RanGAP-Mediated Nuclear Protein Import in Vascular Smooth Muscle Cells Is Augmented by Lysophosphatidylcholine

Abstract

The intracellular mechanism responsible for the mitogenic effects of lysophosphatidylcholine (LPC) is unclear. Import of proteins from the cytoplasm into the cell nucleus is integral to the regulation of gene expression and cell growth. We hypothesized that LPC exerts its intracellular effects through alterations in nuclear protein import. Rabbit aortic smooth muscle cells incubated with LPC induced a significant increase in cell proliferation in both quiescent cells (63.2 Ϯ 6.48% of control) and cells grown in 1% fetal bovine serum (FBS) (28.3 Ϯ 7.35% of control). Vascular smooth muscle cells were preincubated with LPC then microinjected with a marker protein for nuclear import. A significant stimulation of nuclear protein transport was observed. Using a conventional nuclear protein import assay in permeabilized cells, a significant stimulation of import (72.3 Ϯ 5.2% of control) was again observed when the cytosolic nuclear import cocktail was treated with LPC. This effect was not observed with other lysophosphatidyl species. LPC also activated the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) pathway, and this was blocked by 2Ј-amino-3Ј-methoxyflavone (PD98059), which inhibits the activation of ERK 1/2. The stimulation of nuclear import was also blocked by PD98059. LPC-induced MAPK activation augmented GTP hydrolysis by RanGAP, a RanGTPase activating protein and a critical regulatory component of nuclear protein import, and this stimulation was again blocked by PD98059. We conclude that LPC alters gene expression and cell proliferation through striking effects on nuclear protein import via a MAP kinase-induced activation of RanGAP. This may play an important role in cancer and atherosclerosis and other disorders involving accelerated cell growth/proliferation.

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice

“…Conversely, GTP-binding nuclear protein Ran expression is positively associated with VSMC migration (Fuastino et al 2010). GTP-binding nuclear protein Ran expression was upregulated in this experiment.…”

Therapeutic Targeting of Nuclear Protein Import in Pathological Cell Conditions

“…Exposure of VSMCs to LPC stimulates ERK1/2 activity, which in turn increases RanGAP activity (ERK1/2 shares docking sequences with RanGAP). This induces the hydrolysis of Ran-GTP leading to higher levels of RanGDP in the cytosol, which causes an increase in RanGTP in the nucleus, enabling more importins (␣ and ␤) to be recycled to the cytoplasm, which increases nuclear import (Faustino et al, 2007b). In conclusion, it is clear that oxidants are potential triggers for changing the ratio of RanGTP/RanGDP, thereby altering nuclear protein import.…”

“…During atherosclerosis and in cancer, an important mitogenic second messenger is lysophosphatidylcholine (LPC) (Chai et al, 1996;Fang et al, 2000;Faustino et al, 2007b). Exposure of VSMCs to LPC stimulates ERK1/2 activity, which in turn increases RanGAP activity (ERK1/2 shares docking sequences with RanGAP).…”

Ceramide regulation of nuclear protein import

“…VSMCs were cultured from thoracic aorta explants isolated from New Zealand White rabbits as described previously (29,32,33). Cells were seeded on acid-rinsed coverslips at initial seeding densities of 2.0 3 10 5 or 1.9 3 10 5 cells/coverslip and starved for 3 days, then fed with DMEM containing 5% FBS 1 Fig.…”

“…Transport is initiated upon energy-independent NLS recognition by a heterodimeric NLS receptor (17,18) composed of an a subunit (importin-a), which recognizes the NLS (19), and a b subunit (20) (importin-b), which mediates nuclear pore complex docking at the nuclear envelope (21)(22)(23). Translocation of the NLS-receptor assembly through the nuclear pore complex is an energydependent process (24)(25)(26) controlled by a RanGTP/GDP cycle (27)(28)(29). Importin-a is returned to the cytosol by CAS (for Cellular Apoptosis Susceptibility), a nuclear export protein specific for the a subunit (30).…”