IL-6 induction of hepatocyte proliferation through the Tmub1-regulated gene pathway

Abstract

The expression of transmembrane and ubiquitin-like domain containing 1 (Tmub1) is upregulated during liver regeneration, however, the function and underlying molecular mechanisms responsible for Tmub1 action remain to be determined. This study utilized BRL-3A rat liver cells for Tmub1 shRNA lentivirus infection and IL-6 stimulation. Semi-quantitative RT-PCR and western blot analysis were used to detect mRNA and protein expression levels, respectively. A [3H]thymidine incorporation assay was performed to assess changes in cell proliferation rates. Laser scanning confocal microscopy and immunoprecipitation-western blotting were used to assess the interaction between Tmub1 and calcium-modulating cyclophilin ligand (CAML) protein. The effect of Tmub1 on calcium ion influx into BRL-3A cells was measured by inverted fluorescence microscopy. The data showed that IL-6 treatment induced proliferation of rat hepatocytes and expression of Tmub1 mRNA and protein, while Tmub1 shRNA knocked down Tmub1 expression at both the mRNA and protein levels. Furthermore, compared to the negative control, Tmub1 shRNA-infected BRL-3A cells were highly proliferative with or without IL-6 stimulation. Tmub1 is colocalized with CAML in the hepatocellular cytoplasm, whereas knockdown of Tmub1 expression upregulated expression of CAML protein. Influx of Ca2+ into rat liver cells was also affected after Tmub1 knockdown. The data from the current study demonstrate that Tmub1 plays a negative role in IL-6-induced hepatocyte proliferation, and indicate that the interaction between Tmub1 and CAML may mediate the function of Tmub1 in hepatocytes.