Role of nuclear factor-??B in the expression by tumor necrosis factor-? of the human polymeric immunoglobulin receptor ( pIgR ?) gene

Abstract

We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-alpha. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-alpha stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-alpha was decreased by pyrrolidinedithiocarbamate and L-1-4'-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-kappaB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5'-flanking region of the pIgR gene. In the upstream region, we found two NF-kappaB-binding motifs (named kappaB1 and kappaB2 from the 5' region). An electrophoretic mobility shift assay indicated that two components of the NF-kappaB/Re1 family, p50 and p65, bound with higher affinity to the KB2 element than to the kappaB1 element. We also analyzed plgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-alpha significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-alpha. The activation of promoter activity by TNF-alpha was abolished when a mutation was inserted into kappaB1 or kappaB2. These data indicated that pIgR gene expression induced by TNF-alpha is transcriptionally regulated via activation of NF-kappaB. In addition, there is a possibility that another factor may act in concert with NF-kappaB.

De Novo Synthesized RelB Mediates TNF-Induced Up-Regulation of the Human Polymeric Ig Receptor

“…The best-characterized mediator of TNF action is the NF-B family of latent, rapidly activated transcription factors (TFs) (11,12). Up-regulation of pIgR mRNA by TNF is sensitive to NF-B inhibitors (13). We have recently demonstrated that TNF-mediated up-regulation of pIgR in the human intestinal adenocarcinoma cell line HT-29.m3 required cooperation between DNA elements located in both an enhancer region of intron 1 and the proximal promoter (14).…”

“…The role of RelB as an activator of pIgR transcription differentiates the regulation of this gene with that of classical proinflammatory genes expressed upon TNF stimulation of epithelial cells such as IL-8 (35). However, activation of p65/ p50 is presumably also critical for pIgR activation because it is required for activation of the RelB gene itself (13,31). It is tempting to speculate that this dimer also binds to target elements in the pIgR gene at early time points after TNF stimulation and is exchanged for RelB-containing dimers at a later time point to achieve a sustained activation.…”

“…We and others have reported that NF-B can also bind to a target site at position Ϫ450 in the human pIgR promoter (13,14), but deletion or mutation of this site did not reduce TNF responsiveness of the pIgR-derived reporter constructs (14). An ISRE in exon 1 has furthermore been shown by deletional and mutational analysis to contribute positively to TNF responsiveness (14,15), particularly when the intronic NF-B site is missing or mutated (14).…”

A Novel NF-κB/Rel Site in Intron 1 Cooperates with Proximal Promoter Elements to Mediate TNF-α-Induced Transcription of the Human Polymeric Ig Receptor

“…Next, we investigated the role of two previously reported putative TNF-␣-responsive elements: an ISRE in exon 1 (26,27) and an NF-B/Rel site centered around position Ϫ450 in the upstream promoter (18,25). The former is 100% conserved between human, rat, and murine sequence, although the position relative to the transcriptional start site differs somewhat (Fig.…”

“…3 and 24). TNF-␣-mediated transcriptional up-regulation of pIgR/SC was shown to depend on NF-B/Rel activation (25) and an IFN-stimulated response element (ISRE) located in exon 1 (26,27). However, the effect mediated by described DNA elements could not account for the degree of up-regulation indicated by the increased level of mRNA and by nuclear run-on experiments (18).…”