Senescent human diploid fibroblasts do not respond to growth factors like epidermal growth factor (EGF), although they have a normal level of receptors and downstream signaling molecules. To examine the mechanism of signaling attenuation, we investigated Erk activation after EGF stimulation in senescent cells. 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 EGF receptor. Electron microscopic analysis demonstrated an increased number of caveolae structures in senescent cells. More interestingly, brain, spleen, and lung from 26-month-old rats showed substantial increases of caveolin proteins. However, in the case of p53-induced senescence, caveolin-1 was not induced, and EGF stimulation phosphorylated Erk-1/2 as much as young control cells. Finally, we overexpressed caveolin-1 in young human diploid fibroblasts in which the activation of Erk-1/2 upon EGF stimulation was significantly suppressed. These results suggest that the unresponsiveness of senescent fibroblasts to EGF stimulation may be due to the overexpression of caveolins, which seems to be independent of growth arrest and other aging phenotypes.Normal human diploid cells show cellular senescence in vitro after a finite number of population doublings (1). Although senescent cells can maintain their metabolic activity, the loss of proliferation capacity may be due to a diminished response to growth factors (2-4). In aged human diploid fibroblasts, the decreased response to growth factors is suggested to be associated with a repression of c-fos expression, a reduced AP-1 DNA binding, and diminished DNA synthesis. Senescent cells in culture or tissues have been reported to have normal numbers of EGF 1 receptors (EGFRs) (5, 6), and the binding capacity of EGF to EGFR is normal (7). However, there is no mitogenactivated protein kinase activation upon EGF stimulation in senescent cells, and the mechanism for the age-related modulation of EGFR in response to the EGF stimulation is not yet fully resolved. Caveolae are vesicular invaginations of the plasma membrane with a diameter of 50 -100 nm (8, 9) and regulate signal transduction, potocytosis, and transcytosis (10, 11). Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. The stable expression of the caveolin-1 or -3 gene to the mammalian cells without caveolin induced the formation of caveolae structures (12). Caveolin functions as a scaffolding protein within the caveolae membrane and interacts with signaling proteins, namely EGFR, G-proteins, Src-like kinases, Ha-Ras, protein kinase C, endothelial nitric-oxide synthase, integrin and so on (13-18). A short cytosolic N-terminal region of caveolin is involved in the formation of oligomers and mediates the interaction with these signal molecules, which results in the inactivation of signaling (11). The suppression of these signaling m...

Hyporesponsiveness to growth factors is one of the fundamental characteristics of senescent cells. We previously reported that the up-regulation of caveolin attenuates the growth factor response and the subsequent downstream signal cascades in senescent human diploid fibroblasts. Therefore, in the present experiment, we investigated the modulation of caveolin status in senescent cells to determine the effect of caveolin on mitogenic signaling efficiency and cell cycling. We reduced the level of caveolin-1 in senescent human diploid fibroblasts using its antisense oligonucleotides and small interfering RNA, and this resulted in the restoration of normal growth factor responses such as the increased phosphorylation of Erk, the nuclear translocation of pErk, and the subsequent activation of p-Elk upon epidermal growth factor stimulation. Moreover, DNA synthesis and the re-entry of senescent cells into cell cycle were resumed upon epidermal growth factor stimulation concomitantly with decreases in p53 and p21. Taken together, we conclude that the loss of mitogenic signaling in senescent cells is strongly related to their elevated levels of caveolin-1 and that the functional recovery of senescent cells at least in the terms of growth factor responsiveness and cell cycle entry might be achieved simply by lowering the caveolin level.Caveolae are vesicular organelles that represent a subdivision of the plasma membrane (1, 2). They are most abundant in terminally differentiated cell types, i.e. adipocytes, endothelial cells, and muscle cells. Moreover, it has been suggested that caveolae may function as subcellular compartments for the storage of inactive signaling molecules to regulate activation and to facilitate cross-talk between distinct signaling cascades (3, 4).Caveolin, a 21-24-kDa integral membrane protein, is the principal component of caveolae, and the caveolin gene family consists of caveolin-1, -2, and -3. Caveolin-1 and -2 are coexpressed, form a hetero-oligomer in the plasma membrane, and exist in many cell types (5-7), whereas the expression of caveolin-3 is muscle-specific (8). Moreover, recent studies have suggested a regulatory role for caveolin in addition to its structural function. For example, the stable expressions of the caveolin-1 or -3 genes in caveolin-deficient mammalian cells induced the formation of caveolae structures (9).The caveolin functions as scaffolding protein within the caveolae membrane and interacts with signaling proteins such as EGFR, 1 G-proteins, Src-like kinases, Ha-Ras, protein kinase C, endothelial nitric-oxide synthase, and integrin (10 -15). In addition, a short cytosolic N-terminal region of caveolin is involved in the formation of oligomers and mediates the interaction with signaling molecules, which result in the their inactivation (4). The targeted down-regulation of caveolin-1 is sufficient to drive cell transformation and hyperactivates the Erk kinase cascade (16). And caveolin levels in most tumor tissues are significantly lower than in normal tissues, suggesting ...

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