Isolation and characterization of a B lymphocyte mutant with altered signal transduction through its antigen receptor.

A receptor surface Ig (sIg) signaling variant of WEHI-231 was constructed to investigate components and linkages between various signaling events associated with signal transduction through sIg. Unlike the wildtype, crosslinking of sIgM on VS2.12-cl.2 did not result in downregulation of proliferation. Similarly, receptor crosslinking was uncoupled from inositol phospholipid (PI) hydrolysis and upregulation of c-fos expression in the variant. The signaling defect in VS2.12-cl.2 appears to be proximal to phospholipase C activation as direct G protein activation by A1F4- triggers PI hydrolysis and bypassing PI hydrolysis using phorbol diester stimulation of protein kinase C restores the inhibitable phenotype and the ability to upregulate c-fos. Even more interesting, sIg-linked Ca2+ responses by VS2.12-cl.2 are equivalent to these observed in the wildtype WEHI-231. These latter results suggest that contrary to current thought, sIg-generated signals may not be coupled to Ca2+ fluxes entirely via inositol phospholipid hydrolysis. Thus, VS2.12-cl.2 is a new and powerful tool with which to analyze signaling through sIg at the molecular level.

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction.

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.

Differential expression of a zinc finger-encoding gene in response to positive versus negative signaling through receptor immunoglobulin in murine B lymphocytes.

Egr-1 is a murine early growth factor-inducible gene that encodes a protein with zinc fingers and that is believed to be involved in transcriptional regulation. Expression of this gene was investigated in murine B lymphocytes stimulated through their receptor for antigen (surface immunoglobulin [sIg]) with antireceptor antibodies (anti-sIg). Rapid (by 15 min) up-regulation of Egr-1 mRNA expression was observed after sIg cross-linking at a dose of anti-sIg sufficient to drive the majority of cells into cell cycle. Interestingly, signaling through sIg on the murine B-lymphoma cell line WEHI-231 did not up-regulate Egr-1 expression even though similar signaling pathways, including up-regulation of c-fos expression, are associated with this receptor in these cells. Importantly, cell growth and proliferation of WEHI-231 cells were inhibited by anti-sIg stimulation, which suggested a relationship between Egr-1 expression and differential processing of receptor immunoglobulin signals with respect to cellular growth responses. This notion was further supported by the finding that murine B lymphomas whose proliferation was not inhibited by anti-sIg showed receptor immunoglobulin-coupled Egr-1 expression. In further support of this association are results showing that under conditions in which Egr-1 expression was induced in WEHI-231 cells in response to stimulation by anti-sIg, a concomitant change was observed in the growth response of these cells. These results, then, indicate a potential role for Egr-1 expression in the translation of receptor-generated signals into cellular activation or induced unresponsiveness.

Transcriptional repression by the proto-oncogene BCL-6.

In up to 45% of reported cases of the non-Hodgkin's lymphoma, diffuse large cell lymphoma, there are translocations of the BCL-6 gene, which are presumed to deregulate its expression. The BCL-6 protein, which is unmutated in these lymphomas, contains six Krüppel-like zinc fingers at its carboxy terminus and a 121 amino acid domain at its amino terminus, termed the POZ domain, which bears homology with amino terminal domains in a subset zinc finger transcription factors. In this study, we tested whether BCL-6 regulates transcription and if the POZ domain has a role in this function. The BCL-6 POZ domain, when fused to the GAL4 DNA binding domain, strongly repressed transcriptional activation initiated from several different promoters including the SV40 enhancer/promoter. Repression was also observed when the fusion protein was bound at a distance of 200 bp 5' of the promoter. When the GAL4/BCL6 POZ domain fusion protein was expressed in yeast, it was able to homodimerize in the nucleus. Nevertheless, in contrast with mammalian cells, the fusion protein did not repress transcription. To test the ability of the full length BC1-6 protein to repress transcription when bound to DNA through its zinc finger DNA binding domain, high affinity BCL-6 binding sites were selected from a pool of random oligonucleotides. Full length BCL-6 was able to strongly repress transcription when bound to its cognate site cloned upstream of the thymidine kinase promoter. This repression was mediated, in large measure, by the POZ domain, although a variant of BCL-6 lacking the POZ domain was able to repress transcription modestly. The ability of BCL-6 to function as a transcriptional repressor may contribute to its ability to transform B lymphocytes in diffuse large cell lymphoma.

Studies of surface immunoglobulin-dependent B cell activation.

Studies from a number of laboratories have firmly established the potential of surface immunoglobulin-generated signals in B lymphocyte activation. While clearly there are multiple ways of activating B lymphocytes, some of which may not involve surface immunoglobulin, it is clear that crosslinking of surface immunoglobulin whether by antigen or antireceptor antibody can generate signals relevant to B cell activation. Although considerable insight into the mechanism of transduction of mIg-generated signals across the plasma membrane has been realized, a molecular explanation for linking inositol phospholipid hydrolysis to changes within the cytoplasm and nucleus of the B cell is still speculative. A more rigorous definition of the PKC and calcium components of the mIg signal transduction pathway are critical for a thorough understanding of the mechanism of signal transduction by this receptor. The use of tumor cell models allowing selection of mutants within the signalling pathway(s) will be invaluable to fully defining the critical molecular and biochemical events involved in B cell activation.

Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation.

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.