The
pyrrolizidine alkaloid monocrotaline (MCT) initiates
pulmonary hypertension by inducing a "megalocytosis" phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of
monocrotaline (
MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the "Golgi blockade" hypothesis) may represent the subcellular mechanism leading to
MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells.
MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding
protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane
caveolin-1 and
E-cadherin, cortical actin together with a circumnuclear accumulation of
clathrin heavy chain (CHC) and
alpha-tubulin. Flotation analyses revealed losses/alterations in the association of
caveolin-1,
E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1alpha and
glucose regulated
protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of
PERK kinase and
protein disulfide isomerase (PDI). These data further support the hypothesis that an
MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis.