Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis

Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1^−/− hosts repopulated with Bim^−/− conventional CD4⁺ T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1^−/− hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.     

Bim-dependent apoptosis follows IGFBP-5 down-regulation in neuroblastoma cells

The insulin-like growth factor (IGF) axis is frequently activated in neuroblastoma (NB) tumors and cell lines. We show that silencing endogenous expression of IGF Binding Protein-5 (IGFBP-5) in NB cells by using microRNA and siRNA causes mitochondrial apoptosis that is characterized by: (a) release of cytochrome C in the cytoplasm and activation of caspase 9; (b) Erk1 and Erk2 inhibition; and (c) upregulation of pro-apoptotic proteins Bim and Bax. Bim upregulation is caused, at least in part, by protein stabilization that may depend on inhibition of Erk1 and Erk2. Of interest, Bim knock-down by siRNA decreases apoptosis in IGFBP-5-interfered cells. Thus, inhibition of endogenously produced IGFBP-5 is associated with Bim-dependent apoptosis in NB cells.     

A novel cell death pathway that is partially caspase dependent, but morphologically non-apoptotic, elicited by proteasomal inhibition of rat sympathetic neurons

Proteasomal dysfunction has been linked to neurodegeneration. Pharmacological proteasomal inhibitors may have pro-survival or pro-death effects in neuronal cells. We have previously found that application of such agents to mouse sympathetic neurons leads to activation of the intrinsic apoptotic pathway. We show here that in rat sympathetic neurons proteasomal inhibition leads to a form of death that is morphologically non-apoptotic, with features of autophagy. The intrinsic apoptotic pathway is activated in a delayed fashion compared with mouse neurons, and is in part responsible for death, as evidenced by the partial protective effects of bcl-xL and the general caspase inhibitor Boc-aspartyl-fluoromethylketone. Death is accompanied by induction of Bim and caspase activation, but caspase 3 activation is lacking; 3-methyl-adenine inhibits macroautophagy, but has a relatively small pro-survival effect. We conclude that a complex array of pro- and anti-apoptotic effects elicited by proteasomal inhibition in rat sympathetic neurons leads to partial engagement of the intrinsic apoptotic pathway and a morphologically non-apoptotic, autophagic form of death. The species difference with mouse neurons is underscored by the fact that proteasomal inhibitors are protective against apoptosis elicited by nerve growth factor deprivation in rat, but not mouse, sympathetic neurons. The type of death described herein may be relevant to neurodegenerative diseases, where morphological evidence for apoptosis has been scant.     

Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis

The mitogen-activated protein kinases (MAPKs) ERK1/2, p38, and JNK are thought to determine survival-versus-death fate in developing thymocytes. However, this view was challenged by studies using 'MEK1-ERK1/2-specific' pharmacological inhibitors, which block both positive and negative selection. Recently, these inhibitors were also shown to affect MEK5, an upstream activator of ERK5, another class of MAPK with homology to ERK1/2. To define the contribution of the MEK5-ERK5 pathway in T-cell development, we retrovirally expressed dominant-negative or constitutively activated form of MEK5 to inhibit or activate the MEK5-ERK5 pathway. We demonstrate that MEK5 regulates apoptosis of developing thymocytes but has no function in positive selection. ERK5 activity correlates with the levels of Nur77 family members but not that of Bim, two effector pathways of thymocyte apoptosis. These results illustrate the critical involvement of the MEK5-ERK5 pathway in thymocyte development distinct from that of ERK1/2 and highlight the importance of the MAPK network in mediating differential effects pertaining to T-cell differentiation and apoptosis.     

Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET

Increased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initiation, progression and resistance to traditional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to chemotherapy. Recently, we applied fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pattern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the corresponding BimEL mutant showed no decrease in binding to these anti-apoptotic proteins. Therefore, in MCF-7 cells, Bim has unique binding properties compared with other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics.     

The fox and the fat

Comment on: Wilk A, et al. Cell Cycle 2012; 11:2660-71.     

HBI-8000诱导成人T细胞白血病/淋巴瘤的细胞凋亡与Bim和NLRP3活化的相关性

本研究通过评估HBI-8000对日本ATL患者ATL细胞系和原代细胞的作用,来研究HBI-8000诱导成人T细胞白血病/淋巴瘤的细胞凋亡与Bim和NLRP3活化的相关性。选取若干侵袭型ATL患者作为研究对象,口服新型HBI-8000,跟踪并分析药物效果。研究表明,在大多情况下,HBI-8000在原代ATL细胞和细胞系中诱导细胞凋亡。Bim活化、NLR族、NLRP3炎症体通道对HBI-8000诱导ATL细胞死亡具有重大作用。实验为临床研究HBI-8000对ATL患者的疗效提供了理论基础。NLRP3可用于癌症新型治疗策略研究,但对ATL细胞死亡的作用仍有待验证。     

Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim

The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy.