Insulin-like growth factor binding protein-4 interacts with centrosomes and microtubules in primary astrocytes

Insulin-like growth factor-1 and -2 (IGFs) are important for CNS development and have implications in pathological situations of the brain. Insulin-like growth factor binding protein-4 (IGFBP-4) regulates the biological effects of IGFs. We examined the expression of IGFBP-4 in primary rat and human astrocytes. IGFBP-4 mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression was verified by Western blotting of cell lysates as well as conditioned culture medium. When astrocytes were immunostained for IGFBP-4 we detected an intracellular structure that did not derive from organelles involved in cellular trafficking. Ingestion of fluorescein isothiocyanate-labeled transferrin excluded detection of IGFBP-4 within vesicles of endosomal nature, in which the protein might have been incorporated. Double staining with gamma-tubulin demonstrated co-localization of IGFBP-4 with centrosomes of these cells. Treatment with nocodazole resulted in absence of IGFBP-4 signal on centrosomes, indicating a dependency on intact microtubules. Immunoelectron microscopy revealed IGFBP-4 localization not only at the centrioles but also a direct interaction with microtubules. There was no binding of IGFBP-4 to centrioles in primary rat oligodendrocytes, microglia or meningeal cells. The association of IGFBP-4 with centrioles and microtubules in astrocytes suggests an involvement of this molecule in microtubule functions of these cells.