Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species

Abstract

Tumour-stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGFβ (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGFβ1, is involved in the fibroblast-to-MF switch. The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGFβ1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a 'stromal therapy' which was described earlier.

Keywords: MAPK; NAD(P)H oxidase; TGFβ1; myofibroblast; reactive oxygen species; tumour–stroma interaction.

Figure 1. TGFβ1-mediated transition of fibroblasts to MFs

Subconfluent HDF were either mock-treated (CM^HDF), treated with rTGFβ1 (10 ng/ml) for 48 h (CM^HDF,TGF) and in CM of squamous carcinoma cells SCL-1 (CM^SCL−1). (A) The amount of αSMA protein was immunostained for αSMA and (B) determined by Western blot analysis. The densitometric values represent the fold increase over control, which was set at 1.0. The data represent means±S.E.M. of three independent experiments. CM, conditioned medium.

Figure 2. TGFβ1-mediated expression of αSMA

(A) Subconfluent HDFs were either mock-treated or pretreated for 24 h with allopurinol (10 μM) before addition of rTGFβ1 (10 ng/ml). TGFβ1 and the allopurinol were present for an additional 48 h. The level of αSMA protein was determined by Western blot. α-tubulin was used as loading control. Three independent experiments were performed. (B) HDF monolayer cultures were cultured in CM^HDF containing apocynin (1 mM) for 1 h or DPI (5 μM) for 24 h before treatment with TGFβ1 (10 ng/ml) for further 48 h. The level of αSMA protein was determined by Western blot. α-tubulin was used as loading control. The experiments were performed in triplicate. (C) Subconfluent HDF were preincubated for 1 h with apocynin (1 mM) in serum-free medium and then TGFβ1 (10 ng/ml) treated for 24 h. Steady-state mRNA levels of αSMA were analysed by real-time RT-PCR. Data are given as means of three independent experiments±S.E.M.

Figure 3. Apocynin inhibits the ROS production and the oxidative damage in HDF

(A) Subconfluent HDFs were preincubated with apocynin (1 mM) for 1 h (closed circles) before treatment with rTGFβ1 or H₂O₂ (1 mM) for the indicated time. Increase of DCF fluorescence was followed over 20 min versus mock-treated controls (open circle). The experiments were performed in triplicate. Arrows indicate addition of rTGFβ1 or H₂O₂.(B) H₂O₂was detected by amperometric determination. The data represent the mean±S.E.M. of three independent experiments. (C) HDF cells were exposed to rTGFβ1 for 24 h or preincubated with apocynin (1 mM) before oxidized proteins were detected by Western blot analysis via derivatization with DNP hydrazine. H₂O₂ was used as positive control. Three independent experiments were performed.

Figure 4. rTGFβ1 activates the NADH oxidase in dermal fibroblasts

(A) Rates of NADH consumption by ct and time course of the rates from HDF following rTGFβ1 (10 ng/ml) treatment. TPA was used as a positive control. In presence of 250 μM NADH, subconfluent HDF were either mock-treated or treated with rTGFβ1. The consumption of NADH was measured spectrophotometrically. data represent the mean±S.E.M. (B) Subconfluent HDF were preincubated for 1 h with apocynin (1 mM) in the serum-free medium and then rTGFβ1 (10 ng/ml) treated for various time points. p67^phox and NOX4 mRNA expression were analysed by RT–PCR. HPRT1 was used as housekeeping gene. Three independent experiments were performed. (C) Subconfluent HDFs were either mock-treated, treated with rTGFβ1 (10 ng/ml) for 48 h or incubated with apocynin for 1 h or starting 4, 8 and 16 h after rTGFβ1 treatment. The level of αSMA protein was determined by Western blot. Coomassie Brilliant blue staining was used as loading control. Three independent experiments were performed.

Figure 5. Involvement of p38 kinase in TGFβ1/ROS-dependent expression of αSMA

(A) Subconfluent HDFs were preincubated with MAPK inhibitors U0126, SP600125 or SB202190 before treatment with rTGFβ1. Expression of αSMA was detected by Western blots. The densitometric analysis describes protein expression as fold increase over control, which was set at 1.0. The data represent the mean±S.E.M. of three independent experiments. (B) Subconfluent HDF were preincubated for 1 h with SB202190 (10 μM) in the serum-free medium and then rTGFβ1 (10 ng/ml) treated for 24 h. αSMA mRNA levels were analysed by real-time RT-PCR. Data are given as means of three independent experiments±S.E.M. (C) Subconfluent HDFs were either mock-treated, treated with rTGFβ1 (10 ng/ml) for 48 h or incubated with SB 202190 for 48 h or starting 4, 8 and 16 h after rTGFβ1 treatment. The level of αSMA protein was determined by Western blot. α-tubulin was used as loading control. Three independent experiments were performed. (D) Subconfluent HDFs were either mock-treated or pretreated for 1 h with apocynin (1 mM) before addition of rTGFβ1 (10 ng/ml). TGFβ1 and apocynin were present for an additional 12 h. Anisomycin (0.5 μg/ml) was used as technical control and incubated for 20 min. The level of phospho-p38 MAPK protein was determined by Western blot. α-tubulin was used as loading control. Two independent experiments were performed.

Figure 6. Scheme of TGFβ1-mediated signalling

Tumour cells release growth factors, e.g. TGFβ1, which generates ROS due to NAD(P)H oxidase, which is responsible for the downstream signalling resulting in ROS-triggered activation of the stress kinase p38 and expression of αSMA. Both can be inhibited by the specific NAD(P)H oxidase inhibitor apocynin.