Impairment of ribosome biogenesis leads to p53 induction and cell cycle arrest, a checkpoint involved in human disease. Induction of p53 is attributed to the binding and inhibition of human double minute 2 (Hdm2) by a subset of ribosomal proteins (RPs): RPS7, RPL5, RPL11, and RPL23. However, we found that only RPL11 or RPL5, in a mutually dependent manner, elicit this response. We show that depletion of RPS7 or RPL23, like depletion of other RPs, except for RPL11 and RPL5, induces a p53 response and that the effects of RPS7 and RPL23 on p53 induction reported earlier may be ascribed to inhibition of global translation. Moreover, we made the surprising observation that codepletion of two essential RPs, one from each subunit, but not the same subunit, leads to suprainduction of p53. This led to the discovery that the previously proposed RPL11-dependent mechanism of p53 induction, thought to be caused by abrogation of 40S biogenesis and continued 60S biogenesis, is still operating, despite abrogation of 60S biogenesis. This response leads to both a G1 block and a novel G2/M block not observed when disrupting either subunit alone. Thus, induction of p53 is mediated by distinct mechanisms, with the data pointing to an essential role for ribosomal subunits beyond translation.

Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.

Cellular senescence affects many physiological and pathological processes and is characterized by durable cell cycle arrest, an inflammatory secretory phenotype and metabolic reprogramming. Here, by using dynamic transcriptome and metabolome profiling in human fibroblasts with different subtypes of senescence, we show that a homoeostatic switch that results in glycerol-3-phosphate (G3P) and phosphoethanolamine (pEtN) accumulation links lipid metabolism to the senescence gene expression programme. Mechanistically, p53-dependent glycerol kinase activation and post-translational inactivation of phosphate cytidylyltransferase 2, ethanolamine regulate this metabolic switch, which promotes triglyceride accumulation in lipid droplets and induces the senescence gene expression programme. Conversely, G3P phosphatase and ethanolamine-phosphate phospho-lyase-based scavenging of G3P and pEtN acts in a senomorphic way by reducing G3P and pEtN accumulation. Collectively, our study ties G3P and pEtN accumulation to controlling lipid droplet biogenesis and phospholipid flux in senescent cells, providing a potential therapeutic avenue for targeting senescence and related pathophysiology.

Genetic evidence in living organisms from yeast to plants and animals, including humans, unquestionably identifies the Target Of Rapamycin kinase (TOR or mTOR for mammalian/mechanistic) signal transduction pathway as a master regulator of growth through the control of cell size and cell number. Among the mTOR targets, the activation of p70 S6 kinase 1 (S6K1) is exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant flies and mice reveals that S6K1 predominantly regulates cell size versus cell proliferation. Here we review the putative mechanisms of S6K1 action on cell size by considering the main functional categories of S6K1 targets: substrates involved in nucleic acid and protein synthesis, fat mass accumulation, retrograde control of insulin action, senescence program and cytoskeleton organization. We discuss how S6K1 may be involved in the observed interconnection between cell size, regenerative and ageing responses.

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of ‘senescence-associated-ß-galactosidase’, the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

Ependymal cells are multiciliated glial cells lining the ventricles of the mammalian brain. Their differentiation from progenitor cells involves cell enlargement and progresses through centriole amplification phases and ciliogenesis. These phases are accompanied by the sharp up-regulation of mTOR Complex 1 activity (mTORC1), a master regulator of macromolecule biosynthesis and cell growth, whose function in ependymal cell differentiation is unknown. We demonstrate that mTORC1 inhibition by rapamycin preserves the progenitor pool by reinforcing quiescence and preventing alternative cell cycle progression for centriole amplification. Overexpressing E2F4 and MCIDAS circumvents mTORC1-regulated processes, enabling centriole amplification despite rapamycin, and enhancing mTORC1 activity through positive feedback. Acute rapamycin treatment in multicentriolar cells during the late phases of differentiation causes centriole regrouping, indicating a direct role of mTORC1 in centriole dynamics. By phosphoproteomic and phosphomutant analysis, we reveal that the mTORC1-mediated phosphorylation of GAS2L1, a centrosomal protein that links actin and microtubule cytoskeletons, participates in centriole disengagement. This multilayered and sequential control of ependymal development by mTORC1, from the progenitor pool to centriolar function, has implications for pathophysiological conditions like aging and hydrocephalus-prone genetic diseases.