Down‐regulation of amphiphysin‐1 is responsible for reduced receptor‐mediated endocytosis in the senescent cells

Abstract

Senescent cells have been generally characterized to have improper responsiveness to external stimuli and inefficient uptake of materials compared with presenescent cells, probably by downregulation of receptor-mediated endocytosis. Using transferrin-uptake assay and Western blot of endocytosis-related proteins, we found that a significant decrease of amphiphysin-1 is strongly related to the reduction of receptor-mediated endocytosis in both human diploid fibroblasts of multipassages and hydrogen peroxide-induced premature senescence. We reconfirmed the pivotal role of amphiphysin-1 in senescence by showing that transfection of dominant negative amphiphysin-1 reduces transferrin uptake in presenescent cells and that microinjection of wildtype amphiphysin-1 into senescent cells restores the ability of the transferrin uptake nearly up to that of the presenescent cell. We conclude that the loss of endocytotic activity of senescent cells is directly related to the down-regulation of amphiphysin-1, and its functional restorability can be induced by simple microinjection of amphiphysin-1 gene in the senescent cells.Key words: amphiphysin-1 • endocytosis • senescence • human diploid fibroblast ormal human diploid fibroblasts (HDFs) do not divide indefinitely and eventually lead to an arrest of cell division by a process termed cellular, or replicative, senescence (1, 2). Irreversible growth arrest of senescent cells is suggested to be strongly related to the attenuated response to growth factors (3,4). However, in the case of the epidermal growth factor (EGF), the kinetics for binding in senescent cells still remains active as in early-passaged presenescent cells (5,6). Recently, we reported that up-regulation of caveolin in the senescent cells is partly responsible for the attenuated response to growth factors through the regulation of the downstream signal pathway (7). However, for the receptor-mediated endocytosis, the clathrin-dependent endocytosis system plays the more active and dominant role over the caveolae system. Therefore, it would be worthwhile to monitor the efficiency of clathrindependent receptor-mediated endocytosis in the senescent cells in order to elucidate the exact mode of the attenuated response to growth factors in the senescent cells. N The functional capacity of clathrin-dependent receptor-mediated endocytosis in the senescent HDF can be illustrated by the transferrin uptake model system (8). To reveal the molecular mechanism for the altered endocytosis of the senescent cells, the expression of proteins related to the clathrin-mediated endocytosis was analyzed and the functional recovery of the receptormediated endocytosis in the senescent cells was attempted. MATERIALS AND METHODS AntibodiesMonoclonal antibodies against clathrin heavy chain (C68220), dynamin (D25520), α-adaptin (A43920), β-adaptin (A35620), and transferrin receptor (CD71: SC-7088) were purchased from Cell culture and induction of senescenceHuman foreskin fibroblasts were isolated from a 7-year-old Korean male (10) in our labor...

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