Expression of the insulin-like growth factor-I gene and its products: complex regulation by tissue specific and hormonal factors

DOMESTIC ANIMAL ENDOCRINOLOGY

Vol. 8(2):165-178, 1991

EXPRESSION OF THE INSULIN-LIKE GROWTH FACTOR-I GENE AND ITS PRODUCTS: COMPLEX REGULATION BY TISSUE SPECIFIC AND HORMONAL FACTORS 1,2 F.A. Simmen Dairy Science Department University of Florida Gainesville, FL 32611-0701 Received December 28, 1990

INTRODUCTION Insulin-like growth factor I (IGF-I) is a single chain, 70 amino acid peptide with mitogenic and differentiative properties in vivo and in vitro (1, 2, 3). A large body of data have substantiated the importance of IGF-I in regulation of embryogenesis and fetal development, and in postnatal growth, hematopoiesis, ovarian function and other physiological processes (1, 2, 3, 4). In view of this, it is perhaps not surprising that IGF-I production is regulated in an exceedingly complex fashion. In this article, I summarize the current data as regards the tissuespecific and hormonal regulation of IGF-I biosynthesis. In so doing, I have attempted to integrate results of published studies from all animal models to illustrate those aspects of IGF-I expression that are unique to a particular species, as well as those that are evolutionarily conserved. It is the view of this author that studies of rat IGF-I expression, while of obvious importance, may not by themselves fully define regulatory mechanisms underlying IGF-I expression in other mammalian and nonmammalian species. In particular, more extensive use of domestic species as models for such studies seems warranted. IGF-I Expression - General Aspects. Circulating IGF-I is synthesized as a larger precursor protein, containing NH2- and COOH-extension peptides (signal and extension (E) peptides, respectively) (Figure 1). During biosynthesis, the preprotein segments are post-translationally cleaved in an as yet unknown manner to generate mature IGF-I (1). Molecular cloning and sequencing of a large number of cDNAs encoding IGF-I precursors (Table 1) revealed intra- and inter-species sequence heterogeneity within the 5'-untranslated regions (5'UTRs) and putative signal (leader) peptides (Figure 1A). Heterogeneity in 5'-UTRs among IGF-I mRNAs results from differential splicing of RNA precursors and perhaps from differential use of multiple promoters of the IGF-I gene by RNA polymerase II. Two types of COOH-terminal E peptides (designated -IA, -IB) were identified by sequencing of molecularly cloned human and rodent IGF-I cDNAs (Figure IC and 1D, Table 1). The human E peptide variants (IA, IB) are translated from distinct mRNAs generated by differential use of genomic exons, whereas the rodent E peptide variants (IA, IB) result from distinct mRNAs characterized by the presence or absence of a 52 base pair sequence insertion (1). In rat IGF-I cDNAs, there are at least 3 different 5'-UTR sequences represented (denoted as classes A, B, and C), each of which exhibit upstream, in-frame putative initiation codons (ATGs) (5). Heterogeneous 5'-UTRs probably exist in IGF-I mRNAs of other species as well (Figure 1A). Interestingly, in all IGF-I 5'-UTR sequences elucidated to date, there are a minimum of two in-frame initiation codons upstream of the IGF-I peptide coding region which, in theory, could function in vivo (Figure IA). The met -48 codon functions preferentially over met -25 and -22 codons when in vitro translation of human IGF-I mRNA is performed (6). A DNA construct containing the mouse mammary tumor virus promoter and gene regulatory sequences fused to porcine IGF-I cDNA containing the met -25 codon, but lacking the upCopyright © 1991 by Domendo, Inc.

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0739-7240/91/$3.00

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SIMMEN

stream sequences (i.e., met -48 codon) is functional with respect to synthesis and secretion of IGF-I when transfected into animal cells (E A. Simmen, unpublished observations). It is at present unclear whether use of multiple initiation codons occurs in viw~ and, if so, how this might relate to synthesis, intracellular routing and posttranslational processing of IGF-I precursor proteins of differing length. The IGF-IA and IGF-IB precursor proteins arise by different molecular mechanisms in human versus rodent tissues (1, 7, 8, 9, 10). The IGF-IA E peptide sequences elucidated to date are highly conserved (Figure IC). This includes IGF-IA sequences from mammalian, avian and piscine species. IGF-IB E peptide sequences, deduced from cloned cDNAs, are currently only available for human, rat and mouse (Figure 1C). Significant sequence and length differences are apparent for the human and rodent IGF-IB E peptide sequences (Figure IC), reflecting the different modes of generation. In all known IGF-IA type E peptides, there exists a potential N-linked glycosylation site (Asn-X-Ser/Thr) centered at residue 94 (11 ; Figure 1C). Rodent IGF-IA type E peptides contain a second possible glycosylation site centered at residue 102 (11, Figure 1C). hz vitro, these sites can be glycosylated (11). IGF-IB E peptides, in contrast, do not exhibit consensus glycosylation sites (11 ). Thus, differential glycosylation, if it occurs, provides a mechanism whereby nascent IGF-I precursor proteins might be distinguished within the cellular contexts of synthesis and secretion ( 11 ). Circulating IGF-I concentration is a heritable characteristic ( 12, 13, 14, 15). In mice, a heritability of 0.4 _+ .27 for plasma IGF-! concentration at postnatal day 35 was estimated (12). High and low IGF-! secreting lines of mice, selected over seven generations, had 85 _+2 or 58 _+ 2 ng/ml IGF-I, respectively at day 42, postnatal (13). These studies strongly suggest that variation in plasma IGF-I concentrations among individuals has a genetic basis. This likely represents differences in IGF-I gene activity, IGF-I protein synthesis and secretion and/or differences in circulating concentrations of specific IGF-binding proteins among individuals. I G F - I Gene Expression. The human IGF-I gene resides on chromosome 12, in the genomic region 12q22 ~ q24. I (16, 17, 18, 19, 20). The chromosomal IGF-I gene in humans and rats is relatively large (at least 90 Kb), discontinuous in structure (8, 2 I, 22, 23, 24) and is physically linked to the phenylalanine hydroxylase gene (20). Current effort is aimed at identifying the locations and sequences of the one or more promoters, transcriptional initiation sites, and flanking sequences of the rat and human genes. The bovine IGF-I gene is located on bovine chromosome 5 (25). This bovine IGF-I gene region is evolutionarily conserved with the corresponding region of human chromosome 12 (25). Restriction fragment length polymorphisms (RFLPs) have been described lbr the IGF-I locus in humans and cows (8, 17, 18, 20, 25, 26). Attempts to link particular IGF-I RFLPs to differences in growth rate, growth disorders (e.g., Laron dwarfism, Pygmy condition) or to particular carcass characteristics are ongoing (25, 26). The 1GF-I gene is transcribed into a complex array of mRNA species it2 vivo (Fig. 2). In adult rats, the order of IGF-I mRNA abundance (per ug total RNA) in tissues (summed for all IGF-I specific transcripts) is liver > uterus > ovary > lung > kidney > heart > skeletal muscle > testes, brain > mammary gland (27). Thus, in adult rats, it is reasonable to conclude that liver is the major source of circulating IGF-I (27). In growing and adult swine, liver IGF-I mRNA abundance typically is exceeded by that of skeletal and cardiac muscle (28), uterus (29) and adipose tissue (T.G. Ramsay; personal communication). The respective contributions of nonhepatic tissues to circulating IGF-I remains to be determined. In this regard, fetal liver IGF-I mRNAs are expressed at low or undetectable levels (30, 31, 32, 33), despite the presence of physiologically significant amounts of IGF-I in sera (34). Tissue-specific regulation of expression is evident for the 5' UTRs and -IA and -IB sequences in IGF-1 mRNAs (35, 36). Class C 5' UTRs are the most abundant in rat tissues. Class A 5' UTRs are found in moderate abundance in liver, are low in abundance in kidney, lung, testes and stomach and are undetectable in muscle, heart and brain (35L while Class B

COMPLEX REGULATION OF IGF-I EXPRESSION

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IGF-I Leader Seouences

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Fig. 1. Phylogenetic comparison of IGF-I precursors. A) Amino-terminal IGF-I leader (signal) sequences aligned for maximum homology. Putative initiator methionines are boxed and the conserved alanine residue flanking the amino terminus of the mature IGF-I peptide (Panel B) is denoted as -1. B) The entire 70 amino acid sequence of human IGF-I is presented and amino acid substitutions in IGF-I peptides of the other species are indicated. G (I) and A (70) represent amino- and carboxy-tenrtinal glycine and alanine residues, respectively. C) IGF-IA carboxy-terminal extension peptides. Amino acid substitutions relative to the human sequence are indicated. The arrow indicates where a 27 amino acid region in the salmon sequence has been omitted (70). Brackets indicate sites for glycosylation. D) IGF-IB carboxy-terminal extension peptides. Amino acid substitutions are indicated.

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5' UTR sequences are found only in liver (35). In rat liver, IGF mRNAs encoding IGF-IB precursor proteins comprise 13% of the total IGF-1 transcripts (36). In other tissues, these sequences are typically at a much lower proportion (2-5%) (36). Comparable data for other TABLEI. ISOt.ATION,:)[GENOMI("ANI)COMPLEMENTARYDNA (cDNA) SEQtTENCESENCODINGVI-RTEBRAFEIGF-I PRECURSORPROTEINS. Species

DNA

Tissue source for cDNA laver Leiomyosarcoma

References

Human Human Human

cDNA eDNA Genomic

Bovine

eDNA

Liver

112

Ovine

cDNA

Liver

113

Porcine Porcine

Genomic eDNA

Liver

114 29, 114

Mouse

eDNA

Liver

9

Rat Rat Rat

Genomic cDNA cDNA

Liver Testis

23 5. 10, 115, 116 I 17

Xenopus laevis

Genomic

I 18

Chicken Chicken

Genomic cDNA

45 119

Sahnon

eDNA

Liver

7, 20, 110, I I I 104 8, 20, 2 I. 22, 24

70

species is unavailable. Within a particular size class of IGF-I mRNAs (Figure 2), there exists marked sequence heterogeneity as reflected by the presence of different 5'-UTRs and -IA and -1B peptide encoding segments (30, 35, 36). I G F - I Expression During Development. IGF-I is strongly implicated in the control of embryonic, fetal and postnatal development and in fetal-maternal interactions essential to prenatal development (1, 4, 33, 34, 37, 38, 39). In pregnant women, serum IGF-I concentrations rise to a maximum during the last trimester (40, 41,42). There is then a striking decline in IGF-I concentrations immediately postpartum (40, 41, 42). In one study, maternal IGF-I concentrations during the last trimester were highly correlated with serum concentrations of a placental growth hormone variant (41). Placental tissue is characterized by abundant expression of IGF-I mRNAs (43); however, human placental IGF-I mRNA levels are maximal during the first and second trimesters (43). The temporal disparity in placental IGF-1 mRNA expression and maternal serum IGF-I concentration suggests a role for placental IGF-I in fetal-placental growth rather than in maternal endocrine function. Regulatory mechanisms associated with placental IGF-I expression in any species have not been clearly defined. Uterine IGF-1 synthesis may contribute to the maternal serum IGF-I pool as well as support uterine, embryonic and fetal growth (4, 27, 29, 38). In adult, nonpregnant female rats, the uterus is second only to liver in IGF-I mRNA abundance (27). Similarly, uterine endometrium and myometrium of domestic species (e.g., pigs, cows and sheep) express IGF-I mRNAs at easily detectable levels (38, 44, R.D. Geisert, personal communication, F.A. Simmen, unpublished observations). In pigs, endometrial IGF-I mRNA abundance is maximal and exceeds that of most other maternal tissues during early pregnancy (38, 44, F.A. Simmen, unpublished observations). These results indicate enhanced and temporally regulated IGF-I gene expression in utero during pregnancy. Preimplantation pig blastocysts express IGF-I mRNAs (44), as do blastoderm and gastrula stage chicken embryos and early postimplantation mouse embryos (45, 46). In addition, IGF-

COMPLEX REGULATION OF IGF-I EXPRESSION

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I m R N A s are expressed at low abundance in multiple tissues of rat (30, 31, 37, 47, 48, 49), mouse (39), pig (33), chicken (45) and h u m a n (50) fetuses. IGF-I m R N A abundance in many, but not all fetal tissues is developmentally regulated. In the rat, IGF-I m R N A s in liver,

TOP a

b

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Fig. 2. IGF-I messenger RNAs: Schematic of a typical Northern blot- hybridization of rat liver IGF-I mRNAs ( 10, 30, 35, 36, 57, 65, 120, 121). The four size classes of IGF-I mRNAs commonly observed are labeled in order of decreasing molecular weight. Band width reflects the typical autoradiographic intensity observed. Band a represents IGF-I mRNAs of 7.0 to 7.5 kilobases (Kb) in length, containing an unusually long (6.2-6.7 Kb) Y-UTR. This size class displays heterogeneity in 5' UTR sequences and encodes both IGF-IA and IGF-IB protein precursors. It is speculated that the unusually long 3' UTR renders these transcripts more unstable in vivo allowing for preferential and more rapid turnover (120, 121). b represents a minor mRNA class of 4.7 Kb in length, c represents 1GF-I mRNAs of 1.5 to 1.9 Kb containing 3'-UTRs of 1.2 to 1.4 Kb. d represents mRNAs of 0.8 to 1.2 Kb with 3'-UTRs of 0.2 to 0.5 Kb. The IGF-I mRNAs of 2.0 Kb or less in length are characterized by heterogeneity in 5'-UTRs and carboxy-terminalextension peptides. The largest class of rat IGF-I transcripts is also observed in human, cow, sheep, pig and mouse tissues, but not in salmon or chicken liver (70, 119). The smaller size classes are also evident in tissues of the domestic species and humans. heart, and kidney are low in a b u n d a n c e at birth and increase postnatally, whereas IGF-I m R N A levels in stomach, muscle, brain and testis are highest in the late fetal/early neonatal period and exhibit a subsequent decline postnatally (48, 49). L u n g IGF-I m R N A levels, in contrast, r e m a i n c o n s t a n t throughout rat d e v e l o p m e n t (47). In the h u m a n fetus, I G F - I m R N A s were localized to connective tissues or to cells of mesenchymal origin in m a n y organ systems (50). Serum IGF-I concentrations exhibit characteristic and unique changes during pre- and postnatal development. In sheep, fetal serum IGF-I doubles from mid- to late-gestation, then further increases to a m a x i m u m during the neonatal period (51, 52, 53). IGF-I levels in neonatal sheep serum exceed that for fetal, pubertal and adult sheep. In contrast, no gestational changes in serum IGF-I were detected in fetal guinea pigs (54). In pigs, fetal serum IGF-I increases 3-fold during the second half of gestation and an additional 6-fold during the first 6 weeks o f postnatal life (34). In fetal sheep, IGF-I levels are probably only partially dependent on fetal pituitary G H secretion (53, 55). The relevance of a functional hypothalamic-pituitary axis (see below) to fetal IGF-I production in species other than sheep is presently unclear.

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The peri-pubertal surge in serum IGF-I in rats has been temporally associated with onset of production of gonadal steroids (reviewed in 1). However, paradoxically, castration of prepubertal rats results in increased rather than decreased pubertal serum IGF-I (56). In addition, treatment of newborn rats with monosodium glutamate (hypothalamic neurotoxin) abolishes the subsequent pubertal rise, whereas treatment on day 5 postnatal is without this effect (56). These results point to the critical involvement of early postnatal events in the subsequent surge of circulating IGF-I in rats (56). The rise in serum IGF-I is concordant with the postnatal increase in abundance of rat liver IGF-I mRNAs (57). Pituitary Control of IGF-I Production. IGF-I is inhibitory to Growth Hormone Releasing Hormone (GHRH) elicited Growth Hormone (GH) release from pituitary gland (58, 59) and to GHRH release from hypothalamus (60). The negative regulation of GH by IGF-I provides a feedback loop to tightly control serum GH and IGF-I concentrations. Thyroid hormones do not directly affect 1GF-I synthesis, but by virtue of their role in maintenance of GH secretion, indirectly affect IGF-I mRNA and protein levels (61,62). A plethora of papers have described the role of GH as an inducer of IGF-I production at the level of its mRNAs ( 10, 63, 64, 65, 66). GH induction of tissue IGF-I mRNA steady-state levels is observed in pituitary intact and hypophysectomized animals and in cell culture. Tissues responsive to GH in this way include rat liver, kidney, heart, lung, skeletal and cardiac muscle, ovary, uterus, testis, brain and pancreas (10, 63, 64, 65, 66, 67, 68, 69) and salmon liver (70) in vivo, and porcine preadipocyte and granulosa cells in vitro (71,72). The inductive effect of GH has been localized in part to the level of nuclear transcription of the IGF-1 gene (63). GH binding to cell-surface receptors leads to an activation of IGF-I transcription, thus enhancing IGF-1 mRNA levels (63). Hypophysectomy results in reduced IGF-I mRNA levels in multiple tissues (64, 65). 1GF-I mRNAs containing different 5' UTRs appear to be independently regulated in a tissue-specific fashion by GH (35). Similarly, levels of IGF-IA and IGF-IB encoding mRNAs were coordinately increased by GH in kidney, lung and heart, whereas IGF-IB transcripts were preferentially induced by GH in liver (36). The above studies suggest complex regulation by GH at the levels of IGF-I transcription and RNA processing. The known lactational effects of GH are probably mediated in part by mammary expressed IGF-I. GH receptor RNA transcripts are manifest in mammary epithelial and stromal cells (73, 74) and IGF-I mRNAs are expressed in the mammary stroma (74, 75). By analogy with other tissues, a local induction of mammary IGF-I by circulating GH may contribute to enhanced growth and differentiation of mammary secretory cells, a known target cell for IGF-I action. In addition, temporally regulated expression of mammary IGF-I mRNAs probably underlies normal mammary development during puberty and pregnancy (76, 77). Nonetheless, published studies providing direct evidence of such linkages do not currently exist. I G F - I Regulation by Steroid Hormones. Like GH, steroid hormones are potent regulators of IGF-I biosynthesis in reproductive tissues and liver. In uterus, estrogens and progesterone induce IGF-I mRNA abundance and tissue IGF-I protein content (68, 78, 79, 80, 81). Induction of IGF-I mRNA and protein synthesis by estrogen is rapid and relatively specific for uterus and ovary (78, 81, 82). IGF-I mRNA levels are increased by estrogen in all compartments (i.e., myometrium, endometrial stroma and epithelium) of the rat uterus (80). In developing ovarian follicles, IGF-I mRNAs are primarily expressed in the mitotically active granulosa cells (82, 83). Administration of estrogen to immature or immature, hypophysectomized rats results in 1.5 to 2-fold induction of ovarian IGF-I mRNA steady state levels (82, 83). In vitro, estrogen stimulates release of immunoreactive IGF-I by cultured porcine granulosa cells (84). In contrast to effects of estrogen and progesterone, dexamethasone, a synthetic glucocorti-

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coid, inhibits IGF-I gene expression. In cultured neonatal rat neuronal and glial cells, dexamethasone caused a rapid (within 3-6 hr) reduction (50%) in levels of all sequence classes of IGF-I mRNAs (85). Similarly, in intact rats, dexamethasone treatment led to reduced levels of tissue IGF-I mRNAs (86). Like most other regulatory phenomena associated with the IGF-I locus, dexamethasone inhibition of IGF-I gene expression varies by tissue and hormonal status (86). IGF-I Regulation by Growth Factors and Other Hormones. A limited number of in vitro studies have demonstrated regulation of IGF-I production by other growth factors. In cultures of human fibroblasts, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) induced production (synthesis and/or secretion) of immunoreactive IGF-I (87). Somewhat different results were obtained using cultured pig granulosa cells, in which EGF and transforming growth factor (TGF)-et were stimulatory, whereas PDGF and FGF had no effect by themselves (88). TGF 13-1 stimulated IGF-I production by osteoblasts and inhibited IGF-I release from chondrocytes (89). Basic FGE in contrast, is stimulatory to IGF-I production by chondrocytes (89). Other factors implicated as positive regulators of IGF-I production include placental lactogen (40) and vitamin A (90). Knowledge of the levels at which these factors exert their effects (i.e., IGF-I mRNA synthesis, mRNA processing, protein synthesis and secretion) in the various systems is lacking. Nutritional Regulation of IGF-I. Energy and nutrient intake are important determinants of circulating IGF-I concentrations and of tissue IGF-I mRNA levels (91). Fasted rats exhibit decreased expression of IGF-I mRNAs in liver (92, 93) and other tissues (93). Fasting causes a coordinate reduction in all of the hepatic IGF-I mRNA transcript size classes, whereas refeeding leads to rapid and coordinate induction of all hepatic IGF-I mRNAs (92). Interestingly, the effects of a 48-hr fast on IGF-I mRNA abundance may vary by tissue (93). Specifically, IGF-I mRNA levels are more affected in rat liver and lung than in heart. Similarly, fasting of juvenile swine causes disproportionate reductions in steady-state levels of muscle, liver and heart IGF-I mRNAs (28). The amount of protein intake can also affect IGF-I mRNA levels (94). Low protein intake is associated with a reduction in IGF-I gene transcription. Furthermore, the coupling of protein intake and hepatic IGF-I production is age-dependent. Specifically, younger rats are more affected in this regard than are older rats (95). Maternal starvation caused a decline in fetal plasma IGF-I in sheep, an effect reversible by glucose infusion of the dam (96). Diabetes. Streptozotocin-induced diabetes in animal models has provided a means to explore the interrelationships of IGF-I and diabetes-associated growth retardation. Streptozotocin-treated rats have lowered hepatic IGF-I mRNA and serum IGF-I levels (97, 98). IGF-I mRNA levels are also suboptimal in other tissues of diabetic rats (99). In diabetic animals, GH evidently cannot induce IGF-I mRNAs (99), whereas insulin therapy restores liver IGF-I mRNA levels to normal (99, 100). In juvenile swine, streptozotocin treatment caused a 50% reduction in levels of muscle and liver IGF-I mRNAs and a 70% reduction in levels of heart IGF-I mRNAs (28). As for rats, insulin therapy restored IGF-I mRNA levels to normal (28). The molecular basis for this effect of insulin has not been elucidated. IGF-I Expression During Tissue Regeneration. Rats exhibiting compensatory renal hyperplasia, after the unilateral nephrectomy procedure, have enhanced kidney IGF-I mRNA abundance (101). This augmented expression is local in nature since elevated levels of liver IGF-I mRNAs or serum GH and IGF-I are not evident (101). IGF-I protein content in kidney also rises during compensatory hyperplasia (102). In rat skeletal muscle undergoing growth after injury, a rapid increase in muscle IGF-I mRNA abundance is similarly observed (103). Thus, regulatory mechanisms exist whereby IGF-1 expression is induced specifically in regenerating or growing tissues. The increased tissue IGF-I content, in all likelihood, acts locally to stimulate tissue growth and/or differentiation.

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SIMMEN

IGF-I Expression in Neoplasia. Levels of IGF-I mRNAs are enhanced in benign leiomyoma and malignant leiomyosarcoma tumors of smooth muscle origin (104). Further, cells derived from a subset of colon carcinomas and liposarcomas exhibit altered abundance of specific IGF-I mRNA classes when compared to normal counterpart tissues (105). The nature of the molecular mechanisms underlying augmented IGF-I mRNA expression in certain tumor cells is unknown. Similarly, the involvement if any, of IGF-I in cellular transformation and tumorigenesis remains to be elucidated. Nature of Regulatory Pathways Affecting IGF-I Production. IGF-I biosynthesis is characterized by an unusual degree of complexity. Recent results highlight the potential regulation of IGF-I production at multiple levels by a variety of endocrine, paracrine, developmental, and other factors (Figure 3). Many endocrine and paracrine factors work through Level of Regulation

?. . . . . .

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Fig. 3. Regulated IGF-I expression. The diagram illustrates steps at which IGF-I biosynthesis is possibly regulated. The actions of the regulatory agents as indicated are for the most part hypothetical.

generation of intracellular second messengers which subsequently lead to activation of gene transcription and/or posttranscriptional processes (106). The nucleotide sequence of the IGF-I gene promoter(s) and 5'-flanking region(s) is not known. Nonetheless, one can speculate that this region of the gene will be richly endowed with DNA sequence elements that bind multiple classes of nuclear, transcriptional factors. Consensus DNA sequence elements for binding of estrogen, progesterone and glucocorticoid receptors, interspersed among binding sites for other nuclear transcriptional activators such as members of the c-Jun/c-Fos protein family (growth factor inducible) and the cAMP-response element binding proteins (CREBs) (106) are likely to flank the IGF-I promoter. The transcriptional activation of this gene by GH probably occurs via the intermediate involvement of one or more second messengers. In addition, DNA enhancer sequences and tissuespecific enhancer-binding proteins may subserve the relatively high IGF-I gene expression in tissues such as liver and uterus, and in specific cell types such as ovarian granulosa cells dur-

COMPLEX REGULATION OF IGF-I EXPRESSION

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ing particular developmental periods. DNA sequence elements, when closely linked, can exhibit synergistic or opposing effects on gene expression (107, 108). Further, different families of transcriptional activators (e.g. steroid receptors and c-Jun/c-Fos proteins) can interact prior to DNA binding to modify subsequent gene activity (109). Thus, the possibility exists for coupling of a variety of steroid and protein hormonal stimuli with tissue-specific nuclear proteins at the level of IGF-I gene transcription. For example, GH induces IGF-I mRNA production in liver and uterus, whereas estrogen is an IGF-I mRNA inducer only in uterus (68, 78, 79). Combined GH and estrogen treatment affects uterine and liver IGF-I mRNA production in a differential manner (68). The nature of the mechanisms of IGF-I regulation posttranscriptionally (Figure 3) remain obscure. Thus, the continued elucidation of the molecular biology of IGF-I should provide new and important insights that may be applicable to other developmentally regulated and tissue-specific genes. In addition, comparative studies of IGF-I genes and of IGF-I expression during embryogenesis, fetal and postnatal development, and reproductive tissue function may suggest new avenues to augment pre- and postnatal growth and development of domestic animals. ACKNOWLEDGEMENTS/FOOTNOTES Uoumal Series No. R-01394from the Universityof Florida AgriculturalExperimentStation. 2Ithank R.C.M. Simmen, R.D. Geisert and EW. Bazerfor helpful critiquesof the manuscript,C. Feinstein for assistance with sequencecompilationsand M. E. Hissemfor expert preparationof the manuscript.Researchin my laboratory is supported by USDA grants 87-CRCR-I-2532and 89-37265-4545and by a grant from the Florida Milk CheckoffProgram.

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