title: AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells.
abstract: AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity. In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues. To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl(-) absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the overexpression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level.
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Chiara Stella
authors.initials: CS
authors.lastname: Di Stadio
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Altieri
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Miselli
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Ausilia
authors.initials: A
authors.lastname: Elce
authors.affiliation: Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Severino
authors.affiliation: Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy; IRCCS Multimedica, Milan, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.affiliation: Laboratory of Biotechnology, Department of Anesthesia, Surgical and Emergency Sciences, Second University of Naples, Via Costantinopoli 16, I-80138, Naples, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Quagliariello
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Villano
authors.affiliation: Hospital Antonio Cardarelli, Naples, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: de Dominicis
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy. Electronic address: emilia.rippa@unina.it.
authors.forename: Emilia
authors.initials: E
authors.lastname: Rippa
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy; CEINGE, Advanced Biotechnology Scarl, Via Gaetano Salvatore 486, I-80145, Naples, Italy. Electronic address: paolo.arcari@unina.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Arcari
mesh_terms: D045744:Cell Line, Tumor; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics* / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D036361:Peptide Hormones / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism*; D000076988:Sulfate Transporters
chemical_list: D027963:Chloride-Bicarbonate Antiporters; C477000:GKN1 protein, human; D036361:Peptide Hormones; C081011:SLC26A3 protein, human; D000076988:Sulfate Transporters
keywords: Antrum mucosal protein; Brichos domain; Down-Regulated in Adenoma; Gastric cancer