leupeptins and Scleroderma--Systemic

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Topics: Animals; Bleomycin; Boronic Acids; Bortezomib; Cells, Cultured; Collagen Type I; Fibrosis; Hydroxyproline; Leupeptins; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Pyrazines; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Tissue fibrosis results when dysregulation of extracellular matrix (ECM) turnover favors deposition of collagen and other ECM proteins over degradation. Fibrosis may then lead to organ dysfunction and pathology as observed in systemic sclerosis (SSc). In the present study, we investigated the antifibrotic properties of proteasome blockade. A dose- and time-dependent reduction in type-I collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) production was observed in normal fibroblasts exposed to proteasome inhibitors (PI). In the same culture conditions, metalloproteinase-1 (MMP-1) protein and the collagenolytic activity on type I collagen was increased. The steady-state mRNA levels of COL1A1, TIMP-1, and MMP-1 paralleled protein levels. These effects were dominant over the profibrotic properties of TGF-beta and were observed with fibroblasts generated from normal and SSc skin. PI decreased type I collagen mRNA levels with kinetics similar to those observed with DRB, a specific RNA polymerase II inhibitor, thus indicating transcriptional inhibition. Of interest, PI induced c-Jun phosphorylation and c-Jun nuclear accumulation. The specific N-terminal Jun-kinase inhibitor SP-600125 selectively abrogated c-Jun phosphorylation and, in a dose-dependent fashion, the up-regulated synthesis of MMP-1 induced by PI. Finally, PI did not affect fibroblast viability. Thus, the coordinated down-regulation of collagen and TIMP-1 and up-regulation of MMP-1 renders proteasome blockade an attractive strategy for treating conditions as SSc, characterized by excessive fibrosis.

Topics: Acetylcysteine; Anthracenes; Boronic Acids; Bortezomib; Collagen Type I; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Matrix; Fibroblasts; Fibrosis; Genes, jun; Humans; JNK Mitogen-Activated Protein Kinases; Leupeptins; Matrix Metalloproteinase 1; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-jun; Pyrazines; RNA Polymerase II; RNA, Messenger; Scleroderma, Systemic; Skin; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Up-Regulation