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. Author manuscript; available in PMC: 2010 Oct 5.
Published in final edited form as: Gastroenterology. 2010 Jun 1;139(4):1375–1384.e4. doi: 10.1053/j.gastro.2010.05.074

Figure 2. NOX2 activation in HSC results in superoxide production and upregulation of collagen I mRNA by inducing its transcriptional activation.

Figure 2

(A) Primary rat HSC were treated with either scrambled siRNA or NOX2 siRNA and exposed to AB. After 6 hours superoxide production was assessed by the lucigenin assay. In the scrambled siRNA-transfected HSC, phagocytosis induced superoxide production, and this was inhibited in the NOX2 siRNA-transfected cells (NT: non-transfected, C: control, not exposed to AB, *p<0.05, average of 4 experiments, bars indicate 1 SED, shown in fold expression). (B) Collagen IA1 expression was assessed by real-time PCR. Collagen IA1 expression was upregulated in the scrambled siRNA-transfected cells after AB exposure (shown in fold expression, **p≤0.01), and it decreased significantly in the NOX2 siRNA-transfected primary HSC (##p≤0.0001).

(C) Primary wild type or NOX2-/- HSC were transfected by constructs containing the truncated collagen promoter Col1A2 P1-Luc (-378/+58) or with a construct where the peroxide-responsive area is intact [Col1A2 P1-Luc (-2900/+58)], or the empty vector. The cells were exposed to AB in the presence or absence of the reducing agent GSH or catalase. Phagocytosis resulted in a significant induction of the COLIA2 promoter activity in the Col1A2 P1-Luc (-2900/+58)-transfected cells. This was abrogated in the Col1A2 P1-Luc (-378/+58)-transfected cells, or decreased after exposure to catalase, or GSH. In wt cells the collagen promoter activity thus resulted from NOX2-mediated peroxide production following phagocytosis. In the NOX2-/- cells the luciferase activity was significantly blunted after exposure to AB (C: control cells, AB: apoptotic bodies, cata:catalase, GSH: glutathione *p<0.05, **p≤0.01).