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Identification of insulin receptor substrate proteins as key molecules for the TßR-V/LRP-1-mediated growth inhibitory signaling cascade in epithelial and myeloid cells

Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

¹Correspondence: Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard St. Louis, MO 63104, USA. E-Mail: huangss@slu.edu or huangjs{at}slu.edu

SPECIFIC AIMS

The type V TGF-ß receptor/IGFBP-3 receptor (TßR-V) mediates IGF-independent growth inhibition by IGFBP-3 and mediates growth inhibition by TGF-ß₁ in concert with other TGF-ß receptor types. TßR-V was recently found to be identical to LRP-1. This has disclosed novel growth regulatory functions of LRP-1. The aim of this study was to identify key molecules for the TßR-V/LRP-1-mediated growth inhibitory signaling cascade.

PRINCIPAL FINDINGS

1. Insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I antagonize growth inhibition by IGFBP-3
To test the possibility that epithelial cell growth factors (insulin, IGF-I, aFGF, bFGF, and EGF) may modulate IGFBP-3-induced growth inhibition in mink lung epithelial cells (Mv1Lu cells), we examined the effects of these growth factors on IGFBP-3-induced growth inhibition in these epithelial cells stimulated by fetal calf serum (FCS). Since IGF-I is capable of antagonizing IGFBP-3 by forming IGFBP-3 complexes, which render IGFBP-3 unable to bind to TßR-V, (Q³A⁴Y¹⁵L¹⁶) IGF-I (an IGF-I analog with low affinity for IGFBP-3 but high affinity for the IGF-I receptor) was used in the experiments. Among these growth factors, only insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I were capable of reversing IGFBP-3-induced growth inhibition. As shown in Fig. 1 , increasing concentrations of insulin quantitatively antagonized IGFBP-3-induced inhibition of DNA synthesis (stimulated by FCS) in Mv1Lu cells (Fig. 1A ). Insulin completely reversed IGFBP-3-induced inhibition with an ED₅₀ of 2.0 nM (Fig. 1A ). Like insulin, (Q³A⁴Y¹⁵L¹⁶) IGF-I completely reversed IGFBP-3-induced growth inhibition with an ED₅₀ of 0.3 nM (Fig. 1B ). In this experiment, (Q³A⁴Y¹⁵L¹⁶) IGF-I at 1 nM stimulated DNA synthesis by 5–10% which was statistically insignificant when compared with DNA synthesis in cells treated without the growth factor. Since a 100-fold higher concentration (100 nM) is known to be required for insulin to bind to the IGF-I receptor, these results suggest that insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I reverse IGFBP-3-induced growth inhibition through interaction with their cognate receptors.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effects of insulin (A) and (Q3A4Y15L16) IGF-I (B) on DNA synthesis in Mv1Lu cells treated with IGFBP-3. Cells were treated with 0.2 or 0.5 µg/mL (5.7 or 14 nM, respectively) of IGFBP-3 in the presence of increasing concentrations (as indicated) of insulin (A) or (Q3A4Y15L16) IGF-I (B) in DMEM containing 0.2% FCS. After 18 h at 37°C, DNA synthesis was determined by measuring [methyl-3H] thymidine incorporation into cellular DNA. [Methyl-3H] thymidine incorporation (19,131 ± 454 cpm/well) in cells stimulated with FCS in the absence of (Q3A4Y15L16) IGF-I and IGFBP-3 was taken as 100% of DNA synthesis (B). Each data point is the mean ±SD of quadruplicate determinations in 4 independent experiments.

2. IGFBP-3 induces specific dephosphorylation of IRS-1 or IRS-2 in Mv1Lu cells
Since insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I are both able to reverse IGFBP-3-induced growth inhibition, we hypothesized that signaling molecules shared by the insulin receptor and IGF-I receptor signaling cascades are involved in reversal of IGFBP-3-induced growth inhibition by insulin or (Q³A⁴Y¹⁵L¹⁶) IGF-I. Because insulin receptor substrate proteins IRS-1 and IRS-2 are two major signaling and molecule-docking proteins shared by both the insulin receptor and IGF-I receptor signaling cascades, both are candidates for playing a role in IGFBP-3-induced growth inhibition. To test this hypothesis, we studied effects of IGFBP-3 with or without insulin or (Q³A⁴Y¹⁵L¹⁶) IGF-I on the phosphorylation status of IRS-1 or IRS-2 in Mv1Lu cells. In these cells stimulated with FCS, IRS-1 and IRS-2 mainly underwent serine-specific phosphorylation, which was required for cell growth. IGFBP-3 treatment of Mv1Lu cells appeared to induce serine-specific dephosphorylation of IRS-1 and IRS-2, which was inhibited by insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I through interaction with their cognate receptors. IGFBP-3-induced dephosphorylated IRS-2 exhibited increased electrophoretic mobility on SDS-PAGE, referred to as IGFBP-3-induced specific dephosphorylation of IRS-2. IGFBP-3-induced specific dephosphorylation of IRS-2 was prevented by cotreatment of cells with TßR-V/LRP-1 antagonists (RAP and TGF-ß peptantagonist) and okadaic acid (a phosphatase inhibitor) but not EGF, FGF, TGF-ß_1, or other known phosphatase inhibitors. TGF-ß₁ stimulated rather than inhibited serine-specific phosphorylation of IRS-2, suggesting that the mechanisms by which TßR-V/LRP-1 mediates growth inhibition by IGFBP-3 and TGF-ß are different. The magnitude of IGFBP-3-induced specific dephosphorylation of IRS-2 positively correlated with the degree of IGFBP-3-induced growth inhibition in Mv1Lu cells and mutant cells derived from Mv1Lu cells. These results suggest that IGFBP-3 induces growth inhibition by stimulating specific dephosphorylation of IRS proteins and that IGFBP-3-induced specific dephosphorylation is prevented by insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I.

3. IRS proteins are required for IGFBP-3-induced growth inhibition
As described above, insulin and (Q³A⁴Y¹⁵L¹⁶) IGF-I, but not other growth factors and cytokines, prevent IGFBP-3-induced specific dephosphorylation of IRS proteins and reverse IGFBP-3-induced growth inhibition. This suggests that IRS proteins may be involved in insulin- or IGF-I-induced reversal of growth inhibition by IGFBP-3. To test this possibility, we examined the effects of IGFBP-3 on cell growth (as determined by measurement of DNA synthesis) of 32D cells stably transfected with IRS cDNAs, insulin receptor cDNA, or vector only. 32D cells are murine myeloid cells which do not express endogenous IRS proteins. They express very low levels of the insulin receptor and high levels of TßR-V. In addition, 32D cells do not exhibit a significant growth inhibitory response to IGFBP-3. For these reasons, the 32D cell system should be appropriate for defining the roles of IRS proteins in IGFBP-3 growth inhibition. As shown in Fig. 2 , IGFBP-3 inhibited DNA synthesis of 32D cells transfected with vector only minimally or not at all (Fig. 2A and B ). However, 32D cells expressing either IRS-1 or IRS-2 (32D/IRS-1 or 32D/IR/IRS-2 cells) exhibited a potent growth inhibitory response to IGFBP-3 (Fig. 2A ). Insulin was capable of reversing IGFBP-3-induced growth inhibition in 32D cells stably expressing the insulin receptor and IRS-2 (32D/IR/IRS-2 cells) (Fig. 2B ). These results are similar to those obtained using Mv1Lu cells as an assay cell system (Fig. 1A ) and suggest that IRS proteins are involved in IGFBP-3-induced growth inhibition.

View larger version (21K): [in this window] [in a new window]   Figure 2. Growth inhibition induced by IGFBP-3 in 32D/IRS-1 and 32D/IR/IRS-2 cells but not in 32D cells (A) and insulin reversal of growth inhibition by IGFBP-3 in 32D/IR/IRS-2 cells (B). Cells were treated with increasing concentrations of IGFBP-3 in the absence (A) or presence (B) of insulin (10 nM). After 18 h at 37°C, DNA synthesis was determined by measuring [methyl-3H] thymidine incorporation into cellular DNA. [Methyl-3H] thymidine incorporation in cells treated without IGFBP-3 was taken as 100% of DNA synthesis. Each data point is the mean ± SD of quadruplicate determinations in 4 independent experiments.

CONCLUSIONS AND SIGNIFICANCE

The molecular mechanism by which TßR-V/LRP-1 mediates growth inhibition via IRS proteins is unknown. IGFBP-3 appears to induce specific dephosphorylation of IRSs by activating an okadaic acid-sensitive phosphatase. The ability of TßR-V/LRP-1 to mediate such dephosphorylation upon IGFBP-3 binding is intriguing. TßR-V/LRP-1 may form complexes with plasma membrane proteins (e.g., co-receptors) and cytoplasmic protein phosphatases which become activated upon IGFBP-3 binding of TßR-V/LRP-1, resulting in dephosphorylation of IRS proteins. LRP-1 has been shown to be a component in signaling complexes containing protein phosphatases or kinases. Based on results described here and published results, we proposed a simplified model for the insulin/(A⁴Q⁵Y¹⁵L¹⁶) IGF-I reversal of growth inhibition by IGFBP-3 and cross-talk of the TßR-I/TßR-II, TßR-V (or TßR-V/TßR-I), insulin receptor, IGF-I receptor, integrin, and c-Met signaling cascades (Fig. 3 ). In this model, cell growth of responsive cells (e.g., epithelial cells) is stimulated by FCS. The IRS proteins (IRS-1 and IRS-2) undergo serine-specific phosphorylation (at "positive sites") that is required for cell growth. IGFBP-3 induces dephosphorylation of IRS-1 and IRS-2 at these specific phosphorylation sites ("positive sites") by activation of a cytoplasmic okaidic acid-sensitive phosphatase through interaction with TßR-V/LRP-1. This leads to growth inhibition. Insulin and (A⁴Q⁵Y¹⁵L¹⁶) IGF-I antagonize IGFBP-3-induced growth inhibition by stimulating tyrosine-specific phosphorylation of IRS proteins through interaction with their cognate receptors. This tyrosine-specific phosphorylation confers resistance of IRS proteins to IGFBP-3-induced serine-specific dephosphorylation. On the other hand, TGF-ß stimulates serine-specific phosphorylation of IRS proteins at "negative sites," presumably via interaction with TßR-V/TßR-I heterocomplex or TßR-V/LRP-1/cytoplasmic protein kinase complexes. TßR-I is a serine-specific protein kinase. This, in concert with the TßR-I/TßR-II heterocomplex/Smad2/3/4 signaling cascade, leads to growth inhibition. The integrin signaling cascade initiated by extracellular matrix proteins (e.g., fibronectin) induced by TGF-ß impairs the ability of insulin to attenuate the TGF-ß-stimulated TßR-V/LRP-1-mediated signaling cascade by down-regulating insulin-stimulated tyrosine phosphorylation of IRS proteins. This impairment can be partially reversed by disrupting interaction between fibronectin and 5ß1 integrin using 5ß1 integrin antagonists (e.g., a cyclic RGD peptide and anti-5ß1 integrin antibodies). Cross-talk between the TßR-V/LRP-1, insulin receptor, and integrin signaling cascades is supported by the observations: 1) insulin alone does not affect TGF-ß-induced growth inhibition in Mv1Lu cells but is capable of partially reversing TGF-ß-induced growth inhibition of these cells in the presence of 5ß1 integrin antagonists; and 2) insulin alone partially reverses TGF-ß-induced growth inhibition in cells which do not express 5ß1 integrin. The c-Met signaling cascade induced by c-Met ligand hepatocyte growth factor (HGF) down-regulates TßR-V/LRP-1 and TßR-I/TßR-II-mediated growth inhibitory signaling at sites of cell cycle regulation in the nucleus. The finding that insulin-induced signaling antagonizes TßR-V/LRP-1-mediated growth inhibitory signaling has potential clinical implications. Insulin or insulin signaling defects may up-regulate TßR-V/LRP-1-mediated growth inhibitory activity generated in wounds, resulting in attenuation of wound re-epithelialization and healing and generated in affected kidney glomeruli, resulting in acceleration of diabetic glomerulopathy, which are common clinical problems often observed in diabetic patients. TGF-ß and IGFBPs have been implicated in these two pathological processes.

View larger version (22K): [in this window] [in a new window]   Figure 3. A model for reversal of IGFBP-3 growth inhibition by insulin or IGF-I and cross-talk of the TßR-I/TßR-II, TßR-V (or TßR-V/TßR-I), insulin receptor, IGF-I receptor, integrin, and c-Met signaling cascades.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1872fje;

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This Article Full Text (PDF) Erratum All Versions of this Article: 18/14/1719 04-1872fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HUANG, S. S. Articles by HUANG, J. S. Search for Related Content PubMed PubMed Citation Articles by HUANG, S. S. Articles by HUANG, J. S.