Prohibitin Facilitates Cellular Senescence by Recruiting Specific Corepressors To Inhibit E2F Target Genes

Abstract

Prohibitin is a growth regulatory gene that has pleiotropic functions in the nucleus, mitochondria, and cytoplasmic compartments. Earlier studies had proposed a role for prohibitin in modulating cellular senescence, but the underlying mechanisms remain unknown. Here we show that senescence induced by DNAdamaging agents causes the localization of prohibitin to specific heterochromatic foci. Prohibitin could bind to heterochromatin protein 1 (HP1) family proteins and colocalized with HP1␥ in senescence-associated heterochromatic foci. Further, HP1␥ could synergize with prohibitin to repress E2F1-mediated transcriptional activity. The depletion of prohibitin by small interfering RNA or antisense techniques led to a reduction in the senescent phenotype, correlating with a reduced expression of senescence-associated ␤-galactosidase and fewer numbers of senescence-associated heterochromatic foci. Chromatin immunoprecipitation assays showed that prohibitin is needed for the recruitment of HP1␥ to E2F1-regulated proliferative promoters, leading to their repression. The ablation of prohibitin prevented the recruitment of HPI␥, but not Suv39H, to the promoters upon senescence. Prohibitin-mediated recruitment of HP1␥ occurred in only senescent cells, not in quiescent cells; thus, there is a dichotomy in the recruitment of different corepressors by prohibitin, depending on the type of growth arrest. These studies show that prohibitin plays a vital role in inducing cellular senescence.Primary mammalian cells in culture undergo a period of rapid proliferation; however, cell growth eventually decelerates and the cells enter a form of permanent cell cycle arrest termed senescence (20). While normal senescence limits the replicative potential of cells by telomere shortening (41), cells can undergo a similar permanent G 1 growth arrest in response to treatment with drugs like etoposide or adriamycin or in response to oncogenes like Ras (15,45,52,54,55). This growth arrest, also called STASIS (stress-or aberrant signaling-induced senescence), is similar to replicative senescence in that cells are unable to divide under mitotic stimulation even though they remain metabolically active. They also show changes in morphology that are typical of senescence (12,14,27). It has been shown that cultures of immortal cell lines can also undergo permanent cell cycle arrest in response to DNAdamaging agents, suggesting that senescence may act as a natural barrier to cancer progression (6,7,17,46,59,67).A senescent cell typically has a flattened morphology and increased granularity. At the biochemical level, senescence is accompanied by changes in metabolism and characterized by the induction of senescence-associated ␤-galactosidase activity (5, 13, 53), while at the genetic level, alterations in chromatin structure and gene expression patterns are observed (2,25,37,49,57). In senescing cells, the chromatin undergoes remodeling and senescence-associated heterochromatic foci (SAHF) can be detected (37). Heterochromatin is responsible for epigenet...

Cellular senescence and chromatin organisation

“…Thus, p16/Rb links SAHF formation and cell cycle gene silencing. Consistent with this, heterochromatin markers are accumulated on the promoters of the cell cycle genes during senescence Rastogi et al, 2006). It is particularly worth noting that similar heterochromatinisation of some E2F-target genes was observed in a differentiation model (Ait-Si-Ali et al, 2004).…”

A Small Molecule Disruptor of Rb/Raf-1 Interaction Inhibits Cell Proliferation, Angiogenesis, and Growth of Human Tumor Xenografts in Nude Mice

“…One microgram of RNA was DNase treated using RQ1 DNase (Promega), followed by first-strand cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). A fraction (1/20) of the final cDNA reaction volume was used in each PCR (20). Primer sequences are as follows: 5 ¶-CTG CCA GCT GTA CCA GAG AT-3 ¶ (TS forward primer), 5 ¶-ATG TGC ATC TCC CAA AGT GT-3 ¶ (TS reverse primer), 5 ¶-CCC CAT GAT TGT GTT GGT AT-3 ¶ (Cdc6 forward primer), 5 ¶-TTC AAC AGC TGT GGC TTA CA-3 ¶ (Cdc6 reverse primer), 5 ¶-CTC AAC ACG GGA AAC CTC AC-3 ¶ (18S forward primer), and 5 ¶-AAA TCG CTC CAC CAA CTA AGA A-3 ¶ (18S reverse primer).…”

MicroRNA dysregulation in gastric cancer: a new player enters the game

“…This transcriptional co-regulator has been reported to enhance the transcriptional activity of p53 (Fusaro et al, 2003), but have opposite effects on E2F-1 (Rastogi et al, 2006) and NF-kB (Theiss et al, 2009). MiR-27a, an miRNA upregulated in gastric cancer, has been shown to directly bind to the 3 0 -UTR of prohibitin mRNA and downregulate its protein expression.…”