TOPOGRAPHY OF AMPHIREGULIN EXPRESSION IN CULTURED HUMAN KERATINOCYTES- COLOCALIZATION WITH THE EPIDERMAL GROWTH-FACTOR RECEPTOR AND CD44

Much of the autonomous growth of cultured keratinocytes is attributabl e to tile signaling of amphiregulin, a heparin-binding autocrine growt h factor, through the epidermal growth factor receptor. Emerging evide nce suggests, moreover, that the membrane proteoglycan, CD44, is a cof actor for the interaction of heparin-binding ligands with their recept ors. This model was evaluated by characterizing the patterns of the im munolabeled molecules in cultured human neonatal keratinocytes, to tes t the hypothesis that involvement in a common function results in coor dinate segregation within or on the cell. The molecules were localized by double immunofluorescence labeling to detect amphiregulin and eith er the epidermal growth factor receptor or CD44, and the immunostained products were imaged by scanning laser confocal microscopy. Both amph iregulin and the epidermal growth factor receptor segregated to a peri nuclear distribution and to intercellular contacts. In addition, amphi regulin localized to the outer leading edge of colonies and focally to intranuclear sites. Metabolic blockade of proteoglycan sulfation with sodium chlorate inhibited growth of the cells and concurrently enhanc ed the nuclear; but decreased the outer leading edge, labeling Tor amp hiregulin. There was no nuclear or perimeter labeling for the epiderma l growth factor receptor Cultures co-immunolabeled for CD44 and amphir egulin exhibited variable perinuclear staining for both, but otherwise CD44 was distributed to intercellular contacts. The intercellular loc alizations of CD44 with amphiregulin and of amphiregulin with tile epi dermal growth factor receptor were strongly concordant. These data are consistent with a concerted function at intercellular contacts, where cytokine signaling is mediated via receptor binding and possibly regu lated by the CD44 proteoglycan as cofactor The intranuclear and perime ter labeling of amphiregulin, however, suggests that this cytokine has additional functions, both in the nucleus and as a matrix receptor.