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Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

ABSTRACT: Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A 1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.

SUBMITTER: Nunes P

PROVIDER: S-EPMC3627670 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

Nunes Paula P Ernandez Thomas T Roth Isabelle I Qiao Xiaomu X Strebel Déborah D Bouley Richard R Charollais Anne A Ramadori Pierluigi P Foti Michelangelo M Meda Paolo P Féraille Eric E Brown Dennis D Hasler Udo U

Autophagy 20130204 4

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and P ...[more]

PMID: 23380587

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Project description:Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Project description:Autophagy is reported to be involved in the formation of skin hypertrophic scar (HTS). However, the role of autophagy in the process of fibrosis remains unclear, therefore an improved understanding of the molecular mechanisms associated with autophagy may accelerate the development of effective therapeutic strategies against HTS. The present study evaluated the roles of autophagy mediated by transcription factor EB (TFEB), a pivotal regulator of lysosome biogenesis and autophagy, in transforming growth factor‑β1 (TGF‑β1)‑induced fibroblast differentiation and collagen production. Fibroblasts were treated with TGF‑β1, TGF‑β1 + tauroursodeoxycholic acid (TUDCA) or TGF‑β1 + TFEB‑small interfering RNA (siRNA). TGF‑β1 induced phenotypic transformation of fibroblasts, as well as collagen synthesis and secretion in fibroblasts in a dose‑dependent manner. Western blotting and immunofluorescence analyses demonstrated that TGF‑β1 upregulated the expression of autophagy‑related proteins through the endoplasmic reticulum (ER) stress pathway, whereas TUDCA reversed TGF‑β1‑induced changes. Reverse transcription‑quantitative PCR (RT‑qPCR), western blotting and RFP‑GFP‑LC3 double fluorescence analyses demonstrated that knockdown of TFEB by TFEB‑siRNA decreased autophagic flux, upregulated the expression of proteins involved in the apoptotic pathway, such as phosphorylated‑α subunit of eukaryotic initiation factor 2, C/EBP homologous protein and cysteinyl aspartate specific proteinase 3, and also downregulated the expression of α‑smooth muscle actin and collagen I (COL I) in fibroblasts. Immunofluorescence confocal analyses and enzyme‑linked immunosorbent assay indicated that TGF‑β1 increased the colocalization of COL I with lysosomal‑associated membrane protein 1 and Ras‑related protein Rab‑8A, a marker of secretory vesicles, in fibroblasts, as well as the secretion of pro‑COL Iα1 in culture supernatants. Meanwhile, these effects were abolished by TFEB knockdown. The present results suggested that autophagy reduced ER stress, decreased cell apoptosis and maintained fibroblast activation not only through degradation of misfolded or unfolded proteins, but also through promotion of COL I release from the autolysosome to the extracellular environment.

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Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

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Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.

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