal ki- nase 1 (JNK1) activity while, conversely, mature IGF-I triggered oxidative stress in the heart and did not affect SirT1 activity (138,139). Moreover, it has been shown that although both mature IGF-I and IGF-IEa propeptide triggered the phos- phorylation of IGF-IR, its downstream canonical PI3K/Akt/mTOR signaling pathway was not induced in the trans- genic mice overexpressing IGF-IEa (138,140). Instead, this specific IGF-I propeptide activated alternate signaling intermediates 3-phosphoinositide- dependent protein kinase-1 (PDK1) and serum/glucocorticoid regulated kinase 1 (SGK1), as well as SirT1. Thus, it was suggested that this downstream of IGF-I receptor(s) signaling activated by IGF- IEa employs novel pathways and that the divergent signaling mechanisms be- tween the two IGF-I forms (that is, ma- ture IGF-I and IGF-IEa propeptide) may account for their opposing effects on the heart (136,138,140). Considering particularly the bioactiv- ity of the E-peptides, in vitro studies have suggested that the E-peptides of the human IGF-I precursors may act as inde- pendent growth factors, since their syn- thetic analogs, generated from unique re- gions within the E domains, were demonstrated to possess mitogenic (20,27,34,35,73,141), angiogenic (142) and migratory activity (28,141,143), and regu- late cell differentiation (27,28) in various human cells or cell lines. Antitumor ac- tivity of human Eb-peptide also has been reported in some cancer cells (144). Similar to the results derived from human cell lines, studies using animal cell lines models have shown that exoge- nous administration or overexpression of synthetic peptides, generated from dif- ferent regions within the E domain of human (Ec) (28,31,33,145,146), trout (Ea) (144,147) or rodent (Ea and Eb) se- quences (38) in human, rodent or porcine cells in culture showed unique though inconsistent effects in promoting cell pro- liferation and migration, and in delaying or inhibiting cell differentiation. In par- ticular, actions associated with peptides derived from mammalian Ea domain have only recently been established, with rodent Ea-peptide reported to possess bioactivity (38). Furthermore, it was shown that murine Ea- and Eb-peptides increase cell entry of IGF-I from the media, providing evidence that, in addi- tion to having independent activity, they may modulate IGF-I (30). 210 | PHILIPPOU ET AL. | MOL MED 20:202-214, 2014 IGF-I SPLICING, MODIFICATION AND BIOACTIVITY The differential biological effects of the synthetic Ec-peptide compared with ma- ture IGF-I peptide, such as cell prolifera- tion versus differentiation, and the lack of suppression of the synthetic E-peptide bioactivity after blocking (mature) IGF-I signaling with IGF-IR neutralizing anti- bodies, makes it tempting to postulate that the Ec-peptide acts via a different re- ceptor (28,135,145). However, concerns have been raised about the effectiveness of the IGF-IR neutralizing antibodies to block IGF-I signaling, since they could internalize and, in this way, even activate the receptor, or change its localization, thus facilitating an E-peptide action (38). Nevertheless, the mature IGF-I bioactiv- ity appeared to be suppressed in those cells where IGF-IR neutralizing antibod- ies were used. Interestingly, an autonomous, IGF-IR- independent bioactivity has been re- ported for specific regions of the human Eb domain of IGF-I; the synthetic analog of the EB 1 peptide (see above: IGF-I Pro- cessing, Secretion and Glycosylation) was found not only to possess mitogenic ac- tivity in human bronchial epithelial cells but also to bind to specific high-affinity receptors on those cells. Furthermore, neither ligand binding was inhibited by recombinant IGF-I or recombinant in- sulin, nor did a monoclonal antibody an- tagonist to the IGF-IR suppress the prolif- erative response induced by the synthetic EB 1 peptide. It was suggested that IB1 is a growth factor that mediates its effect through a specific receptor (20). Similarly, in accordance with the well established notion that the molecular action of a bioactive peptide initiates through its specific binding to a cell surface receptor, it was demonstrated that the human Eb- peptide binds to common cell surface molecules on human neuroblastoma cells (27). Again, the saturation of mature IGF-I or insulin did not displace the bind- ing of the human Eb-peptide, suggesting the existence of distinct putative receptor components on those cells (148). Further evidence for distinct, IGF-IR- independent bioactivity of the human Ec domain was provided from its divergent signaling compared with mature IGF-I. Our group (31,33,34,65) and others (39) have shown that a synthetic analog of the human Ec peptide possesses distinct sig- naling compared with IGF-IR ligand. Spe- cifically, to distinguish any unique biolog- ical effect of the Ec domain from a potential bioactivity possessed by the common part (that is, the first 16 residues) of the IGF-I E domains, we utilized a syn- thetic Ec-peptide corresponding to the region beyond that common sequence, that is, a peptide similar to the C-terminal 24-residues, of which 16 are encoded by exon 5 and the last 8 by exon 6. It was documented that this sequence of the human Ec domain possesses distinct sig- naling since, in contrast to mature IGF-I, it activates only ERK1/2 and not Akt (31,33,34,65). Moreover, the selective acti- vation of only one of the two main signal- ing pathways downstream of IGF-I/IGF- IR and the IGF-IR- and IR- independent bioactivity of this synthetic part of the Ec domain was further documented by using the siRNA knock-out model in various human cell lines (34,35,73). More recently, synthetic E-peptides corresponding to the rodent Ea and Eb domain sequences were utilized in a ki- nase receptor activation (KIRA) assay to test IGF-I-dependent and -independent activation of IGF-IR by these E-peptides, showing that they do not directly induce IGF-IR phosphorylation in mouse fibrob- lasts (P6 cells) (38). Interestingly, how- ever, the presence of either of those E- peptides increased IGF-IR activation by IGF-I in the murine C2C12 cell line, sug- gesting that they may modulate IGF-I ac- tivity (38). Moreover, from the findings of that model, it was concluded that E- peptides signaling, as well as mitogenic and motogenic effects are dependent upon a functional IGF-IR, and their activ- ity plausibly reflects actions of pro-IGF-I (38). Further evidence has been provided recently, suggesting that IGF-I splice variants may exert their actions through mature IGF-I and not the E-peptides (149), or by supporting the bioactivity of pro-IGF-I forms (97). Furthermore, there is controversial evidence regarding the role of proforms (103,136,149) or E-peptides (38) of the murine IGF-I iso- forms, particularly in cell differentiation. Whether it reflects a differential mode of action of the free E-peptides compared with the pro-IGF-I isoforms, or potential differences between their exogenous ad- ministration and overexpression (or nat- ural endogenous production), possibly should be addressed. Collectively, the in vitro models utiliz- ing the sequences of the rodent IGF-I E domains have provided specific infor- mation regarding the bioactivity and the mode of action of those E-peptides in murine cell lines. The findings of these studies suggest that the E-peptides have little or no independent activity and, in- stead, they modulate mature IGF-I (lig- and) activity and signaling (see review in [150]). Nevertheless, since these do- mains are very variable and much less conserved, it has been indicated that the species specificity must clearly be taken into account when assessing the activity of the human IGF-I Eb and Ec domains, from which peptides with important bi- ological activities have been reported (23). Thus, it remains to be further eluci- dated whether the autonomous, IGF-IR- independent and IR-independent bio - activity, and the specific high-affinity receptor binding observed for human Eb- and Ec-peptides in various human cell lines (20,34,35,73,148) reflect an alter- native, species-specific ligand/ receptor mechanism of action for these human E domains. Species-specific models, in terms of utilizing peptide sequences that correspond to the IGF-I E domains of the species or the cells used in the model, are of particular importance to investigate physiological mechanisms of actions, such as a ligand/receptor mechanism. Moreover, it is still an elusive fact where the E-peptides signaling diverges from that of mature IGF-I (given the dis- tinct activation of ERK1/2 but not Akt pathway by the E-peptide). It has been proposed that the E-peptides may in- crease the phosphorylation of ERK1/2 by increasing the IGF-IR internalization, REVIEW ARTICLE MOL MED 20:202-214, 2014 | PHILIPPOU ET AL. | 211 which would act in favor of the MAPK arm of the IGF-IR downstream signaling (38). However, since the possibility that the E-peptides also activate ERK1/2 through an IGF-IR independent mecha- nism cannot be excluded, it remains to be further elucidated whether they af- fect the ERK1/2 pathway only at the level of the IGF-IR per se (that is, possi- bly by increasing its internalization, or by affecting its conformational change after its binding to IGF-I and, thus, acti- vating exclusively a specific pathway) or if the E-peptide–induced activation of ERK1/2 occurs at a level downstream of the IGF-IR, or even upstream, in that the E-peptide utilizes another “receptor” or cellular uptake mechanism, given the activation of ERK1/2 after exogenous administration of the E-peptide. The in- tracellular signaling pathways initiated by IGF-IR ligation may interact with signaling via G protein-coupled recep- tors or other mediators, modulating some responses (151). However, the ex- istence of such a putative, canonical or noncanonical receptor or internalization mechanism for the E-peptide remains to be determined and characterized. Alter- natively, the exclusive phosphorylation of ERK1/2 has been proposed to be a re- sult of a possible tuning of the IGF-IR signaling cascade by the E-peptide to- ward MAPK (38). The identification of the signaling pathways and effectors upstream and downstream of the E-peptide–induced activation of the extracellular-regulated kinases ERK1 and ERK2, the best- characterized members of the MAP ki- nase family (152), would provide infor- mation of particular importance regarding the mechanisms of E-peptide bioactivity. Furthermore, it is tempting to speculate that the selective activation of MAPK/ERK1/2 pathway up to a critical level by the E-peptide might further exert an inhibitory regulation of its com- petitive PI3K/Akt pathway, since it has been shown that the activity of one path- way might inactivate portions of the other (153,154), thus consisting of a regu- latory mechanism of competitive biologi- cal actions such as cell proliferation and differentiation. It should be also mentioned that, given the subcellular as well as differential local- ization of the E-peptides (94,141,155,156), an intracrine signaling mechanism that mediates E-peptides bioactivity, as pro- posed for some peptide growth factors (157), cannot be excluded.

IGF-I regulation occurs at multiple lev- els due to the alternative splicing of its pre-mRNA both at the 5′and 3′ end, and the isoform-specific co- and posttransla- tional modifications, which appear to play key roles in modulating the bio - availabilty and bioactivity of this growth factor. New evidence supports the con- cept of IGF-I isoform– or IGF-I various peptides–specific functions, possibly via specific and divergent signaling. Never- theless, it remains to be determined where the distinct E-peptide signaling diverges from that of mature IGF-I, or whether this signaling is induced uniquely and au- tonomously by the E-peptides or syner- gistically with the mature IGF-I bioactive molecule, and in the form of processed or pro-IGF-I molecules. Thus, the regulatory mechanism(s) of a potentially specific and apportioned bioactivity of the various IGF-I peptides is of particular interest within the context of a revisited character- ization of the multiple IGF-I actions. DISCLOSURE The authors declare that they have no competing interests as defined by Molecu- lar Medicine, or other interests that might be perceived to influence the results and

reported in this paper.

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