Downregulation of the NHE3-Binding PDZ-Adaptor Protein PDZK1 Expression during Cytokine-Induced Inflammation in Interleukin-10–Deficient Mice

<div><h3>Background</h3><p>Impaired salt and water absorption is an important feature in the pathogenesis of diarrhea in inflammatory bowel disease (IBD). 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.</p> <h3>Methodology/Principal Findings</h3><p>Gene and protein expression were analyzed by real-time reverse transcription polymerase chain reaction (<em>q</em>RT-PCR), <em>in situ</em> 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. 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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

Short-Term Regulation of Murine Colonic NBCe1-B (Electrogenic Na⁺/HCO₃⁻ Cotransporter) Membrane Expression and Activity by Protein Kinase C

<div><p>The colonic mucosa actively secretes HCO₃⁻, and several lines of evidence point to an important role of Na⁺/HCO₃⁻ cotransport (NBC) as a basolateral HCO₃⁻ import pathway. We could recently demonstrate that the predominant NBC isoform in murine colonic crypts is electrogenic NBCe1-B, and that secretagogues cause NBCe1 exocytosis, which likely represents a component of NBC activation. Since protein kinase C (PKC) plays a key role in the regulation of ion transport by trafficking events, we asked whether it is also involved in the observed NBC activity increase. Crypts were isolated from murine proximal colon to assess PKC activation as well as NBC function and membrane abundance using fluorometric pH_i measurements and cell surface biotinylation, respectively. PKC isoform translocation and phosphorylation occurred in response to PMA-, as well as secretagogue stimulation. The conventional and novel PKC inhibitors Gö6976 or Gö6850 did not alter NBC function or surface expression by themselves, but stimulation with forskolin (10⁻⁵ M) or carbachol (10⁻⁴ M) in their presence led to a significant decrease in NBC-mediated proton flux, and biotinylated NBCe1. Our data thus indicate that secretagogues lead to PKC translocation and phosphorylation in murine colonic crypts, and that PKC is necessary for the increase in NBC transport rate and membrane abundance caused by cholinergic and cAMP-dependent stimuli.</p></div

An Extended ΔCT-Method Facilitating Normalisation with Multiple Reference Genes Suited for Quantitative RT-PCR Analyses of Human Hepatocyte-Like Cells

<div><p>Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of <i>in vitro</i> cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies <i>PSMB6, MDH1</i> and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells <i>in vitro</i>. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG - here identified suited for human hepatocyte-like cells - for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.</p></div

NBCe1 membrane expression during secretagogue stimulation and PKC inhibition with Gö6850 or Gö6976.

<p>In cell surface biotinylation experiments, neither Gö6850 (A; 5 μM) nor Gö6976 (B; 5 μM; continuous incubation for 20 min [+] vs. vehicle [−]) caused changes in NBCe1 surface expression. When forskolin (10⁻⁵ M) or carbachol (10⁻⁴ M) were added after 10 min, NBCe1 surface expression significantly decreased (40 and 59% for Gö6850, and 8 and 31% for Gö6976, respectively; n = 5–7 preparations from separate mice in each group, *:p<0.05, ANOVA for correlated samples followed by Tukey's HSD).</p

Gene expression by <i>in situ</i> RT-PCR of proinflammatory cytokines, iNOS, and procaspase 3 in the colonic mucosa.

<p>(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.</p

PKC translocation in response to PMA and secretagogues.

<p>PKC membrane expression as assessed by surface biotinylation and probing with a general PKC antibody significantly increased after exposure to PMA (100 nM), forskolin (10⁻⁵ M), or carbachol (10⁻⁴ M, 10 min each), which was paralleled by a decrease in cytosolic PKC expression [ODI: optical density integrated; *:p<0.05 vs. unstimulated, n =  6 preparations from separate mice in each group, ANOVA for correlated samples followed by Tukey's HSD (Tukey's honestly significant difference test), values expressed as % of the unstimulated control for cytosol and membrane, respectively].</p

Immunohistochemical staining of NBCe1 and PKC isoforms in murine colonic tissue sections.

<p>NBCe1 (A) is expressed in the basolateral membrane of colonic crypts. Cytoplasmatic expression was observed for PKCα (C), δ (E) and ε (G) isoforms, with an apparent slight accumulation of the signal in the vicinity of the cell membrane for PKC α and PKC ε. Control experiments without the secondary antibody (NBCe1, B) and blocking peptides (PKC isoforms, D/F/H) showed no specific signal. Size of the scale bar is 50 μm.</p

Double immunofluorescence staining and densitometric quantification of NHE3, NHERF2, and PDZK1 in enterocytes of the inflamed colon.

<p>(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.</p

<b>Table 1.</b> Gene expression profile of the proinflammatory cytokines IL-1β, TNF-α, IFN-γ and iNOS and the cell death marker procaspase 3 in the colonic mucosa of WT and IL-10^−/− mice.

<p>Results are expressed as the mean normalized expression and fold change of IL-10^−/− mice against WT controls. mRNA was quantified in relation to β-actin. Data are mean values ± SEM (from 5–6 experiments in each group).</p>*<p>p<0.05 versus control.</p

pH-microfluorometrical determination of NBC activity in the presence of PKC inhibitors with and without forskolin stimulation.

<p>NBC transport rates were determined as the Na⁺- and CO₂/HCO₃⁻-dependent, DMA-insensitive proton flux rates during pH_i recovery from an acid load. A: Average pH_i trace (n = 5) illustrating the pH_i recovery protocol, where crypts were acidified with an NH₄ “prepulse” to a pH of 6.4±0.2 in Na⁺-free buffer (TMA-Cl) and let to recover after Na⁺ re-addition in the presence of 700 μM DMA to block all Na⁺/H⁺ exchanger isoforms. In the absence of CO₂/HCO₃⁻, no significant pH_i recovery was observed, but in its presence, there was a steady pH_i increase representing Na⁺/HCO₃⁻ cotransport (NBC; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092275#pone.0092275-Bachmann1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092275#pone.0092275-Bachmann3" target="_blank">[8]</a>). B: Left side: Incubation with Gö6976 (5 μM, 10 min prior to stimulation, light grey bars) or Gö6850 (5 μM, 10 min prior to stimulation, dark grey bars) did not alter the control proton flux rates (solid bars; p = n.s.). Right side: Stimulation with forskolin led to the previously observed significant stimulation of NBC (10.2±0.8 vs. 5.3±0.4 mM/min; *:p<0.05). Preincubation with either of the PKC inhibitors completely reversed this effect (n = 5–7 experiments from separate mice, p = n.s., ANOVA for independent samples followed by Tukey's HSD).</p