Intracellular localization of hyaluronan in proliferating cells

Hyaluronan is a high molecular weight glycosaminoglycan found in the extrac ellular matrix of many tissues, where it is believed to promote cell migrat ion and proliferation. It was recently shown that hyaluronan-dependent peri cellular matrix formation is a rapid process that occurs as cells detach du ring mitosis. Growing evidence for intracellular hyaluronan in tissues in v ivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyalur onan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-lab eled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyal uronan staining was seen in the cytoplasm in a diffuse, network-like patter n and in vesicles. Nuclear hyaluronan staining was observed and confirmed b y confocal microscopy and was often associated with nucleoli and nuclear cl efts. After serum stimulation of 3T3 cells, there was a dramatic increase i n cytoplasmic hyaluronan staining, especially during late prophase/early pr ometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan b inding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluo rescein-labeled hyaluronan was taken up avidly into vesicles in growing cel ls but was localized distinctly compared to endogenous hyaluronan, suggesti ng that hyaluronan in cells may be derived from an intracellular source. Th ese data indicate that intracellular hyaluronan may be involved in nucleola r function, chromosomal rearrangement, or other events in proliferating cel ls.