title: An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex.
abstract: Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form ubiquitinated protein aggregates that suppressed 20 S proteasome function instead of being degraded by 20 S proteasome. Keratinocytes with mutant K14-induced phosphorylation of the stress-activated kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicates induction of endoplasmic reticulum stress. HaCaT cells were susceptible to apoptosis by activation of caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-alpha (TNFalpha) in the culture medium was increased in keratinocytes with mutant K14 compared with wild K14, and the addition of neutralizing anti-TNFalpha antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFalpha release and the subsequent activation of the TNFalpha receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFalpha receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 because of impairment of the cytoprotective mechanism mediated by K14-TRADD interaction.
authors.affiliation: Department of Dermatology, Akita University School of Medicine, Akita 010-8543, Japan. kyoneda@doc.med.akita-u.ac.jp
authors.forename: Kozo
authors.initials: K
authors.lastname: Yoneda
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Furukawa
authors.forename: Ya-Juan
authors.initials: YJ
authors.lastname: Zheng
authors.forename: Takashi
authors.initials: T
authors.lastname: Momoi
authors.forename: Ichiro
authors.initials: I
authors.lastname: Izawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Inagaki
authors.forename: Motomu
authors.initials: M
authors.lastname: Manabe
authors.forename: Nobuya
authors.initials: N
authors.lastname: Inagaki
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004396:Coloring Agents / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004195:Disease Models, Animal; D000091342:Endoplasmic Reticulum Chaperone BiP; D016110:Epidermolysis Bullosa Simplex / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D053547:Keratin-14; D015603:Keratinocytes / Q000378:metabolism*; D007633:Keratins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D047990:TNF Receptor-Associated Factor 1; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D017304:Annexin A5; D002352:Carrier Proteins; D004396:Coloring Agents; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C507540:KRT14 protein, human; D053547:Keratin-14; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D014409:Tumor Necrosis Factor-alpha; D007633:Keratins; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex