title: Disruption of ZO-1/claudin-4 interaction in relation to inflammatory responses in methotrexate-induced intestinal mucositis.
abstract: OBJECTIVE
  Methotrexate (MTX)-induced intestinal mucositis limits the use of the drug. We previously reported that MTX-dependent production of reactive oxygen species is an initiating signal leading to neutrophil migration and intestinal barrier dysfunction. Moreover, alterations of zonula occludens (ZO)-1, an integral component of tight junctions (TJs), contribute to its dysfunction. This study aimed to clarify the identity of inflammatory mediators in the intestine of MTX-treated rats and to evaluate MTX-stimulated alterations in the expression of TJ proteins other than ZO-1 (e.g., occludin and claudins).
  
  
  METHODS
  Male Wistar rats were administrated MTX (15 mg kg(-1)) orally once daily for 4 days. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant-2, Toll-like receptor 4 (TLR4), and occludin were determined by real-time RT-PCR. Expression, distribution, and interactions of TJ proteins were evaluated by Western blotting, immunohistochemistry, and immunoprecipitation.
  
  
  RESULTS
  MTX increased the mRNA levels of TNF-α, IL-1β, MIP-2, and TLR4 in the small intestine, as well as the protein expression of claudin-2. Increased claudin-2 and decreased claudin-4 immunostaining were also observed. Occludin mRNA levels were significantly diminished by MTX administration, whereas occludin protein levels and the interaction between ZO-1 and occludin were unaltered; however, the interaction between ZO-1 and claudin-4 was significantly compromised.
  
  
  CONCLUSIONS
  These results indicate that elevated levels of inflammatory cytokines and chemokines in the small intestine of MTX-treated rats may contribute to the inhibition of ZO-1/claudin-4 binding, and that inhibition of ZO-1/claudin-4 binding may in turn lead to a reduction in claudin-4 expression.
authors: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan.
authors: Kazuma
authors: K
authors: Hamada
authors: Naoko
authors: N
authors: Kakigawa
authors: Shuichi
authors: S
authors: Sekine
authors: Yoshihisa
authors: Y
authors: Shitara
authors: Toshiharu
authors: T
authors: Horie
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity; D018925:Chemokines / Q000378:metabolism; D062506:Claudin-4 / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007249:Inflammation / Q000139:chemically induced* / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D007421:Intestine, Small / Q000187:drug effects / Q000473:pathology; D008297:Male; D008727:Methotrexate / Q000633:toxicity*; D052016:Mucositis / Q000139:chemically induced* / Q000473:pathology; D062793:Occludin / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism / Q000473:pathology; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D018925:Chemokines; D062506:Claudin-4; D016207:Cytokines; D018836:Inflammation Mediators; D062793:Occludin; D012333:RNA, Messenger; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D008727:Methotrexate