Regulation of memory B-cell survival by the BH3-only protein Puma.

Apoptosis is crucial for immune system homeostasis, including selection and survival of long-lived antibody-forming cells and memory cells. The interactions between proapoptotic and pro-survival proteins of the Bcl-2 family are critical for this process. In this report, we show that expression of the proapoptotic BH3-only Bcl-2 family member Puma was selectively up-regulated on in vitro activation with antigens or mitogens of both human and mouse B cells. Puma expression coincided in vivo, with the prosurvival Bcl-2 family member Mcl-1 within the germinal centers and its expression correlates with the germinal center like phenotype of Burkitt lymphoma. Experiments performed in Puma-deficient mice revealed that Puma is essential for apoptosis of mitogen-activated B cells in vitro and for the control of memory B-cell survival. In conclusion, using both human and murine models, our data show that Puma has a major role in the T cell- dependent B-cell immune response. These data demonstrate that Puma is a major regulator of memory B lymphocyte survival and therefore a key molecule in the control of the immune response.

Caspase-3 triggers a TPCK-sensitive protease pathway leading to degradation of the BH3-only protein puma.

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.

BimL upregulation induced by BCR cross-linking in BL41 Burkitt's lymphoma results from a splicing mechanism of the BimEL mRNA.

B lymphocyte receptor-mediated apoptosis is associated with increased expression of the BimL isoform of Bim. The mechanisms involved in the regulation of BimL protein expression are still unknown. We report that BimL expression following BCR activation is not associated with a specific increase of BimL mRNA but rather to the intron retention structure of the BimEL mRNA. Indeed, expression of a BimEL cDNA leads in Hela cells leads to the production of both BimEL and BimL proteins. Mutation of the intron-splicing GT sequence present in the exon 3 results in the production of only BimEL protein. Ectopic expression of BimEL cDNA resulted in a large increase of BimL expression upon BCR-stimulation, whereas cells transfected with the GT/AA mutated form of BimEL only produced BimEL proteins upon BCR-activation. These data showed that BimL expression induced by BCR activation may result from the splicing of BimEL mRNA independently of Bim promoter regulation.

EBV infection of human B lymphocytes leads to down-regulation of Bim expression: relationship to resistance to apoptosis.

EBV infects a large proportion of the human population worldwide and is one of the major viruses with human B lymphocyte tropism. It can immortalize human B lymphocytes and controls their resistance to apoptosis. EBV infection is associated with several lymphomas, including Burkitt's lymphoma. In this report we show that EBV infection leads to the post-transcriptional down-regulation of expression of the proapoptotic protein Bim. This process involves the phosphorylation of BimEL by the constitutive EBV-activated kinase ERK1/2, followed by its degradation through the proteasome pathway. We also show that ectopic expression of BimEL in EBV-positive Burkitt's lymphoma cells can enhance the sensitivity of these cells to serum deprivation-dependent apoptosis. Thus, EBV-mediated resistance to growth factor deprivation in human B lymphocytes is dependent on BimEL expression. Our data suggest that this regulatory pathway is an important contributor to the oncogenic potential of EBV.

B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression.

Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-mu Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS.