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. 2014 Apr:35:82-90.
doi: 10.1016/j.matbio.2013.12.010. Epub 2014 Jan 2.

Decorin differentially modulates the activity of insulin receptor isoform A ligands

Alaide Morcavallo  1 , Simone Buraschi  2 , Shi-Qiong Xu  3 , Antonino Belfiore  4 , Liliana Schaefer  5 , Renato V Iozzo  6 , Andrea Morrione  7
Affiliations

Decorin differentially modulates the activity of insulin receptor isoform A ligands

Alaide Morcavallo et al. Matrix Biol. 2014 Apr.

Abstract

The proteoglycan decorin, a key component of the tumor stroma, regulates the action of several tyrosine-kinase receptors, including the EGFR, Met and the IGF-IR. Notably, the action of decorin in regulating the IGF-I system differs between normal and transformed cells. In normal cells, decorin binds with high affinity to both the natural ligand IGF-I and the IGF-I receptor (IGF-IR) and positively regulates IGF-IR activation and downstream signaling. In contrast, in transformed cells, decorin negatively regulates ligand-induced IGF-IR activation, downstream signaling and IGF-IR-dependent biological responses. Whether decorin may bind another member of the IGF-I system, the insulin receptor A isoform (IR-A) and its cognate ligands, insulin, IGF-II and proinsulin, have not been established. Here we show that decorin bound with high affinity insulin and IGF-II and, to a lesser extent, proinsulin and IR-A. We utilized as a cell model system mouse embryonic fibroblasts homozygous for a targeted disruption of the Igf1r gene (designated R(-) cells) which were stably transfected with a human construct harboring the IR-A isoform of the receptor. Using these R(-)/IR-A cells, we demonstrate that decorin did not affect ligand-induced phosphorylation of the IR-A but enhanced IR-A downregulation after prolonged IGF-II stimulation without affecting insulin and proinsulin-dependent effects on IR-A stability. In addition, decorin significantly inhibited IGF-II-mediated activation of the Akt pathways, without affecting insulin and proinsulin-dependent signaling. Notably, decorin significantly inhibited IGF-II-mediated cell proliferation of R(-)/IR-A cells but affected neither insulin- nor proinsulin-dependent mitogenesis. Collectively, these results suggest that decorin differentially regulates the action of IR-A ligands. Decorin preferentially inhibits IGF-II-mediated biological responses but does not affect insulin- or proinsulin-dependent signaling. Thus, decorin loss may contribute to tumor initiation and progression in malignant neoplasms which depend on an IGF-II/IR-A autocrine loop.

Keywords: Decorin; IR-A; Proliferation; Signaling.

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Figures

Fig. 1
Fig. 1
Decorin protein core binds to the IR-A, IGF-II, insulin and proinsulin. [A–D] soluble decorin protein core binds to immobilized human recombinant IR-A and IR-A ligands, IGF-II, insulin and proinsulin in a saturable fashion. Solid phase ELISA assays were performed as described in details in Experimental Procedures. Values represent the mean ± SEM of three independent experiments run in triplicates.
Fig. 2
Fig. 2
Decorin does not affect IR-A phosphorylation. [A] Serum-starved R−/IR-A cells were preincubated for 1h with decorin (200 nM) and then stimulated with decorin alone (200 nM), IGF-II, insulin or proinsulin (10 nM) or decorin supplemented with growth factors for 10 min. Phosphorylation of the IR-A was determined by immunoblot using anti-phospho-IGF-IRβ (Tyr1135/1136)/IRβ Tyr1150/1151) rabbit monoclonal antibodies (Cell signaling Technology). Total IR-A levels were detected using anti-IR polyclonal antibodies, which recognize the β subunit of the IR-A. [B] To test the effect of decorin core protein on ligands, IGF-II, insulin and proinsulin were preincubated with 200 nM decorin core and then supplemented to R−/IR-A cells. IR-A phosphorylation and total levels were determined as above. Blots are representative of three independent experiments. [C, D] Densitometric analysis was performed using the ImageJ program (rsbweb.nih.gov/ij/) and the values are reported as relative densitometric arbitrary units. Values represent the mean ±SD of three independent experiments run in duplicates.
Fig. 3
Fig. 3
[A] Decorin enhances IGF-II-dependent IR-A downregulation. Serum-starved R−/IRA cells were incubated with either decorin alone (200 nM), IGF-II, insulin, proinsulin (10 nM) or ligands supplemented with decorin for 24 h and IR-A levels were detected by immunoblot using anti-IR polyclonal antibodies, which recognize the β subunit of the IR-A. Protein loading was monitored using anti-GAPDH monoclonal antibodies. [B] Densitometric analysis was performed using the ImageJ program (rsbweb.nih.gov/ij/) and the values are reported as relative densitometric arbitrary units. Values represent the mean ±SD of three independent experiments run in duplicates and we present the statistically significant differences between critical samples (*p<0.05). [C] Cell surface IR-A levels were measured by ELISA assay in unstimulated (basal cell surface level), decorin alone (200 nM) or ligand-stimulated cells (IGF-II, insulin and proinsulin at 10 mM) supplemented or not with decorin core (200 nM). Densitometric analysis was performed using the ImageJ program (rsbweb.nih.gov/ij/) and the values are reported as relative densitometric arbitrary units. Values represent the mean ±SD of three independent experiments run in duplicates (***p<0.001).
Fig. 4
Fig. 4
Decorin affects IGF-II-induced signaling. [A] Western immunoblotting of serum-starved R−/IR-A treated for 10 and 30 min with the various ligands as indicated at the bottom. The various bands were visualized using the PathScan Multiplex Western Cocktail I (Cell Signaling Technology). ElF4e protein provides a control to monitor protein loading. Blots are representative of three independent experiments. [B–D] Quantification of three independent experiments similar to that shown in panel A. Densitometric analysis was performed using the ImageJ program (rsbweb.nih.gov/ij/) and the values are reported as relative densitometric arbitrary units. Values represent the mean ±SD of three independent experiments run in duplicates and we present the statistically significant differences between critical samples (*p<0.05). The PathScan includes also antibodies for activated p90RSK, which was not detectable in these experimental conditions (data not shown).
Fig. 5
Fig. 5
Decorin inhibits IGF-II-induced proliferation in R−/IR-A cells. Quantification of R−/IR-A cell growth was performed as previously described (Morcavallo et al., 2012) and detailed in Experimental Procedures. Cells were incubated with decorin core alone (200 nM), IGF-II, insulin, proinsulin (10 nM) or ligands supplemented with decorin core protein. Cells were counted after 48 h in a hemocytometer. Values represent the mean ±SD of three independent experiments run in triplicates (*p<0.05, **p< 0.01, ***p<0.001).
Fig. 6
Fig. 6
Schematic drawing summarizing decorin effects on the regulation of ligand-dependent IR-A activity. Decorin binds IGF-II, insulin, proinsulin and the IR-A and enhances IGF-II-induced IR-A downregulation, Akt activation and IGF-II-induced cell proliferation. In addition decorin may regulate IR-A levels at the cell surface. For more information consult the Discussion.
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