Cross-talk between transcription factors NF-kappa B and C/EBP in the transcriptional regulation of genes.

The study of the acute phase response has attracted substantial interest, not only for its medical implication, but also its provision as an excellent system with which to elucidate the molecular mechanisms involved in the modulation of gene expression. Our previous data suggest that the synergistic induction of the major acute phase reactant serum amyloid A2 (SAA2) expression by interleukin-1 (IL-1) and interleukin-6 (IL-6) is mediated by two families of transcription factors, namely NF-kappa B and C/EBP. To understand the molecular mechanisms of this synergy, we have undertaken a molecular dissection of the factors involved in the formation of the regulatory complex. Electrophoretic mobility shift analysis indicates that NF-kappa B p65 (RelA) and p50, but not p52 or c-Rel, bind specifically to the NF-kappa B site of the SAA2 promoter in response to IL-1 stimulation. In addition, C/EBP beta and C/EBP delta, but not C/EBP alpha, bind specifically to the C/EBP site of SAA2 in response to IL-6 stimulation. Transient co-transfection analysis indicates that co-operative association of NF-kappa B p65 with C/EBP beta and, in particular, with C/EBP delta, results in synergistic transcriptional activation of the SAA2 promoter. When incubated together, NF-kappa B p65 and C/EBP beta form a ternary complex by direct protein/protein interaction. Mutational analysis demonstrates that the C-terminus region of the Rel homology domain (RHD) and the C-terminus of the activation domain of p65 are important for its interaction with C/EBP beta. These results suggest the NF-kappa B and C/EBP may form a new complex of transcription factors that mediates the synergistic induction of SAA2 by IL-1 and IL-6.

The role of NF-kappa B and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression by interleukin-1 and interleukin-6.

To understand the mechanisms by which large increases in serum amyloid A (SAA) occur during the acute phase response, human hepatoma cells were transfected with SAA2 gene reporter plasmids and stimulated with combinations of cytokines. Although interleukin-1 (IL-1) and interleukin-6 (IL-6) stimulated transcription from this promoter individually, addition of both mediators produced a response between two and nine times greater than the expected additive response. This synergistic activation was dependent on the integrity of at least two cis-acting sequences in the SAA2 enhancer. The SAA2 NF-kappa B site was required functionally for the response to both IL-1 and IL-6 alone as well as for synergistic activation; however, IL-6 did not directly induce binding of nuclear proteins to the NF-kappa B sequence. A NF-IL6 site was required for full induction by IL-1 and IL-6, and also mediated strong transactivation by recombinant NF-IL6. Furthermore, transfected NF-IL6 synergized strongly with co-transfected NF-kappa B, particularly with RelA (p65). However synergy between IL-1 and IL-6 was only partly reduced by mutation of the NF-IL6 site, indicating further levels of interaction in addition to the NF-kappa B/NF-IL6 cooperativity.

Constitutive and NF-kappa B-like proteins in the regulation of the serum amyloid A gene by interleukin 1.

Serum amyloid A (SAA) is a major acute-phase protein whose chronic production by the liver can lead to the fatal disorder of secondary amyloidosis. Control of SAA is mediated by several inflammatory cytokines, including interleukin 1 (IL-1). To study the cis-acting regulatory elements responsible for constitutive and IL-1-induced expression, DNA constructs containing varying lengths of the promoter region from the human SAA2 beta gene 5' to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT), were generated and transfected into human hepatoma cells, HepG2. Both positive and negative regulatory elements were found in the 5' flanking region of the human SAA2 beta gene. The more proximal region contains an IL-1 enhancer sequence GGGACTTTCC (SAA kappa B1; between -82 and -91), the binding site for the ubiquitous transcription factor NF-kappa B. IL-1 induction of the binding of nuclear factor to this sequence is maximal between 5 min and 30 min after incubation with IL-1 and negative in cells incubated for 60 min or longer. Mutation of the SAA kappa B1 sequence to a nonbinding form of NF-kappa B (CTCACTTTCC) abolishes the IL-1 effect. The SAA 5' region also contained an upstream repressor element, shown by transfection experiments. Within this element, a second NF-kappa B binding site (SAA kappa B2; -626 to -635) was found, and mutation of SAA kappa B2 to a non-NF-kappa B-binding form results in an increase in both constitutive + IL-1 stimulated SAA transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kappaB-like transcription factor.

We have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). We found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, we placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by R. Sen and D. Baltimore (Cell 46:705-716, 1986) as the binding site for the nuclear factor NF kappa B. In a cotransfection competition experiment, we could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF kappa B-binding sequence. The same long-terminal-repeat DNA containing mutant NF kappa B-binding sequences (G. Nabel and D. Baltimore, Nature [London] 326:711-713, 1987) did not affect CAT expression, which suggested that binding by an NF kappa B-like factor is required for increased SAA transcription.

Anderson Fabry disease, a close linkage with highly polymorphic DNA markers DXS17, DXS87 and DXS88.

Anderson Fabry disease is an X-linked lysosomal storage disorder caused by alpha-galactosidase A deficiency. Hemizygous males and some heterozygous females develop renal failure and cardiovascular complications in early adult life. We have investigated six large UK families to assess the possible linkage of five polymorphic DNA probes to the Anderson Fabry locus, previously localised to Xq21-24. No recombination was found between Anderson Fabry disease and DXS87, DXS88 and DXS17, which gave lodmax = 6.4, 6.4 and 5.8 respectively at theta = 0.10, (upper confidence limit 0.10). DXS3 gave lodmax 2.9 at theta = 0.10 (upper confidence limit 0.25). DXYS1 was excluded from linkage. The best fit map (DXYS1/DXS3) theta = 0.192 (DXS17/DXS87/DXS88/Anderson Fabry locus) provided no information about the order of loci in parentheses due to the absence of recombinants. The close linkage of DXS17, DXS87 and DXS88, together with alpha-galactosidase A estimation, can be used for antenatal diagnosis and carrier detection until the application of a gene specific probe has been evaluated.

Anderson-Fabry disease--family linkage studies using two polymorphic X-linked DNA probes.

Anderson-Fabry disease is an X-linked lysosomal storage disorder due to alpha-galactosidase A deficiency. In affected males there is a high mortality in early adult life due to renal failure and cardiovascular complications. We describe our preliminary results from genetic linkage studies in five families using two polymorphic DNA probes, DXS17 and DXYS1, mapping to an area on the long arm of the X chromosome between Xq13-22. DXS17 identified a Taql polymorphism closely linked to the disease locus in three families (lodmax Z = 4.23. at a recombination fraction decreases theta = 0.0). Restriction fragment length polymorphisms detected by DXYS1 were not linked.