Abstract
A family of six high affinity IGF-binding proteins (IGFBPs 1-6) plays an important role in modulating IGF activities. Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. However, very little is known about possible IGF-independent actions of IGFBP-6. We have generated a non-IGF-binding IGFBP-6 mutant by substituting Ala for four amino acid residues (Pro(93)/Leu(94)/Leu(97)/Leu(98)) in its N-domain IGF-binding site. A >10,000-fold loss of binding affinity for IGF-I and IGF-II was observed using charcoal solution binding assay, BIAcore biosensor, and ligand blotting. Wild-type and mutant IGFBP-6, as well as IGF-II, induced cell migration in RD rhabdomyosarcoma and LIM 1215 colon cancer cells. Cell migration was mediated by the C-domain of IGFBP-6. Transient p38 phosphorylation was observed in RD cells after treatment with IGFBP-6, whereas no change was seen in phospho-ERK1/2 levels. Phospho-JNK was not detected. IGFBP-6-induced cell migration was inhibited by SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of ERK1/2 MAPK activation. In contrast, SP600125, a JNK MAPK inhibitor, had no effect on migration. Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration.
Highlights
A family of six high affinity IGF-binding proteins (IGFBPs 1– 6) plays an important role in modulating IGF activities
Knockdown of p38 MAPK using short interfering RNA blocked IGFBP-6-induced migration of RD cells. These results indicate that p38 MAPK is involved in IGFBP-6-induced IGF-independent RD cell migration
We present evidence suggesting that IGFBP-6 activates p38 MAPK, which in turn is involved in IGFBP-6-stimulated migration of human RD rhabdomyosarcoma cells
Summary
Recent studies suggest that some IGFBPs may have IGF-independent effects, including induction of apoptosis and modulation of cell migration. Some IGFBPs have IGF-independent effects, including inhibition of growth, and modulation of cell migration and apoptosis [1]. A recent crystal structure of a complex comprising the N-domain of IGFBP-4 and IGF-I confirmed this binding site [4] Substitution of these hydrophobic residues in IGFBP-3 and -5 decreased their IGF-I binding affinities Ͼ1000-fold and diminished their effects on IGF actions [5, 6]. Because of its binding preference, IGFBP-6 inhibits IGF-II-induced cell proliferation, differentiation, and survival [12,13,14,15,16], but it has marginal or no effect on IGF-I actions. We present evidence suggesting that IGFBP-6 activates p38 MAPK, which in turn is involved in IGFBP-6-stimulated migration of human RD rhabdomyosarcoma cells
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