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

Abstract

PB. Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 294: L449-L468, 2008. First published December 14, 2007 doi:10.1152/ajplung.00377.2007.-Lung vascular lesions in pulmonary arterial hypertension (PAH) are characterized by enlarged, vacuolated ("megalocytotic") pulmonary arterial endothelial (PAEC) and smooth muscle cells (PASMC). We have recently proposed that dysfunction of vesicle tethers, soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), and SNAP receptors (SNAREs), leading to disruptions of intracellular trafficking in the Golgi to plasma membrane (centrifugal) and the plasma membrane to cell interior (centripetal) directions is a key causal mechanism in this disease. In PAH, there was a reciprocal relationship between loss of caveolin-1 (cav-1) in PAECs and increased expression of "activated" tyrosine-phosphorylated STAT3 (PY-STAT3) associated with a block in centrifugal trafficking to/through the Golgi organelle. In the present study, we investigated 1) whether centripetal trafficking of STAT3 and PY-STAT3 in PAECs and PASMCs was membrane-associated, and 2) whether this might be affected in PAH. Immunofluorescence and live cell imaging studies showed that, in both PAEC and PASMC, STAT3 was associated with cytoplasmic vesicles partially colocalizing with markers of the endolysosomal compartments (clathrin, EEA1, Rab5, Rab11, and LAMP1). Overexpression of cav-1 increased the targeting of STAT3 to lysosomes and inhibited STAT3 transcriptional activity. Exposure of PAECs to monocrotaline (MCT) pyrrole, which causes PAH in the rat, led to a loss of caveolar STAT3 with increased sequestration of STAT3 and PY-STAT3 in endosomes. In vivo, marked cytoplasmic sequestration of activated PY-STAT3 was a common feature in PAEC in the rat/MCT model and in cells in the proliferative arterial and plexiform lesions in PAH in humans. These data highlight the epigenetic regulation of centripetal cytokine and growth-factor signaling pathways and its modulation in PAH.caveolin-1; signaling endosome; monocrotaline PULMONARY ARTERIAL HYPERTENSION (PAH) is characterized by the reduction of the lumen of medium-sized pulmonary arteries by proliferating, enlarged, vacuolated, and apoptosis-resistant ("megalocytotic") pulmonary arterial endothelial (PAEC) and smooth muscle (PASMC) cells (42,43). We (24, 26 -28, 41-43, 47) recently proposed that dysfunction of vesicle tethers, soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), and SNAP receptors (SNAREs), leading to disruptions of intracellular trafficking from the Golgi to plasma membrane (centrifugal) and the plasma membrane to cell interior (centripetal) directions might represent a key subcellular mechanism leading to the changes observed in endothelial and vascular smooth muscle cells in PAH. We (24, 26 -28, 41-43, 47) have previously provided evidence for disrupt...

Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging

“…A single exposure of confluent bovine PAEC cultures to MCTP for as short a time as 2 min leads to apoptosis of a small subset of cells with the development of marked megalocytosis of cells in the surviving, but still confluent, cell sheet 18 -24 h later (18,38,48,49,68, 73). As with the in vivo observations (27, 34 -36), the cell culture studies showed dramatic increases in cell size, in Golgi stacks, and in the endoplasmic reticulum; increased DNA synthesis; and a unique premitotic cell cycle arrest despite the continued DNA synthesis resulting in hyperploidy of the affected cells (18,25,33,36,(39)(40)(41)48,49,55,57,61,66; reviewed in Refs. 53,54).…”

“…Neointima development, thrombotic obliteration of arterial lumen, and development of plexiform lesions, reminiscent of human PAH, have also been reported with MCT when combined with partial pneumonectomy (44, 65 and citations therein). Additionally, perivascular/adventitial infiltrative cells and their cytokine contributions (such as IL-6) and transcription factors activated by such cytokines (STAT3 and NF-B) have been posited as contributory factors in the development of disease in this model (2,14,15,30,41,64).Over the last two decades bovine PAECs and PASMCs in culture exposed to MCTP have been used as experimental models to investigate some of the cellular, subcellular, and biochemical changes elicited by MCTP (18,25,26,30,[38][39][40][41][42]48,49,[53][54][55]57,61,66). A single exposure of confluent bovine PAEC cultures to MCTP for as short a time as 2 min leads to apoptosis of a small subset of cells with the development of marked megalocytosis of cells in the surviving, but still confluent, cell sheet 18 -24 h later (18,38,48,49,68, 73).…”

“…Over the last two decades bovine PAECs and PASMCs in culture exposed to MCTP have been used as experimental models to investigate some of the cellular, subcellular, and biochemical changes elicited by MCTP (18,25,26,30,[38][39][40][41][42]48,49,[53][54][55]57,61,66). A single exposure of confluent bovine PAEC cultures to MCTP for as short a time as 2 min leads to apoptosis of a small subset of cells with the development of marked megalocytosis of cells in the surviving, but still confluent, cell sheet 18 -24 h later (18,38,48,49,68, 73).…”

“…Neointima development, thrombotic obliteration of arterial lumen, and development of plexiform lesions, reminiscent of human PAH, have also been reported with MCT when combined with partial pneumonectomy (44,65 and citations therein). Additionally, perivascular/adventitial infiltrative cells and their cytokine contributions (such as IL-6) and transcription factors activated by such cytokines (STAT3 and NF-B) have been posited as contributory factors in the development of disease in this model (2,14,15,30,41,64).…”

Nuclear protein extraction from frozen porcine myocardium

“…In recent years, evidence has emerged that phosphorylation of STAT3 does not necessarily lead to nuclear translocation [21,23]. It is therefore necessary to demonstrate the nuclear accumulation of PY-STAT3 when studying PY-STAT3-dependent gene expression.…”