Induction of fibrosis-related genes in TK-173 fibroblasts by nanomolar levels of tacrolimus.

<p>Exposure to increasing concentrations of tacrolimus (0–1000 nM) induced mRNA expression in a concentration-dependent manner after three days for Nox4 (A), transgelin (B), tropomyosin-1 (C), procollagen α1(V) (D), and transforming growth factor β-1 (E). a-smooth muscle actin mRNA expression (F) was not affected. RT-qPCR results were standardized to 18S rRNA, linearized, and normalized to the control group (without tacrolimus). (*) indicates significance from untreated cells p≤0,05.</p

SMAD2 phosphorylation, and NOX4 expression and activity.

<p>Basal SMAD2 phosphorylation in untreated cells (Ctrl) is increased after 1 h, 2,5 h, and 5 h exposure to 100 nM tacrolimus (Tac). Preincubation with the TGF-β1-RI kinase inhibitors LY364947, or SB-431542 before exposure to tacrolimus (LY+Tac, and SB+Tac, resp.) ablates phosphorylation of SMAD2. 10 ng/ml TGF-β1 (TGF) served as a positive control. pSMAD2 band appeared at approx. 55 kD (A). Western blot for NOX4 protein in TK-173 fibroblasts treated over three days with 100 nM tacrolimus (Tac), 10 ng/ml TGF-β1 (TGF), and untreated control cells (Ctrl). A specific Nox4 band at 60 kD is present after stimulation of the cells with tacrolimus or TGF-β1, but undetectable in control cells (B). Intracellular H₂O₂ levels in TK-173 cells, expressed as relative DCF (dichlorofluorescin) fluorescence, were increased by tacrolimus in a concentration-dependent manner, following a sigmoid curve (C). Co-application of the TGF-β1-RI kinase inhibitor LY364947 significantly reduced intracellular H₂O₂ concentrations to control levels in cells treated with 100 nM tacrolimus (TAC), or 10 ng/ml TGF-β1 (D). Results were normalized to untreated control cells.</p

Time course of mRNA expression in TK-173 cells cells treated with 100 nM tacrolimus.

<p>Expression of mRNA for NAD(P)H-oxidase 4 (A), transgelin (B), procollagen α1(V) (C), and alpha-smooth muscle actin (D) in untreated cells (open columns), and cells treated with 100 nM tacrolimus (filled). Cells were switched to serum-free medium on day -1, and were then exposed to 100 nM tacrolimus starting at day 0. Cell samples were collected from day 0 to day 5. All results were normalized to day 0. Cells showed a significant response to tacrolimus after one day (Nox4 and transgelin) or two days (tropomyosin-1), respectively. Alpha-smooth muscle actin mRNA showed a slow up-regulation in both treated and untreated cells, most likely as a reaction to serum deprivation. (*) denotes significant (p≤0,05) difference between tacrolimus-treated and control cells at the same timepoint.</p

Effect of siRNA-mediated Nox-4 knock-down in human TK-173 fibroblasts.

<p>Cells were transfected with Nox4-targeted siRNA (siNOX4), or non-target siRNA (si(-)) as a control, resp., and exposed to 100 nM tacrolimus, or control conditions, for three days. Transfection with Nox4-targeted siRNA reduced Nox4 mRNA expression by 61% (untreated control), and 64% (100 nM tacrolimus), resp., compared to non-target siRNA transfected cells (A). Nox4 knock-down did not have significant effects on transgelin (B), tropomyosin-1 (C), and alpha-smooth muscle actin (D) mRNA expression, but induced a significant up-regulation of mRNA for procollagen α1(V) in untreated cells, and a down-regulation to control levels in tacrolimus-treated cells (E). Results were normalized to control-transfected, untreated cells. (*) denotes significant (p≤0,05) differences between control-transfected and siRNA-transfected cells.</p

Effects of TGF-β1 signaling blockade.

<p>TK-173 cells exposed to 100 nM tacrolimus (TAC), or 10 ng/ml TGF-β1 (TGF) for three days showed increased expression of Nox4 (A), transgelin (B), and tropomyosin-1 (C). Blockade of TGF signaling by TGF-β1 RI inhibitor LY364947 inhibited both the reactions to TGF-β1 and tacrolimus [p≤0,05 (*), and p≤0,001 (**), resp.]. In contrast, application of anti-TGF-β antibody to the medium left the reaction to tacrolimus unaffected (E), but prevented the effects of 5 ng/ml TGF-β1 on the expression of procollagen α1(V) (Col5a1), Nox4, transgelin (Tagln), and tropomyosin-1 (Tmp1) almost completely (D). Antibodies had no effect in unstimulated control cultures (F). All values were normalized to untreated control cultures.</p

Comparison of the immunosuppressants cyclosporine A, tacrolimus, and rapamycin.

<p>Both tacrolimus (TAC; 100 nM) and rapamycin (RAPA, 100 nM) induced mRNA for Nox4, transgelin, and tropomyosin-1 after three days, although with a slightly different pattern. Cyclosporine A (CSA; 1 µM) had no effect on mRNA levels. All values normalized to untreated control. (*) denotes significance from untreated cells p≤0,05.</p