Toll-like Receptor 2 Signalling and the Lysosomal Machinery in Barrett's Esophagus.
OBJECTIVE
Inflammation plays an important role in the development of esophageal adenocarcinoma and its metaplastic precursor lesion, Barrett's esophagus. Toll-like receptor (TLR) 2 signalling and lysosomal function have been linked to inflammation-associated carcinogenesis. We examined the expression of TLR2 in the esophagus and the effect of long-term TLR2 activation on morphological changes and expression of factors involved in lysosomal function in a Barrett's esophagus epithelium cell line.


METHODS
TLR2 expression in normal squamous esophagus, reflux esophagitis, Barrett's esophagus and esophageal adenocarcinoma biopsies was assessed with Q-RT-PCR, in situ hybridization and immunohistochemistry. Barrett's esophagus epithelium cells (BAR-T) were incubated with acid and bile salts in the presence or absence of the TLR2 agonist Pam3CSK4 for a period up to 4 weeks. Morphological changes were assessed with electron microscopy, while Q-RT-PCR was used to determine the expression of lysosomal enzymes (Cathepsin B and C) and factors involved in endocytosis (LAMP-1 and M6PR) and autophagy (LC3 and Rab7).


RESULTS
TLR2 was expressed in normal squamous esophagus, reflux esophagitis, Barrett's esophagus but was most prominent in esophageal adenocarcinoma. Long-term TLR2 activation in acid and bile salts exposed BAR-T cells resulted in more and larger lysosomes, more mitochondria and increased expression of LAMP-1, M6PR, Cathepsin B and C when compared to BAR-T cells incubated with acid and bile salts but no TLR2 agonist. Factors associated with autophagy (LC3 and Rab7) expression remained largely unchanged.


CONCLUSIONS
Activation of TLR2 in acid and bile salts exposed Barrett epithelium cells resulted in an increased number of mitochondria and lysosomes and increased expression of lysosomal enzymes and factors involved in endocytosis.
Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands. r.verbeek@antoniusziekenhuis.nl.
Romy E
RE
Verbeek
Department of Gastroenterology and Hepatology, University Medical Center Utrecht; Department of Gastroenterology and Hepatology, Radboud University Medical Center, Nijmegen, The Netherlands.
Peter D
PD
Siersema
Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands.
Frank P
FP
Vleggaar
Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Fiebo J
FJ
Ten Kate
Department of Cell Biology, Center for Electron Microscopy, University Medical Center Utrecht, Utrecht, The Netherlands;
George
G
Posthuma
Department of Medicine, University of Texas Southwestern Medical Center, VA North Texas Health Care System, Dallas, Texas, USA.
Rhonda F
RF
Souza
Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands.
Judith
J
de Haan
Department of Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands.
Jantine W P M
JW
van Baal
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000368:Aged; D000369:Aged, 80 and over; D001471:Barrett Esophagus / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001647:Bile Acids and Salts / Q000494:pharmacology; D016022:Case-Control Studies; D002401:Cathepsin B / Q000378:metabolism; D020789:Cathepsin C / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004942:Esophagitis, Peptic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004947:Esophagus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D055666:Lipopeptides / Q000494:pharmacology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051195:Toll-Like Receptor 2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1
D001647:Bile Acids and Salts; D002401:Cathepsin B; D020789:Cathepsin C; D055666:Lipopeptides; D051907:Lysosomal Membrane Proteins; D017527:Receptor, IGF Type 2; D051195:Toll-Like Receptor 2; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C508075:Pam(3)CSK(4) peptide; C495342:TLR2 protein, human; C034304:cation-dependent mannose-6-phosphate receptor; C533918:CTSC protein, human; C533912:CTSB protein, human