NF-kappaB regulates the expression of human complement receptor 2 gene

Authors:
Tolnay M, Vereshchagina LA and Tsokos GC
In:
Source: J Immunol
Publication Date: (2002)
Issue: 169: 6236-6243
Research Area:
Immunotherapy / Hematology
Cells used in publication:
B cell, human
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Complement receptor 2 (CR2) is a key regulator of the B cell response to antigens. CR2 on B cells serves as a co-receptor for the Ag receptor. NF-kappaB is involved in the regulation of numerous genes activated during inflammatory and immune responses. The authors studied the role of NF-kappaB in the regulation of the CR2 promoter activity. Experiment 1: Resting B cells were transiently co-transfected with CAT reporter plasmid driven by wild-type (WT) CR2 promoter and NF-kappaB p50 or p60 expression plasmids. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promotor in primary B cells, suggesting a direct role for NF-kappaB in regulating promotor activity. Experiment 2: Resting B cells were nucleofected with a CAT reporter plasmid driven by the wild-type CR2 promoter and stimulated with known activators of NF-kappaB (PMA or LPS). After 15 hours cells were assayed for CAT activity. PMA enhanced CR2 promoter activity by ~3-fold, whereas LPS enhanced it by 50%.
Abstract
CR2 is a key regulator of the B cell response to Ag. Here we show that NF-kappaB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-kappaB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-kappaB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-kappaB p50 and p65. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-kappaB in regulating promoter activity. Importantly, mutation of the NF-kappaB element or the X box/E box rendered the promoter unresponsive to NF-kappaB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-kappaB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-kappaB element or the X box/E box. Finally, stimuli that activate NF-kappaB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-kappaB signaling pathway enhances the expression of the CR2 gene, as a result of NF-kappaB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.