Regulation of cell function and identity by cellular senescence.

During aging and in some contexts, like embryonic development, wound healing, and diseases such as cancer, senescent cells accumulate and play a key role in different pathophysiological functions. A long-held belief was that cellular senescence decreased normal cell functions, given the loss of proliferation of senescent cells. This view radically changed following the discovery of the senescence-associated secretory phenotype (SASP), factors released by senescent cells into their microenvironment. There is now accumulating evidence that cellular senescence also promotes gain-of-function effects by establishing, reinforcing, or changing cell identity, which can have a beneficial or deleterious impact on pathophysiology. These effects may involve both proliferation arrest and autocrine SASP production, although they largely remain to be defined. Here, we provide a historical overview of the first studies on senescence and an insight into emerging trends regarding the effects of senescence on cell identity.

Cholesterol biosynthetic pathway induces cellular senescence through ERRα.

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate pathway-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes the transcriptional activity of ERRα that could lead to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis or level in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

Loss of Pla2r1 decreases cellular senescence and age-related alterations caused by aging and Western diets.

Cellular senescence is induced by many stresses including telomere shortening, DNA damage, oxidative, or metabolic stresses. Senescent cells are stably cell cycle arrested and they secrete many factors including cytokines and chemokines. Accumulation of senescent cells promotes many age-related alterations and diseases. In this study, we investigated the role of the pro-senescent phospholipase A2 receptor 1 (PLA2R1) in regulating some age-related alterations in old mice and in mice subjected to a Western diet, whereas aged wild-type mice displayed a decreased ability to regulate their glycemia during glucose and insulin tolerance tests, aged Pla2r1 knockout (KO) mice efficiently regulated their glycemia and displayed fewer signs of aging. Loss of Pla2r1 was also found protective against the deleterious effects of a Western diet. Moreover, these Pla2r1 KO mice were partially protected from diet-induced senescent cell accumulation, steatosis, and fibrosis. Together these results support that Pla2r1 drives several age-related alterations, especially in the liver, arising during aging or through a Western diet.

NF-κB-dependent secretome of senescent cells can trigger neuroendocrine transdifferentiation of breast cancer cells.

Cellular senescence is characterized by a stable proliferation arrest in response to stresses and the acquisition of a senescence-associated secretory phenotype, called SASP, composed of numerous factors including pro-inflammatory molecules, proteases, and growth factors. The SASP affects the environment of senescent cells, especially during aging, by inducing and modulating various phenotypes such as paracrine senescence, immune cell activity, and extracellular matrix deposition and organization, which critically impact various pathophysiological situations, including fibrosis and cancer. Here, we uncover a novel paracrine effect of the SASP: the neuroendocrine transdifferentiation (NED) of some epithelial cancer cells, evidenced both in the breast and prostate. Mechanistically, this effect is mediated by NF-κB-dependent SASP factors, and leads to an increase in intracellular Ca2+ levels. Consistently, buffering Ca2+ by overexpressing the CALB1 buffering protein partly reverts SASP-induced NED, suggesting that the SASP promotes NED through a SASP-induced Ca2+ signaling. Human breast cancer dataset analyses support that NED occurs mainly in p53 WT tumors and in older patients, in line with a role of senescent cells and its secretome, as they are increasing during aging. In conclusion, our work, uncovering SASP-induced NED in some cancer cells, paves the way for future studies aiming at better understanding the functional link between senescent cell accumulation during aging, NED and clinical patient outcome.

Generation of a conditional transgenic mouse model expressing human Phospholipase A2 Receptor 1.

The Phospholipase A2 Receptor 1 (PLA2R1) was first identified for its ability to bind some secreted PLA2s (sPLA2s). It belongs to the C-type lectin superfamily and it binds different types of proteins. It is likely a multifunctional protein that plays a role i) in inflammation and inflammatory diseases, ii) in cellular senescence, a mechanism participating in aging and age-related diseases including cancer, and iii) in membranous nephropathy (MN), a rare autoimmune kidney disease where PLA2R1 is the major autoantigen. To help study the role of PLA2R1 in these pathophysiological conditions, we have generated a versatile NeoR-hPLA2R1 conditional transgenic mice which will allow the specific expression of human PLA2R1 (hPLA2R1) in relevant organs and cells following Cre recombinase-driven excision of the NeoR-stop cassette flanked by LoxP sites. Proof-of-concept breeding of NeoR-hPLA2R1 mice with the ubiquitous adenoviral EIIa promoter-driven Cre mouse line resulted in the expected excision of the NeoR-stop cassette and the expression of hPLA2R1 in all tested tissues. These Tg-hPLA2R1 animals breed normally, with no reproduction or apparent growth defect. These models, especially the NeoR-hPLA2R1 conditional transgenic mouse line, will facilitate the future investigation of PLA2R1 functions in relevant pathophysiological contexts, including inflammatory diseases, age-related diseases and MN.

High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System.

The identification of soluble, folded domains of proteins is a recurring task in modern molecular biology. We detail a protocol for identifying compact soluble protein domains using a self-assembling two-part split-GFP comprised of a detector fragment (GFP ß-strands 1 through 10, or GFP1-10) and a tagging fragment (GFP ß-strand 11, or GFP11). The assay is performed in E. coli cells and in cell extracts. A selection step insures the protein fragments are in frame and contain no stop codons, while an inverse PCR is used to enrich protein fragment libraries containing a specific target sequence.