Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism.
Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis").
Shah
Mehul
M
Dept. of Cell Biology and Anatomy, New York Medical College, 201 Basic Sciences Bldg., Valhalla, NY 10595, USA.
Patel
Kirit
K
Sehgal
Pravin B
PB
D000818:Animals; D001324:Autoantigens; D015153:Blotting, Western; D051242:Caveolin 1; D022461:Caveolins / Q000187:drug effects / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D016686:Monocrotaline / Q000031:analogs & derivatives* / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects; D050796:STAT3 Transcription Factor; D013552:Swine; D015534:Trans-Activators / Q000187:drug effects / Q000378:metabolism
D001324:Autoantigens; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D004268:DNA-Binding Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C014114:monocrotaline pyrrole; D016686:Monocrotaline; D048049:Extracellular Signal-Regulated MAP Kinases