leupeptins and Pulmonary-Fibrosis

Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, plays an important anti-tumor role and is involved in various other biological processes, such as, neuroprotection by prevention of aggregation of misfolded proteins generated because of genetic defects. Surfactant protein A2 mutations (G231V and F198S) have been identified to be associated with pulmonary fibrosis and lung cancer, and these mutations cause protein aggregation, instability as well as secretion deficiency. The present study focused on investigating the inhibitory effects of EGCG on aggregation of mutant SP-A2 and elucidating the potential mechanisms underlying this action.. Wild-type and mutant SP-A2 were transiently expressed in CHO-K1 cells. The aggregated and soluble proteins were separated into NP-40-insoluble and NP-40-soluble fractions. Protein stability was validated by chymotrypsin limited proteolysis assay. Western blot and RT-PCR were used to determine the protein and mRNA expression level, respectively.. Mutant SP-A2 alone or wild-type SP-A2 co-expressed with G231V formed NP-40-insoluble aggregates in CHO-K1 cells. EGCG significantly suppressed this aggregation and alleviated mutant SP-A2 accumulation in the ER. When combined with 4-PBA, EGCG treatment completely blocked mutant SP-A2 aggregate formation. Though secretion of mutant protein was not affected, EGCG facilitated protein instability in both wild-type and mutant protein. Importantly, MG132, a proteasome inhibitor, reversed EGCG-induced aggregate reduction.. EGCG inhibits aggregation of misfolded SP-A2 via induction of protein instability and activation of proteasomal pathway for aggregate degradation.

Topics: Animals; Butylamines; Catechin; CHO Cells; Cricetulus; Cysteine Proteinase Inhibitors; Detergents; Gene Expression; Leupeptins; Mutation; Octoxynol; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Stability; Proteolysis; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein A; Recombinant Proteins; Solubility

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.

Topics: A549 Cells; Animals; CHO Cells; Cricetulus; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; Mutation; Phenylbutyrates; Protein Aggregates; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein A

Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

Topics: Amino Acid Substitution; Animals; Biomarkers; Bronchoalveolar Lavage Fluid; Cell Extracts; Cell Line, Tumor; Detergents; Dogs; Endoplasmic Reticulum; Female; Humans; Leupeptins; Male; Mutant Proteins; Mutation; Pedigree; Protein Processing, Post-Translational; Protein Stability; Protein Structure, Quaternary; Pulmonary Alveoli; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein A; Solubility; Stress, Physiological

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

To investigate the role of iron and active oxygen species (AOS) in asbestos-induced fibrosis, we loaded increasing amounts of Fe(II)/Fe(III) onto the surface of amosite asbestos fibers and then applied the fibers to rat tracheal explants. Explants were harvested after 7 d in air organ culture. Asbestos by itself doubled procollagen gene expression, and a further increase was seen with increasing iron loading; actual collagen content measured as hydroxyproline was increased in a similar pattern. Iron loading also increased gene expression of platelet-derived growth factor (PDGF)-A and transforming growth factor (TGF)-beta(1). Neither asbestos alone nor iron-loaded asbestos affected gene expression of PDGF-B, tumor necrosis factor-alpha, or TGF-alpha. The AOS scavenger tetramethylthiourea or treatment of fibers with the iron chelator deferoxamine prevented asbestos-induced increases in procollagen, PDGF-A, and TGF-beta gene expression, whereas glutathione had no effect. The proteasome inhibitor MG-132 abolished asbestos-induced increases in procollagen gene expression but did not affect increases in PDGF-A or TGF-beta(1) expression, whereas the extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 had exactly the opposite effect. We conclude that surface iron as well as the iron-catalyzed generation of AOS play a role in asbestos-induced matrix (procollagen) production and that this process is driven in part through oxidant-induced nuclear factor kappa B activation. Surface iron and AOS also play a role in PDGF-A and TGF-beta gene expression, but through an ERK-dependent mechanism.

Topics: Animals; Asbestos, Amosite; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Flavonoids; Gene Expression; Iron; Leupeptins; Male; MAP Kinase Signaling System; NF-kappa B; Platelet-Derived Growth Factor; Procollagen; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Trachea; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha