Evidence for a causal link between adaptor protein PDZK1 downregulation and Na⁺/H⁺ exchanger NHE3 dysfunction in human and murine colitis.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

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

BACKGROUND: 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. 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-1ß 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. 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.