Regulation of TRIB1 abundance in hepatocyte models in response to proteasome inhibition

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. In view of the key metabolic role of TRIB1 and the impact of proteasome inhibition on hepatic function, we continue our exploration of TRIB1 regulation in two commonly used human hepatocyte models, transformed cell lines HuH-7 and HepG2. In both models, proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was unaffected by MAPK inhibitors while ER stress was a weaker inducer. Suppressing proteasome function via PSMB3 silencing was sufficient to increase TRIB1 mRNA expression. ATF3 was required to sustain basal TRIB1 expression and support maximal induction. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitylation, proteasome inhibition delayed but did not prevent TRIB1 loss upon translation block. Immunoprecipitation experiments indicated that TRIB1 was not ubiquitylated in response to proteasome inhibition. A control bona fide proteasome substrate revealed that high doses of proteasome inhibitors resulted in incomplete proteasome inhibition. Cytoplasm retained TRIB1 was unstable, suggesting that TRIB1 lability is regulated prior to its nuclear import. N-terminal deletion and substitutions were insufficient to stabilize TRIB1. These findings identify transcriptional regulation as a prominent mechanism increasing TRIB1 abundance in transformed hepatocyte cell lines in response to proteasome inhibition and provide evidence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.

Automated summary

Tribbles related homolog 1 (TRIB1) is involved in the regulation of lipid and glucose metabolism by promoting the degradation of target cargos through the proteasome. In this study, it was found that proteasome inhibitors significantly increased the levels of TRIB1 mRNA and protein in transformed hepatocyte cell lines HuH-7 and HepG2. Further experiments showed that suppressing proteasome function via PSMB3 silencing upregulated TRIB1 mRNA expression. Although proteasome inhibition delayed TRIB1 degradation, it did not completely prevent it, suggesting the presence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.