V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

Abstract

V(D)J [variable-(diversity)-joining] recom-bination is regulated developmentally, being restricted to cells of the early B-and T-lymphocyte lineages. In this report we show that recombination activity can also be regulated in response to chemical effectors. Compounds that increase intracellular cAMP increase V(D)J recombination of extrachromosomal substrates in pre-B-cell lines as much as 10-fold. In contrast, V(D)J recombination is reduced 5-to 8-fold in response to phorbol 12-myristate 13-acetate or to the calcium ionophore A23187. The effect of cAMP agonists on recombination appears to reflect an increase in cellular recombination activity, as indicated by the caffeine-induced rise in the level of mRNA from the recombination-activating genes RAG) and RAG2. Our data demonstrate that intracellular second messengers modulate recombination activity in lymphoid cell lines, implying that recombination activity can be regulated by these signals in developing B and T lymphocytes.The assembly of functional immunoglobulin and T-cell receptor genes is carried out by a series of site-specific recombination events known as V(D)J [variable-(diversity)-joining] recombination (1). In this process, specific signal sequences that flank each of the possible coding regions act as targets for the recombination event. The recombination activity is limited to cell lines representing precursors of B and T lymphocytes (2,3) and is absent in mature cells that produce immunoglobulin or T-cell receptor molecules. The proteins involved in V(D)J recombination have not been characterized biochemically, but recently two lymphoidspecific genes have been identified as playing a crucial role in the process. The products of the RAGI and RAG2 genes, when expressed in fibroblasts, can activate V(D)J recombination in those cells and may well be directly involved in the reaction (4, 5).Lymphoid cells have been shown to be responsive to many different extracellular factors such as lipopolysaccharides, lymphokines, neurotransmitters, histamine, and growth factors (6). Various cell lines respond to these factors by making new surface markers (7), producing lymphokines (8), transcribing specific genes (9), and activating DNA-binding proteins (10). In several cases, the intracellular second messengers of extracellular factors have been determined. Abraham et al. (11) have shown that interleukin 2 production in a T-cell line is induced by interleukin 1 and phytohemagglutinin, and this induction can be mimicked by factors that stimulate protein kinase C (PKC) and increase intracellular calcium. Similarly, K light-chain enhancer binding activity and lightchain transcription can be induced by interleukin 1 or agents that increase intracellular cAMP concentration in certain B-cell lines (10). Thus, many enzyme systems in lymphocytes are influenced by changes in various second-messenger pathways.In this study we have tested agents that affect secondmessenger pathways for their influence on V(D)J recombination activity of several lymphoid cell lines. ...

Impaired Light Chain Allelic Exclusion and Lack of Positive Selection in Immature B Cells Expressing Incompetent Receptor Deficient of CD19

“…Those aim to transduce signals to the nucleus and to activate transcription factors regulating V(D)J recombination (67,68). Several studies have shown that agents activating protein kinase A or increasing cAMP also elevate V(D)J recombination in B cells (50,51). How BCR or pre-BCR signal for allelic exclusion is not known, nor have the ligands, if any, that transduce these signals been identified.…”

“…V(D)J recombination and RAG gene expression are increased upon induction of intracellular second messengers such as protein kinase A and cAMP (50,51).…”

Coordinate Transcription and V(D)J Recombination of the Kappa Immunoglobulin Light-Chain Locus: NF-κB-Dependent and -Independent Pathways of Activation

“…Previous results have suggested that within a given cell line, the V(D)J recombinase activity correlates well with the expression of RAG-1 and RAG-2 over a broad range (1,26). It was possible that induction of RAG-1 or RAG-2 by LPS and IFN-␥ accounted for the observed induction of recombination.…”

The Effects of Cyclosporin A on V(D)J Recombination Activity

“…These signals are mediated through the tyrosine/serine-threonine pathway and the mobilization of intracellular Ca 2+ . The up-regulation of RAG gene expression and V(D)J recombination can be mimicked in vitro by cAMP-derivatives and caffeine which inhibits the cAMP degrading enzyme adenylate cyclase [16,17]. The down-regulation of RAG gene expression and V(D)J recombination can also be mimicked by the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which activates the protein kinase C (PKC), and the Ca 2þ mobilizer thapsigargin [16,17].…”

“…The up-regulation of RAG gene expression and V(D)J recombination can be mimicked in vitro by cAMP-derivatives and caffeine which inhibits the cAMP degrading enzyme adenylate cyclase [16,17]. The down-regulation of RAG gene expression and V(D)J recombination can also be mimicked by the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which activates the protein kinase C (PKC), and the Ca 2þ mobilizer thapsigargin [16,17]. The antagonistic regulation of V(D)J recombination by the PKA pathway on the one hand and the PKC pathway and intracellular Ca 2þ on the other hand makes V(D)J recombination an ideal system to study substances which influence these pathways.…”