Evidence that IGF binding protein-5 functions as a ligand-independent transcriptional regulator in vascular smooth muscle cells

Insulin- like growth factor binding protein ( IGFBP)- 5 is a conserved protein synthesized and secreted by vascular smooth muscle cells ( VSMCs). IGFBP- 5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP- 5 also stimulates VSMC migration through an IGF- independent mechanism, but the molecular basis underlying this ligand- independent action is unknown. In this study, we show that endogenous IGFBP- 5 or transiently expressed IGFBP- 5- EGFP, but not IGFBP- 4- EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP- 4/ 5 chimeras and IGFBP- 5 points mutants, we demonstrated that the IGFBP- 5 C- domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP- 5 nuclear localization, suggesting the nuclear IGFBP- 5 is derived from the secreted protein. When added exogenously, I-125- or Cy3- labeled IGFBP- 5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor( s) that interact with IGFBP- 5, a human aorta cDNA library was screened by a yeast two- hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP- 5, no known transcription factors were found. Further motif analysis revealed that the IGFBP- 5 N- domain contains a putative transactivation domain. When fused to GAL- 4 DNA dinging domain and tested, the IGFBP- 5 N- domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF- I has little effect on transactivation activity. These results suggest that IGFBP- 5 is localized in VSMC nucleus and possesses transcription- regulatory activity that is IGF independent. The full text of this article is available online at http:// circres. ahajournals. org.