Andreas Mykoniatis

Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-κB 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-1β increased the expression of the NK-1R gene at the mRNA and protein levels. Because IL-1β function involves nuclear factor κB (NF-κB) activation, these data suggest that this increase in the expression of the NK-1R gene is mediated by the NF-κB transcription factor. An earlier report noted that the promoter region of the human NK-1R gene contains a putative binding site for NF-κB [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-κB-binding site, and that NF-κB 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-κB-dependent IL-1β induction of the human NK-1R gene is regulated via a transcriptional event through this NF-κB 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-1β or overexpression of NF-κB cDNAs resulted in a significant increase in the amount of luciferase activity that was diminished greatly in cells transfected with IκBα, the NF-κB inhibitor. These results directly implicate NF-κB 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.

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

The basolateral Na+-K+-2Cl− cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ϵ) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCϵ- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCϵ-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCϵ, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

884 Regulation of NKCC1 Surface Expression By PKC Involves Dynamin II and Clathrin-Dependent Endocytic Machinery

G A A b st ra ct s before and after medication were analyzed for small intestinal injuries. NSAID treatment significantly increased the mean number of mucosal breaks and reddened lesions per subject in the Control-group, from 0.1±0.3 to 15.8±71.5 and from 4.0±3.9 to 10.0±10.3 respectively; but only up from 0.1±0.3 to 4.2±7.8 and from 5.4±6.3 to 9.2±6.6 in the Rebamipide-group. The percentage of subjects with at least one mucosal break at post-treatment was also higher in the Control-group (63%) than in the Rebamipide-group (47%); (NS). The percentage of subjects with post-treatment increases in reddened lesions ≥ 10 was significantly higher in the Control-group (46%) than in the Rebamipide-group (14%); (P=0.04). Conclusion: Rebamipide administration reduced the incidence of small intestinal lesions induced by twoweek administration of diclofenac sodium. Further studies are warranted to determine whether a higher rebamipide dosage can further protect against NSAID-induced small intestinal mucosal breaks.