Regulation of TGF beta sub(1)-mediated Collagen Formation by LOX-1: Studies Based on Forced Overexpression of TGF beta sub(1) in Wild-Type and Lox-1 Knock-Out Mouse Cardiac Fibroblasts

Transforming growth factor beta sub(1) (TGF beta sub(1)) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGF beta sub(1)-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGF beta sub(1) was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGF beta sub(1) moiety (AAV/TGF beta super(ACT) sub(1)). Transfection of WT mouse cardiac fibroblasts with AAV/TGF beta super(ACT) sub(1) markedly enhanced the expression of NADPH oxidases (p22 super(phox), p47 super(phox), and gp91 super(phox) subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGF beta sub(1)-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGF beta sub(1)-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGF beta sub(1)-reactive oxygen species-LOX-1 cascade.