Changes in the expression of methionine adenosyltransferase genes and S-adenosylmethionine homeostasis during hepatic stellate cell activation

Abstract

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Methionine adenosyltransferase (MAT) catalyzes biosynthesis of S-adenosylmethionine (SAMe), the principle methyl donor. SAMe metabolism generates two methylation inhibitors, methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). Liver cell proliferation is associated with induction of two nonliver-specific MATs: MAT2A, which encodes the catalytic subunit alpha2, and MAT2beta, which encodes a regulatory subunit beta that modulates the activity of the MAT2A-encoded isoenzyme MATII. We reported that MAT2A and MAT2beta genes are required for liver cancer cell growth that is induced by the profibrogenic factor leptin. Also, MAT2beta regulates leptin signaling. The strong association of MAT genes with proliferation and leptin signaling in liver cells led us to examine the role of these genes during HSC activation. MAT2A and MAT2beta are induced in culture-activated primary rat HSCs and HSCs from 10-day bile duct ligated (BDL) rat livers. HSC activation led to a decline in intracellular SAMe and MTA levels, a drop in the SAMe/SAH ratio, and global DNA hypomethylation. The decrease in SAMe levels was associated with lower MATII activity during activation. MAT2A silencing in primary HSCs and MAT2A or MAT2beta silencing in the human stellate cell line LX-2 resulted in decreased collagen and alpha-smooth muscle actin (alpha-SMA) expression and cell growth and increased apoptosis. MAT2A knockdown decreased intracellular SAMe levels in LX-2 cells. Activation of extracellular signal-regulated kinase and phosphatidylinositol-3-kinase signaling in LX-2 cells required the expression of MAT2beta but not that of MAT2A.

Conclusion: MAT2A and MAT2beta genes are induced during HSC activation and are essential for this process. The SAMe level falls, resulting in global DNA hypomethylation.

Fig. 1. Expression of MAT2A and MAT2β genes is induced during in vitro HSC activation

Quiescent HSCs were isolated from rat liver as described in Methods. HSCs (1x10⁶ cells) were cultured on 6 cm plastic dishes and allowed to activate till day 7. (A) RNA was isolated from HSCs at different days in culture and the expression of MAT2A, MAT2β , Col1A2 and α-SMA mRNA was assessed by real-time RT-PCR. Results represent Mean±SE from four HSC preparations; *p<0.05, †p<0.005 vs. day 1. (B) Total cellular protein was extracted from HSCs as described in Methods and subjected to Western blotting for detection of MAT2A, MAT2β , type I collagen and α-SMA. Representative images and densitometric analysis (Mean±SE) from 3 to 4 HSC preparations is shown; *p<0.005, †p<0.05 vs. day 1.

Fig. 2. Expression of MAT2A and MAT2β genes is induced during in vivo HSC activation

Rats were subjected to BDL surgery or sham operation for a period of 10 days. HSCs were isolated from BDL or sham control rat livers and 1x10⁶ cells were plated on 6 cm plastic dishes for 16 hours. (A) RNA was isolated from BDL or sham HSCs and the expression of MAT2A, MAT2β and Col1A2 mRNA was assessed by real-time RT-PCR. Results represent Mean±SE from four BDL or four sham HSC preparations; *p<0.005 vs. sham control. (B) Total cellular protein was extracted from BDL or sham HSCs as described in Methods and subjected to Western blotting for detection of MAT2A, MAT2β and type I collagen. Representative images and densitometric analysis (Mean±SE) from four BDL or sham preparations is shown; *p<0.05, †p<0.005 vs. sham control.

Fig. 3. Effect of MAT2A gene silencing on HSC activation and proliferation

Knockdown of MAT2A gene in primary rat HSCs was performed as described in Methods. (A) Total RNA from MAT2A knockdown HSCs was subjected to real-time PCR and expression of MAT2A, Col1A2 and α-SMA was compared to control and scrambled RNAi. Results represent Mean±SE from five HSC preparations; *p<0.005, †p<0.05 vs. scrambled RNAi. (B) Total cellular protein from MAT2A knockdown HSCs was subjected to Western blotting for detection of MAT2A, type I collagen and α-SMA and compared to control and scrambled RNAi. Representative images and densitometric analysis (Mean±SE) from four HSC preparations is shown; *p<0.005, †p<0.05 vs. scrambled RNAi. (C) Knockdown of MAT2A was done for 48 or 80 hours and BrDU incorporation in RNAi-treated cells was compared to control or scrambled RNAi. Mean±SE of two HSC preparations in quadruplets is shown; *p<0.005 vs. scrambled RNAi.

Fig. 4. Effect of MAT2A and MAT2β knockdown on activation, proliferation and apoptosis in human LX-2 cells

Knockdown of MAT2A and MAT2β genes in LX-2 cells was performed as described in Methods. (A) Total RNA isolated from MAT2A or MAT2β knockdown cells was subjected to real-time RT-PCR analysis and expression of MAT2A, MAT2β , Col1A2 and α-SMA was compared to control or scrambled RNAi. Results represent Mean±SE from three experiments in duplicates for MAT2A, MAT2β , Col1A2 and four experiments for α-SMA; **p<0.05, †p<0.005 vs. control, *p<0.005 vs. scrambled RNAi. (B) Total cellular protein from MAT2A or MAT2β knockdown cells was subjected to Western blotting for detection of MAT2A, MAT2β, type I collagen and α-SMA and compared to control and scrambled RNAi. Representative images and densitometric analysis (Mean±SE) from three experiments is shown; *p<0.05 vs. scrambled RNAi. (C) Knockdown of MAT2A or MAT2β was done for 24, 48 or 72 hours and BrDU incorporation in RNAi-treated cells was compared to control or scrambled RNAi-treated cells. Results represent Mean±SE of three experiments in duplicates; *p<0.05 vs. control, †p<0.005 vs. scrambled RNAi. (D) Apoptotic cells in control or RNAi-treated samples were detected after 24, 48 or 72 hours using Hoechst staining as described in methods. Result represent Mean±SE of two experiments in duplicates; *p<0.005 vs. control, †p<0.05, ¥p<0.005 vs. scrambled RNAi.

Fig. 5. Effect of MAT2A and MAT2β knockdown on signaling in LX-2 cells

Silencing of MAT2A and MAT2β genes in LX-2 cells was performed and phosphorylation of ERK1/2 (A) or AKT (for PI-3K signaling) (B) was checked by Western blotting as described in Methods. Subsequently the membranes were stripped and reprobed with antibodies against total ERK2 or AKT to verify protein levels. Representative images from 3 to 4 independent experiments are shown and results of densitometric analysis (Mean±SE) are shown below each blot; *p<0.005 vs. control, †p<0.005, **p<0.05 vs. scrambled RNAi.