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The biological activity of the transcription factor NF-B is differentially controlled by three IB proteins, IB␣, IB␤, and IB. We have examined the molecular basis for the differential inhibitory strengths of IB proteins by constructing hybrid IBs and found that the first ankyrin repeat of IB␣ is responsible for its strong inhibitory effect. Swapping a putative ␤-turn within the first ankyrin repeat between the strong IB␣ and the weak IB␤ inhibitors switches their in vivo inhibitory activity on NF-B. The qualitatively distinct contacts made by this ␤-turn in IB␣ and IB␤ with NF-B determine the efficiency by which IBs sequester NF-B to the cytoplasm, thus explaining their distinct effects on gene activity.The transcription factor NF-B orchestrates the activation of numerous genes involved in the control of cell activities in the immune system and is also vital for craniofacial, liver, and limb development in higher eukaryotic organisms. NF-B exists in virtually all cell types in the form of dimeric complexes consisting of different members of the Rel family of proteins. In mammals, there are five Rel proteins, p50, p52, p65, c-Rel, and RelB, all of which share an amino-terminal 300 amino acid conserved region known as Rel Homology Region. This region is responsible for DNA binding, dimerization, and nuclear localization. Unlike most transcriptional activators, NF-B resides in the cytoplasm and must therefore translocate to the nucleus to function. Association with the inhibitory IB proteins tightly regulates the activity of NF-B. These interactions have two functional consequences. First, NF-B͞IB complexes are sequestered in the cytoplasm, because IBs mask the nuclear localization signal (NLS) of NF-B, presumably by means of direct protein-protein interactions, and secondly, IBs can inhibit NF-B DNA binding. In response to a large variety of extracellular stimuli, the IB proteins, while still bound to NF-B, are phosphorylated, ubiquitinated, and finally degraded by the proteasome. The free NF-B translocates to the nucleus, where it activates gene transcription (reviewed in refs. 1-5).The IB family consists of three members, IB␣, IB␤, and IB (6-10). Importantly, the carboxyl-terminal regions of the precursors for p50 and p52, p105, and p100, respectively, can also function as IBs. Each member of the family contains six copies of a 33 amino acid module known as ankyrin repeat, which functions as a protein-protein interaction domain. The region carboxyl-terminal to the ankyrin repeats contains a proline (P), glutamate (E), serine (S), and threonine (T) (PEST) sequence regulating basal level protein turnover and is also required for inhibition of DNA binding, whereas the amino-terminal region is the signal responsive domain (2, 5). Despite their extensive structural similarities, IB␣, IB␤, and IB exhibit substantial differences in vivo (10-13). Depending on the cell type and on the stimulus, IBs respond differentially to NF-B-inducing signals. In general, IB␣ is rapidly degraded, whereas IB␤ and IB are degrade...

The biological activity of the transcription factor NF-B is mainly controlled by the IB proteins IB␣ and IB␤, which restrict NF-B in the cytoplasm and enter the nucleus where they terminate NF-B-dependent transcription. In this paper we describe the cloning and functional characterization of mouse IB. Mouse IB contains 6 ankyrin repeats required for its interaction with the Rel proteins and is expressed in different cell types where we found that it is up-regulated by NF-B inducers, as is the case for IB␣ and human IB. IB functions as a bona fide IB protein by restricting Rel proteins in the cytoplasm and inhibiting their in vitro DNA binding activity. Surprisingly, IB did not inhibit transcription of genes regulated by the p50͞p65 heterodimer efficiently, such as the human interferon-␤ gene. However, IB was a strong inhibitor of interleukin-8 expression, a gene known to be regulated by p65 homodimers. In addition, IB appears to function predominantly in the cytoplasm to sequester p65 homodimers, in contrast with the other two members of the family, IB␣ and IB␤, which also function in the nucleus to terminate NF-B-dependent transcriptional activation.The transcription factor NF-B plays a major role in the activation of numerous genes involved in the function and development of the immune system, in the recruitment of leukocytes from the circulation into extravascular space, and in inflammatory and acute responses, etc. (reviewed in refs. 1-4). NF-B consists of homo-and heterodimeric proteins that belong to the Rel family of transcription factors. In mammals there are five Rel proteins, p50, p52, p65 (RelA), c-Rel, and RelB, all of which contain a so-called Rel homology region (RHR) that includes DNAbinding and dimerization domains and a nuclear localization signal (NLS). The Rel proteins are present in most cell types in an inactive cytosolic form. The cellular partitioning of NF-B is tightly regulated by the IB proteins, which are complexed with NF-B in the cytoplasm (5). The NF-B DNA binding activity can be induced by a large variety of extracellular signals, all of which culminate in the proteolytic degradation of IBs by the proteasome, thereby freeing NF-B to translocate to the nucleus, where it activates gene transcription (1-7).There are two known types of Rel complexes in the cytoplasm. The first type consists of heterodimers containing the p65 or c-Rel proteins associated with the precursors for p50 or p52 (p105 and p100, respectively). The second type consists of p65 and c-Rel homo-or heterodimers (with p50 or p52) associated with a member of the IB family. A common characteristic of the IB proteins is the presence of multiple copies of a motif, the ankyrin repeat, which interacts with the RHR. This interaction has two functional consequences. First, NF-B-IB complexes are sequestered in the cytoplasm, because the IBs mask the NLS through direct protein-protein interactions; and second, IBs can prevent NF-B from binding to the DNA in vitro and in vivo. The IB family consists of IB␣ (5 ankyrin rep...

Interaction of the neuropeptide substance P (SP) and its neurokinin-1 receptor (NK-1R) plays an important role in the pathophysiology of intestinal inflammation. SP is known to stimulate production of interleukin (IL)-6 and IL-8 in the U-373-MG human astrocytoma cell line via activation of p38 MAPK (mitogen-activated protein kinase) and nuclear factor (NF)-kappaB, respectively. However, the signalling mechanisms by which SP-NK-1R interaction induces NF-kappaB activation and IL-8 expression are still not clear. In this study we demonstrate that SP stimulates IL-8 secretion and IL-8 promoter activity in the NCM460 non-transformed human colonic epithelial cell line transfected with NK-1R cDNA. Our results indicate that inhibition of endogenous Rho family proteins (RhoA, Rac1 and Cdc42) by their respective dominant negative mutants significantly decreases SP-induced IL-8 secretion and IL-8 promoter activity. We also demonstrate that SP rapidly activates RhoA, Rac1 and Cdc42 and that co-expression of the dominant negative mutants of RhoA, Rac1 and Cdc42 in NK-1R cDNA-transfected NCM460 cells significantly inhibits SP-induced NF-kappaB-dependent gene expression. These results demonstrate that Rho family small GTPases RhoA, Rac1 and Cdc42 are novel signal transducers for SP-stimulated IL-8 expression.

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor B (NF-B)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-B activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10 Ϫ7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKC␦, PKC, and PKC⑀, followed by I-B kinase, IB␣, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 M) blocked SP-induced IB␣ and p65 phosphorylation and IL-8 production. The PKC␦ inhibitor rottlerin at a low concentration (1 M), but not pseudosubstrate PKC and PKC⑀ inhibitors (10 M), significantly reduced IL-8 secretion. PKC␦ silencing by RNA interference reduced PKC␦ protein expression and SP-induced PKC␦ phosphorylation that was associated with diminished IL-8 promoter and NF-B luciferase activities in response to SP. Moreover, overexpression of wildtype PKC␦ increased SP-induced IL-8 promoter-and NF-Bdriven luciferase activities that were rottlerin-sensitive. We conclude that PKC␦ plays an important role in SP-induced proinflammatory signaling in human colonocytes.

We report here that human monocytic͞macrophage THP-1 cells express the neurokinin 1 receptor (NK-1R), and that exposure of these cells to the proinflammatory cytokine IL-1␤ increased the expression of the NK-1R gene at the mRNA and protein levels.

We have previously shown that colonic epithelial cells are a major site of MIP-3␣ production in human colon and that enterocyte MIP-3␣ protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3␣ gene transcription in Caco

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