Up-regulation of the expression of leucine-rich alpha(2)-glycoprotein in hepatocytes by the mediators of acute-phase response

Abstract

Leucine-rich alpha(2)-glycoprotein (LRG) is a plasma protein in which leucine-rich repeats (LRRs) were first discovered. Although the physiological function of LRG is not known, increases in the serum level of LRG have been reported in various diseases. In this study, we found that LRG was induced by recombinant human IL-6 in human hepatoma HepG2 cells. The induction of LRG by IL-6 was up-regulated synergistically with either IL-1beta or TNFalpha in a pattern similar to those for type 1 acute-phase proteins. We also found that lipopolysaccharide (LPS) administered intraperitoneally to mice enhanced dose-dependently the expression of LRG mRNA in the liver as well as those for mouse major acute-phase proteins. These results strongly suggest that LRG was a secretory type 1 acute-phase protein whose expression was up-regulated by the mediator of acute-phase response.

Fig. 1

Time course study on the LRG mRNA expressions in HepG2 cells stimulated with proinflammatory cytokines. HepG2 cells were stimulated with (A) none, (B) 10 ng/ml IL-1β alone, (C) 10 ng/ml TNFα alone, (D) 10 ng/ml IL-6 alone, (E) 10 ng/ml IL-6 and 10 ng/ml IL-1β, or (F) 10 ng/ml IL-6 and 10 ng/ml TNFα. The expressions of LRG and GAPDH mRNA were measured by real-time quantitative RT-RCR at 0, 2, 4, 6, 12, 24, and 48 h after the cytokine stimulation. The expression levels of LRG mRNA were corrected with that of GAPDH mRNA. Values are means ± SD of two independent determinations.

Fig. 2

Synergistic effects of IL-1β and TNFα on LRG induction by IL-6 in HepG2 cells. (A) Western blot analysis of the culture media of HepG2 cells at 48 h after stimulation with 10 ng/ml IL-6 alone, 10 ng/ml IL-6 and 10 ng/ml IL-1β, and 10 ng/ml IL-6 and 10 ng/ml TNFα. (B) Comparison of the expressions of LRG, SAA, and PLA₂ mRNAs in HepG2 cells at 4 h after stimulations with 10 ng/ml IL-6 alone, 10 ng/ml IL-6 and 10 ng/ml IL-1β, and 10 ng/ml IL-6 and 10 ng/ml TNFα. The amounts of the respective mRNAs were measured by real-time quantitative RT-RCR. The relative expression levels of each mRNA were corrected with that of GAPDH mRNA and then compared with that for an unstimulated control sample. Values are means ± SD of two independent determinations.

Fig. 3

The expressions of (A) LRG, (B) SAA, and (C) SAP mRNAs in the liver of mouse at 6 h after intraperitoneal injection of various concentrations of LPS. The amounts of the respective mRNAs were measured by real-time quantitative RT-PCR. The relative expression levels of each mRNA were corrected with that of GAPDH mRNA. Horizontal bars represent the mean ± SEM.