Human Insulin-like Growth Factor I Gene Research Articles

Control of the timing and dosage of IGF-I delivery from encapsulated cells

We report here on the development and characterization of a cell-based system for the regulated delivery of bioactive insulin-like growth factor I (IGF-I). A stable mammalian cell line, CHO-K1 Tet-IGFI, was genetically modified to have tetracycline-induced transcription of the human IGF-I gene. Cells were activated to express IGF-I in the presence of doxycycline (DOX), a tetracycline derivative, while expression was inactivated in the absence of DOX. Temporal, or on-off, release of IGF-I from cells encapsulated within Ca²⁺-alginate hydrogels was demonstrated in a pilot study over the course of 10 days in culture. Released growth factor was bioactive, exhibiting a proliferative effect comparable to recombinant purified IGF-I protein. The dosage levels and temporal control of IGF-I release from encapsulated cells meet the requirements of orthopedic wound repair, making this approach an attractive means for the controlled synthesis and delivery of growth factors in situ for wound healing.

Acceleration of articular cartilage repair by combined gene transfer of human insulin-like growth factor I and fibroblast growth factor-2 in vivo

Improving the biochemical and structural qualities of the new tissue that fills deep osteochondral defects is critical to enhance articular cartilage repair. We developed a novel molecular therapy to increase articular cartilage repair based on a combined strategy to stimulate chondrogenesis by co-transfection of the human insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) in a xenogenic transplantation model. NIH 3T3 cells were transfected with expression plasmid vectors containing a cDNA for the E. coli lacZ gene (lacZ implants), the human IGF-I gene (IGF-I implants) or both the human IGF-I and FGF-2 genes (IGF-I/FGF-2 implants). The expression patterns of the transgenes were monitored in vitro for 21 days. LacZ, IGF-I and IGF-I/FGF-2 implants were transplanted into osteochondral defects in the trochlear groove of rabbits. At 3 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. Both IGF-I and IGF-I/FGF-2 implants secreted increased levels of the corresponding recombinant proteins in vitro. In vivo, transplantation of the co-transfected IGF-I/FGF-2 implants increased the DNA content of the repair tissue, accelerated the formation of the subchondral bone and improved articular cartilage repair in a magnitude that was larger than with IGF-I alone or when compared to lacZ implants. These results suggest that gene delivery of a combination of IGF-I and FGF-2 to cartilage defects may be more beneficial than application of IGF-I alone.

Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences

BackgroundAdeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV) vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I).ResultsWe constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC) eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC), that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter.ConclusionTaken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements may serve to augment the therapeutic value of rAAV vectors.

Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I)

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2+/-22.3 ng/10(7) cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.

Identification of a Distal STAT5-binding DNA Region That May Mediate Growth Hormone Regulation of Insulin-like Growth Factor-I Gene Expression

Growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression through signal transducer and activator of transcription 5b (STAT5b) and STAT5a. The objective of this study was to identify the cis-regulatory DNA region involved in this process. By cotransfection analyses of shotgun DNA fragments of a bacterial artificial chromosome sequence containing the entire human IGF-I gene and a large 5'-flanking region, a approximately 700-bp DNA region approximately 75 kb 5' to the IGF-I gene was found to have the ability to enhance gene expression from both heterologous and homologous promoters in the presence of constitutively active STAT5a or STAT5b. This 700-bp DNA region contains two closely located consensus STAT5-binding sites, and its sequence appears to be evolutionarily conserved. Electrophoretic mobility shift assays verified the ability of the two putative STAT5-binding sites to bind to STAT5a and STAT5b. Cotransfection analyses confirmed that both STAT5-binding sites were necessary for the 700-bp DNA region to mediate STAT5a or STAT5b activation of gene transcription. Chromatin immunoprecipitation assays demonstrated that the chromosomal region containing these two STAT5-binding sites was bound by constitutively active STAT5b protein in HepG2 cells and that the binding was accompanied by increased expression of IGF-I mRNA. In reconstituted GH-responsive cells, this 700-bp DNA region was able to mediate GH-induced STAT5a or STAT5b activation of gene expression. These results together suggest that this STAT5-binding site-containing distal 5'-flanking region of IGF-I gene may be an enhancer mediating GH-induced STAT5 activation of IGF-I gene transcription.

Protein Kinase C-dependent, CCAAT/Enhancer-binding Protein β-mediated Expression of Insulin-like Growth Factor I Gene

The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBP beta in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBP beta activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBP beta-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBP beta chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBP beta binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBP beta to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBP beta binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells.

The Hepatocyte Nuclear Factor 3β Stimulates the Transcription of the Human Insulin-like Growth Factor I Gene in a Direct and Indirect Manner

Promoter 1 (P1) of the human insulin-like growth factor I (IGF-I) gene is most active in adult liver. In this study we show that HNF-3beta, a member of the winged helix protein family of liver-enriched transcription factors, has a strong stimulatory effect on the activity of P1. Transient transfection experiments in combination with bandshift and DNase I footprinting analysis revealed the presence of two HNF-3 binding sites in the proximal promoter region of P1. Both binding sites, which are well conserved in evolution, are required for maximal transactivation. Studies employing HNF-3 mutant constructs indicated that IGF-I expression is also regulated indirectly by HNF-3beta as a consequence of enhanced expression of HNF-1alpha. This liver-enriched transcription factor has previously been shown to transactivate P1. Thus, HNF-3beta regulates the expression of the human IGF-I gene via two distinct mechanisms.

Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells.

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.

Transforming Growth Factor-β and Forskolin Increase All Classes of Insulin-like Growth Factor-I Transcripts in Normal Human Osteoblast-like Cells

In this study, we examined regulation of insulin-like growth factor I (IGF-I) gene expression and transcript splicing in normal human osteoblast-like cells. Previous studies in rat osteoblastic cells have indicated that transforming growth factor-β (TGF-β) inhibits IGF-I expression, whereas inducers of intracellular cAMP stimulate IGF-I expression. However, in human osteoblast-like cells both TGF-β and forskolin increased IGF-I mRNA levels in a time- and dose-dependent manner. All 4 classes of IGF-I transcript that can result from alternate leader exon usage and splicing of the human IGF-I gene were induced proportionally. Although human osteoblasts increase IGF-I mRNA in response to important skeletal regulatory factors, some responses may be species-specific.

The human IGF-I gene contains two cell type-specifically regulated promoters

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.

Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon.

Tissue distribution and potential alternative splicing of insulin-like growth factor I (IGF-I) messenger RNA were studied using reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA from several tissues at various stages of the life cycle of coho salmon (Oncorhynchus kisutch). DNA sequence analysis of RT-PCR products revealed three IGF-I mRNA transcripts, designated Ea-1, Ea-2, and Ea-3, which code for three distinct prohormones, IGF-IA-1, IGF-IA-2, and IGF-IA-3, respectively. The E-domain of proIGF-IA-1 is 35 amino acids long and shares 77% sequence identity with the E-domain of human proIGF-IA, which is also 35 amino acids long. The proIGF-IA-2 and proIGF-IA-3 E-domains are homologous to the proIGF-IA-1 E-domain but contain 27 and 39 amino acid inserts, respectively, between Lys86 and Glu87. In the human IGF-I gene Lys86 is coded by exon 4 and Glu87 is coded by exon 6. This suggests that Ea-2 and Ea-3 transcripts may be the result of alternative splicing during pre-mRNA processing. All three transcripts were readily detectable using a solution hybridization/RNase protection assay. Furthermore, RT-PCR and DNA sequencing analysis indicate the presence of three IGF-I prohormones in another member of the Salmonidae family, the Atlantic salmon (Salmo salar). An analysis of IGF-I and -II E-domains from several vertebrates suggests that certain chemical and physical properties of the molecule are well conserved despite wide variations in primary structure. Ea-1, Ea-2, and Ea-3 transcripts were found in whole embryos, and liver, muscle, and brain of juvenile and adult salmon. At least one IGF-I transcript was found in heart, kidney, testes, ovary, adipose tissue, and spleen of juvenile salmon. These results indicate that IGF-I is expressed during embryonic development of fish, and that most tissues are capable of IGF-I mRNA production. These data also indicate that pre-mRNA transcripts can be alternatively spliced to yield at least three prohormones.

Structure and Function of a Human Insulin-like Growth Factor-I Gene Promoter

We have identified and characterized a promoter regulatory region for the human insulin-like growth factor-I (IGF-I) gene. The 5'-ends of IGF-I mRNAs were first mapped to a series of sites within a 158-nucleotide portion of exon 1 that was found to be structurally similar to the recently delineated transcription start region of the chicken IGF-I gene. In both species multiple initiation sites are probably a reflection of the absence of typical transcriptional regulatory signals, such as a TATA or CCAAT box, in the proximal promoter, although neither gene sequence resembles a GC-rich housekeeping promoter, which also controls a dispersed initiation region. To test promoter function, hybrid genes were constructed linking human IGF-I DNA to a promoterless reporter plasmid. Fusion genes containing from 385-4300 nucleotides of the IGF-I 5'-flanking region enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line. A plasmid with 1630 nucleotides of 5'-DNA gave maximal activity and directed accurate initiation of the hybrid gene while the same promoter fragment inserted in the reverse orientation was inert. Although cognate recognition sequences were identified for several transcription factors in the 1630 nucleotides 5' to the transcription start region, no sites were found that resembled the putative GH response element recently mapped to the proximal promoter of the rat Spi2.1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of multiple transcription start sites in the human insulin-like growth factor-I gene

We have localized four transcription initiation sites in the human insulin-like growth factor-I (IGF-I) gene. Two transcription start sites were identified which result in a longer and shorter version of the leader derived from the known exon 1 of the IGF-I gene. Transcription starting at the upstream transcription initiation site results in a leader exon 1 of about 1155 nucleotides (nt), whereas transcription starting at the downstream initiation site results in a leader of about 240 nt. The majority of the transcripts initiate at the latter site. We further identified a region in the human IGF-I gene between exons 1 and 2, which shows a high degree of homology with the rat IGF-I leader exon 1B. By means of the polymerase chain reaction (PCR) we detected human IGF-I mRNAs containing this novel leader. The corresponding exon was designated exon 1B according to the rat IGF-I gene terminology. PCR and RNase protection analyses identified two transcription start sites within this alternative leader exon 1B. Transcription initiated at the most upstream start site results in a leader of about 750 nt, whereas transcription starting at the downstream site is heterogeneous, resulting in leaders of 65–75 nt long. No consensus TATA-box or AT-rich regions are present immediately upstream of all four transcription start sites identified, nor are these regions particularly GC-rich. The IGF-I gene is known to be expressed differentially in a tissue- and development-specific fashion. Differential activation of multiple promoters could very well play a crucial role in IGF-I gene regulation.

A Novel Human Insulin-Like Growth Factor I Messenger RNA is Expressed in Normal and Tumor Cells

We have identified and characterized a novel human insulin-like growth factor I (IGF-I) precursor from the transplantable T61 human breast cancer xenograft and from normal liver. The mRNA encoding this precursor contains a 5'-untranslated region that is 83% identical to the corresponding region of a previously described variant rat IGF-I. The nucleotide sequence of the cloned cDNA predicts an IGF-IA protein precursor of 137 amino acids, including a 32 residue signal peptide, 70 amino acid IGF-I, and a 35 residue COOH-terminal extension or E peptide. The exon encoding this variant maps in the genome between IGF-I exons 1 and 2, in a similar location to the homologous rat exon 1a. The rat and human exons 1a are 59% identical over 1443 nucleotides, with DNA sequence conservation occurring in a mosaic pattern. Human IGF-I mRNAs encoding this novel exon are expressed in liver, T61 tumor cells, and in an ovarian carcinoma cell line, NIH OVCAR3. These studies demonstrate that as in the rat, the human IGF-I gene contains six exons that are variably processed into multiple IGF-I mRNAs. The mechanisms responsible for generating different IGF-I mRNAs thus appear to be conserved among mammalian species.

Low Prevalence of Insulin-Like Growth Factor-I Gene Mutations in Human Growth Disorders*

In an attempt to identify genetic lesions contributing to human growth disorders, we evaluated a prospectively recruited group of children with growth failure for mutations in the insulin-like growth factor-I (IGF-I) gene. Two complementary approaches were used: Southern blot analysis to examine the large scale organization of the gene, and a solution hybridization, nuclease protection assay to identify small alterations, such as point mutations. From a total of 61 subjects studied, 52 had no organic basis for their short stature. Analysis of chromosomal DNA from these individuals failed to reveal any variation in the IGF-I gene except for a HindIII site polymorphism which maps near the 3' end of the last IGF-I exon. No single nucleotide substitutions were found within IGF-I-coding regions. Since the frequency of the length polymorphism was the same for both normal-sized and short individuals, it is unlikely to be associated with growth abnormalities. Our results suggest that there is minimal DNA sequence variability in the human IGF-I gene and that mutations in IGF-I exons are infrequent causes of growth failure.

Mosaic evolution of the insulin-like growth factors. Organization, sequence, and expression of the rat insulin-like growth factor I gene.

Insulin-like growth factor I (IGF-I) plays a major role in mammalian growth and regenerative processes as a mediator of many of the biological effects of growth hormone. We have demonstrated recently that the human IGF-I gene is transcribed and processed into distinct messenger RNA molecules, each of which directs the synthesis of unique IGF-I-containing peptides. As a means to determine whether a similar model of IGF-I gene organization and expression is the paradigm in mammals and as an initial step in devising experimental approaches to the study of regulation of IGF-I biogenesis, we have isolated and characterized the rat IGF-I gene. The rat gene, like its human counterpart, is very large, extending over at least 73 kilobases, and is composed of five exons subdivided by four introns. As in the human example, the rat IGF-I gene hybridizes to several messenger RNAs: 0.8-1.2, 1.6-2.1, and 7.8 kilobases. There is extensive nucleotide and amino acid sequence conservation between the two genes. The predicted mature rat IGF-I protein is identical to the human peptide in 67 of 70 residues. A comparably high degree of amino acid sequence identity is also found for both the amino- and carboxyl-terminal extension peptides, suggesting that, like mature IGF-I, the extension molecules may have physiological function.

Organization and sequence of the human insulin-like growth factor I gene. Alternative RNA processing produces two insulin-like growth factor I precursor peptides.

Insulin-like growth factor I (IGF-I), a 70-amino acid basic polypeptide, plays a fundamental role in postnatal mammalian growth as a major mediator through which growth hormone exerts its biological effects. We have recently identified two human IGF-I cDNAs which predict distinct peptide precursors of 153 and 195 amino acids. In the present study, both cDNAs were used to isolate and characterize the human IGF-I gene from genomic libraries. The IGF-I gene extends over at least 45 kilobase pairs and contains five exons interrupted by four introns. The DNA sequence of exons 1 through 4 encodes the 195-amino acid precursor, while exons 1, 2, 3, and 5 code for the 153-residue peptide, confirming the hypothesis that at least two IGF-I mRNAs are generated by alternative RNA processing of the primary gene transcript. The structure of the IGF-I gene resembles that of its companion somatomedin, IGF-II, as judged by the analogous location of two introns and considerable nucleotide and amino acid sequence similarity, but appears more distantly related to other members of the insulin gene family. Restriction endonuclease polymorphisms in the IGF-I gene, which map near exon 5 as determined by Southern blot analysis, will be useful in defining the genetics of familial growth failure.

Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe.

A single synthetic oligonucleotide was employed as hybridization probe to detect and enable isolation of the human insulin-like growth factor I (IGF-I) gene from a human genomic DNA library. The synthetic oligonucleotide probe coded for the B-chain of IGF-I and was designed for expression in Escherichia coli. Despite numerous interspersed mismatches, the synthetic probe hybridized specifically with seven recombinant lambda phage containing almost the entire B-chain region of the human IGF-I gene. The usefulness of this approach was further demonstrated by the detection of lambda phage containing human preproinsulin, using A and B chain synthetic oligonucleotides, 90 and 63 nucleotides in length, as hybridization probes. The nucleotide sequence of the human IGF-I exon suggests that IGF-I is synthesized as a larger precursor molecule.