What is the role of circulating IGF-I?

Postnatal growth and development are coordinated by genetic and environmental influences and numerous growth factors. The growth hormone-insulin-like growth factor-I (GH-IGF-I) axis plays an essential role in these processes. Although the GH-IGF-I axis is a closely coordinated system, both GH and IGF-I have independent actions, many of which have become apparent more recently following the characterization of clinical syndromes and the development of mouse models. Genetic manipulation of mice has enabled investigators to re-examine many of the established hypotheses regarding the GH-IGF-I axis. Results gleaned from a mouse model created by tissue-specific gene deletion of liver IGF-I has enabled investigators to re-evaluate the original 'somatomedin hypothesis'.

Mechanical distention modulates alveolar epithelial cell phenotypic expression by transcriptional regulation.

The development of a normal pulmonary alveolar epithelium, essential for gas exchange, is critical for the successful adaptation to extrauterine life. From observations of natural and experimental developmental abnormalities, it has been hypothesized that mechanical factors may play a role in regulating differentiation of the pulmonary alveolar epithelium. To test this hypothesis directly, we have investigated the in vitro effects of mechanical distention on the expression of specific markers for the type I and type II cell phenotypes. Fetal rat lung (18-d) explants were mechanically distended in culture for 18 h. Mechanical distention caused an increase in RTI 40 messenger RNA (mRNA), a marker of the type I cell phenotype, of 10.6 times (n = 3, P < 0.05) that of undistended controls. In contrast, mechanical distention resulted in a decrease in mRNA content of two markers of the type II cell phenotype, surfactant protein (SP)-B and SP-C. SP-B was reduced to 10 +/- 9% (n = 3, P < 0.005) and of SP-C to 12 +/- 7% (n = 3, P < 0.0001) of undistended controls. Mechanical distention had no effect on content of mRNA for SP-A or 18S ribosomal RNA. Examined by nuclear run-on assays, mechanical distention caused changes in transcriptional rates of RTI 40, SP-B, and SP-C. These data show that mechanical distention stimulates expression of a type I cell marker and inhibits expression of markers for the type II phenotype; these effects occur at least in part at the transcriptional level. These studies support the hypothesis that mechanical distention of fetal lung tissue stimulates expression of the type I cell phenotype and inhibits expression of the type II phenotype.

Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors.

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-gamma, tumor necrosis factor-alpha, and transforming growth factor (TGF)-beta in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-gamma and TGF-beta in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-gamma and TGF-beta had the most potent and consistent effects in both systems.

Apoptosis in the development of rat and human fetal lungs.

The establishment of an effective pulmonary alveolar-capillary interface occurs during mid to late gestation. This requires an expansion of endothelial, epithelial, and air space compartments with relative thinning of the interstitial compartment. Traditionally, these changes have been attributed to differences in the rate of cell growth in the respective compartments. We hypothesized that apoptosis also participates in this lung remodeling. Using light and electron microscopy, the nucleosomal ladder pattern of DNA digestion, and the detection of apoptotic cells in situ by the TUNEL method (Gavrieli, et al. J. Cell Biol. 1992;119:493-501), we demonstrated the occurrence of apoptosis in fetal lungs in vivo and in explant culture. In the rat fetal lung (RFL) in vivo we detected apoptosis from 16 through 22 d gestation. There was variation in the amount of DNA digestion between fetal lungs, but no correlation with gestational age. The findings in human fetal lungs (HFL) from 15 through 24 wk gestation were similar to those of the RFL; the apoptotic indices for both were about 2 apoptotic cells per thousand, suggesting that a significant percentage of cells are eliminated by this mechanism. In the HFL explant culture system, a rapid and massive wave of apoptosis occurred. In all samples of RFL and HFL examined, apoptosis was restricted to interstitial cells. This work has demonstrated for the first time that apoptosis is a feature of normal fetal lung development and that the process is accelerated in lung explant culture.

Coverslip mounted-immersion cycled in situ RT-PCR for the localization of mRNA in tissue sections.

An improved method was developed for in situ reverse transcription-polymerase chain reaction (RT-PCR) to detect and localize mRNA in tissue sections. The coverslip mounted-immersion cycled (COSMIC) in situ RT-PCR technique combines the advantages of solution-phase PCR with the tissue immobilization necessary for in situ analysis. The tissue specimen is mounted on an AES-silane-coated coverslip, excess glass is removed and the sample is immersed in reaction mixture in a PCR tube and subjected to thermal cycling. Processing the section on the coverslip is efficient, the thin glass withstands the high temperature cycling and the tissue adheres securely through the process. The specimen is fully exposed to the reagents, and is heated uniformly and accurately according to temperatures programmed into the thermal cycler. An application is described for the detection and localization of the mRNA for surfactant protein A (SP-A) in fetal rat lung tissue.

Crocidolite asbestos induces apoptosis of pleural mesothelial cells: role of reactive oxygen species and poly(ADP-ribosyl) polymerase.

Mesothelial cells, the progenitor cells of the asbestos-induced tumor mesothelioma, are particularly sensitive to the toxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in mesothelial cells are not known. We asked whether asbestos induced apoptosis in mesothelial cells and whether reactive oxygen species were important. Rabbit pleural mesothelial cells were exposed to crocidolite asbestos or control particles (1-10 micrograms/cm2) over 24 hr and evaluated for oligonucleosomal DNA fragmentation, loss of membrane phospholipid asymmetry, and nuclear condensation. Asbestos fibers, not control particles, induced apoptosis in mesothelial cells by all assays. Induction of apoptosis was dose dependent; crocidolite (5 micrograms/cm2) induced apoptosis (15.0 +/- 1.1%, mean +/- SE; n = 12) versus control particles (< 4%), as measured by appearance of nuclear condensation. Apoptosis induced by asbestos, but not by actinomycin D, was inhibited by extracellular catalase, superoxide dismutase in the presence of catalase, hypoxia (8% oxygen), deferoxamine, and 3-aminobenzamide (an inhibitor of the nuclear enzyme, poly(adenosine diphosphate-ribosyl) polymerase). We conclude that asbestos induces apoptosis in mesothelial cells via reactive oxygen species. We speculate that escape from this pathway could allow the abnormal survival of mesothelial cells with asbestos-induced mutations.

Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species.

Mesothelial cells, the progenitor cell of the asbestos-induced tumor mesothelioma, are particularly sensitive to the toxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in mesothelial cells are not known. We asked whether asbestos induced apoptosis in mesothelial cells and whether reactive oxygen species were important. Pleural mesothelial cells (rabbit or human) were exposed to asbestos (crocidolite, amosite, or chrysotile) or control particles at moderate doses (1-10 microg/cm2) over 24 h and evaluated for oligonucleosomal DNA fragmentation, loss of membrane phospholipid asymmetry, and nuclear condensation. Asbestos fibers, not control particles, induced apoptosis in mesothelial cells by all assays and induction of apoptosis was dose dependent for all types of asbestos, with crocidolite (5 microg/cm2) inducing 15.0+/-1.1% (mean+/-SE; n = 12) apoptosis versus control particles < 4%. Apoptosis induced by asbestos, but not by actinomycin D, was inhibited by extracellular catalase, superoxide dismutase in the presence of catalase, hypoxia (8% oxygen), deferoxamine, 3-aminobenzamide [an inhibitor of poly(ADP-ribosyl) polymerase], and cytochalasin B. Only catalase and cytochalasin B decreased fiber uptake. We conclude that asbestos induces apoptosis in mesothelial cells via reactive oxygen species. Escape from this pathway could allow the abnormal survival of mesothelial cells with asbestos-induced mutations.

Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells.

To better understand the role of insulin-related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)--an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at 10(-8) M, increased the proportion of PM1-positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted-local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF's role, local IGFBPs expression can contribute to the fine modulation.

A novel chicken homeobox-containing gene expressed in neurulating embryos.

We have isolated a cDNA clone from chicken embryo that contains a homeobox sequence (CHox-cad2). Analysis at the nucleotide and amino acid levels revealed closest similarity to the Xenopus Xcad1 and to other homeoboxes related to Drosophila caudal. RNA blot analysis showed hybridization of CHox-cad2 to two transcripts of 2.6 and 1.5 kb, present at day 1 of embryogenesis (E1). Using the highly sensitive polymerase chain reaction (PCR) to amplify cDNAs from embryonic RNAs from E0 (unincubated blastoderm) to E4, we confirmed the restricted expression of this homeobox sequence to the period of neurulation (E1).

Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex.

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.

Insulin-like growth factor-I and insulin as growth and differentiation factors in chicken embryogenesis.

The avian embryo has been a useful model system for studies on the role of insulin and its close relative insulin-like growth factor-I (IGF-I) in development. The unfertilized chicken egg contains both peptides from maternal origin, and the embryo expresses insulin and IGF-I before the major organs are formed. Insulin receptors and IGF-I receptors are found in the blastoderm and in all tissues examined during organogenesis. When exogenous insulin or IGF-I are added to the embryo, growth and differentiation events are stimulated. By contrast, insulin antibodies and insulin receptor antibodies retard embryo development. In embryos cultured ex ovo, in which growth is impaired, the levels of serum IGF-I are decreased.

Genes encoding receptors for insulin and insulin-like growth factor I are expressed in Xenopus oocytes and embryos.

Insulin and insulin-like growth factor I (IGF-I) initiate their metabolic, growth, and differentiation effects through binding to the insulin receptor and the IGF-I receptor, two members of the tyrosine kinase family of receptors. To study the role of these peptides and receptors in early development, we used the polymerase chain reaction and embryo-derived RNA to generate partial cDNA sequences of the insulin receptor and IGF-I receptor from the amphibian Xenopus laevis. Three unique tyrosine kinase-related sequences were obtained. Two of the nucleotide sequences, XTK 1a and XTK 1b, corresponded to peptide that share 92% amino acid identity, and each is 89% identical to the human insulin receptor. The third sequence, XTK 2, corresponds to a peptide that has 92% amino acid identity with the human IGF-I receptor but only 80% identity with XTK 1a and XTK 1b. On the basis of these similarities, the pattern of conserved amino acids, and the tetraploid nature of the Xenopus genome, we suggest that XTK 1a and XTK 1b most likely represent the product of two different nonallelic insulin receptor genes, while XTK 2 may be one of the probable two Xenopus IGF-I receptor genes. By reverse transcription-polymerase chain reaction and gene-specific hybridization, expression of the three XTK sequences was detected in the oocyte, unfertilized egg, and embryos through gastrulation, neurulation, and tailbud stages. Competition binding assays with Xenopus membrane preparations demonstrated insulin receptors and IGF-I receptors in older tadpoles. IGF-I receptors were also present in oocytes, eggs, and gastrula embryos. By contrast, insulin binding was present but atypical in oocytes and was barely detected in eggs and gastrula embryos. The expression of receptors for insulin and IGF-I in early Xenopus embryos and their apparent distinct developmental regulation suggest that these molecules and their ligands may be important in early Xenopus development.

Genes for the insulin receptor and the insulin-like growth factor I receptor are expressed in the chicken embryo blastoderm and throughout organogenesis.

The early expression of insulin and insulin-like growth factor I (IGF-I) in the chicken embryo suggests that these peptides play an important role in early development. The receptors for insulin and IGF-I, however, had not been studied at the molecular level in this model. We report two chicken sequences that, by comparison with known tyrosine kinases, appear to correspond to the tyrosine kinase domain of the insulin receptor homologue (CTK-1) and the IGF-I receptor homologue (CTK-2). Using reverse-transcription of RNA, amplification with the polymerase chain reaction (RT-PCR), and gene-specific hybridization, we demonstrate that the two genes, CTK-1 and CTK-2, are expressed in embryos at least as early as the blastoderm (Day 0), during neurulation (Day 1), and in early (Days 2-3) and late (Day 9) organogenesis.

Insulin-like growth factor I activity is stored in the yolk of the avian egg.

Growth factors of maternal origin may be incorporated into the vertebrate egg and play a role in early phases of embryo growth and differentiation. An avian insulin-like growth factor I (IGF-I) activity from unfertilized chicken egg-yolk has been partially purified by HPLC. The material is slightly more hydrophobic than recombinant human IGF-I. It reacts in a human IGF-I radioimmunoassay and is specifically depleted by anti-human IGF-I antibodies. Like authentic IGF-I, the extracts enriched in IGF-I activity stimulated the accumulation of delta-crystallin mRNA in epithelial cells from chick embryo lens with a potency approximately equivalent to its IGF-I immunoactivity.