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Decorin affects endothelial cells by interacting with IGF‐I and its receptor
Author(s) -
Schönherr Elke
Publication year - 2004
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/j.0959-9673.2004.369ai.x
Subject(s) - decorin , microbiology and biotechnology , proteoglycan , receptor , tyrosine kinase , chemistry , kinase insert domain receptor , angiogenesis , biology , biochemistry , extracellular matrix , vascular endothelial growth factor a , vascular endothelial growth factor , cancer research , vegf receptors
Endothelial cells (ECs) undergoing angiogenesis start to synthesize decorin, a member of the family of small proteoglycans with leucine‐rich repeats. Previously, we could show that decorin synthesis in ECs leads to capillary formation and survival of ECs in an angiogenesis model (Schönherr et al . 1999). In this model, decorin enhanced the phosphorylation of protein kinase B (Akt) and subsequently induced the cyclin‐dependent kinase inhibitor p21 (Schönherr et al . 2001). Materials and methods Cell culture : ECs of the permanent cell line EA.hy 926 were cultured on collagen type‐I‐coated dishes in Waymouth MAB 87/3 medium containing 1% heat‐inactivated fetal calf serum and antibiotics. Decorin purified from fibroblast cultures and/or the respective inhibitors were added. Binding studies: Decorin and IGF‐I were labelled with 125 I‐iodine. (1) Decorin was run on an SDS‐PAGE and transferred to nitrocellulose. Blots were probed with 125 I‐IGF‐I and exposed to X‐ray films. (2) A Sepharose CL‐4B gel filtration column was equilibrated with or without decorin containing buffer and 125 I‐IGF‐I (500.000 cpm) was applied. The elution profiles were monitored. (3) IGF‐receptor was immunoprecipitated from EA.hy 926 cells with the antibody (sc‐713) against the β‐chain of the receptor. The immune complex was incubated with labelled or unlabelled decorin or IGF‐I (as indicated). Immune complexes without receptor were used as controls. Results To analyse how decorin activates Akt, inhibitors of different receptor tyrosine kinases were tested. The EGF‐receptor inhibitor tyrphostin AG1478 (10 µm) had no effect on decorin‐induced Akt posphorylation, but preincubation with tyrphostin AG1024 (10 µm), an inhibitor of the insulin and the IGF‐I receptor tyrosine kinases, inhibited Akt phophorylation and p21 expression. Combined addition of IGF‐I and decorin to ECs in culture had an additive effect on Akt phosphorylation. Binding experiments with 125 I‐IGF‐I to decorin immobilized on nitrocellulose revealed that both the core protein and the proteoglycan bind 125 I‐IGF‐I. Binding of 125 I‐IGF‐I to decorin in solution showed the dose‐dependent formation of a high molecular weight complex that eluted in the included volume of the column. Immunoprecipitated IGF‐I receptor bound 125 I‐‐IGF‐I as well as 125 I‐decorin. In addition, 125 I‐IGF‐I bound to the receptor could be displaced by unlabelled decorin. Discussion These results suggest that decorin can bind to IGF‐I and to its receptor and that this interaction enhances Akt phosphorylation in ECs. The effect of decorin on the IGF‐I receptor could be mediated by direct interaction. However, a further possibility is that decorin couples trace amounts of IGF‐I from the medium or the collagen on the dishes to a larger complex, which can more effectively activate the IGF‐I receptor. These possibilities as well as the role of IGF binding proteins will need further investigation.