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...
Human diploid fibroblasts (HDF) do not divide indefinitely and eventually lead to an arrest of cell division by a process termed cellular or replicative senescence. Irreversible growth arrest of senescent cells is strongly related to the attenuated response to growth factors. Recently, we reported that up‐regulation of caveolin in the senescent cells is responsible for the attenuated response to growth factors. Senescent cells did not phosphorylate Erk‐1/2 after EGF stimulation, whereas young cells did. In those senescent cells, we found an increased level of caveolin proteins and strong interactions between caveolin‐1 and EGFR. When we overexpressed caveolin‐1 in young HDF, the activation of Erk‐1/2 on EGF stimulation was significantly suppressed. These results suggest that the hyporesponsiveness of senescent fibroblasts to EGF stimulation might be due to the overexpression of caveolin. In addition, the clathrin‐dependent endocytosis system plays the more active and dominant role over the caveolae system. Therefore, we monitored the efficiency of clathrin‐dependent 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. Using a transferrin‐uptake assay and Western blot analysis of endocytosis‐related proteins, we found a significant decrease of amphiphysin‐1 in human diploid fibroblasts of multipassages. By adjusting the level of amphiphysin, we could modulate the efficiency of receptor‐mediated endocytosis either in young or old cells toward growth factors: that is, a dominant negative mutant of amphiphysin‐1 blocked the endocytosis in the young cells, while microinjection of the gene resumed its activity in the old cells. Taken together, we conclude that the loss of endocytotic activity of senescent cells is directly related to the down‐regulation of amphiphysin‐1 and/or up‐regulation of caveolins. This opens a new field of functional recovery of the senescent cells simply through adjusting the receptor‐mediated endocytosis capacity.
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