Intestinal Epithelial TCPTP Deficiency Reduces Expression of the Tight Junction Protein Tricellulin through MEK/ERK Signaling

Abstract

Genome‐wide association studies identified protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) as a susceptibility gene for inflammatory bowel disease (IBD), Diabetes and Celiac disease, while loss of the PTPN2 protein product T‐cell protein tyrosine phosphatase (TCPTP) compromises intestinal epithelial barrier function. The protein composition of tight junctions (TJ) determines the tightness of the barrier and alterations in select TJ proteins leads to increased paracellular permeability. Tricellulin (also called marvelD2) is a recently discovered tight junction (TJ) protein, which is preferentially localized at tricellular contacts. Tricellulin plays an important role in the maintenance of intestinal epithelial barrier function by restricting paracellular permeability to macromolecules. The aim of the present study was to identify the molecular mechanism underlying barrier defects in TCPTP‐deficient intestinal epithelial cells (IEC). For this purpose, we developed Caco2‐BBe and HT‐29 intestinal epithelial cell lines (IECs) expressing short hairpin RNA for TCPTP using a lentiviral system to stably knock down TCPTP expression (TCPTP‐KD). Western blot analysis indicated that TCPTP expression was significantly reduced (by 45 ± 4%, p<0.01, n=3) in TCPTP‐KD cells. FD4 permeability was increased in KD cells (p<0.001; n=3) compared to control cells. Western blot and immunofluorescence studies revealed that the expression of tricellulin expression was decreased (WB: 44 ± 8.8%, p<0.05; n=3) in unchallenged TCPTP‐KD cells compared to control shRNA transfected cells. This decrease was further enhanced by challenge with the pro‐inflammatory cytokine interferon‐gamma (IFN‐g) (n=2). In previous studies, we observed increased basal phosphorylation of the TCPTP substrate ERK1/2 in TCPTP‐KD cells. Moreover, an inverse relationship exists between mitogen‐activated protein kinase kinase kinase 1 (MEKK1) that lies upstream of ERK1/2, and MarvelD3, a member of the same protein family as tricellulin. To investigate whether tricellulin loss in TCPTP‐KD cells occurs through MEK/ERK signaling, TCPTP‐KD IEC were treated with the ERK1/2 inhibitor U0126 (10mM) for 14 hours. ERK inhibition rescued tricellulin expression in TCPTP‐KD cells. In conclusion, we report that tricellulin expression is down regulated, at least in part, by the MEK/ERK pathway and that decreased tricellulin levels could contribute to the increased macromolecule permeability in cells lacking TCPTP.Support or Funding InformationNIH‐R01‐DK‐091281 and Crohn's & Colitis Foundation of America(CCFA) to Declan F. McCole