G-Protein-Mediated Signaling in Cholesterol-Enriched Arterial Smooth Muscle Cells. 1. Reduced Membrane-Associated G-Protein Content Due to Diminished Isoprenylation of G-γ Subunits and p21ras

Abstract

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Gαi-1, Gαi-2, Gαi-3, Gq/11, and Gαs. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Gγ-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Gαi subunits was found to be post-transcriptional, since the mRNA steady-state levels of Gαi(1−3) were unchanged following cholesterol enrichment. Second, the membrane expression of α and β subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Gαi and Gβ expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Gαi subunits. Fifth, cholesterol enrichment also reduced membrane expression of Gγ-5, Gγ-7upper; these γ subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-γ-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Gγ subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane associationPDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

Cholesterol Depletion of Caveolae Causes Hyperactivation of Extracellular Signal-related Kinase (ERK)

“…No one has studied the MAP kinase activity in caveolae of cholesterolenriched cells, but our results predict the enzyme would be hyporesponsive to growth factors such as EGF. Recently, Pomerantz et al (35) reported that cultured smooth muscle cells with elevated levels of cholesterol exhibit a reduced MAP kinase activity in response to platelet-derived growth factor. At the other extreme, transformed cells have below normal amounts of caveolae cholesterol, which is restored to normal after the cells are transfected with caveolin-1 (10).…”

Cyclic Strain Stress-induced Mitogen-activated Protein Kinase (MAPK) Phosphatase 1 Expression in Vascular Smooth Muscle Cells Is Regulated by Ras/Rac-MAPK Pathways

“…Membrane protein preparation for Ras and Rac analysis was similar to that described by Pomerantz et al (41). Briefly, VSMCs were washed with cold phosphate-buffered saline (4°C), scraped in phosphate-buffered saline, pelleted, and resuspended in 500 l of homogenizing buffer (25 mM Hepes, 1.0 mM EDTA, 1 g/ml leupeptin, 1 g/ml pepstatin A, and 100 M phenylmethylsulfonyl fluoride).…”

Hypercholesterolemia Impairs Transduction of Vasodilator Signals Derived From Ischemic Myocardium

“…Armstead 31 has recently demonstrated that superoxide inhibits the dilation of cerebral vessels mediated by G PTX protein. Furthermore, Pomerantz et al 32 have shown that the G i␣ level of vascular smooth muscle cells is decreased under the condition of cholesterol enrichment by post-transcriptional mechanisms such as diminished isoprenylation of G ␤␥ subunits, which could lead to low anchoring of G i protein.…”