Hint1 Up-Regulates IκBα by Targeting the β-TrCP Subunit of SCF E3 Ligase in Human Hepatocellular Carcinoma Cells

There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-kappa B alpha (I kappa B alpha) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526-1534, 2009). We examined HINT1 and I kappa B alpha expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on I kappa B alpha protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on I kappa B alpha ubiquitination. Next, the interaction between HINT1 and beta-TrCP was investigated in immunoprecipitation and immunofluorescence assays. Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased I kappa B alpha protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of I kappa B alpha, but HINT1 inhibited proteasomal I kappa B alpha degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCF beta-TrCP E3 ligase, which is responsible for I kappa B alpha ubiquitination and degradation. This study provides new evidence that HINT1 is a regulator of I kappa B alpha through SCF beta-TrCP E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.