Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism

Abstract

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").

Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice

“…The mechanism by which FLP works is quite different because MCT damages cells, whereas FLP engages extracellular matrix receptors without causing cell damage (16). Subsequently, transplanted LSECs proliferated in MCT-pretreated mice due to impairment in the survival and proliferation capacity of native LSECs, as shown by our studies, which were in agreement with the propensity of MCT to induce persistent cell-cycle arrest, such as through G2/M block, in ECs (38,39). Consequently, the extent of liver repopulation with LSECs reached 10%-20% of the endothelial mass, judging from the analysis of cell fractions in our studies.…”

Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension

“…PAECs grown in 6-well plates were fixed and processed for immunofluorescence using either the STAT3 pAb alone or STAT3 pAb incubated with a 5-fold excess amount of relevant blocking peptide or an irrelevant blocking peptide [a caveolin-1 (cav-1) blocking peptide]. Incubation of the STAT3 pAb with the blocking peptides was done at room temperature for 1 h as described previously (47). Scale bar ϭ 25 m. STAT3 transcriptional signaling, a significant reduction in cav-1 levels per se was difficult to verify by Western blotting, and a similar enhancement of IL-6/transcriptional signaling was observed using an irrelevant RNAi control oligonucleotide.…”

Dysfunctional Intracellular Trafficking in the Pathobiology of Pulmonary Arterial Hypertension

“…Exposure to MCTP initiates a cascade of changes in affected PAEC culminating in the development of PAH 10-14 d later. In cell culture experiments in endothelial and epithelial cells, the active pyrrolic derivative MCTP is used directly [27,28]). …”

“…Inasmuch as cav-1 traffics to the cell surface plasma membrane to/through the Golgi, the above studies were extended to an investigation of membrane trafficking (27,28,(33)(34)(35). MCTP and hypoxia trapped cav-1 in the Golgi in PAEC in culture (27,34,35).…”

“…MCTP and hypoxia trapped cav-1 in the Golgi in PAEC in culture (27,34,35). This trapping of cav-1 in the Golgi of PAEC ("Golgi blockade") helped focus attention on disruption of intracellular trafficking, and it was discovered that MCTP, hypoxia, and senescence all disrupted the molecular machinery of vesicular trafficking at the level of Golgi tethers, SNAREs, and SNAPs (34, 35) ( Figure 2B).…”