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C T Roberts

Publications and source records attributed to C T Roberts.

At least 163 records · Page 9Linked to original sources
PubMed1992

Structural and functional analysis of the insulin-like growth factor I receptor gene promoter.

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.

Animals↗
PubMed1992

Effect of growth hormone on levels of differentially processed insulin-like growth factor I mRNAs in total and polysomal mRNA populations.

As a result of multiple transcription initiation sites and differential splicing involving the first exons and alternate exon use, many insulin-like growth factor I (IGF-I) mRNA species are produced. In the present study, we have assessed the effect of GH on transcription start site usage and splicing and have determined the apparent in vivo translatability of IGF-I mRNAs with different 5'-untranslated region (UTR) sequences by comparing their abundance in total and polysomal RNA fractions from control, hypophysectomized, and GH-treated hypophysectomized rats. Hypophysectomy decreased the level of all IGF-I mRNA species, but those initiated at start site 3 in exon 1 and the major start site in exon 2 were preferentially reduced. These same variants were preferentially increased by GH treatment. Under all conditions, exon 1 mRNAs with shorter 5'-UTR sequences were enriched in polysomal RNA at the expense of IGF-I mRNAs with long 5'-UTR sequences, in accordance with the scanning model of translation initiation. Exon 2-derived mRNAs, on the other hand, which have short 5'-UTR sequences, were not enriched on polysomes, suggesting that some aspect of the exon 2-derived 5'-UTR other than length influences translation in vivo. These results demonstrate that transcription within exon 1 and between exons 1 and 2 is differentially regulated by GH status and that the variant IGF-I mRNA species resulting from the complex patterns of transcription initiation and splicing in these leader exons are differentially translated in vivo.

Animals↗
PubMed1992

How distinct are the insulin and insulin-like growth factor I signalling systems?

Insulin and insulin-like growth factor I (IGF-I) are closely related peptides. Insulin is primarily involved in regulating carbohydrate, fat and protein metabolism. IGF-I, however, regulates growth and development of the whole organism as well as differentiated functions in specific tissues. Each of these functions are mediated by specific tyrosine kinase receptors expressed on the cell surface. The insulin and IGF-I receptors, though separate gene products, are very similar. Amino acid similarities range between 40 and 85% in different domains, the highest degree of homology being found in the tyrosine kinase domain. Tertiary structure similarities further explain the interactions of each ligand with the heterologous receptor; thus insulin receptors bind insulin with high affinity and IGF-I with lower affinity, and the opposite is true for the IGF-I receptor. Since each ligand can stimulate both receptors and both receptors seem capable of mediating both metabolic and growth activities, what separates these two distinct physiological roles? The interaction of the ligands with their own specific high affinity receptors is facilitated by the presence of IGF-specific binding proteins (BPs) which, however, do not bind insulin. These BPs, found both in the circulation and in tissues, bind all the circulating IGFs and transport the IGFs to their target tissues, thus ensuring that at physiological concentrations IGF-I will only interact with its own receptor. Furthermore, they modulate IGF effects. Since insulin circulates at much lower concentrations compared with the IGFs, this ensures that insulin will only interact with high-affinity insulin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Analysis of the human type I insulin-like growth factor receptor promoter region.

We isolated genomic fragments containing the 5' region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5'-untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5'-flanking region. The region surrounding the transcription start site was similar to a recently described "initiator" sequence. The 5'-flanking and 5'-untranslated regions were highly GC-rich, with numerous potential Sp1 binding sites. A potential AP-2 binding site was identified in the 5'-flanking region and a potential thyroid response element was identified in the 5'-untranslated region. The 5' region of the human gene was very similar to that of the rat gene, with conservation of many of the potential regulatory elements.

Animals↗
PubMed1991

Transcription initiation in the two leader exons of the rat IGF-I gene occurs from disperse versus localized sites.

Rat IGF-I mRNAs contain two different 5'-UTR sequences as a result of alternate splicing of leader exons. Using a combination of solution hybridization/RNase protection and primer extension assays, we have mapped the transcriptional start sites in these leader exons. There appear to be three putative transcription start sites in exon 1 spread over a 140-bp region, the most upstream of which defines a 381 bp-long exon 1. There appear to be three distinct start sites in exon 2, the most upstream of which defines a greater than 770 bp-long exon 2. The two downstream start sites in exon 1 together account for approximately 70% of IGF-I gene expression in adult rat liver. Essentially all of the remaining IGF-I gene expression comes from the second start site in exon 2. Rat IGF-I gene transcription may therefore be regulated by two distinct promoter regions, a disperse promoter for exon 1, with several transcription initiation sites, and a more typical promoter region for exon 2, which controls transcription initiation from a discrete region.

Animals↗
PubMed1991

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.

Animals↗
PubMed1991

Differential association of insulin-like growth factor I mRNA variants with polysomes in vivo.

Expression of the rat insulin-like growth factor I (IGF-I) gene results in a number of mature mRNA species that differ in size primarily at the 3' end due to differential polyadenylation site usage. Additionally, alternate splicing in both 5' and 3' regions produces RNAs which have the capacity to encode different IGF-I precursor peptides. We have analyzed total and polysomal RNAs using Northern blot analyses and solution hybridization/RNase protection assays to assess the in vivo translatability of these various IGF-I mRNA species. The results suggest that all of the known splicing variants are found on polysomes and may, therefore, be translated into a number of IGF-I precursors in vivo. One particular 5'-untranslated (UTR) variant is relatively enriched in polysomal RNA, a finding which suggests that removal of some of the 5'-UTR sequences encoded by exon 1 may enhance translatability. Of the IGF-I mRNAs with different lengths of 3'-UTR, only the shorter species were found on polysomes, suggesting that some aspect of the long 3'-UTR may prevent translation. Thus, differential processing of the primary transcript of the IGF-I gene may serve to generate IGF-I mRNA species which specify different precursors as well as to control their relative translatability.

Animals↗
PubMed1991

Insulin-like growth factor I mRNA levels are developmentally regulated in specific regions of the rat brain.

The expression of mRNAs encoding insulin-like growth factor I (IGF-I) and the IGF-I receptor in the developing rat brain from embryonic day 16 to postnatal day 82 was analyzed using solution hybridization-RNase protection assays. Four distinct developmental patterns in the steady-state levels of IGF-I mRNA were seen. Specifically, the olfactory bulb showed a high perinatal level of IGF-I mRNA which declined dramatically by postnatal day 8. In contrast, cerebral cortex displayed maximal levels of IGF-I mRNA at postnatal day 8 and 13, which subsequently declined to adult levels (P82). A third developmental pattern was seen in the hypothalamus, where IGF-I mRNA increased from E16 up to postnatal day 3 and remained elevated thereafter. Finally, IGF-I mRNA levels in brainstem and cerebellum remained unchanged throughout the time period studied. We conclude that there are specific regional patterns of IGF-I gene expression in the developing rat brain. In contrast, IGF-I receptor gene expression did not exhibit any region-specific developmental changes. The developmental patterns of IGF-I gene expression seen in this study further substantiate the potential role of IGF-I in normal brain development.

Amino Acid Sequence↗
PubMed1991

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.

Base Sequence↗
PubMed1991

Insulin-like growth factors: the ovarian connection.

A large body of information now supports the existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins. Although much remains to be learned, the emerging consensus would suggest that the intraovarian IGF system is concerned largely with the amplification of gonadotrophin hormonal action for the facilitation of follicular growth and development. Future studies are likely to address the central issue of indispensability and the documentation of a meaningful in vivo role for this and related putative intraovarian regulators.

Animals↗
PubMed1991

Insulin-like growth factor I as an intraovarian regulator: basic and clinical implications.

Although much remains to be learned with respect to the possible relevance of IGF-I to ovarian physiology, it may be possible at this time to tentatively formulate possible functions of IGF-I in this connection: 1. Amplification of gonadotropin hormonal action--a key requirement given the exponential nature of follicular development. 2. Integration of follicular development--an essential facet concerned with the coordination of granulosa-theca cooperation. 3. Selection of dominant follicle(s)--a speculative proposition assuming timely and selective activation of the IGF-I system in "chosen" follicles. Aside from its possible role(s) in the course of established follicular cycles, IGF-I (and/or IGF-II) may also participate in the very formation of the follicular apparatus during the late fetal/early neonatal period. Although the ovary is gonadotropin-independent at that time, we previously showed that IGF-I may well interact with VIPergic input now implicated in the morphodifferentiation of the follicular apparatus. Similarly, IGF-I may be concerned with the promotion of juvenile and early pubertal follicular gonadotropin (FSH) levels; ovarian IGF-I may have a bearing on the puberty-promoting effect of growth hormone. Indeed, an association appears to exist between isolated growth hormone deficiency and delayed puberty in both rodents and human subjects, a process reversed by systemic growth hormone replacement therapy. Given that ovarian IGF-I and its receptor may be growth hormone-dependent, it is tempting to speculate that the ability of growth hormone to accelerate pubertal maturation may be due, at least in part, to the promotion of ovarian IGF-I production and reception with the consequent local potentiation of gonadotropin action.

Animals↗
PubMed1991

Hormonal regulation of rat hypothalamic neuropeptide mRNAs: effect of hypophysectomy and hormone replacement on growth-hormone-releasing factor, somatostatin and the insulin-like growth factors.

Hormonal feedback regulation of hypothalamic peptides putatively involved in growth hormone (GH) regulation has been studied by measurement of steady-state mRNA levels in male hypophysectomized rats with or without thyroid hormone, corticosterone, testosterone or GH replacement. Hypothalamic GH-releasing factor (GRF) mRNA levels increased progressively following hypophysectomy to 420% of sham levels after 15 days while hypothalamic insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) mRNA levels decreased to less than 40% of sham levels. Whole hypothalamic somatostatin mRNA levels were not significantly different from sham. One week of continuous GH infusion restored hypothalamic IGF-I mRNA to levels (95%) indistinguishable from those in sham-operated controls but had no effect on either IGF-II or GRF mRNA. Thyroid hormone, corticosterone and testosterone treatment without GH had no effect on the hypophysectomy-induced reduction of either IGF-I or IGF-II mRNA levels but reversed the elevation of GRF mRNA. We conclude that hypothalamic IGF-I may be involved in GH feedback regulation and thus may function as a hypothalamic modulator of GH. In contrast, IGF-II may be regulated by one of the pituitary trophic hormones but not by GH or the target hormones tested. Finally, hypothalamic GRF mRNA regulation appears to be complex and may include target hormone feedback.

Animals↗
PubMed1991

Developmental regulation of insulin-like growth factor-I-stimulated glucose transporter in rat brain astrocytes.

Astrocytic glial cells from 1- and 21-day-old rat brains were established in primary culture to study the expression of insulin-like growth factor-I (IGF-I) receptors and IGF-I-stimulated glucose transporter (Glut-1). Astrocytes from both age groups expressed specific high affinity IGF-I receptors, whose relative affinities for IGF-I, IGF-II, and insulin were comparable. However, the total number of binding sites and IGF-I receptor mRNA levels were 148% and 240% higher in astrocytes from 21-day-old compared with 1-day-old brains. IGF-I caused a dose-dependent stimulation of [3H]2-deoxy-D-glucose [( 3H]dGlc) uptake in astrocytes from 1-day-old brains. This was associated with increases in Glut-1 protein and mRNA levels. In contrast, astrocytes from 21-day-old brains exhibited a 58% decrease in the binding capacity and a 77% decrease in the steady state levels of Glut-1 protein and its mRNA. In addition, IGF-I failed to stimulate the Glut-1 system in these cells. This lack of IGF-I effect is not due to an alteration inherent to the Glut-1 system, since 12-O-tetradecanoyl-phorbol-13-acetate stimulated [3H]dGlc uptake and Glut-1 protein and its mRNA levels. These observations suggest that changes in basal and IGF-I-stimulated Glut-1 system in brain astrocytes may be developmentally regulated.

Animals↗
PubMed1991

Renal IGF-1 mRNA levels are enhanced following unilateral nephrectomy in immature but not adult rats.

The increase in IGF-1 gene expression following unilateral nephrectomy (UNX) in adult rats is controversial. In this study we have examined whether developmental differences exist in the effect of UNX on IGF-1 gene expression. Immature (23 days) and adult (4 months) Wistar rats underwent a sham operation or left UNX, and were sacrificed 24 or 48 hrs later. IGF-1 mRNA levels were determined in left (control) and right (compensated) kidneys using solution hybridization/RNase protection assays. By 48 hrs post-UNX, remnant kidneys had grown 20 +/- 1% in adult rats (P less than 0.05), and 69 +/- 5% in immature rats (P less than 0.05). IGF-1 mRNA levels were not increased in the adult compensated kidneys at either 24 or 48 hrs post-UNX. In contrast, kidneys from immature rats 24 and 48 hrs post-UNX had an average 4-fold increase (P less than 0.05) in exon 1 IGF-1 mRNA levels, and an average 3-fold increase (P less than 0.05) in exon 2 mRNA levels. Thus, these findings suggest that there is an age-dependent difference in the effects of UnX on IGF-1 gene expression, and provide the first evidence that IGF-1 gene expression increases following unilateral nephrectomy in immature rats.

Aging↗
PubMed1991

Expression, action, and steroidal regulation of insulin-like growth factor-I (IGF-I) and IGF-I receptor in the rat corpus luteum: their differential role in the two cell populations forming the corpus luteum.

The overall aim of this investigation was to examine the expression and steroidal regulation of insulin-like growth factor-I (IGF-I) and the IGF-I receptor in the rat corpus luteum and to examine the specificity of IGF-I action in the two luteal cell populations. We first examined whether the corpus luteum expresses the IGF-I and IGF-I receptor genes. Using a solution hybridization/RNase protection assay, IGF-I and IGF-I receptor mRNAs were represented by protected bands 224 and 265 bases in length, respectively. In addition, Northern blot analysis showed that, as in liver, rat IGF-I and IGF-I receptor cDNAs hybridized with 7.5-, 1.8-, and 0.8- to 1.2-kilobase transcripts and an 11-kilobase transcript, respectively. Both IGF-I and IGF-I receptor mRNAs were detected on all days of pregnancy tested (days 5-21). Since the rat corpus luteum increases remarkably in size and steroidogenic capacity at midpregnancy due to estradiol stimulation, we determined whether these developmental changes are accompanied by an increased expression of the IGF-I and/or IGF-I receptor genes. Total RNA was isolated from corpora lutea of day 12 hypophysectomized-hysterectomized rats treated with or without estradiol for 3 days. Estradiol caused a clear and marked reduction in IGF-I and IGF-I receptor mRNA. [125I]IGF-I bound with high specificity and affinity to luteal cell membranes. Large and small cell populations forming corpora lutea of day 3 and 14 pregnant rats were separated by elutriation and used for the determination of binding activity and for cell culture, respectively. IGF-I receptors were found to be localized principally in the large luteal cell population. The small luteal cells had approximately 6.5-fold less IGF-I-binding activity. The difference in binding activity in both cell populations was reflected in the ability of both cell types to respond to IGF-I. IGF-I (25 ng/ml) had a profound effect on the production of progesterone by the large luteal cells. No stimulatory effect of IGF-I on the small luteal cells was observed. Addition of estradiol (10 ng/ml) to the cell culture remarkably enhanced IGF-I stimulation of progesterone biosynthesis by the large luteal cells. In summary, the results of this investigation have revealed that the corpus luteum of the pregnant rat is a major site of expression of both the IGF-I and IGF-I receptor genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗