Osna Natalia A, Clemens Dahn L, Donohue Terrence M
Liver Study Unit, Research Service, The Omaha Veterans Affairs (VA) Medical Center, University of Nebraska Medical Center, Omaha, NE 68105, USA.
Hepatology. 2005 Nov;42(5):1109-17. doi: 10.1002/hep.20909.
We previously showed that IFNgamma signal transduction was suppressed by ethanol in recombinant HepG2 cells (VL-17A cells), which express alcohol dehydrogenase (ADH) and CYP2E1. We examined the mechanisms by which STAT1 phosphorylation is blocked by ethanol treatment in VL-17A cells. Cells were exposed to 0 or 100 mmol/L ethanol for 72 hours. STAT1 phosphorylation was determined by Western blot after 1 hour IFNgamma exposure. Reduction of STAT1 phosphorylation by ethanol was prevented in the presence of 4MP, DAS, or uric acid, indicating that the oxidative products from ethanol metabolism were partly responsible for suppression of STAT1 phosphorylation. Ethanol exposure decreased STAT1 tyrosine phosphorylation, whereas serine phosphorylation on the protein was unchanged. These effects of ethanol were mimicked by the peroxynitrite (PN) donor, SIN-1, which also blocked tyrosine, but not serine phosphorylation, on STAT1. When cells expressing either ADH (VA-13 cells) or CYP2E1 (E-47 cells) were exposed to ethanol, both ADH- and CYP2E1-generated products reduced STAT1 phosphorylation. In addition, SOCS1, a negative regulator of IFNgamma signaling and which is degraded by the proteasome, was stabilized by ethanol treatment, presumably because of inhibited proteasome activity. Furthermore, SIN-1 treatment elevated SOCS1 levels in VL-17A cells, indicating that PN has a role in SOCS1 elevation. In conclusion, under conditions of ethanol-elicited oxidative stress, PN prevents STAT1 phosphorylation by stabilization of SOCS1, and possibly by nitration of tyrosine residues in STAT1 protein.
我们之前发现，在表达乙醇脱氢酶（ADH）和CYP2E1的重组HepG2细胞（VL-17A细胞）中，乙醇可抑制IFNγ信号转导。我们研究了VL-17A细胞中乙醇处理阻断STAT1磷酸化的机制。将细胞暴露于0或100 mmol/L乙醇中72小时。在暴露于IFNγ 1小时后，通过蛋白质印迹法测定STAT1磷酸化情况。在存在4MP、DAS或尿酸的情况下，乙醇对STAT1磷酸化的降低作用被阻止，这表明乙醇代谢产生的氧化产物部分导致了STAT1磷酸化的抑制。乙醇暴露降低了STAT1酪氨酸磷酸化，而该蛋白上的丝氨酸磷酸化未发生变化。过氧亚硝酸盐（PN）供体SIN-1模拟了乙醇的这些作用，它也阻断了STAT1上的酪氨酸磷酸化，但未阻断丝氨酸磷酸化。当表达ADH的细胞（VA-13细胞）或表达CYP2E1的细胞（E-47细胞）暴露于乙醇时，ADH和CYP2E1产生的产物均降低了STAT1磷酸化。此外，SOCS1是IFNγ信号的负调节因子，可被蛋白酶体降解，乙醇处理使其稳定，可能是由于蛋白酶体活性受到抑制。此外，SIN-1处理提高了VL-17A细胞中SOCS1的水平，表明PN在SOCS1升高过程中发挥作用。总之，在乙醇引发的氧化应激条件下，PN通过稳定SOCS1以及可能通过使STAT1蛋白中的酪氨酸残基硝化来阻止STAT1磷酸化。
