Ching-I Pao

Molecular Regulation of Insulin-like Growth Factor-I and Its Principal Binding Protein, IGFBP-3

The insulin-like growth factors (IGFs) have diverse anabolic cellular functions, and structure similar to that of proinsulin. The distribution of IGFs and their receptors in a wide variety of organs and tissues enables the IGFs to exert endocrine, paracrine, and autocrine effects on cell proliferation and differentiation, caloric storage, and skeletal elongation. IGF-I exhibits particular metabolic responsiveness, and circulating IGF-I originates predominantly in the liver. Hepatic IGF-I production is controlled at the level of gene transcription, and transcripts are initiated largely in exon 1. Hepatic IGF-I gene transcription is reduced in conditions of protein malnutrition and diabetes mellitus, and our laboratory has used in vitro transcription to study mechanisms related to diabetes. We find that the presence of sequences downstream from the major transcription initiation sites in exon 1 is necessary for the diabetes-induced decrease in IGF-I transcription. Six nuclear factor binding sites have been identified within the exon 1 downstream region, and footprint sites III and V appear to be necessary for metabolic regulation; region V probes exhibit a decrease in nuclear factor binding with hepatic nuclear extracts from diabetic animals. IGFs in biological fluids are associated with IGF binding proteins, and IGFs circulate as a 150-kDa complex that consists of an IGF, an IGFBP-3, and an acid-labile subunit. Circulating IGFBP-3 originates mainly in hepatic nonparenchymal cells, where IGF-I increases IGFBP-3 mRNA stability, but insulin increases IGFBP-3 gene transcription. Regulation of IGFBP-3 gene transcription by insulin appears to be mediated by an insulin-responsive element, which recognizes insulin-responsive nuclear factors in both gel mobility shift assays and southwestern blots. Studies of mechanisms underlying the modulation of IGF-I and IGFBP-3 gene transcription, and identification of critical nuclear proteins involved, should lead to new understanding of the role and regulation of these important growth factors in diabetes mellitus and other metabolic disorders.

Nutrition and Somatomedin: XXVI. Molecular Regulation of IGF-I by Insulin in Cultured Rat Hepatocytes

Although decreased levels of circulating insulinlike growth factor I (IGF-I) and hepatic IGF-I mRNA in diabetic animal models support a role for insulin in IGF-I regulation, it has been difficult to study the effects of insulin in vitro. Few immortal cell lines exhibit hormone-responsive production of IGF-I, and studies of cultured hepatocytes have often been inconclusive because conventional methods may not reproduce the IGF-I transcripts recognized in vivo. We have modified the hepatocyte isolation and RNA extraction procedures to circumvent this problem, resulting in transcripts of 1, 2, and 7.5 kb. With increasing insulin from 10−10 to 10−6 M, dexamethasone from 10−10 to 10−7 M, and amino acids from 1 to 10 × rat arterial levels, IGF-I release rose by 226, 257, and 447%, respectively (P < 0.05), with correlated changes in IGF-I mRNA (r = 0.75, P < 0.005). In the presence of 5 × amino acids and 10−7 M dexamethasone, insulin-stimulated IGF-I release was dose dependent, increasing 183% at 10−6 M (P < 0.01). Across a broad range of amino acid concentrations (0.25–6.25 ×), insulin provided consistent stimulation of both IGF-I mRNA content and release of IGF-I. Cells cultured for 2 days at 10−10 M insulin and then 2 days at 10−6 M insulin released 66% more IGF-I than cells cultured 4 days at 10−10 M insulin (P < 0.01). Hepatocyte IGF-I release was correlated strongly with content of IGF-I mRNA, both sum of transcripts (r = 0.76, P < 0.001) and individual transcripts. Insulin appears to regulate IGF-I release at the mRNA level in hepatocyte primary culture. This system should be a useful tool for further studies of molecular mechanisms of IGF-I regulation.

Involvement of Sp1 in the transcriptional regulation of the rat insulin-like growth factor-1 gene

UNLABELLED Most insulin-like growth factor-I (IGF-I) transcripts are initiated in exon 1, but mechanisms of regulation are not well understood. Since potential Sp1 sites are found in footprinted regions within approximately 360 bp upstream and downstream from the major initiation sites in exon 1, we explored the involvement of Sp1 and Sp3 in regulation of IGF-1 expression. Gel shift assays showed strong Sp1 binding to the downstream site, but binding to the upstream site was weak; Sp1 bound to a CCTGCCCA sequence in downstream footprint region V, and Sp3 binding was centered on the same sequence. IGF-I basal promoter constructs containing a mutation in the downstream Sp1 site exhibited a 32% decrease in expression in CHO cells and a 75% decrease in HepG2 cells, indicating the importance of Sp1 for expression in vivo. Sp1 and Sp3 expression vectors provided three- to five-fold stimulation of wild-type IGF-I constructs, but had little effect on a construct containing a mutation in the downstream Sp1 site, and Sp1 had comparable effects in Drosophila SL2 cells. IGF-I heterologous promoter constructs exhibited similar responses: in both SL2 cells and CHO cells, stimulation by Sp1 was enhanced with constructs containing downstream region V. Since Sp1 also stimulated expression of concatamers of putative cis-acting sites fused to the SV40 promoter enhancer in pGL3, the results in combination indicate that the presence of IGF-I region V is sufficient to permit stimulation by Sp1. CONCLUSION Sp1 and related factors may play an important role in the regulation of IGF-I gene transcription, through interactions with region V downstream from the major initiation sites in exon 1.

In Vitro Transcription of the Rat Insulin-like Growth Factor-I Gene

Although the liver is the major source of circulating insulin-like growth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between −300 and −60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression.

Identification of core sequences involved in metabolism-dependent nuclear protein binding to the rat insulin-like growth factor I gene.

In the liver, most insulin-like growth factor I (IGF-I) transcripts originate in exon 1, where important cis-regulatory regions are located downstream from the major transcription initiation sites. Within these regions, we have attempted to identify sequences which are involved in the decrease in IGF-I gene transcription associated with diabetes mellitus. The function of different genomic templates was assessed by in vitro transcription, which revealed a consistent 50–80% decrease in the activity of nuclear extracts from streptozotocin-diabetic as compared with normal rats. The disparity in transcriptional activity between normal and diabetic nuclear extracts was reduced with templates containing 11-bp mutations within DNase I protected regions III or V (+42 and +129 bp, respectively, from the major transcription initiation site), but a mutation between regions IV and V had little effect. Within region III, gel mobility shift analysis and methylation interference studies indicated that DNA-protein interac...