title: Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress.
abstract: A number of recent reports have used secreted protein-based reporter assays for monitoring intercellular and intracellular events involved in inflammation. However, we found that these assay systems are critically affected by endoplasmic reticulum (ER) stress. When reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of NF-kappaB were exposed to IL-1beta or TNF-alpha, induction of SEAP activity was markedly reduced under ER stress conditions. Downregulation of SEAP activity was observed regardless of cell types and type of regulatory elements; e.g., when reporter hepatocytes that express SEAP under the control of the dioxin responsive elements were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene, ER stress similarly suppressed the increase in SEAP activity despite its transcriptional upregulation. Activity of constitutively expressed SEAP in various cells was also reduced by ER stress in a magnitude-dependent manner, and it was associated with disturbed subcellular transport of SEAP to the Golgi. Furthermore, interference by ER stress was similarly observed in other reporter assay using secreted luciferase. These results evidenced critical interference by ER stress in secreted protein-based reporter systems. The suppression of reporter responses by ER stress should be considered carefully for experimental design and interpretation of data when secreted protein-based reporter systems are used for investigation.
authors.affiliation: Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Hiramatsu
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kasai
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Hayakawa
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nagai
authors.forename: Takeo
authors.initials: T
authors.lastname: Kubota
authors.forename: Jian
authors.initials: J
authors.lastname: Yao
authors.forename: Masanori
authors.initials: M
authors.lastname: Kitamura
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter; D007249:Inflammation / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D000469:Alkaline Phosphatase