Insulin-like Growth Factor-binding Protein (IGFBP)-3 and IGFBP-5 Share a Common Nuclear Transport Pathway in T47D Human Breast Carcinoma Cells

Abstract

Insulin-like growth factor-binding proteins (IGFBPs) play an integral role in modifying insulin-like growth factor actions in a wide variety of cell types. Recent evidence suggests that IGFBP-3 and IGFBP-5 also have effects on cell growth that are insulin-like growth factor-independent. In investigating possible mechanisms for this effect, the intracellular trafficking of IGFBP-3 and IGFBP-5, both of which contain sequences with the potential for nuclear localization, was studied in T47D cells. Nuclear uptake of fluorescently labeled IGFBP-3 and IGFBP-5 was observed in a proportion of T47D cells that appeared to be rapidly dividing. IGFBP-1 and IGF-BP-2, which do not possess the putative domain for nuclear translocation, were not transported to the nuclei of T47D cells. When T47D cells were preincubated with excess unlabeled IGFBP-3, nuclear localization of labeled IGFBP-3 or IGFBP-5 was not detected, indicating that their nuclear translocation involves a common pathway. Inhibition of receptor-mediated endocytosis did not affect nuclear uptake of IGFBP-3, suggesting that it uses an alternative non-classical import pathway for transport across the plasma membrane. In addition, a variant form of IGFBP-3 with a mutation in the putative nuclear localization sequence was unable to translocate to the nuclei of T47D cells, suggesting that nuclear translocation of IGFBP-3 was dependent on these carboxyl-terminal basic residues.The insulin-like growth factors (IGF-I and IGF-II) 1 are potent mitogens, which stimulate proliferation in many normal and malignant cell types (1). They bind to specific receptors, designated the type I and II IGF receptors (2), although the mitogenic effects of the IGFs are mediated through the type I IGF receptor. The IGFs also have high affinity for a family of six structurally related IGF-binding proteins, IGFBP-1 to IGF-BP-6, which are responsible for regulating the bioavailability of the IGFs in the circulation (3). The IGFBPs also modulate the activity of IGFs at the cellular level, either inhibiting or enhancing IGF action (4), and in this context are believed to be important in regulating IGF-dependent proliferation of many cancers (5).Several reports have described the growth inhibition of breast cancer cells (6) and other cell types (7) by IGFBP-3, which appears independent of activation of the type I IGF receptor. More recently, overexpression of IGFBP-3 in fibroblasts with a targeted disruption of the IGF-I receptor gene was shown to have an inhibitory effect on cell growth (8). Transforming growth factor ␤ (TGF␤) and retinoic acid, which are known to inhibit cell growth and induce apoptosis in a variety of normal and malignant cell types, also induce the expression and secretion of IGFBP-3 (5, 9). Several reports have now shown that the growth inhibitory effects of TGF␤ and retinoic acid (10, 11) and the TGF␤ induction of apoptosis (12) are mediated, at least in part, by IGFBP-3. There are fewer reports on IGF-independent action of IGFBP-5. These include the stimulation of bone ce...

Nuclear Import of Insulin-like Growth Factor-binding Protein-3 and -5 Is Mediated by the Importin β Subunit

“…These include platelet-derived growth factor A (30), acidic fibroblast growth factor (31), and parathyroid hormone-related protein (32). We and others have described the nuclear transport of IGFBP-3 and IGFBP-5 in a number of cell lines (33)(34)(35)(36).…”

“…IGFBP-3 was purified from conditioned media by IGF-I affinity chromatography and reverse-phase high pressure liquid chromatography (38). In studies requiring fluorescently labeled IGFBP-3, the protein was conjugated to dichlorotriazinylaminofluorescein I HCl as described previously for Cy3 (35). The fusion proteins generated by linking ␤-galactosidase to the bipartite NLS derived from the Xenopus laevis phosphoprotein, N1N2 (N1N2 NLS:␤-gal), or the T-ag NLS were expressed, purified, and, where appropriate, fluorescently labeled with 5-iodoacetamidofluorescein as described previously (39).…”

“…Although the cytosol-independent nature of IGF-BP-3 nuclear import implies that a role for Ran is unlikely, other GTP-binding proteins may be involved in nuclear protein import, and the action of these proteins may constitute the basis of the inhibition of IGFBP-3 nuclear import by GTP␥S. (35). However, we have also shown that this IGFBP-3 mutant is unable to bind at the cell surface (38), leading to the speculation that transport to the nucleus may be blocked at the level of the plasma membrane rather than at the level of entry into the nucleus.…”

The Insulin‐Like Growth Factor System

“…A number of potential IGF-independent survival-promoting effects of IGFBP-4 and IGFBP-5 have been reported; however, the mechanisms underlying these effects are not known (Perks et al, 1999a). Finally, IGFBP-3 and IGFBP-5 are translocated into the nucleus via the importin-5 subunit (Schedlich et al, 1998(Schedlich et al, , 2000. The cellular consequences of these actions are unknown.…”

Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions

“…A few studies have investigated structural determinants involved in nuclear receptor-IGFBP-3 interactions, using mutants of both RXRα and IGFBP-3, including the basic domain IGFBP-3 mutant originally characterized for its deficient ALS binding and nuclear translocation Schedlich, et al 1998). Using GST-pulldown assays, IGFBP-3 was found to interact with the DNA-binding domain (C domain) of RXRα.…”