Document resource
Introduction Fibrosis is a major source of morbidity and mortality, yet progress towards an effective treatment has been frustrated by the redundancy and pleiotropy inherent in the signalling pathways involved. Recently, it has emerged that scar formation is an energy- and amino acid-intensive process that requires substantial metabolic reprogramming in fibroblasts. We have shown that this reprogramming is mediated by a combination of inflammatory and growth factor signalling. Growth factors, such as TGFbeta, promote activation of the PI3K-AKT-mTOR axis to activate protein synthesis via 4EBP1 and S6K. Previously, we found that the ATM kinase is the major PI3K downstream of TGFbeta. Here, we also report a link between inflammatory signalling and metabolism via the PIM kinase, a downstream STAT effector.Material and Methods Primary human lung fibroblasts were cultured in DMEM with 10% FBS and serum-starved for 20 h prior to any treatment. For western-blotting and qPCR, the cells were treated with TGFbeta, the PIM inhibitor AZD1208 and/or the ATM inhibitor KU60019 for 20 h. Glycolysis and mitochondrial respiration were assessed with a Seahorse extracellular flux analyzer. For the bleomycin model, mice were dosed with AZD1208 via oral gavage, 5 days a week for 3 weeks starting 1 day before the bleomycin challenge (prophylactic) or for 2 weeks starting at 7 days after bleomycin challenge (therapeutic). For the wound healing assay, the mice were wounded on both shoulders at day zero. KU60019 was administered by peritoneal injection every other day between day 2 and day 12.Results We show that TGFbeta-activated fibroblasts have a highly active energy metabolism, indicating operation under intense ATP demand. SSc patient-derived fibroblasts show a significantly higher mitochondrial respiration coupled to ATP production compared to healthy control cells (n=4, P=0.0296). PIM and ATM inhibitors decrease energy metabolism individually but are more potent when combined (n=4, P=0.0201). PIM and ATM also exhibit an inhibitory effect on 4EBP1 and S6, while downregulating key amino acid biosynthetic enzymes. We show that PIM inhibition can ameliorate experimental fibrosis in the bleomycin mouse model, while ATM inhibition delays wound healing in the skin-punch biopsy model in mice.Conclusions We propose that PIM and ATM act cooperatively to maintain high levels of amino acid and energy production, and both must be blocked to starve fibroblasts of the raw materials and energy needed to continue to lay down and expand fibrotic tissue. Combined PIM and ATM inhibition may be a novel way to slow or stop fibrotic disease progression.