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. 2012;7(7):e40657.
doi: 10.1371/journal.pone.0040657. Epub 2012 Jul 27.

Downregulation of the NHE3-binding PDZ-adaptor protein PDZK1 expression during cytokine-induced inflammation in interleukin-10-deficient mice (V体育平台登录)

Henrike Lenzen  1 , Maria Lünnemann, Andre Bleich, Michael P Manns, Ursula Seidler, Anne Jörns
Affiliations

Downregulation of the NHE3-binding PDZ-adaptor protein PDZK1 expression during cytokine-induced inflammation in interleukin-10-deficient mice

Henrike Lenzen et al. PLoS One. 2012.

"VSports注册入口" Abstract

Background: Impaired salt and water absorption is an important feature in the pathogenesis of diarrhea in inflammatory bowel disease (IBD) VSports手机版. We analyzed the expression of proinflammatory cytokines in the infiltrating immune cells and the function and expression of the Na(+)/H(+) exchanger isoform 3 (NHE3) and its regulatory PDZ-adaptor proteins NHERF1, NHERF2, and PDZK1 in the colon of interleukin-10-deficient (IL-10(-/-)) mice. .

Methodology/principal findings: Gene and protein expression were analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR), in situ RT-PCR, and immunohistochemistry. NHE3 activity was measured fluorometrically in apical enterocytes within isolated colonic crypts. Mice developed chronic colitis characterized by a typical immune cell infiltration composed of T-lymphocytes and macrophages, with high levels of gene and protein expression of the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α. In parallel, inducible nitric oxide synthase expression was increased while procaspase 3 expression was unaffected. Interferon-γ expression remained low. Although acid-activated NHE3 activity was significantly decreased, the inflammatory process did not affect its gene and protein expression or its abundance and localization in the apical membrane V体育安卓版. However, expression of the PDZ-adaptor proteins NHERF2 and PDZK1 was downregulated. NHERF1 expression was unchanged. In a comparative analysis we observed the PDZK1 downregulation also in the DSS (dextran sulphate sodium) model of colitis. .

Conclusions/significance: The impairment of the absorptive function of the inflamed colon in the IL-10(-/-) mouse, in spite of unaltered NHE3 expression and localization, is accompanied by the downregulation of the NHE3-regulatory PDZ adaptors NHERF2 and PDZK1. We propose that the downregulation of PDZ-adaptor proteins may be an important factor leading to NHE3 dysfunction and diarrhea in the course of the cytokine-mediated inflammatory process in these animal models of IBD. V体育ios版.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gene expression by in situ RT-PCR of proinflammatory cytokines, iNOS, and procaspase 3 in the colonic mucosa.
(A) and (B) High expression levels of IL-1β and TNF-α in infiltrating immune cells of IL-10−/− mice. (C) IFN-γ exhibited scant expression in only single immune cells of IL-10−/− mice and healthy WT mice. (D) High expression of iNOS in the immune cells surrounding the epithelial cells and in single immune cells in the connective tissue of the mucosa compared to WT mice. (E) Comparable procaspase 3 expression in WT and IL-10−/− mice. Arrows show representative mononuclear cells. Representative images of three independent experiments.
Figure 2
Figure 2. Double immunofluorescence staining of the proinflammatory cytokines IL-1β and TNF-α in infiltrating CD3 T-lymphocytes and macrophages.
IL-10−/− mice showed signs of inflammation with strong accumulation of CD3 T-lymphocytes and macrophages (Mø) in the mucosal and submucosal layer in the colon with high expression of the proinflammatory cytokines IL-1β and TNF-α. Only single-cytokine–positive CD3 T-lymphocytes and macrophages were observed in the layers of the colon from WT mice. The dashed line separates the epithelial layer from the connective tissue of the submucosal layer. Double immunofluorescence staining presents as mostly white in the overlay (instead of yellow) as an amalgamation of green and red due to the background staining with DAPI (blue). Representative cells are marked with arrows. Shown are representative images of three independent experiments.
Figure 3
Figure 3. Fluorometric assessment of NHE3 activation by acid in colonic enterocytes in IL-10−/− mice.
(A) Exemplary pH curve. (B) Reduced NHE3 activity as measured by acid-activated Hoe642-insensitive proton flux in colonic enterocytes in IL-10−/− mice (black columns) compared to healthy WT mice (open columns). n = 3 pairs of mice; * p<0.05 versus control. (C) Unaltered NHE3 gene expression but downregulation of the PDZ-adaptor proteins NHERF2 and PDZK1 in IL-10−/− mice. The expression of NHERF1 was not affected. Results are expressed as a comparison of fold change in expression of NHE3, NHERF1, NHERF2 and PDZK1 in the inflamed and noninflamed colon (from 5–6 experiments in each group). mRNA was quantified in relation to ß-actin. *p<0.05 versus control.
Figure 4
Figure 4. Gene expression by in situ RT-PCR of NHE3, NHERF2, and PDZK1 in the colonic mucosa.
(A) Unaltered NHE3 expression levels in enterocytes of the epithelial layer of WT and IL-10−/− mice. (B) mRNA expression of NHERF2 and (C) PDZK1 in the epithelial enterocytes were markedly decreased in IL-10−/− mice. The dashed line separates the epithelial layer from the connective tissue of the lamina propria of the mucosa. Representative images of two independent experiments.
Figure 5
Figure 5. Double immunofluorescence staining and densitometric quantification of NHE3, NHERF2, and PDZK1 in enterocytes of the inflamed colon.
(A) and (B) NHE3 protein expression was clearly restricted to the apical region of the enterocytes in colonic mucosa of diseased IL-10−/− mice and WT mice in comparable intensities. (A) NHERF2 protein expression (red) was restricted to the apical region of the enterocytes in a dotted-like fashion in association with the NHE3 staining (green). The number of red dots was markedly reduced in diseased IL-10−/− mice. (B) The PDZK1 immunostaining (green) was localized in the whole cytoplasm of the enterocytes under healthy and diseased conditions while the intensity of PDZK1 was markedly reduced in IL-10−/− mice. NHE3 staining (red) was not reduced in IL-10−/− mice. Nuclei of the cells were stained with DAPI (blue). A and B show representative images of three independent experiments. (C) Densitometric quantification of the NHE3, NHERF2, and PDZK1 protein expression in enterocytes of WT and IL-10−/− mice. Densities are expressed in mean fluorescence intensities per pixel as means ± SEM. n = 4 animals; in each animal, 9–23 enterocytes in the same cell section plane were analyzed. *p<0.01 versus control.
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