Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration

Abstract

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

Figure 1

Alcohol increases Brf1 expression and Pol III gene transcription. (a, b) HepG2-ADH and HepG2-vector cells were grown in 10%FBS/DMEM to 85% confluency and starved in FBS-free DMEM for 4 hours and then treated with 50 mM ethanol for another 2 hours. Total RNA and protein were extracted from the cells. Brf1 mRNA (a) and protein (b) were determined by RT-qPCR and immunoblot assays. A representative immunoblot is shown in (b). (c) The cellular levels of pre-tRNA^Leu (c, left panel) and 5S rRNA (c, right panel). (d, e) HepG2-ADH cells were transfected with mismatch siRNA (MM siRNA as a control) or Brf1 siRNA and then treated with 50 mM ethanol. RT-qPCR were used to determine the levels of pre-tRNA^Leu (d) and 5S rRNA (e). The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. ^∗∗p < 0.01.

Figure 2

Identification of alcohol-induced response fragments of Brf1 promoter. (a) Alcohol increases Brf1 promoter activity. HepG2-ADH cells were transfected with Brf1-Luc reporter construct (p-382/+109bp) and treated with 50 mM ethanol to determine Luc activity. (b) Identifying alcohol-induced response fragment in Brf1 promoter region. HepG2-ADH and HepG2-Vec cells were transfected with the three different length fragments of Brf1-Luc reporter constructs (p-182/+109bp, p-382/+109bp, and p-760/+109bp) and treated with 50 mM ethanol to determine Luc activity. These results indicate that alcohol-induced high-response region of Brf1 promoter locates at p-382/+109bp of Brf1-Luc construct. The bars represent mean ± SE of at least three independent determinations. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 3

Alcohol induces MSK1 phosphorylation to mediate Brf1 expression. (a) Alcohol induces MSK1 activation. HepG2-ADH cells were starved in FBS-free DMEM for 4 hours and then treated with 50 mM ethanol for another 2 hours. MSK1 phosphorylation at serine 376 and tyrosine 581 of resultant cell lysates was determined with the corresponding antibodies. (b) HepG2 cells were pretreated with MSK1 inhibitor, SB-747651A (10 μM), and treated with 50 mM ethanol. The cellular levels of Brf1 proteins were determined by immunoblot assay against Brf1 antibody. A representative immunoblot is shown in (b). (c, d) AML-12 cells and HepG2-ADH cells were pretreated with SB-747651A, and the cellular levels of Brf1 mRNA were determined by RT-qPCR. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 4

Inhibiting MSK1 pathway decreases alcohol-induced Pol III gene transcription. Primary mouse hepatocytes, AML-12 cells, and HepG2-ADH cells were pretreated with different amounts of SB-747651A and then treated with 50 mM ethanol as described above. Total RNAs were extracted from these cells. Resultant RNAs were used to determine the levels of pre-tRNA^Leu and 5S rRNA by RT-qPCR. (a, b) Primary mouse hepatocytes; (c, d) immortalized mouse AML-12 cells; (e, f) engineered HepG2-ADH cells. Left panel: 5S rRNA; right panel: pre-tRNA^Leu. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 5

MSK1 mediates Brf1 promoter activity. (a) Inhibiting MSK1 inhibits Brf1 promoter activity. HepG2-ADH cells were transfected with the Brf1-Luc reporter construct (p-382/+109bp), pretreated with 5 μM SB-747651A, and then treated with 50 mM ethanol to determine Luc activity. (b–d) Inactivating MSK1 reduces Brf1 expression and Pol III gene transcription. HepG2-ADH were transfected with inactivated MSK1 at N-terminal or C-terminal expression constructs for 48 hours. The cells were treated with 50 mM ethanol for another 2 hours. Total RNAs were extracted from the cells to determine the levels of Brf1 mRNA (b), pre-tRNA^Leu (c), and 5S rRNA (d) by RT-qPCR. The fold changes are calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. ^∗∗p < 0.01.

Figure 6

Inhibiting MSK1 pathway caused cellular phenotypic alteration. (a) HepG2-ADH cells were seeded into 6-well plats and grown with different amounts of SB-747651A and 50 mM ethanol. The concentrations of SB-747651A were 0, 2.5, 5, 10, and 20 μM in turn. The pictures were taken under a microscope (Nikon Eclipse TE300). Original magnification ×100. (b, c) The viability and total cell numbers were counted after plated cells. The cells were treated serially by SB-747651A as a dose curve (b) and 50 mM ethanol as indicated. The cells were treated with 10 μM SB-747651A and 50 mM for 1-6 days as a time curve (c). (d, e) The cells were treated as above, and the rates of cell growth were detected by MTT assay. The bars represent mean ± SE of at least three independent determinations. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 7

Brf1 expression was increased in tumor tissues. (a, b) Colony formation. HepG2-ADH cells were seed into 6-well plates and treated with SB-747651A and ethanol as described above. The results indicate that MSK1 inhibitor reduces the rates of colony formation. (c, d) Overexpression of Brf1 in tumor foci of mouse liver. Total RNAs and lysates were extracted from the tissues of normal liver (sample #3), alcohol-intake liver (sample #2), and HCC liver with alcohol consumption (sample #1). The levels of Brf1 mRNA were determined by RT-qPCR (c). The protein levels of Brf1 in the tissues were analyzed by immunoblot (d). (e) Brf1 staining. Immunohistochemistry (IHC) staining of Brf1 in paracarcinoma tissues (e, left) and tumor foci (e, middle) of human HCC. H&E staining of HCC tumor tissue (e, right). A representative Brf1 staining of human HCC samples. Magnification ×200. These results clearly show that Brf1 expression was increased in tumor tissues of human HCC.

Figure 8

Schematic illustration of MSK1-mediated alcohol-associated HCC. Alcohol activates MSK1 to increase transcription of Brf1 and Pol III genes, resulting in cell proliferation and colony formation, eventually causing HCC. Inhibited MSK1 decreases Brf1 expression to reduce the rates of cancer cell growth and colony formation.↑: increase; ↓: decrease.