Diana L. Esposito

Signaling via the Insulin-like Growth Factor-I Receptor: Does it Differ from Insulin Receptor Signaling?

The insulin and insulin-like growth factor (IGF-I) receptors while similar in structure and function serve different physiological functions in vivo. In non-disease states the insulin receptor is primarily involved in metabolic functions whereas the IGF-I receptor mediates growth and differentiation. The separation of these functions is controlled by a number of factors including the tissue distribution of the respective receptors. Modulation of the binding of the ligands insulin or IGF-I and IGF-II to their respective receptors by the local environment of the cell also offers signaling specificity mediated via the receptors. Each ligand bind to its respective receptor with high affinity. This high affinity binding is dictated by the primary sequence of both the ligand and the receptor. Furthermore IGF-binding proteins are specific for IGF-I and IGF-II thereby modulating the binding of the IGFs to the IGF-I receptor. In contrast insulin circulates unbound to any proteins and interacts in the free state with the insulin receptor. It has been postulated that downstream substrates of the activated receptors differ in their specificity for the receptors, thus lending further specificity to the actions mediated by the receptors. While a number of known endogenous substrates such as IRS-1, IRS-2 and She are utilized by both receptors, the structural differences in the beta subunits of the two receptors has lead investigators to suggest that certain substrates may be unique to each receptor. Candidate substrates which show this specificity of action have been and are being described. Full eludication of the specificities of the insulin and IGF-I signaling pathways is of interest of course for a better understanding of intercellular communication. In addition, because the closely related proteins insulin and IGF-I are used clinically, a clear understanding of the pathways activated by these agents is essential if more specific therapeutic modalities are to be developed for use in disease states.

Tyr(612) and Tyr(632) in human insulin receptor substrate-1 are important for full activation of insulin-stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose cells.

To examine contributions of specific YXXM motifs in human insulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3(IR) cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr(612) and Tyr(632) fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr(612) or Tyr(632) alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the absence of insulin) to approximately 80% of the levels seen in insulin-stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1-Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr(612) and Tyr(632) are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2

Background and aims: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. Methods: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. Results: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and β-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of ApcMin/+ and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. Conclusions: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

Autophagy in hematopoietic stem/progenitor cells exposed to heavy metals: Biological implications and toxicological relevance.

The inherent toxicity of many metal compounds, together with their widespread environmental distribution, raises concerns of potential health hazards. Little is known about the impact of these important environmental toxicants on adult stem/progenitor cells, necessary for tissue homeostasis and repair. We recently reported that autophagy is implicated in the response of hematopoietic stem/progenitor cells to toxic concentrations of hexavalent chromium (Cr[VI]) and cadmium (Cd), two well known carcinogenic heavy metal cations. Autophagy may lead to cell death if carried out too extensively, but also acts as a survival pathway in cells under stress. In stem/progenitor cells, an autophagic phenotype could mitigate metal-induced toxicity, contributing to the conservation of tissue renewal capability. Given the key role of toxic damage to adult stem/progenitor cells in cancer, it is necessary to investigate whether autophagic responses modulate the carcinogenic potential of exposure to heavy metals during stem/progenitor cell differentiation. Addendum to: Di Gioacchino M, Petrarca C, Perrone A, Farina M, Sabbioni E, Hartung T, Martino S, Esposito DL, Lotti LV, Mariani-Costantini R. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells. Sci Total Environ. 2008; 392:50-8.

TGF-β1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor β1 (TGF-β1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-β1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-β1 and IGF-I in FRTL-5 cells. TGF-β1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-β1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-β1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-β1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-β1 on the IGF-I-induced mitogenic effect.

Novel insulin receptor substrate 1 and 2 variants in breast and colorectal cancer

The insulin/insulin-like growth factor pathway is involved in breast and colorectal cancer (CRC) development. In the present study, we analyzed the coding region and short intron-exon borders of the insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) genes in 12 cell lines derived from breast cancer (BC), 14 cell lines derived from CRC and 33 primary CRCs. The nucleotide variants identified in BC were 3 in IRS-1, 1 of which (p.Arg267Cys) was novel and with a pathogenic potential as predicted by in silico analysis and 6 in IRS-2. Twenty-one variants in IRS-1 and 18 in IRS-2 were identified in the CRC samples. These included 11 novel IRS-1 variants detected exclusively in CRCs, which included 5 missense (p.Pro559Leu, p.Gln655His, p.Asp1014Gly, p.Asp1181His and pPro1203Ser) with a pathogenic potential as predicted by in silico analysis, 2 frameshifts predicted to generate a truncated protein, 1 splice-site mutation and 3 silent variants. In the CRC samples we also identified 7 novel IRS-2 variants, including 4 missense variants, which included 2 (p.Asp782Asn and p.Gly1230Ser) with a pathogenic potential as predicted by in silico analysis, 2 frame insertion mutations and 1 silent variant. Most of the novel IRS-1 and IRS-2 variants may be involved in the modulation of IRS-1 or IRS-2 functions and could be relevant to breast and colorectal tumorigenesis.

Correction to: Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib

The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.