Korean J Physiol Pharmacol.  2016 Nov;20(6):595-603. 10.4196/kjpp.2016.20.6.595.

The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

• Kim HS
• Kim SJ
• Bae J
• Wang Y
• Park SY
• Min YS
• Je HD
• Sohn UD

• KMID: 2364263
• DOI: http://doi.org/10.4196/kjpp.2016.20.6.595

Abstract

Ribosomal S6 kinase is a family of serine/threonine protein kinases involved in the regulation of cell viability. There are two subfamilies of ribosomal s6 kinase, (p90rsk, p70rsk). Especially, p90rsk is known to be an important downstream kinase of p44/42 MAPK. We investigated the role of p90rsk on ethanol-induced cell proliferation of HepG2 cells. HepG2 cells were treated with 10~50 mM of ethanol with or without ERK and p90rsk inhibitors. Cell viability was measured by MTT assay. The expression of pERK1, NHE1 was measured by Western blots. The phosphorylation of p90rsk was measured by ELISA kits. The expression of Bcl-2 was measured by qRT-PCR. When the cells were treated with 10~30 mM of ethanol for 24 hour, it showed significant increase in cell viability versus control group. Besides, 10~30 mM of ethanol induced increased expression of pERK1, p-p90rsk, NHE1 and Bcl-2. Moreover treatment of p90rsk inhibitor attenuated the ethanol-induced increase in cell viability and NHE1 and Bcl-2 expression. In summary, these results suggest that p90rsk, a downstream kinase of ERK, plays a stimulatory role on ethanol-induced hepatocellular carcinoma progression by activating anti-apoptotic factor Bcl-2 and NHE1 known to regulate cell survival.

Keyword

Bcl-2; Ethanol; Hepatocellular carcinoma; NHE1; p90rsk

MeSH Terms

Blotting, Western
Carcinoma, Hepatocellular
Cell Proliferation*
Cell Survival
Enzyme-Linked Immunosorbent Assay
Ethanol
Hep G2 Cells*
Humans
Phosphorylation
Phosphotransferases
Protein Kinases
Ribosomal Protein S6 Kinases
Ethanol
Phosphotransferases
Protein Kinases
Ribosomal Protein S6 Kinases

Figure

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  Fig. 1 Effect of ethanol on the growth of HepG2 cell line.Serum-starved HepG2 cells were incubated with ethanol for 24 hours at the indicated concentration. The cell viability was estimated using MTT assay. Data are expressed as means±S.E of three experiments (student's t-test; *p<0.05 vs. control).

• 

  Fig. 2 Activation of ERK in HepG2 cell lines after the treatment with ethanol (10~50 mM) for 24 hours.(A) Cell lysates were analyzed by Western blot to detect pERK1. (B) The graph represents fold expression of pERK1 relative to β-actin averaged from four independent experiments. Representative Western immunoblots are presented. Actin expression is shown for a control loading. Data are expressed as means±S.E of four experiments (student's t-test; *p<0.05 vs. control).

• 

  Fig. 3 Activation of p90rsk in HepG2 cell lines after the treatment with ethanol (10~50 mM) for 24 hours.Serum-starved HepG2 cells were incubated with ethanol for 24 hours at the indicated concentration. The concentration of phosphorylated-p90rsk in the cell lysate was determined by ELISA. Data are expressed as means±S.E of four experiments (student's t-test; *p<0.05 vs. control).

• 

  Fig. 4 The effect of inhibitor of ERK on the expression of p90rsk in HepG2 cell lines after the treatment with ethanol (20 mM) for 24 hours.Serum-starved HepG2 cells were incubated with ethanol for 24 hours with or without PD98059 (ERK inhibitor) at the indicated concentration. The concentration of phosphorylated-p90rsk in the cell lysate was determined by ELISA. Data are expressed as means±S. E of four experiments (student's t-test; *p<0.05 vs. control, #p<0.05 vs. ethanol alone).

• 

  Fig. 5 The effect of inhibitor of p90rsk on the ethanol-induced growth of HepG2 cells.Serum-starved HepG2 cells were treated for 24 hours with ethanol (20 mM) alone or in combination with SL0101 (p90rsk inhibitor) at each concentration. Cell viability was assessed with MTT assay. Data are expressed as means±S.E of three experiments (student's t-test; *p<0.05 vs. normal group, #p<0.05 vs. ethanol alone).

• 

  Fig. 6 Activation of NHE1 in HepG2 cell lines after the treatment with ethanol (10~50 mM) for 24 hours.A, serum-starved HepG2 cells were treated with ethanol for 24 hours at the indicated concentration. Identical amounts of lysate proteins were subjected to 7.5% SDS-PAGE and immunoblotted (IB) with anti-NHE1 antibody. β–actin content within lystates is shown as an loading control. B, the graph represents fold expression of NHE1 relative to β–actin averaged from four independent experiments. Data are expressed as means±S.E of four experiments (student's t-test; *p<0.05 vs. control).

• 

  Fig. 7 The effect of inhibitor of p90rsk on the expression of NHE1 in HepG2 cell lines after the treatment with ethanol (20 mM) for 24 hours.A, Serum-starved HepG2 cells were treated for 24 hours with ethanol (20 mM) alone or in combination with SL0101 (p90rsk inhibitor) at each concentration. Identical amounts of lysate proteins were subjected to 7.5% SDS-PAGE and immunoblotted (IB) with anti-NHE1 antibody. β–actin content within lystates is shown as an loading control. B, the graph represents fold expression of NHE1 relative to β–actin averaged from three independent experiments. Data are expressed as means±S.E of four experiments (student's t-test; *p<0.05 vs. control, #p<0.05 vs. ethanol alone).

• 

  Fig. 8 The effect of inhibitor of p90rsk and NHE1 on the expression of Bcl-2 in HepG2 cell lines after the treatment with ethanol (20 mM) for 24 hours.HepG2 cells were treated with 20 mM of ethanol alone or in combination of p90rsk or NHE1 inhibitor for 24 hours. Total RNA was extracted and subjected to quantitative RT-PCR (qRT-PCR) for Bcl-2 mRNA. Data are expressed as means±S.E of three experiments (student's t-test; *p<0.05 vs. control, #p<0.05 vs. ethanol alone).

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