Regulation of NF-kappaB-dependent gene expression by the POU domain transcription factor Oct-1.

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.

The role of neurokinin 1 receptors in the maintenance of visceral hyperalgesia induced by repeated stress in rats.

BACKGROUND & AIMS: The neurokinin 1 receptors (NK(1)Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK(1)Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress. METHODS: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK(1)R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK(1)Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia. RESULTS: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK(1)Rs. Immunohistochemistry showed an increased number of NK(1)R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK(1)Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls. CONCLUSIONS: Stress-induced increased NK(1)R expression on spinal neurons and the inhibitory effect of intrathecal NK(1)R antagonist on visceral hyperalgesia support the key contribution of spinal NK(1)Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression.

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.

Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cdelta activation.

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 kappa B (NF-kappaB)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-kappaB 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(-7) M) induced an early (1 min) phosphorylation of the PKC isoforms PKCdelta, PKC, and PKCepsilon, followed by I-kappaB kinase, IkappaBalpha, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 microM) blocked SP-induced IkappaBalpha and p65 phosphorylation and IL-8 production. The PKCdelta inhibitor rottlerin at a low concentration (1 microM), but not pseudosubstrate PKC and PKCepsilon inhibitors (10 microM), significantly reduced IL-8 secretion. PKCdelta silencing by RNA interference reduced PKCdelta protein expression and SP-induced PKCdelta phosphorylation that was associated with diminished IL-8 promoter and NF-kappaB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCdelta increased SP-induced IL-8 promoter- and NF-kappaB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCdelta plays an important role in SP-induced proinflammatory signaling in human colonocytes.

Leptin mediates Clostridium difficile toxin A-induced enteritis in mice.

BACKGROUND & AIMS: Leptin regulates energy homeostasis and participates in the regulation of the hypothalamic-pituitary-adrenal axis. Although hyperleptinemia is described in experimental colitis, its role in the pathophysiology of enterotoxin-mediated diarrhea and inflammation remains unclear. We examined the role of leptin in the inflammatory diarrhea induced by toxin A from Clostridium difficile, the causative agent of antibiotic-related colitis. METHODS: Toxin A (10 microg) or buffer were administered in ileal loops of leptin-deficient (ob/ob), leptin-resistant (db/db), or wild-type mice and enterotoxic responses were measured. RESULTS: In toxin A-treated wild-type mice, circulating leptin and corticosterone levels were increased compared with buffer-injected animals. Toxin A also stimulated increased mucosal expression of the Ob-Rb at the messenger RNA (mRNA) and protein level. Ob/ob and db/db mice were partially protected against toxin A-induced intestinal secretion and inflammation, and this effect was reversed by leptin administration in ob/ob, but not db/db, mice. Basal- and toxin A-stimulated plasma corticosterone levels in ob/ob and db/db mice were higher compared with toxin A-treated wild-type mice. To assess whether the effect of leptin in intestinal inflammation is mediated by corticosteroids we performed adrenalectomy experiments in db/db and wild-type mice. Our results suggested that the diminished intestinal response to toxin A in db/db mice was related only in part to increased levels of corticosteroids. CONCLUSIONS: Leptin plays an important role in regulating the severity of enterotoxin-mediated intestinal secretion and inflammation by activating both corticosteroid-dependent and -independent mechanisms.

Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-kappa B site on its promoter.

We report here that human monocytic/macrophage THP-1 cells express the neurokinin 1 receptor (NK-1R), and that exposure of these cells to the proinflammatory cytokine IL-1 beta increased the expression of the NK-1R gene at the mRNA and protein levels. Because IL-1 beta function involves nuclear factor kappa B (NF-kappa B) activation, these data suggest that this increase in the expression of the NK-1R gene is mediated by the NF-kappa B transcription factor. An earlier report noted that the promoter region of the human NK-1R gene contains a putative binding site for NF-kappa B [Takahashi, K., Tanaka, A., Hara, M. & Nakanishi, S. (1992) Eur. J. Biochem. 204, 1025-1033]. Here we demonstrate that this is indeed a functional NF-kappa B-binding site, and that NF-kappa B is responsible for regulating the expression of the NK-1R gene by binding to the promoter region of the NK-1R gene. To further substantiate that the observed NF-kappa B-dependent IL-1 beta induction of the human NK-1R gene is regulated via a transcriptional event through this NF-kappa B site on the NK-1R gene promoter, we transfected THP-1 cells with a luciferase promoter-reporter construct containing the 5' promoter region of the human NK-1R gene. Exposure of these cells to IL-1 beta or overexpression of NF-kappa B cDNAs resulted in a significant increase in the amount of luciferase activity that was diminished greatly in cells transfected with I kappa B alpha, the NF-kappa B inhibitor. These results directly implicate NF-kappa B in the regulation of the NK-1R gene and provide a molecular mechanism for the increase in expression of the NK-1R gene in responsive cells.

ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3alpha gene expression in Caco-2 human colonic epithelial cells.

We have previously shown that colonic epithelial cells are a major site of MIP-3alpha production in human colon and that enterocyte MIP-3alpha protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3alpha gene transcription in Caco-2 intestinal epithelial cells. We show that a kappaB element at nucleotides -82 to -93 of the MIP-3alpha promoter binds p50/p65 NF-kappaB heterodimers and is a major regulator of basal and interleukin-1beta (IL-1beta)-mediated gene activation. Scanning mutagenesis of the MIP-3alpha 5'-flanking region also identified two additional binding elements: Site X (nucleotides -63 to -69) and Site Y (nucleotides -143 to -154). Site X (CGCCTTC) bound Sp1 and regulated basal MIP-3alpha gene transcription. Overexpression of Sp1 increased basal luciferase activity, whereas, substitutions in the Sp1 element significantly reduced reporter activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal and cytokine-induced gene activation and bound the Ets nuclear factor ESE-1. Substitutions in the Site Y element markedly reduced inducible MIP-3alpha reporter activity. Conversely, overexpression of ESE-1 significantly up-regulated MIP-3alpha luciferase levels. Taken together, our findings demonstrate that co-ordinate activation and binding of ESE-1, Sp1, and NF-kappaB to the MIP-3alpha promoter is required for maximal gene expression by cytokine-stimulated Caco-2 human intestinal epithelial cells.

Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases.

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.