The Structure of the TNFRSF13C Promoter Enables Differential Expression of BAFF-R during B Cell Ontogeny and Terminal Differentiation2010-07-01 22:09:34
The Journal of Immunology; 2010 July; 185(2):1045-54
Stephen A. Mihalcik, Paul M. Huddleston III, Xiaosheng Wu, and Diane F. Jelinek
B cell-activating factor of the TNF family (BAFF; also called BLyS; TNFSF13B) and its high-affinity receptor, BAFF receptor (BAFF-R; also called BR3; TNFRSF13C), have been shown to mediate a powerful survival signal in transitional and mature B cells. Mice deficient in BAFF, BAFF-R, or A/WySnJ mice with mutant BAFF-R all show very similar defects, in particular a precipitous decline in B cells beginning at the transitional stage, indicating the essential and complementary nature of both the receptor and its ligand to the survival of mature B cells. Recent data have clearly shown that murine developing B cells in the bone marrow (BM) first express BAFF-R on their surface at the immature stage Hardy Fraction E. It is also at this critical stage in B cell development, when the cells express a complete and functional BCR on their surface, that selection must occur, culling B cells that express autoreactive receptors and, as Ig receptor usage studies have suggested, positively selecting a subset of those cells. The complicated nature of BCR signaling, which must be capable of both initiating apoptosis and of promoting survival, is coincident with the crucial survival signal initiated by the ligation of BAFF-R. The ontogenic association of these two signals has raised intriguing questions into the nature of survival of developing B cells and into the connections between these two receptors.
Recent data have suggested that BCR signaling plays a central role in the BAFF-R survival signal. Stadanlick et al. have demonstrated an essential role for tonic BCR signaling in the BAFF-R–mediated survival of murine B cells, in which the BCR’s constitutive baseline signal induction is responsible for generating the NF-κB family member p100, which serves as a substrate for the alternative NF-κB pathway essential for BAFF-R–mediated survival. In a complementary study, Castro et al. suggested that induced signaling of murine B cells through the BCR is additionally responsible for upregulation of the BAFF-R, itself, through a c-Rel–dependent mechanism. The nature of the cross-talk between these two receptors may indeed include multiple complex and interacting pathways. Understanding these systems necessitates a precise examination of each component both in isolation and in context. In this study, we focused on the transcriptional regulation of BAFF-R expression in human B cells. The regulatory elements controlling BAFF-R expression at the genetic level are critically and fundamentally important in understanding the way that B cells use both the BCR and BAFF-R to survive development and to enter the periphery as mature B cells, a feat only 5% of newly generated BM B cells accomplish.
The reports linking BCR-initiated signaling pathways to BAFF-R expression are consistent with a role for NF-κB family member c-Rel in B cells with a mature BCR, because it has been demonstrated that c-Rel becomes a dominant component of nuclear NF-κB complexes only after B cells express a complete BCR, whereas the composition of NF-κB complexes in their immediate precursors, pre-B cells, are dominated by p50/p65 heterodimers. Consistent with this notion, mice deficient in c-Rel and c-Rel redundant molecule RelA showed a deficit of mature B cells. Furthermore, such a B cell phenotype can be rescued by the overexpression of prosurvival factor Bcl-2, suggesting that cell survival is indeed the limiting factor during B cell maturation in the c-Rel/RelA double-knockout mice. TACI-Ig–transgenic mice, which have an inhibited BAFF/BAFF-R axis because of the presence of a soluble BAFF-binding receptor in the circulation, recapitulate a very similar phenotype when their B cells are rescued by transgenic Bcl-2 expression.
Just as the point at which B lineage cells first express BAFF-R provides essential clues to its transcriptional control, so too does the point at which B cells no longer express BAFF-R. It is clear already that the human plasma cell population does not express this BAFF-binding receptor and that the induction of the plasma cell differentiation pathway can downregulate BAFF-R expression in vitro. To our knowledge, the mechanisms that extinguish BAFF-R expression on the surface of plasma cells are unknown, and in these studies, we provide the first evidence that this downregulation is an outcome dependent solely on the transcription factor network.
Because there is mounting evidence that BAFF plays a key role in B cell cancers and in autoimmune illnesses, there is an urgent need to better understand the regulation of BAFF-R expression. This study establishes the developmental regulation of BAFF-R expression in human B cells and uses interspecies homology to identify a regulatory region adjacent to the TNFRSF13C gene that acts as a promoter in response to B cell intrinsic signals. The generation and application of reporter vectors coordinated with in vitro protein-DNA interaction assays, DNase protection and EMSA, identified a core promoter region under the control of B cell constitutive transcription factors and a site within that promoter that is sensitive to a single base pair substitution. The demonstration of in vivo interactions using chromatin immunoprecipitation (ChIP) and in vitro small interfering RNA (siRNA)-mediated gene silencing demonstrated that c-Rel contributes to this purpose.
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