Components Of Insulin-like Growth Factor System Research Articles

Insulin-like growth factor system components and the coupling of bone formation to resorption.

In the adult skeleton, bone formation is regulated by an event referred to as the coupling of bone formation to resorption (i.e. formation is quantitatively linked to resorption), which is thought to be mediated in part by osteogenic growth factor molecules produced by bone cells. Of the various osteogenic growth factors identified in bone, there is sufficient evidence to document an important role for the insulin-like growth factor (IGF) system in mediating this coupling process. Studies on the basic aspects of the IGF system reveal that it is complex and involves a number of components which include the inhibitory IGF binding protein (IGFBP)-4 and stimulatory IGFBP-5 and their corresponding proteases. This complex regulatory scheme of the IGF component system, together with the finding that all of the IGF system components are regulated in bone cells by both local and systemic effectors of bone metabolism, underscores the importance of the IGFs in mediating coupling of bone formation to resorption. In addition, a number of in vitro and in vivo findings also suggest that the IGF system could play a role in the positive (net gain of bone) or negative (net loss of bone) uncoupling of bone formation to resorption.

Insulin-like growth factor (IGF) system components in human prostatic cancer cell-lines: LNCaP, DU145, and PC-3 cells.

Evidence has been accumulating that in many tumors, insulin-like growth factors (IGFs) promote cancer cell growth in an autocrine/paracrine manner via the IGF-I receptor. In an effort to understand the role of IGFs in prostate cancer cell growth, we characterized the IGF system components produced by human prostatic cancer cell-lines, LNCaP, DU145, and PC-3, grown in serum-free medium. IGFs, their receptors, and IGF binding proteins (IGFBPs) produced by the three human prostate cell lines were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR), radioimmunoassay (RIA), Western ligand blot, Western immunoblot, and Northern blot analyses. mRNA for IGF-II and receptors for IGF-I and IGF-II were detected in all three cell-lines by RT-PCR. In contrast to the published study, only LNCaP cells expressed a trace amount of IGF-I mRNA. RIA on conditioned media collected from these cells revealed that all three cell-lines produced measurable IGF-II but not IGF-I. Western Ligand blot, Western immunoblot, and Northern blot analyses revealed that LNCaP, DU145, and PC-3 cells expressed IGFBP-2, IGFBP-2/-3/-4/-6, and IGFBP-2/-3/-4/-5/-6, respectively. IGF-II stimulated [3H]thymidine incorporation into DNA in DU145 and PC-3 cells significantly although the effect was small. DNA synthesis in PC-3 cells but not in LNCaP and DU145 cells was significantly inhibited by the IGF-I receptor-specific monoclonal antibody. Theses results suggest potentially important roles of IGFs and IGFBPs in prostate cancer cell growth, and that in particular, IGF-II may play a critical role in prostate cancer cell growth.

Cellular patterns of insulin-like growth factor system gene expression in murine chondrogenesis and osteogenesis.

In situ hybridization histochemistry was used to map cellular patterns of gene expression for the insulin-like growth factor (IGF) system in developing murine skeleton from embryonic day 15 (E15) through postnatal day 25 (P25). IGF-I receptor and IGF-II receptor messenger RNAs (mRNAs) are both selectively concentrated in developing chondrocytes and osteoblasts. IGF-II and IGF-binding protein-5 and -6(IGFBP-5 and -6) mRNAs are abundant in mesenchymal condensations and chondroblasts on E15. Chondrocyte IGF-II mRNA levels remain high, but IGFBP-5 and -6 mRNAs decline significantly as cartilage matures. Low levels of IGFBP-6 mRNA are detected in postnatal chondrocytes up to at least P25, but IGFBP-5 mRNA is no longer detected in chondrocytes after E18. IGF-I and IGFBP-2, -3, and -4 mRNAs are detected in surrounding mesenchymal tissue, but are not detected in mesenchymal condensations or chondrocytes at any stage of development. IGFBP-3 mRNA is localized in sprouting capillaries invading the perichondrium and periosteum throughout development. IGF-I, IGF-II, and IGFBP-2, -4, -5, and -6 mRNAs are detected in osteoblasts localized in zones of endochondral ossification from E18 to at least P25. IGFBP-1 mRNA is not detected in cartilage or bone cells at any stage of development. These data confirm the recent report by Shinar et al. that IGF-II, but not IGF-I, mRNA is detected in rat chondrocytes in vivo and show that this pattern also applies to the mouse. The present study demonstrates, for the first time, the cell-specific patterns of IGF-I and -II receptor and IGFBP-2 to -6 gene expression during the processes of chondro- and osteogenesis in vivo. Interestingly, IGF-II, both IGF receptors, and IGFBP-5 and -6 are simultaneously coexpressed in chondrocyte precursors early in skeletal development, suggesting functional interactions between these specific factors in chondrogenesis. Both IGFs, both IGF receptors, and IGFBP-2, -4, -5, and -6 are all expressed in osteoblasts, providing evidence for potential local interactions between these IGF system components in osteogenesis. Thus, 9 of 10 known components of the IGF system demonstrate dynamic cell-specific patterns of gene expression during chondro- and osteogenesis, supporting the view that the IGF system has a complex and integral role within the developing skeleton.

Regulation of insulin-like growth factor system components by osteogenic protein-1 in human bone cells

Bone morphogenetic proteins (BMPs) have the unique ability to convert mesenchymal cells into matrix-producing osteoblasts. To understand the mechanism(s) by which a BMP produces a multitude of effects on bone cells, we examined the effects of recombinant human osteogenic protein (OP)-1 (referred to as BMP-7) on the insulin-like growth factor (IGF) regulatory system, an important growth factor system in bone. After 48 h of treatment, OP-1 increased the level of IGF-II (3- and 2-fold, respectively, at 100 ng/ml) in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cells with osteoblastic characteristics, whereas IGF-I levels were low to undetectable in the CM of either cell type. OP-1 treatment had no significant effect on the messenger RNA (mRNA) level for type 1 and type 2 IGF receptors. In TE85 and SaOS-2 cells, 100 ng/ml OP-1 increased the level of IGF binding protein (BP)-3 more than 10-fold, decreased the IGFBP-4 level by 50%, and increased the level of the 29-32.5 kDa IGFBP-5 3-fold in the CM as determined by analysis with Western ligand blot, Western immunoblot, and RIA. The effect of OP-1 on IGFBP production was time and dose dependent. The OP-1 induced changes in the levels of IGFBPs were associated with decreased IGFBP-3 and -5 protease activity (29% and 71%, respectively) and proportional changes in IGFBP mRNA levels. OP-1 increased the level of IGFBP-3 mRNA (2- and 10-fold, respectively, after 4 and 24 h of treatment at 100 ng/ml) and of IGFBP-5 mRNA (more than 5-fold after 24 h of treatment) but decreased the level of IGFBP-4 mRNA (> 50% after 24 h at 100 ng/ml). OP-1 treatment had no effect on IGFBP-4 protease activity. These results collectively demonstrate that OP-1 can act locally by modulating the IGF regulatory system, suggesting that the mitogenic/differentiative effect of OP-1 on human bone cells may in part be mediated via IGF-II by increasing its secretion, and by regulating the balance between the stimulatory (e.g. IGFBP-5) and inhibitory (e.g. IGFBP-4) classes of IGFBPs both at the level of production (mRNA) and at the level of degradation but not by up-regulating the IGF receptor.

Regulation of insulin-like growth factor system components by osteogenic protein-1 in human bone cells.

Bone morphogenetic proteins (BMPs) have the unique ability to convert mesenchymal cells into matrix-producing osteoblasts. To understand the mechanism(s) by which a BMP produces a multitude of effects on bone cells, we examined the effects of recombinant human osteogenic protein (OP)-1 (referred to as BMP-7) on the insulin-like growth factor (IGF) regulatory system, an important growth factor system in bone. After 48 h of treatment, OP-1 increased the level of IGF-II (3- and 2-fold, respectively, at 100 ng/ml) in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cells with osteoblastic characteristics, whereas IGF-I levels were low to undetectable in the CM of either cell type. OP-1 treatment had no significant effect on the messenger RNA (mRNA) level for type 1 and type 2 IGF receptors. In TE85 and SaOS-2 cells, 100 ng/ml OP-1 increased the level of IGF binding protein (BP)-3 more than 10-fold, decreased the IGFBP-4 level by 50%, and increased the level of the 29-32.5 kDa IGFBP-5 3-fold in the CM as determined by analysis with Western ligand blot, Western immunoblot, and RIA. The effect of OP-1 on IGFBP production was time and dose dependent. The OP-1 induced changes in the levels of IGFBPs were associated with decreased IGFBP-3 and -5 protease activity (29% and 71%, respectively) and proportional changes in IGFBP mRNA levels. OP-1 increased the level of IGFBP-3 mRNA (2- and 10-fold, respectively, after 4 and 24 h of treatment at 100 ng/ml) and of IGFBP-5 mRNA (more than 5-fold after 24 h of treatment) but decreased the level of IGFBP-4 mRNA (> 50% after 24 h at 100 ng/ml). OP-1 treatment had no effect on IGFBP-4 protease activity. These results collectively demonstrate that OP-1 can act locally by modulating the IGF regulatory system, suggesting that the mitogenic/differentiative effect of OP-1 on human bone cells may in part be mediated via IGF-II by increasing its secretion, and by regulating the balance between the stimulatory (e.g. IGFBP-5) and inhibitory (e.g. IGFBP-4) classes of IGFBPs both at the level of production (mRNA) and at the level of degradation but not by up-regulating the IGF receptor.

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.

Insulin-like growth factor-II (IGF-II), type 2 IGF receptor, and IGF-binding protein-2 gene expression in rat lung alveolar epithelial cells: relation to proliferation.

Pulmonary alveolar type 2 cells act as a reservoir of stem cells which can be induced to proliferate during periods of lung growth and repair after lung injury. Despite the importance of this process, the mechanisms that regulate type 2 cell proliferation have not been well characterized. We show in this study that insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) accumulates to high levels in culture medium of growth-arrested type 2 cells. This is associated with an increased expression of IGFBP-2 messenger RNA (mRNA). Study of the other components of the IGF system also reveals induction of IGF-II and type 2 IGF receptor mRNA during the process of type 2 cell block of proliferation. When growth-arrested cells are allowed to resume proliferation by the addition of serum, the level of expression of IGFBP-2, type 2 IGF receptor, and IGF-II rapidly decreased. Despite the similarities in the timing of induction, it is likely that these components are not necessarily linked to mediate effects through a single pathway. Indeed, we show that the addition of conditioned medium from growth-arrested cells on proliferative cells results in down-regulation of IGFBP-2 and increased expression of IGF-II and type 2 IGF receptor mRNA. Treatment of the cells with various concentrations of IGF-II affects only the level of expression of type 2 IGF receptor, whereas IGF-I and insulin appear to influence only the expression of IGFBP-2. From the results presented in this study, it can be suggested that IGFBP-2, IGF-II, and type 2 IGF receptor play an important role in the transition of lung alveolar epithelial cells in and out of the cell cycle.

Coordinate expression of insulin-like growth factor system components by neurons and neuroglia during retinal and cerebellar development

The interactions of insulin-like growth factors (IGFs) with the type I IGF receptor are modulated by a family of high-affinity IGF binding proteins (IGFBPs). One of these, IGFBP2, demonstrates a striking spatiotemporal relationship with IGF-I during cerebellar and retinal development. IGF-I mRNA is transiently expressed in large projection neurons--cerebellar Purkinje and retinal ganglion cells--while IGFBP2 mRNA is selectively expressed by contiguous neuroglia--Bergmann glia in the cerebellum and Müller cells and astrocytes of the nerve fiber layer in the retina. IGF-I and IGFBP2 gene expression is not only neuroanatomically coordinated but also temporally synchronized, peaking together during the postnatal maturation of these structures. This pattern of IGF system expression suggests that IGFBP2 is closely related to IGF-I's action in the developing nervous system.