Barbara Valentinis

IGF-I receptor signalling in transformation and differentiation

The type 1 insulin-like growth factor receptor (IGF-IR) sends several signals, some of which are contradictory. When the concentrations of insulin receptor substrate 1 (IRS-1), a major substrate of the IGF-IR, are high, the signal is mitogenic, anti-apoptotic, and can even cause malignant transformation. However, in the absence of IRS-1, the IGF-IR sends a differentiation signal, which leads to granulocytic differentiation in haemopoietic cells. The mitogenic signal of the IGF-IR/IRS-1 combination depends largely, but not exclusively, on the activation of the phosphatidylinositol-3 kinase (PI3K).

Growth and Differentiation Signals by the Insulin-like Growth Factor 1 Receptor in Hemopoietic Cells Are Mediated through Different Pathways

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

mGrb10 Interacts with Nedd4

We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-APcarboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca2+-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.

Insulin receptor substrate-1, p70S6K and cell size in transformation and differentiation of Hemopoietic cells

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70S6K, which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70S6K and an increase in cell size. Phosphorylationin vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70S6K seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70S6K in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70S6K activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

Anti-apoptotic signaling of the IGF-I receptor in fibroblasts following loss of matrix adhesion.

The type 1 insulin-like growth factor receptor (IGF-IR) is known to protect cells from a variety of apoptotic injuries. In several instances, the anti-apoptotic effect of the wild type IGF-IR is more evident under conditions of anchorage-independence than in cells in monolayer cultures. We have investigated IGF-IR signaling in cells in anoikis, a form of apoptosis that occurs when cells are denied attachment to the extra-cellular matrix. IGF-I protects mouse embryo fibroblasts (MEF) from anoikis caused by withdrawal of growth factors. Survival is dependent on the concentration of IGF-I and a sufficient number of functional IGF-I receptors. In this model, IGF-I protection correlates best with ras activation and cell-to-cell aggregation, while PI3-kinase, Akt and MAP kinases seem to play a lesser, alternative role.

The role of mGrb10alpha in insulin-like growth factor I-mediated growth.

Several isoforms of Grb10 are known to interact with either the insulin receptor or the insulin-like growth factor I (IGF-I) receptor, or both. Inasmuch as the data in the literature on the function of Grb10 are not always concordant, we have investigated the role of one of these isoforms, mGrb10α, in cell proliferation. For this purpose, a plasmid expressing mGrb10α was stably transfected into p6 cells and other mouse embryo fibroblast cell lines. An overexpressed mGrb10α inhibits IGF-I-mediated growth, causes a delay in the S and G2 phases of the cell cycle, and partially reverses the transformed phenotype. In contrast, it has no effect on insulin stimulation of cell proliferation. These studies indicate that this isoform of Grb10 has an inhibitory effect on IGF-I signaling of cell proliferation.

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.

Insulin-like growth factor-I-mediated survival from anoikis: role of cell aggregation and focal adhesion kinase.

Anoikis is a form of cell death that occurs when cells are denied attachment to the extra‐cellular matrix. Using p6 cells, that are 3T3 cells overexpressing the type 1 insulin‐like growth factor receptor (IGF‐IR), we show that these cells undergo apoptosis when seeded on polyHEMA plates in serum‐free medium (SFM). IGF‐I protects p6 cells from anoikis, without inducing mitogenesis or DNA synthesis. In the surviving p6 cells in suspension cultures, the focal adhesion kinase (FAK) is tyrosyl phosphorylated by IGF‐I, although this phosphorylation occurs only after several hours. The importance of FAK in protection from anoikis is confirmed by v‐src‐transformed R‐cells, in which FAK is constitutively phosphorylated, that survive even in SFM. Surviving cells, whether p6 or v‐src transformed, tend to form large cell aggregates, whose appearance precedes the phosphorylation of FAK. These and other findings suggest that FAK phosphorylation in the case of IGF‐I is a mediated effect rather than a direct one. When p6 cells are plated on polyHEMA dishes, IGF‐I induces cell aggregation and this aggregation correlates with survival and the eventual phosphorylation of FAK. J. Cell. Physiol. 176:648–657, 1998.

The Role of the IGF-I Receptor in Apoptosis

Publisher Summary This chapter examines the role of the insulin-like growth factor-I receptor (IGF-IR) in apoptosis. As cellular proliferation and transformation are clearly related to apoptosis, the role of the IGF-IR in mitogenesis is examined and in transformation, and its role in apoptosis is also focused. The IGF-IR is a tyrosine kinase receptor with a 70% homology to the insulin receptor (IR). It is synthesized as a single precursor polypeptide of 1367 amino acids, including a 30-amino-acid signal peptide. The organization of the preproreceptor is NH2–signal peptide–α subunit–β subunit–COOH. After removal of the signal peptide, the proreceptor is cleaved at the tetrapeptide RKRR after residue 706, to form the α and β subunits, linked by disulfide bonds. The α subunit, entirely extracellular, is involved in ligand binding; the transmembrane β subunit has a tyrosine kinase domain that is entirely intracellular. When activated by its ligands, the IGF-IR transmits a signal to its major substrates, insulin receptor substrate-1 (IRS-11, insulin receptor substrate-2 (IRS-2), and Shc, a signal that is subsequently transduced by the common signal-transducing pathway, through Ras and Raf, all the way to the nucleus.

Dissociation between resistance to apoptosis and the transformed phenotype in IGF-I receptor signaling.

Programmed Cell Death (PCD) is known to play an important role in both the development and the growth rate of human tumors. It has in fact been suggested that suppression of the apoptotic pathway is a requirement for the establishment of the transformed phenotype. In order to elucidate the relationship between resistance to apoptosis and transformation, we have asked in this investigation whether or not the two processes can be directly correlated. For this purpose, we have used mouse embryo fibroblasts (MEF) expressing either the wild‐type or several mutants of the type 1 insulin‐like growth factor receptor (IGF‐IR). The wild‐type IGF‐IR has both transforming and anti‐apoptotic activities, and we have asked whether these two activities can be or not separated in mutant receptors. Using this well‐defined system, our results show that certain mutants of the IGF‐IR that have strong anti‐apoptotic and mitogenic activities, are incapable of transforming MEF (colony formation in soft agar). We have, instead, a good correlation between mitogenic and anti‐apoptotic activities, suggesting the possibility that the two processes may share similar signaling pathways from the IGF‐IR. On the other hand, our results indicate that transformation requires an additional signal, above and beyond the mitogenic and survival signals. Our conclusion is that, at least in this system, the establishment of the malignant phenotype and resistance to apoptosis can be dissociated, implying the possibility of separate targeting. J. Cell. Biochem. 72:294–310, 1999.