Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.     

A-Myb up-regulates Bcl-2 through a Cdx binding site in t(14;18) lymphoma cells

In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.     

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.     

Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes.

Cell death is a prominent feature of B cell development. For example, a large population of B cells dies at the pre-B cell stage presumably due to the failure to express a functional immunoglobulin receptor. In addition, developing B cells expressing antigen receptors for self are selectively eliminated at the immature B cell stage. The molecular signals that control B cell survival are largely unknown. The product of the bcl-2 proto-oncogene may be involved as its overexpression inhibits apoptotic cell death in a variety of biological systems. However, the physiological role of the endogenous Bcl-2 protein during B cell development is undetermined. Here we show a striking developmental regulation of the Bcl-2 protein in B lymphocytes. Bcl-2 is highly expressed in CD43+ B cell precursors (pro-B cells) and mature B cells but downregulated at the pre-B and immature B cell stages of development. We found that Bcl-2 expressed by B cells is a long-lived protein with a half-life of approximately 10 h. Importantly, susceptibility to apoptosis mediated by the glucocorticoid hormone dexamethasone is stage-dependent in developing B cells and correlates with the levels of Bcl-2 protein. Furthermore, expression of a bcl-2 transgene rescued pre-B and immature B cells from dexamethasone-induced cell death, indicating that Bcl-2 can inhibit the apoptotic cell death of progenitors and early B cells. Taken together, these findings argue that Bcl-2 is a physiological signal controlling cell death during B cell development.     

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.     

Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population.

We characterized the basis for the follicular lymphoproliferation in transgenic mice bearing a Bcl-2-immunoglobulin (Bcl-2-Ig) minigene representing the t(14;18) of human follicular lymphoma. Discriminatory S1 nuclease protection assays revealed that the Bcl-2-Ig transgene was overexpressed relative to endogenous mouse Bcl-2 in spleen and thymus. Western (immunoblot) analysis demonstrated the overproduction of the human 25-kilodalton Bcl-2 protein, which arose from the transgene, in spleen, thymus, and the expanded B-cell subset. Despite the generalized lymphoid pattern of deregulation, two-color flow cytometry and density gradient centrifugation indicated that the expanded lymphocytes were predominantly small, resting B cells coexpressing B220, immunoglobulin M (IgM), IgD, Ia, and kappa. Cell cycle analysis confirmed that about 97% of these expanded B cells reside in G0/G1. An extensive characterization of transgenic lines revealed a fourfold excess of IgM-IgD-expressing B cells in spleen and dramatically increased numbers in bone marrow. While resting, these cells proliferated in response to lipopolysaccharide and anti-IgM and demonstrated normal B-cell colony formation in soft agar. Moreover, these B cells, which demonstrated an extended survival in vitro even in the absence of stroma, were also resting in G0, yet were capable of proliferative responses. These findings provide consistent evidence that the accumulation of B cells after Bcl-2 overproduction is secondary to prolonged cell survival and not increased cell cycling. This suggests a unique role for Bcl-2 as a proto-oncogene that enhances cell survival independent of promoting cell division.     

Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.     

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.