Up-regulation of Caveolin Attenuates Epidermal Growth Factor Signaling in Senescent Cells

Abstract

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...

Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology

“…Senescent cells are resistant to mitogeninduced proliferation (Aggarwal et al, 1995;Park et al, 2000), express senescence-associated b-galactosidase (SAb-gal) (Dimri et al, 1995), have a characteristic enlarged, flattened morphology (Wagner et al, 2001), and show altered gene expression (Smith and Pereira-Smith, 1996). RNA differential displays and the serial assessment of gene expression (Welle et al, 2001) have been applied to explore senescence-associated genes to gain an insight into the molecular mechanisms underlying senescence.…”

Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

“…Cellular senescence has also been characterized by increases in the levels of some senescence‐associated proteins, such as P‐ERK1/2 (Lim et al ., 2000), caveolin‐1 (Park et al ., 2000), and cell cycle regulators like p53, p16 ink4a , and p21 waf1 (Yeo et al ., 2000b). Therefore, to confirm PPKO‐induced cellular senescence, we examined levels of these proteins by Western blot analysis after treating HDFs with 1 mM PPKO for 3–7 days.…”

Caveolar vesicles generate DNA damage and perpetuate cellular aging

“…Interestingly, a close examination of the EM of caveolar structures ( Figure 6 of Bai's paper [6]), revealed that they are caveolar vesicles and not cave like structures at the membrane. These pinched off vesicles may represent a unique misregulation of caveolae in senescent cells that could explain both the functional differences and the increase in caveolar structures observed previously [7,8]. This misregulation may also explain why PTRF leads to DNA damage.…”

“…The original investigations into the relationship between caveolae and senescence showed a unexpected upregulation of the proteins caveolin-1 and 2 during replicative senescence [7,8]. It was unclear what caused this upregulation, and now Bai et al demonstrated that it is due to the regulatory cavin protein, PTRF, which is known to drive the biogenesis of caveolae [9].…”

“…Previous studies were at odds as to whether caveolin expression generated bona-fide functional caveolar structures [7] or represented a differential regulation of lipid rafts [8]. Interestingly, a close examination of the EM of caveolar structures ( Figure 6 of Bai's paper [6]), revealed that they are caveolar vesicles and not cave like structures at the membrane.…”