Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Abstract

, tandem fluorescent microtubule-associated protein 1 light chain 3 A/B (green/red fluorescent proteins chimera); TNFRSF1A/TNFR1, tumor necrosis factor receptor superfamily member 1A; TNFRSF1B/TNFR2, tumor necrosis factor receptor superfamily member 1B.Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other ironbinding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

Autophagy Improves the Immunosuppression of CD4+ T Cells by Mesenchymal Stem Cells Through Transforming Growth Factor-β1

“…Autophagy has long been recognized as an important intracellular process used to degrade cellular contents through lysosomes [31]. Previous studies have demonstrated that autophagy has an intimate connection with immunity [32].…”

Reappraisal of metallothionein: Clinical implications for patients with diabetes mellitus

“…Whereas OS impairs autophagy, MT helps maintain it. Lysosomal delivery of MT protects cells against apoptosis by modulation of autophagy . Metallothionein has been reported to inhibit biological membrane lipid peroxidation injury and to improve recovery after transient myocardial ischemia–reperfusion‐induced injuries …”

“…Lysosomal delivery of MT protects cells against apoptosis by modulation of autophagy. 145 Metallothionein has been reported to inhibit biological membrane lipid peroxidation injury and to improve recovery after transient myocardial ischemia-reperfusion-induced injuries. 146 Extracellular MT and anti-inflammatory and immune function Extracellular MTs have been confirmed and their functions are gradually being determined, [31][32][33][37][38][39][40] including immune regulation 36 and neuronal protection.…”

Metallothionein 1 family profiling identifies MT1X as a tumor suppressor involved in the progression and metastastatic capacity of hepatocellular carcinoma

“…In humans, MTs are encoded by 17 genes, with four defined subtypes (MT1‐MT4). MTs are increased during oxidative stress states in order to protect cells against cytotoxicity, radiation, and DNA damage . MT1 family (MT1s) is comprised of eight functional members: MT1A, MT1B, MT1E‐H, MT1M, and MT1X …”

“…MT1s are involved in diverse functions, such as metal homeostasis, metal donation to different enzymes, angiogenesis, apoptosis, cell differentiation, radiation cell damage as well as carcinogenesis . Given their broad regulatory properties, MT1s have also been suggested to play important roles in cell senescence and various pathological processes, such as neurodegeneration, osteoarthritis, metabolic disorders, and certain immune processes .…”