NAC deactivaties PSC by interfering with TGFß1 induced MAPK signaling

Aims: Progressive fibrosis is a characteristic feature of chronic pancreatitis of various etiologies. Activated pancreatic stellate cells (PSC) are the effector cells of pancreatic fibrosis. The molecular mechanisms leading to PSC activation are currently under investigation. No effective treatment to inhibit pancreatic fibrosis has been identified up to now. However, recently we were able to demonstrate a deactivation of activated PSC by culturing the cells on a layer of matrigel and concomitant treatment with N-Acetylcysteine. In this study we analysed how this effect is mediated.

Methods: Immortalized human pancreatic stellate cells (RLT-PSC) were starved overnight by incubation in DMEM without FCS. Then the cells in part were preincubated with 2,5 mM N-Acetylcysteine (NAC) for 90min before they were stimulated with TGFß1 for 40min (for protein analysis) or 2 hours (for RNA analysis). Equal amounts of protein were resolved by SDS-PAGE and blotted onto NC membranes. Subsequently the blots were analysed using the following phosphospecific antibodies: p-SMAD2, pp38, p-ERK and pJNK. RNA was isolated from the cells using the RNeasy mini kit. RNA was reverse transcribed by the RevertAid Kit and amplified using intron spanning primers for genes implicated in the activation of pancreatic stellate cells.

Results: On the RNA level stimulation with TGFß1 induced the expected upregulation of the genes PAI-1, SMAD7, αSMA, Col I and TGFß1. Preincubation with NAC had no effect on the TGFß1 induced upregulation of the genes PAI-1 and SMAD7. In contrast induction of αSMA, Col I and TGFß1 expression by TGFß1 was abrogated by preincubation with NAC. On the protein level TGFß1 induced phosphorylation of all signalling proteins analysed (SMAD2, p38, JNK and ERK). Preincubation with NAC had no effect on basal phosphorylation of these proteins exept for ERK, which was phosphorylated by the preincubation. Moreover, NAC preincubation had no effect on the phosphorylation of SMAD2 and p38 induced by TGFß1. In contrast, it abrogated the phosphorylation of JNK and it further increased ERK phosphorylation induced by TGFß1.

Conclusions: We were able to demonstrate that NAC interferes with MAPK signalling induced by TGFß1, whereas signalling via the SMAD pathway was not disturbed.