Platelet-derived growth factor negatively regulates the insulin-like growth factor signaling pathway through the coordinated action of phosphatidylinositol 3-kinase and protein kinase C beta I.

We recently described that epidermal and fibroblast growth factors (EGF and FGF) regulate the IGF-I signaling pathway at the level of IRS-1 through the cooperative action of two independent signaling pathways; one dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and the other on protein kinase D1 (PKD1) (Karam et al. [22]). To determine whether this mechanism could be generalized to another tyrosine kinase receptor-dependent growth factor, the effect of platelet-derived growth factor (PDGF) on the IGF-I signaling pathway was studied. PDGF inhibited IGF-I-stimulated IRS-1 tyrosine phosphorylation and subsequent IGF-I-induced PI 3-kinase activity, and stimulated IRS-1 serine 307 phosphorylation. These effects were mediated through a PI 3-kinase-dependent but extracellular signal-regulated kinase (ERK)-independent signaling pathway. However, PDGF-induced IRS-1 serine 307 phosphorylation was not sufficient per se to inhibit the IGF-I signaling but required another independent pathway. Noteworthy, although acutely stimulated by PDGF, and contrary to what we previously described (Karam et al. [22]), PKD1 did not associate with IRS-1and did not inhibit the IGF-I signaling in response to PDGF. However, we identified PKCßI as a new regulatory partner of PI 3-kinase for PDGF-induced inhibition of the IGF-I signaling pathway. Therefore, our results reinforce the idea that a coordinated action of two independent pathways seems absolutely necessary to negatively regulate IRS-1. Moreover, they also demonstrated that, depending of the cross-talk considered, subtle and specific regulatory mechanisms occur at the level of IRS-1 and that a unique regulatory model is not conceivable.

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway.

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.

Growth factor-specific regulation of insulin receptor substrate-1 expression in MCF-7 breast carcinoma cells: effects on the insulin-like growth factor signaling pathway.

IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway. FGF and, to a lesser extent, EGF were found to increase IRS-1 protein, whereas PDGF had no effect. This indicates that growth factors can specifically modulate IRS-1 protein content. The increases provoked by EGF and FGF were dependent on the MAPK signaling pathway but independent of phosphatidylinositol 3-kinase (PI 3-kinase) signaling and required de novo protein synthesis. We noted that the kinetics of MAPK activation was continuous in response to FGF but transient in response to EGF. In addition, transfection of cells with a constitutively active form of MAPK kinase, which results in continuous MAPK activity, increased IRS-1 expression. Taken together, these results suggest that stimulation of IRS-1 expression was therefore stronger when MAPK activity was sustained. Pretreatment of cells with EGF, FGF, or PDGF for 24 h reduced IGF-I-induced tyrosine phosphorylation per molecule of IRS-1. However, IGF-I-induced PI 3-kinase activity was decreased by 24 h of pretreatment with EGF or PDGF but not with FGF. Our results therefore demonstrate that different growth factors are capable of specifically modulating the IGF-I signaling via IRS-1. They further suggest that the FGF-induced increase in IRS-1 counterbalances the inhibition of IRS-1 tyrosine phosphorylation to allow normal stimulation of IGF-I-induced PI 3-kinase activity.

Insulin-like growth factor binding protein-3 increases intracellular calcium concentrations in MCF-7 breast carcinoma cells.

Insulin-like growth factor binding protein-3, IGFBP-3, specifically binds to IGFs with high affinity, but it is also capable of modulating the IGF-I signalling pathway or inducing apoptosis independently of its binding to IGFs. The molecular mechanisms underlying the action of IGFBP-3 have not been elucidated. In this study, we have demonstrated that binding of IGFBP-3 to a cell surface receptor in MCF-7 breast carcinoma cells induces a rapid and transient increase in intracellular free calcium. This increase was mediated via a pertussis toxin-sensitive pathway, indicating that the IGFBP-3 receptor may be specifically coupled to a Gi protein. The effect of IGFBP-3 on calcium concentrations was dose-dependent and also occurred when IGFBP-3 was complexed with either IGF-I or heparin, suggesting that the receptor binding site is probably located in the least conserved central domain of IGFBP-3. Neither IGFBP-1, nor IGFBP-5 (structurally the closest to IGFBP-3) altered intracellular calcium concentrations. These results provide evidence that a specific intracellular signal is triggered by IGFBP-3 binding to a cell surface receptor.