The NF-kappa B-binding site mediates phorbol ester-inducible transcription in nonlymphoid cells.

Abstract

The mouse immunoglobulin K light-chain enhancer can interact with at least three independent nuclear proteins. One of these proteins, NF-KB, is constitutively present only in nuclear extracts derived from B cells and plasma cells. A DNA-binding protein with the same sequence specificity (and therefore presumed to be NF-KB itself) can be induced in pre-B cells, T cells, and nonlymphoid cells by phorbol 12-acetate-13-myristate (PMA); however, it is not clear whether the induced factor can activate transcription in nonlymphoid cells as NF-KB does in B cells. In this paper we show that multimerization of a fragment of the mouse K enhancer that carried only the binding site for NF-KB behaved like a B-cell-specific regulatory element. Furthermore, this unit served to activate transcription in nonlymphoid cells after treatment with PMA (but not with cyclic AMP derivatives), and the kinetics of transcription activation correlated well with the kinetics of factor induction.Thus, the induced DNA-binding activity appeared to be functionally indistinguishable from that of NF-KB.B-cell-specific transcription of the immunoglobulin genes is governed in part by tissue-specific enhancers located in the heavy-chain (4,11,12,22,24) and the kappa (K) light-chain loci (26,27). Most of the activity of the heavychain enhancer can be located in a 220-base-pair (bp) Hinfl fragment that lies within the originally identified 700-bp XbaI-EcoRI fragment. The smaller fragment has the ability to interact with at least four independent nuclear factors (3,25,30,35), of which three are found in extracts derived from a wide variety of cell types. One factor (NF-A2) that binds to a conserved octanucleotide sequence (ATTTGCAT) appears to be restricted in expression to B and T cells only (18,33) and is therefore a good candidate to confer the observed tissue specificity of the enhancer. The K light-chain enhancer is organized in a similar fashion and can interact with tissue-specific and nonspecific factors. Deletion analysis of the K enhancer has shown that 30% of the activity is located in a 200-bp DdeI fragment within the JK-CK intron (28). Analysis of nuclear protein-binding sites by the electrophoretic mobility shift assay has shown that this segment binds at least three proteins in a sequence-specific manner (30). One of these proteins, NF-KB, binds to the B site located in the 5' DdeI-HaeIII fragment of the K enhancer and is constitutively present only in extracts derived from cells that represent the B-cell and plasma cell stages of B-lymphocyte differentiation (30 Sweden. gous to that by NF-KB. We show here that this is indeed the case, making it very likely that the induced protein is the same as that found constitutively in B lymphocytes. MATERIALS AND METHODSCell lines and culture. HeLa is a human cervical carcinoma cell line. Stably transfected HeLa cell lines KCAT-1, KCAT-2, KCAT-4, and SPCAT were grown in Dulbecco modified Eagle (DME) medium supplemented with 10% calf serum. HAFTL is a pre-B-cell line grown in RPMI medium containi...

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