Insulin-Like Growth Factor-I

SUBCHAPTER 19B

Insulin-Like Growth Factor-I Munetaka Shimizu

Abbreviation: IGF-I Additional names: somatomedin C, non-suppressible insulinlike activity soluble in acid/ethanol (NSILA-S) IGF-I is a multifunctional polypeptide structurally related to proinsulin, IGF-I promotes cell proliferation, differentiation, growth, migration, and survival through autocrine/paracrine and endocrine pathways. It mediates part of the growth hormone actions and is essential for normal prenatal and postnatal growth.

Discovery In 1957 a “sulfation factor” that mediates the action of growth hormone (GH) on the incorporation of [35S] sulfate into cartilage segment was discovered in rats, and named as somatomedin, now known as IGF-I [1]. IGF-I was also identified in 1963 as non-suppressible insulin-like activity soluble in acid/ ethanol (NSILA-S). In 1978, IGF-I was isolated from the Cohn fraction of plasma proteins together with IGF-II.

Structure Structural Features IGF-I is a single chain polypeptide sharing high structural homology with proinsulin (about 50%) and IGF-II (about 70%) (Figure 19B.1) [1,2]. Three disulfide bonds that are involved in structural maintenance of the insulin family peptides are well conserved. PreproIGF-I is composed of a signal peptide and five domains; B, C, A, D, and E domains (Figure 19B.2). The E domain is proteolytically cleaved before secretion.

Primary Structure The aa sequence of IGF-I is highly conserved in vertebrates (B80% identical; E-Figure 19B.2).

Properties Mr B7,500, pI 8.5. Lyophilized peptide should be reconstituted in 10-mM HCl. Lyophilized peptide can be stored at 2 4 C for at least 2 years.

Synthesis and Release Gene, mRNA, and Precursor The human IGF-I gene, IGF1, location 12q22 q23, consists of six exons [2,3]. There are multiple transcript variants that differ in promoter use, transcription start site, splicing, and/or

polyadenylation (E-Figure 19B.1) [2,3]. These IGF-I mRNAs are classified based on the alternative splicing of exons encoding the E domain. In this regard, human has two IGF-I mRNAs, IGF-IA and IGF-IB. Both types encode the signal peptide of 48 or 33 aa residues depending on the transcription start site on exon 1 (class 1) or 2 (class 2), the same mature protein of 70 aa residues, and different E domains (35 aa residues for IGF-IA and 77 aa residues for IGF-IB) [2,3]. Salmon have four transcript variants alternatively spliced at the E domain. Two non-allelic genes for IGF-I have been identified in Xenopus, zebrafish, and salmon [4]. In zebrafish and tilapia, a gonad-specific igf-3 (or igf-1b) has been identified.

Distribution of mRNA In postnatal animals, the liver is the major site of expression. The IGF-I gene is also expressed widely by connective tissue cell types such as stromal cells.

Tissue and Plasma Concentrations Tissue: Tissue levels of IGF-I are much lower than serum levels, being 6 9% equivalent in rats [1]. Plasma: The characteristics, tissue distribution, and plasma concentration of IGF-I in vertebrates can be seen in Table 19B.1. The levels are approximately 50 ng/ml (childhood), 400 ng/ml (puberty), and 100 200 ng/ml (.25 years old) in humans. It is essential to extract IGFs before assay to avoid interference by IGF-binding proteins (IGFBPs) [5]. Sizeexclusion chromatography under acidic conditions is the gold standard; however, acid/ethanol extraction is most commonly used for IGF extraction due to its simplicity. After extraction, an excess amount of IGF-II is often added to saturate residual IGFBPs.

Regulation of Synthesis and Release Mammalian IGF-I genes have two promoters (P1 and P2) that lack TATA and CAAT elements [2,3]. P1 is the potent, major promoter, and is conserved widely in vertebrates. The proximate promoter region of the IGF-I gene contains binding sites for liver-enriched transcription factors such as HNF-1α, C/EBPα, and C/EBPβ. GH is the primary hormone regulating the synthesis and release of IGF-I in the liver after birth. The action of GH is mediated chiefly by the JAK2/Stat5b pathway. Several GH-inducible Stat5b binding sites have been found in introns and distal regions of the Igf1 loci. IGF-I is also regulated at the transcription level by other hormones, such as insulin, cortisol, and sex steroids, and by developmental stage independently of GH action. Nutritional status regulates IGF-I

Y. Takei, H. Ando, & K. Tsutsui (Eds): Handbook of Hormones. DOI: http://dx.doi.org/10.1016/B978-0-12-801028-0.00149-5 © 2016 Elsevier Inc. All rights reserved.

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P A R T I Peptides and Proteins in Vertebrates

Figure 19B.1 Comparison of amino acid sequence of human mature IGF-I with that of proinsulin (ProIns). Consensus amino acid residues are shaded.

Figure 19B.2 Structure of the human preproinsulin and two preproIGF-Is. Ex., exon.

Table 19B.1 Characteristics, Tissue Distribution, and Plasma Concentration of IGF-I in Vertebrates Species

# of aa

Mr

Human

70

Rat Mouse

# of Exon

Tissue (Prenatal)

Tissue (Postnatal)

Blood (ng/ml) (Prenatal)

Blood (ng/ml) (Postnatal)

7,649 12

6

liver, muscle, bone, cartilage

10 60

150 400

70 70

7,687 7 7,677 10

6 6

400 1,000 400 900

70

7,738 1

4

liver, kidney, lung, testis liver, kidney, spleen, lung, pancreas, testis liver, muscle

160 240 low

Chicken

5 20

30 50

Xenopus (I”)

70

7,796 ?

?

brain, stomach, placenta lung, stomach liver, lung, brain, kidney pancreas, brain, eye, muscle heart

?

?

Zebrafish (Ia) 70

7,747 LG4

5

liver, lung, heart, kidney, perioneal fat liver, brain, eye

?

?

Salmon

7,705 ?

5

liver, brain, kideny, gill, gonad, intestine, spleen

?

5 100

70

Gene Location

anterior part of embryo 1 (whole embyro)

mRNA at the post-transcriptional level by affecting mRNA processing and stability.

Receptors Structure and Subtype The receptor of IGF-I (type I IGF receptor, IGF-IR) belongs to a family of the receptor tyrosine kinase (RTK) containing a single transmembrane domain, and shares high sequence homology with the insulin receptor (60%). The human IGF-IR gene is located on chromosome 15q25 26, and consists of 21 exons encoding an extracellular α-subunit (706 aa residues), which contains a ligand binding domain, and a transmembrane β-subunit (627 aa residues), which contains tyrosine kinase activity (E-Figures 19B.3, 19B.4) [6]. The α- and β-subunits are synthesized as a single chain prepropeptide and cleaved after translation, bridged by a disulfide bond to form the IGF-I half-receptor (αβ). Two half-receptors dimerize to form a functional IGF-IR (α2β2). The IGF-I halfreceptor can also form a hybrid receptor with the insulin half-receptor to bind mainly IGF-I. Teleosts have two paralogs of IGF-IR.

Signal Transduction Pathway Human IGF-IR binds IGF-I and IGF-II with a high affinity (0.1 1 nM) but with a 100-fold lower affinity for insulin. Ligand binding to the α-subunit induces autophosphorylation of the intracellular domain of the β-subunit, which in turn activates the RTK. The activated RTK phosphorylates several signaling elements such as insulin receptor substrates (IRSs), which provide docking sites for signaling effectors mainly 162

through the SH domain, and those effectors branch into downstream signal pathways. The two main pathways are the PI3K/Akt pathway, which induces many metabolic responses, and the RAS/RAF/MAPK pathway, which generally controls cell growth and proliferation. IGF-IR is also capable of translocating into the nucleus and acts as a transcription factor.

Agonists IGF-II, insulin, Des [1 3] IGF-I, Long R3 IGF-I (reduced binding to IGFBPs), and LL-37 are agonists.

Antagonists IGFBPs, JB1 and JB3 (12-aa synthetic peptides), and M1557 (D domain analog) are antagonists.

Biological Functions Target Cells/Tissues and Functions IGF-I acts on most tissues, but liver may not be a major target. IGF-I is involved in growth and metabolism at the organismal level, and in cell proliferation, migration, differentiation, and survival at the cellular level. IGF-I inhibits apoptosis. An important role of circulating IGF-I is to regulate GH synthesis/secretion at the pituitary and hypothalamus through a negative feedback loop [7]. A single IGF-I allele is responsible for small size in dogs. In euryhaline fish, IGF-I is involved in the development of hypo-osmoregulatory ability by acting on osmoregulatory organs such as gills.

S U B C H A P T E R 1 9 B Insulin-Like Growth Factor-I Phenotype in Gene-Modified Animals The growth of Igf1-null mice is severely retarded (30% of wild adult size) and they become infertile. Null mutants for the Igf-1r die shortly after birth. Studies using conditional Igf1 knockout mice, in which the hepatic expression of Igf1 was inactivated, showed that liver-derived IGF-I is the principal source of endocrine IGF-I (about 75%) but is not required for normal postnatal growth [7,8]. An important contribution of endocrine IGF-I to postnatal longitudinal bone growth (about 30%) has been revealed by knocking-in an Igf1 cDNA into Igf-1 null mice [8].

Pathological Implications Clinical Implications IGF-I deficiency is related to Laron syndrome (short stature due to GH resistance or insensitivity), liver cirrhosis, and agerelated cardiovascular and neurological diseases [9]. Epidemiologic studies suggest relationships between IGF-I and cancer risks such as prostate, colon, and breast cancers.

Use of Diagnosis and Treatment IGF-I levels are routinely used for diagnosis in patients with suspected acromegaly or GH/IGF-I deficiency. The US Food and Drug Administration has approved recombinant human IGF-I for treatment of IGF-I deficiency patients. Due to its anti-apoptotic and proliferative actions, inhibition of the IGF-I

signaling using antagonists or antibodies is a potential therapy for cancers and atherosclerosis [10]. References 1. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrine Rev. 1989;10:68 91. 2. Potwein P. Molecular biology of IGF-I and IGF-II. In: Rosenfeld RG, Roberts CT, eds. The IGF System: Molecular Biology, Physiology, and Clinical Applications. Totowa NJ: Humana Press; 1999:19 35. 3. Oberbauer AM. The regulation of IGF-1 gene transcription and splicing during development and aging. Front Endocrinol. 2013;4:39. 4. Wood AW, Duan C, Bern HA. Insulin-like growth factor signaling in fish. Int Rev Cytol. 2005;243:215 285. 5. Rikke H, Frystyk J. Determination of IGFs and their binding proteins. Best Practice Res Clin Endocrinol Metab. 2013;27:771 781. 6. Chitnis MM, Yuen JSP, Protheroe AS, et al. The type 1 insulin-like growth factor receptor pathway. Clin Cancer Res. 2008;14: 6364 6370. 7. LeRoith D, Bondy C, Yakar S, et al. The somatomedin hypothesis: 2001. Endocrine Rev. 2001;22:53 74. 8. Ohlsson C, Mohan S, Sjgren K, et al. The role of liver-derived insulin-like growth factor-I. Endocrine Rev. 2009;30:494 535. 9. Puche JE, Castilla-Corta´zar I. Human conditions of insulin-like growth factor-I (IGF-I) deficiency. J Trans Med. 2012;10:224. 10. Clemmons DR. Modifying IGF1 activity: an approach to treat endocrine disorders, atherosclerosis and cancer. Nat Rev Drug Discov. 2007;6:821 833.

Supplemental Information

E-Figure 19B.1 Gene and precursor structures of the human IGF-I. Human IGF-I: IGF-I, location 12q22 q23. P, promoter; C, class; Ex., exon; Sp, signal peptide. Arrows on exons indicate transcription start sites. Asterisks indicate polyadenylation cites. †, “Muscle-type” peptide, also called mechano-growth factor (MGF). Accession numbers: gene, 3479; cDNA transcript variant 1, NM_001111283; transcript variant 2, NM_001111284; transcript variant 3, NM_001111285; transcript variant 4, NM_000618.

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P A R T I Peptides and Proteins in Vertebrates

E-Figure 19B.2 Mature hormone sequences of various animals. Domain names are indicated above; consensus/major amino acid residues are shaded. The sequences of mature IGF-Is are from the following accession numbers: human, AAA52538/AAA52539; rat, AAA41386; mouse, NP_034642; chicken, NP_001004384; frog, NP_001156865; zebrafish, NP_571900; salmon, AAA49410; tilapia, CAA71789; shark, CAA90412.

E-Figure 19B.3 Gene, mRNA, and precursor structures of the human IGF-IR. Human type I IGF receptor: IGF-IR, location 15q25 26. Sp, signal peptide. Accession numbers: gene, 3480; cDNA, NM_000875.

e19B-2

S U B C H A P T E R 1 9 B Insulin-Like Growth Factor-I

E-Figure 19B.4 Primary structure of the human IGF-IR. Asterisks indicate cysteine residues important for the IGF binding; an open box indicates the proteolytic cleavage site (CS) between α- and β-subunits. Signal peptide and transmembrane domain are underlined.

e19B-3

P A R T I Peptides and Proteins in Vertebrates E-Table 19B.1 Accession Numbers of Genes, cDNAs, and Proteins for IGF-I and IGF-IR Species

Type

Gene/cDNA Accession#

Human Human

Gene cDNA

3479 NM_001111283 NP_001104753

cDNA

NM_001111284 NP_001104754

cDNA

NM_001111285 NP_001104755

cDNA

NM_000618

NP_000609

Human

IGF-I IGF-IC (transcript variant 1) IGF-IIA (transcript variant 2) IGF-IB (transcript variant 3) IGF-IA (transcript variant 4) IGF-IA

cDNA

AAA52538

Human Rat Rat

IGF-IB IGF-I IGF-Ia

cDNA cDNA cDNA

Rat

IGF-Ib

cDNA

Rat

IGF-IIC (isoform a) IGF-IIA (isoform b) IGF-IB (isoform c) IGF-IA (isoform d) IGF-IB (isoform 1) IGF-I (isoform 2) IGF-IIB (isoform 3) IGF-IA (isoform 4) IGF-IIA (isoform 5) IGF-I IGF-IA IGF-1 (IGF-1a) IGF-3 (IGF-1b) IGF-I IGF-I

cDNA

M12659 M14153 M14154 M14156 M11568 M17335 M15647 M15648 M15649 M15651 M15647 M15648 M15649 M15650 NM_001082477

NP_001075946

cDNA

NM_178866

NP_849197

cDNA

NM_001082478 NP_001075947

cDNA

NM_001082479 NP_001075948

cDNA

NM_010512

NP_034642

cDNA

NM_184052

NP_908941

cDNA

NM_001111274 NP_001104744

cDNA

NM_001111275 NP_001104745

cDNA

NM_001111276 NP_001104746

cDNA cDNA cDNA

NM_001004384 NP_001004384 NM_001163393 NP_001156865 NM_131825 NP_571900

cDNA

NM_001115050 NP_001108522

cDNA cDNA

M32792 Y10830

AAA49410 CAA71789

IGF-I IGF IGF-IR IGF-IR IGF-IR IGF-IR IGF-IR IGF-IR IGF-IRa IGF-IRb

cDNA cDNA Gene cDNA cDNA cDNA cDNA cDNA cDNA cDNA

Z50081 AB081462 3480 NM_000875 NM_052807 NM_010513 NM_205032 NM_001088265 NM_15298 NM_152969

CAA90412 BAC15764

Human

Human Human

Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Chicken Xenopus Zebrafish Zebrafish Coho salmon Mozambique tilapia Spiny dogfish Sea lamprey Human Human Rat Mouse Chicken Xenopus Zebrafish Zebrafish

e19B-4

Protein#

AAA52539 AAA41386 AAA41215

AAA41214

NP_000866 NP_434694 NP_034643 NP_990363 NP_001081734 NP_694500 NP_694501