Several lines of evidence indicate that caveolin-1 may function as a general negative regulator to inhibit the basal activity of many signaling proteins. One would predict that down-regulation of caveolin-1 leads to increased basal activity for a number of signaling pathways and subsequent cell transformation. Numerous independent but complementary data support this hypothesis. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-Abl and H-ras (G12V); caveolae are absent from these cell lines. In addition, induction of caveolin-1 expression in v-Abl- and H-ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae (Engelman et al., 1997
). Moreover, antisense-mediated reduction of caveolin-1 protein expression in NIH 3T3 cells is sufficient to drive oncogenic transformation and constitutively activate the p42/44 mitogen-activated protein kinase cascade (Galbiati et al., 1998
). Thus, down-regulation of caveolin-1 expression and caveolae organelles may be critical for maintaining the transformed phenotype.
Consistent with this idea, we have recently demonstrated that overexpression of caveolin-1 in MEFs induces cell cycle arrest in the G0
phase of the cell cycle, a reduction in cell proliferation, and a reduction in the DNA replication rate (Galbiati et al., 2001b
). We have also shown that a p53/p21-dependent pathway mediates this caveolin-1-induced cell cycle arrest (Galbiati et al., 2001b
). Given the role of caveolin-1 in mediating cell cycle arrest, one might predict a possible role for caveolin-1 in promoting cellular senescence. In fact, cell cycle arrest is an essential step in achieving the senescent phenotype. In support of this hypothesis, up-regulation of caveolin-1 was recently observed during the serial passaging of normal human diploid fibroblasts (Park et al., 2000
). However, whether up-regulation of caveolin-1 is a necessary step in promoting cellular senescence remains unknown.
In the present study, we directly investigated the importance of caveolin-1 protein in mediating cellular senescence in vivo. We demonstrated that MEFs overexpressing caveolin-1 show premature irreversible growth arrest, have a senescence-like cell morphology, and are enriched in senescence-associated acid β-galactosidase activity, which is typical of senescent cells. In addition, the senescent phenotype is characterized by induction of cell cycle inhibitory proteins (Dimri et al., 1995
; Dumont et al., 2000
; Frippiat et al., 2001
). Importantly, we have previously demonstrated that overexpression of caveolin-1 activates a p53/p21-dependent pathway in MEFs (Galbiati et al., 2001b
). Taken together, these results indicate that overexpression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype.
Tumor development is initiated by a multiplicity of genetic abnormalities. Moreover, tumor cells need to escape barriers that limit uncontrolled cell proliferation. One of these barriers is represented by cellular senescence. Cancer cells need to overcome this obstacle to produce a clinically relevant tumor mass. For these reasons, cellular senescence represents a natural tumor suppressor mechanism. In recent years, several independent lines of evidence have emerged that suggest that caveolin-1 functions as a “tumor suppressor protein” in mammalian cells. In fact, modification and/or inactivation of caveolin-1 expression appears to be a common feature of the transformed phenotype. For example, caveolin-1 protein expression has been demonstrated to be absent in several transformed cell lines derived from human mammary carcinomas, including MT-1, MCF-7, ZR-75–1, T47D, MDA-MB-361, and MDA-MB-474 (Sager et al., 1994
). We show here that caveolin-1 expression is critical in achieving the senescent phenotype. These results suggest that cellular senescence may represent one of the molecular mechanisms through which caveolin-1 acts as a tumor suppressor protein.
Cellular senescence is spontaneously achieved by somatic cells. However, many external and internal cellular stimuli can accelerate the acquisition of the senescent phenotype. Oxidative stress, for example, has been widely demonstrated to be responsible for premature senescence (Chen and Ames, 1994
; Chen et al., 1995
; Dimri et al., 1995
; von Zglinicki et al., 1995
; Dumont et al., 2000
; Frippiat et al., 2001
). Understanding at the molecular level the intracellular pathways affected by cellular stresses will improve our knowledge of the more complicated aging process. In this report, we demonstrated that overexpression of caveolin-1 induces premature senescence. As a consequence, we next asked whether premature senescence induced by oxidative stress is associated with increased endogenous caveolin-1 expression. H2
has been previously demonstrated to induce premature senescence in human diploid fibroblasts (Chen and Ames, 1994
; Chen et al., 1995
; von Zglinicki et al., 1995
). We demonstrate here that treatment of NIH 3T3 cells with subcytotoxic doses of H2
induces premature senescence and up-regulation of caveolin-1 at the transcriptional level and at the protein level. Interestingly, 3 d after subcytotoxic stimulation with H2
, caveolin-1 expression was up-regulated and remained elevated up to 11 d, whereas senescence-associated β-galactosidase activity was first observed only after 7 d post-H2
stimulation (our unpublished results) and remained elevated up to 11 d. These results indicate that up-regulation of caveolin-1 precedes the onset of the senescent phenotype, suggesting that caveolin-1 expression may be necessary to initiate and maintain cellular senescence.
The maintenance of a “physiological redox tone” is essential to prevent the degenerative processes associated with aging. Dietary antioxidants are believed to prevent and/or contain oxidative damages induced by oxidative stress (Halliwell, 1996
; Palmer and Paulson, 1997
). We demonstrated that quercetin, a flavanoid found in foods of plant origin, and vitamin E prevented the premature senescence phenotype and the up-regulation of caveolin-1 induced by H2
. We also found that quercetin directly negatively regulates caveolin-1 protein expression. These results support the idea of a tight correlation between the senescence phenotype and up-regulation of caveolin-1. This data is supported by studies showing that endogenous antioxidants such as reduced glutathione decrease with age (Hu et al., 2000
). Interestingly, caveolin-1 protein expression has been demonstrated to increase with age (Park et al., 2000
). Because we demonstrated in this report that oxidative stress up-regulates caveolin-1, whereas the dietary antioxidant quercetin down-regulates caveolin-1 protein expression, we may speculate that up-regulation of caveolin-1 occurring with the aging process may be due in part to the accumulation of oxidants and the reduction of endogenous antioxidants.
Using an antisense-based approach, we previously generated and characterized NIH 3T3 cells that express substantially reduced levels of caveolin-1 (Galbiati et al., 1998
). These cells are characterized by a transformed phenotype. In fact, they form foci in petri dishes, exhibit anchorage-independent growth in soft agar, and form tumors in immunodeficient mice (Galbiati et al., 1998
). If caveolin-1 expression is a key element in promoting cellular senescence, NIH 3T3 cells harboring caveolin-1 antisense should be protected against SIPS. We demonstrate in this report that Cav-1-AS cells express significantly lower levels of acid β-galactosidase activity when stimulated with subcytotoxic levels of H2
as compared with normal control NIH 3T3 cells. Interestingly, the ability of these cells to express high levels of senescence-associated β-galactosidase activity is recovered when caveolin-1 levels are restored. Taken together, these data indicate that caveolin-1 may be a fundamental player in the intracellular pathway that leads to premature senescence.
The role of caveolin-1 in mediating apoptosis remains contradictory. On one hand, caveolin-1 has been shown to promote ceramide-induced apoptosis in diploid fibroblasts (Zundel et al., 2000
). Moreover, Lisanti and colleagues (Liu et al., 2001
) have demonstrated that caveolin-1 sensitizes fibroblasts and epithelial cells to staurosporine-induced programmed cell death and that caveolin-1 antisense cells are resistant to staurosporine-induced apoptosis. In addition, we have shown that transgenic expression of caveolin-1 in MEFs sensitizes these cells to staurosporine-induced programmed cell death (Galbiati et al., 2001b
). On the other hand, caveolin-1 has been shown to act as a suppressor of c-myc-induced apoptosis in LNCaP cells, a human epithelial prostate cancer-derived cell line (Timme et al., 2000
). Thus, evidence has been presented that caveolin-1 is both a facilitator and a suppressor of programmed cell death in different contexts. In the present study, we show that cells expressing low levels of caveolin-1 (Cav-1-AS cells) preferentially undergo apoptosis when stimulated with subcytotoxic levels of H2
as compared with normal control NIH 3T3 cells and Rev-Cav-1-AS cells, as verified by cell and nuclear morphology. This result is not surprisingly because it has been previously reported that subcytotoxic concentrations of H2
can induce apoptosis in human fibroblasts (Hampton and Orrenius, 1997
; Macho et al., 1997
; Chen et al., 2000
). In addition, this result is consistent with the idea that the role of caveolin-1 in mediating apoptosis may be different depending on the nature of the extracellular apoptotic stimulus. We can speculate that diploid fibroblasts react differently to oxidative stress depending on the level of endogenous caveolin-1. Cells with normal levels of caveolin-1 react to oxidative stress by choosing premature senescence through up-regulation of caveolin-1, whereas cells in which caveolin-1 expression is kept low (by the antisense vector in the case of Cav-1-AS cells) preferentially undergo programmed cell death.
It is interesting to point out that ionizing radiation and chemotherapeutic drugs, which induce apoptosis in cancer cells, represent sources of reactive oxygen species. We know that caveolin-1 protein expression is down-regulated during cell transformation. Also, caveolin-1 has been shown to be up-regulated in multidrug-resistant cancer cells (Lavie et al., 1998
; Yang et al., 1998
). We speculate that differences in caveolin-1 protein expression may facilitate or prevent the efficacy of specific anticancer treatments. In addition, the dual role of caveolin-1 in promoting senescence and apoptosis is not uncommon. In fact, the tumor suppressor protein p53 is directly involved in both cell cycle arrest/senescence and programmed cell death (Amundson et al., 1998
; el-Deiry, 1998
; Sionov and Haupt, 1998
; Bates and Vousden, 1999
). Lisanti and colleagues (Razani et al., 2000
) have demonstrated that caveolin-1 gene transcription is induced by p53. We have recently shown that caveolin-1 expression can increase the activity of p53 (Galbiati et al., 2001b
). Thus, caveolin-1 and p53 may act synergistically in their dual role of promoting senescence and apoptosis in different cellular contexts.
Because transgenic expression of caveolin-1 in MEFs induces cellular senescence and SIPS correlates with increased endogenous caveolin-1 expression, we speculate that caveolin-1 transgenic mice may represent an interesting mouse model for the study of the aging process and the characterization of the molecular mechanisms underlying degenerative diseases. However, additional experiments are necessary to directly test this hypothesis.