IgM receptor-mediated transactivation of the IgH 3' enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.

The function of the temporally regulated B lymphocyte-specific immunoglobulin heavy chain (IgH) 3' enhancer has been linked to the IgH class switch machinery, but the physiological mechanism(s) of activation has not been discerned. Following crosslinking of the IgM receptor, we demonstrate that the enhancer is transactivated in the B lymphoma cell line BAL-17. In both induced primary B lymphocytes and BAL-17 cells, the enhancer activation is concomitant with the recruitment of a novel DNA binding complex, nuclear factor of activated B cells (NFAB). NFAB contains the tissue-restricted Ets protein Elf-1 and the AP-1 factors Jun-B and c-Fos, which bind to a novel 3' enhancer ETS-AP-1 motif. IgM receptor-mediated activation or stimulation by phorbol-ester in BAL-17 cells demonstrates that the ETS-AP-1 motif, when linked to a heterologous gene, can confer a ligand/receptor-dependent response. In NIH 3T3 cells, Elf-1 expression is required for efficient ETS-AP-1 promoter activity in response to stimulation by 12-O-tetradecanylphorbol 13-acetate. Our results suggest a biological role for Elf-1 in the regulation of IgH gene expression, attribute a functional role for receptor-induced AP-1 proteins in B lymphocytes and provide evidence for a direct link between IgM receptor-mediated signalling and 3' enhancer activation.     

Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly in the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a "downstream" signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered "physiologically" through sIg.     

Regulation of the rat interstitial collagenase promoter by IL-1β, c-Jun,and ras-dependent signaling in growth plate chondrocytes

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5′-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1β–inducible reporter gene activity. Cells stimulated by IL-1β (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1β regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation. J. Cell. Biochem. 67:92–102, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.     

Suppression of glutathione transferase P expression by glucocorticoid.

A strong enhancer element, GPEI, of the glutathione transferase P gene (GST-P) gene is composed of two phorbol 12-O-tetradecanoate 13-acetate (TPA) responsive element (TRE)-like sequences at opposite orientation. Unlike TRE sequences of other genes, GPEI exhibits a strong enhancer activity in F9 cells, which contains little AP-1. GPEI bound to AP-1 In vitro and GST-P expression was activated by TPA and exogenously introduced c-jun gene in a rat fibroblast cell line. Both the stimulated expression of GST-P gene by TPA and that by over-expressed c-Jun were suppressed to the basal level by dexamethasone, an inhibitor of AP-1. Basal expression of GST-P gene, however, was not inhibited by dexamethasone. Transfected chloramphenicol acetyltransferase (CAT) gene having GPEI also behaved as the endogenous GST-P gene. These results indicate that the GPEI is activated by AP-1 but constitutive activity of this enhancer in a rat fibroblast cell line 3Y1 cells is due to some unknown mechanism other than AP-1.