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Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localisation and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localisation is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we demonstrated that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs displayed reduced 1,4,5-inositol trisphosphate receptor (IP3R)-mediated ER calcium release downstream of Phospholipase C activation. Collectively, our data indicate that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the level of the ER, in addition to its well-known role in regulating apoptosis through the mitochondria., (© 2024. The Author(s).)

Non-lethal caspase activation (NLCA) has been linked to neurodevelopmental processes. However, how neurons control NLCA remains elusive. Here, we focused on Bcl-xL, a Bcl-2 homolog regulating caspase activation through the mitochondria. We generated a mouse model, referred to as ER-xL, in which Bcl-xL is absent in the mitochondria, yet present in the endoplasmic reticulum. Unlike bclx knockout mice that died at E13.5, ER-xL mice survived embryonic development but died post-partum because of altered feeding behavior. Enhanced caspase-3 activity was observed in the brain and the spinal cord white matter, but not the gray matter. No increase in cell death was observed in ER-xL cortical neurons, suggesting that the observed caspase-3 activation was apoptosis-independent. ER-xL neurons displayed increased caspase-3 activity in the neurites, resulting in impaired axon arborescence and synaptogenesis. Together, our findings suggest that mitochondrial Bcl-xL finely tunes caspase-3 through Drp-1-dependent mitochondrial fission, which is critical to neural network design., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s).)

Unlabelled: The involvement of the renin-angiotensin-aldosterone system (RAAS) and cortisol in increased cardiovascular risk is well known. If numerous relationships between RAAS and corticosteroids have been described, their interactions within the arterial wall, especially during the transdifferentiation of vascular smooth muscle cells (VSMCs) and the atheroma formation, are not established. Here, we clarified the relationships between mRNA levels of corticosteroid and angiotensin system components using cortisol, fludrocortisone, and angiotensin II treatments of cultured VSMCs maintained in a contractile phenotype or induced to a lipid storing phenotype. We then determined the quantitative relationships between the mRNA content of these components measured with reverse transcription polymerase chain reaction (RT-PCR), in the atheroma plaque and nearby macroscopically intact tissue (MIT) from 27 human carotid endarterectomy samples. In both VSMC phenotypes, cortisol markedly increased both angiotensinogen (AGT) and AT1-receptor (AT1R) mRNA levels. These effects of cortisol were mediated via glucocorticoid receptor-α (GRα) without any illicit activation of the mineralocorticoid receptor (MR). Angiotensin II increased GRα, 11βHSD1, CYP11B1, as well as CYP11B2 mRNAs and decreased AT1R in contractile VSMC; only GRα and CYP11B2 were increased in lipid storing VSMCs, while MR and AGT mRNAs decreased. In endarterectomy specimens, positive correlations between mRNA levels of AGT and aldosterone synthase or 11βHSD1 in MIT and of AT1R and MR in atheroma were detected. The arterial tissue angiotensin system is a target for local glucocorticoids and arterial glucocorticoids for angiotensin II. Both systems appear activated in lipid storing VSMCs and strongly correlated in vivo, and their mutual amplification may contribute to the development of atheroma., Key Message: Cortisol increases angiotensin II signaling in VSMCs via GRα. Angiotensin II stimulates cortisol signaling through increased GRα and 11β-HSD1. Corticoid and angiotensin receptors are strongly correlated in the arterial wall. These correlations are maintained at different stages of atheroma development. An auto-amplification loop between angiotensin and cortisol signaling favors atherogenesis.

High cortisol and aldosterone levels increase cardiovascular risk, but the respective roles of each hormone within the arterial wall remain controversial. We tested the hypothesis that cortisol production within the arterial wall may contribute to atherosclerotic remodeling and act through illicit activation of the mineralocorticoid receptor (MR). Gene expression studies of the corticoid system components and marker genes of the atherosclerotic process in human carotid atheroma plaque and nearby macroscopically intact tissue (MIT) were considered together with clinical data and compared with pharmacological stimulations of human vascular smooth muscle cells (VSMCs) in contractile or lipid-storing phenotypes. The components of corticoid production and action were present and active within the human carotid wall and VSMCs. Atheroma plaque and lipid-storing VSMCs expressed 11β-hydroxysteroid deshydrogenase-1 (11β-HSD1) at two- to tenfold higher levels than MIT or contractile VSMCs. The 11β-HSD1 expression was stimulated by cortisol and cortisone, especially in lipid-storing VSMCs. MR mRNA level was lower in atheroma and lipid-storing VSMCs and downregulated via MR by fludrocortisone and cortisol. Cortisol upregulated collagen1 and MCP-1 mRNAs via the glucocorticoid receptor (GRα), in both VSMC phenotypes, whereas fludrocortisone stimulated the collagen1 expression only in lipid-storing VSMCs. The GRα mRNA level in MIT was higher in patients with previous stroke and correlated positively with the collagen1 mRNA but negatively with diastolic blood pressure. Local cortisol production by 11β-HSD1, and its action via high parietal GRα could be relevant from the first step of atherosclerotic remodeling and auto-amplify with transdifferentiation of VSMCs during atheroma progression., (© 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Société Française de Pharmacologie et de Thérapeutique.)

p21WAF1 and 14-3-3sigma, which are both transcriptional products of p53, have been reported to play a role in the G2 DNA damage checkpoint in mammalian cells. Human colon carcinoma cells, isogenic except for the presence or absence of either p21WAF1 or 14-3-3sigma (T. A. Chan et al., Genes Dev., 14: 1584-1588, 2000), are useful models for analysis of the role of these proteins in checkpoint control. Here, we have examined mitotic behavior within a single cell cycle after DNA damage in these cell lines. Our results show that p21WAF1, but not 14-3-3sigma, imposes a significant G2 delay after DNA damage. After G2 delay, we found that all isogenic cells, including those competent for both p21WAF1 and 14-3-3sigma, adapt to the DNA damage checkpoint and progress into mitosis, where they undergo incomplete chromosome segregation and reenter G1 with a tetraploid DNA content. Strikingly, our results show that p21WAF1, but not 14-3-3sigma, activates a checkpoint in response to DNA damage that prevents continued cycling of the tetraploid cells that result from a mitotic catastrophe characterized by failure to complete cell division. These results demonstrate that a tetraploid DNA content is not a reliable criterion to establish that arrest occurs in G2. Also, the DNA damage checkpoint mediated by p53-dependent induction of p21WAF1 assures neither G2 arrest nor DNA repair sufficient to enable accurate chromosome segregation in human colon carcinoma cells. We conclude that p21WAF1, but not 14-3-3sigma, has a unique role in the induction of G1 arrest in tetraploid cells that results from mitotic catastrophe after DNA damage.

A "spindle assembly" checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21(WAF1) and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity.