Analysis of the molecular mechanism of claudin-1 expression in SCCKN cells, oral squamous cell carcinoma cell line

Abstract

Objectives

Expression of human double minute-2 (HDM2), a p53 ubiquitin ligase, increases the nuclear translocation of β-catenin and expression of claudin-1 (CLD1), a major component protein of tight junctions. Ligand of numb protein X1 (LNX1), an E3 ubiquitin ligase, binds to CLD1 and promotes its degradation by lysosomes in canine renal tubular epithelial cells. However, effect of LNX1 on CLD1 expression in oral squamous cell carcinoma (OSCC) remains unknown. Therefore, in this study, we aimed to elucidate the molecular mechanisms by which LNX1 affects CLD1 expression in OSCC cells.

Methods

We investigated the role of the β-catenin/TCF pathway in CLD1 expression to determine the relationship between LNX1 and CLD1 expression in SCCKN cell line, an OSCC cell line, via co-immunoprecipitation and mammalian two-hybrid assays. Additionally, we evaluated the effect of HDM2 on LNX1 expression in SCCKN cells.

Results

CLD1 expression was regulated via the β-catenin pathway, indicating that nuclear translocation of β-catenin upon HDM2 upregulation elevated the CLD1 levels in SCCKN cells. Inhibitor assays revealed that CLD1 protein in SCCKN cells was degraded by proteasomes or lysosomes. Co-immunoprecipitation and mammalian two-hybrid assays showed that CLD1 bound to LNX1 in SCCKN cells. Furthermore, LNX1p80 bound to HDM2 and underwent proteasomal or lysosomal degradation in SCCKN cells.

Conclusions

Our results suggest that HDM2-induced increase in LNX1p80 degradation suppresses CLD1 degradation by the proteasomes and lysosomes, thereby increasing the CLD1 levels in SCCKN cells.