Dataset Information

Lysosomal adaptation: How cells respond to lysosomotropic compounds.

ABSTRACT: Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

SUBMITTER: Lu S

PROVIDER: S-EPMC5354416 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Lysosomal adaptation: How cells respond to lysosomotropic compounds.

Lu Shuyan S Sung Tae T Lin Nianwei N Abraham Robert T RT Jessen Bart A BA

PloS one 20170316 3

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and ...[more]

PMID: 28301521

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Project description:Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.

Project description:Oxidative stress and Th17 cytokines are important mediators of inflammation. Treatment with beta-adrenoceptor (ADRB) antagonists (beta-blockers) is associated with induction or aggravation of psoriasis-like skin inflammation, yet the underlying mechanisms are poorly understood. Herein, we identify lysosomotropic beta-blockers as critical inducers of IL23A in human monocyte-derived Langerhans-like cells under sterile-inflammatory conditions. Cytokine release was not mediated by cAMP, suggesting the involvement of ADRB-independent pathways. NFKB/NF-κB and MAPK14/p38 activation was required for propranolol-induced IL23A secretion whereas the NLRP3 inflammasome was dispensable. MAPK14 regulated recruitment of RELB to IL23A promoter regions. Without affecting the ubiquitin-proteasome pathway, propranolol increased lysosomal pH and induced a late-stage block in macroautophagy/autophagy. Propranolol specifically induced reactive oxygen species production, which was critical for IL23A secretion, in Langerhans-like cells. Our findings provide insight into a potentially crucial immunoregulatory mechanism in cutaneous dendritic cells that may explain how lysosomotropic drugs regulate inflammatory responses.AbbreviationsATF: activating transcription factor; DC: dendritic cell; ChIP: chromatin immunoprecipitation; gDNA: genomic DNA; IL: interleukin; LAMP1: lysosomal associated membrane protein 1; LC: Langerhans cell; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MoDC: monocyte-derived DC; MoLC: monocyte-derived Langerhans-like cell; mtDNA: mitochondrial DNA; NAC: N-acetyl-L-cysteine; NLRP3: NLR family pyrin domain containing 3; PBMC: peripheral blood mononuclear cell; PI: propidium iodide; PYCARD/ASC: PYD and CARD domain containing; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TLR: Toll-like receptor; TRAF6: TNF receptor associated factor 6; TNF: tumor necrosis factor; Ub: ubiquitin.

Dataset Information

Lysosomal adaptation: How cells respond to lysosomotropic compounds.

Publications

Lysosomal adaptation: How cells respond to lysosomotropic compounds.

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