BCR-mediated apoptosis associated with negative selection of immature B cells is selectively dependent on Pten.

The molecular basis of B cell receptor (BCR)-induced apoptosis during the negative selection of immature B cells is largely unknown. We use transitional immature B cells that are highly susceptible to BCR-induced apoptosis to show that Pten is selectively required for BCR-mediated initiation of the mitochondrial death pathway. Specifically, deleting Pten, but not other pro-apoptotic molecules, abrogates BCR-elicited apoptosis and improves viability in wild-type immature B cells. We further identify a physiologically and significantly higher intracellular Pten level in immature B cells, as compared to mature B cells, which is responsible for low AKT activity and the propensity towards death in immature B cells. Restoration of AKT activity using a constitutive form of AKT or reduction of Pten to a level comparable with that seen in mature B cells rescues immature B cells from BCR-induced apoptosis. Thus, we provide evidence that Pten is an essential mediator of BCR-induced cell death, and that differential regulation of intracellular Pten levels determines whether BCR ligation promotes cell death or survival. Our findings provide a valuable insight into the mechanisms underlying negative selection and clonal deletion of immature B cells.

NF-kappaB inducible genes BCL-X and cyclin E promote immature B-cell proliferation and survival.

B-cell receptor (BCR) ligation induces proliferation and survival in mature B-cells but conversely, can lead to apoptosis in immature B-cells. We have previously shown that c-Rel, a member of the NF-kappaB transcription factor family, is essential for mature B-cell survival and proliferation via regulation of the anti-apoptotic molecule Bcl-X and cell cycle genes E2F3a and cyclin E. Here, we report that c-Rel-deficient mature B-cells are rendered sensitive to BCR-induced growth arrest and apoptosis in a manner that strongly resembles the phenotypic response of immature B-cells to BCR signaling. We further demonstrate that BCR-stimulated immature B-cells are defective in NF-kappaB activation, but that introduction of two downstream c-Rel target genes, Bcl-X and cyclin E, can restore survival and proliferation to these cells. Our studies therefore suggest that specific blockade of NF-kappaB activation may be responsible for the growth arrest and apoptosis of BCR-activated immature B-cells.

Cyclin E and Bcl-xL cooperatively induce cell cycle progression in c-Rel-/- B cells.

Aberrant overexpression of the c-rel protooncogene is associated with lymphoid malignancy, while c-rel deletion produces severe lymphoproliferative defects and immunodeficiency. To investigate the mechanism of c-rel-induced proliferation and cell cycle progression in B lymphocytes, we have compared signaling events elicited through the BCR in c-rel-/- and wild-type B cells. BCR stimulation of c-rel-/- B cells fails to induce proper cyclin expression, resulting in G1 phase arrest, but it is unclear whether these defects are in fact secondary events of decreased B-cell survival, since c-rel deletion also affects the expression of antiapoptotic genes such as bcl-xL. Here, we use the bcl-xL transgene to correct the viability of c-rel-deficient B cells, and show that the inhibition of apoptosis does not necessarily confer hyperproliferation of B cells activated through the BCR. c-rel-/- B cells still fail to enter the S phase despite improved survival by bcl-xL overexpression, suggesting that c-Rel-associated cell cycle progression is dependent on more than just enhanced cell viability. Overexpression of cyclin E protein, however, can cooperate with Bcl-xL to restore cell cycle progression to c-rel-/- B cells via induction of the cyclin-CDK/Rb-E2F pathway. Furthermore, we show that c-Rel can directly regulate transcription of the e2f3a promoter/enhancer, which is then likely to lead to transcriptional activation of the cyclin E promoter by E2F3a. Hence, these studies provide clear evidence that control of lymphocyte proliferation via c-Rel is linked to a cyclin-dependent process, and suggest that c-Rel not only activates antiapoptotic signaling but also the induction of cell cycle progression.