title: PKCδ-dependent signaling mediates ethambutol-induced toxic effects on human retinal pigment cells.
abstract: OBJECTIVE
  Our previous report demonstrated that ethambutol (EMB) might induce cytoplasmic vacuolization and reduce the uptake of photoreceptor rod outer segments (ROS) in retinal pigment epithelium (RPE) cells, which are mediated via a protein kinase C (PKC)-dependent pathway. In the present study, we sought to identify the PKC isozyme(s) involved.
  
  
  METHODS
  EMB-induced cytoplasmic vacuolization and uptake of ROS were observed under a phase contrast microscope. Western blots were performed to observe the membrane translocation of PKC isozymes and cytoplasmic release of cathepsin D. Quantitative PCR were performed to analyze gene expression of PKCδ. Human RPE cell line RPE50 and ARPE19 cells were pretreated with specific inhibitors or transfected with shRNAs of various PKC isozymes, including PKCα, β, ε, γ, and δ, to examine whether EMB-induced toxic effects were prevented.
  
  
  RESULTS
  In RPE50 cells, gene expression of PKCδ on both mRNA and protein levels was induced by EMB within 30 min to 3 h. EMB-induced cytoplasmic vacuolization in both RPE50 and ARPE19 cells was prevented by pretreating the cells with a specific inhibitor of PKCδ, Rottlerin, or depletion of PKCδ by shRNA. EMB-triggered reduction of ROS uptake was also significantly suppressed by pretreatment with Rottlerin, or depletion of PKCδ by shRNA technology. In contrast, pretreatment of the cells with specific inhibitors of PKCα, β, ε, or γ, or depletion of PKCα or β didn't influence the aforementioned EMB-triggered toxic effects. In addition, in RPE50, EMB induced the release of lysosomal enzyme cathepsin D into cytosol within 30 min to 6 h, which was also prevented by Rottlerin.
  
  
  CONCLUSIONS
  EMB-induced vacuole formation, cytoplasmic release of cathepsin D, and reduction of phagocytosis in RPE are intimately correlated and regulated by the PKCδ signal pathway.
authors: Department of Ophthalmology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan.
authors: Rong Kung
authors: RK
authors: Tsai
authors: Ming Shan
authors: MS
authors: He
authors: Zih-Yao
authors: ZY
authors: Chen
authors: Wen Chen
authors: WC
authors: Wu
authors: Wen Sheng
authors: WS
authors: Wu
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000995:Antitubercular Agents / Q000009:adverse effects*; D001578:Benzopyrans / Q000494:pharmacology; D015153:Blotting, Western; D002402:Cathepsin D / Q000032:analysis; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004977:Ethambutol / Q000009:adverse effects*; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008853:Microscopy; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012374:Rod Cell Outer Segment / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000098:Acetophenones; D000995:Antitubercular Agents; D001578:Benzopyrans; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D004977:Ethambutol; C085746:rottlerin; D051745:Protein Kinase C-delta; D002402:Cathepsin D