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1 Cell Biology & Anatomy, New York Medical College, Valhalla, NY, USA
2 Cell Biology & Anatomy, New York Medical College, Valhalla, NY, USA; Medicine, New York Medical College, Valhalla, NY, USA
* To whom correspondence should be addressed. E-mail: pravin_sehgal{at}nymc.edu.
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 megalocytosis phenotype in target endothelial and parenchymal cells. Although this phenomenon has been known for over 50 years, the subcellular mechanism has remained elusive. A hit-and-run type of exposure to the bioactive pyrrolizidine derivative results by 2-3 days in enlarged cells with enlarged nuclei, Golgi and endoplasmic reticulum, while the cells remain in G2/M block (karyocytomegaly). Megalocytosis of endothelial cells is characterized by stimulation of DNA synthesis and hypertetraploidy, while that of hepatocytes by inhibition. In monocrotaline pyrrole (MCTP)-treated pulmonary arterial endothelial cells (PAEC), there is an inverse relationship between loss of caveolin-1 (cav-1)/rafts and stimulation of DNA synthesis. In the present study, we recapitulated MCTP-induced megalocytosis in cultures of bovine PAEC, extended these observations to cultured 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. MCTP-induced megalocytosis led to a reduction in 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 a heavy to a lighter density into fractions enriched for hypo-oligomeric cav-1 and for BiP (GRP78) indicating dysfunctional cav-1 trafficking through the Golgi. Immunofluorescence studies confirmed the trapping of cav-1 in a GM130-positive Golgi compartment. There was an increase in 25Ser-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 M upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly following nocodazole-induced fragmentation. Moreover, following exposure to 2-ME, the non-mitotic cells in both untreated and MCTP-treated PAEC cultures showed equivalent microtubule and actin organization. Taken together, these data suggest that a disruption of the trafficking and mitosis-sensor functions of the Golgi organelle (the Golgi blockade hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis.
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