Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI

Abstract

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPAR␣-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPAR␣ Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPAR␣-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPAR␣-dependent degradation of SR-BI in hepatocytes.Anti-atherosclerotic properties of HDL and the major HDL apolipoprotein, apoA-I, are believed to include their ability to induce signaling events that promote cholesterol export from peripheral cells to the liver for disposal. However, the signal transduction pathways that contribute to stimulate reverse cholesterol transport in macrophages and hepatocytes are not fully understood (1-3). HDL binding to receptors such as SR-BI 6 activates various cellular processes, including endothelial nitric-oxide synthase activation in endothelial cells (1-3) and proliferation in smooth muscle cells (4) but also cell surface localization of SR-BI in hepatocytes (5). Downstream targets of HDL include Src family kinases, phospholipase C and D, Ras, phosphatidylinositol 3-kinase (PI3K), Akt, the mitogen-activated protein kinase (MAPK) pathway (Mek1/2 and Erk1/2), and Rac/Rho GTPases (1-8). Other kinases implicated in HDL-or apoAI-inducible cholesterol transport include protein kinase C (PKC), protein kinase A (PKA), c-Jun N-terminal kinase (JNK), and p38 MAPK (9 -14).Alternatively, signaling pathways can modulate the activity of nuclear receptors, including PPAR and LXR, to alter the ability of cells to transport cholesterol (15-18). Indeed, post-translational phosphorylation via various kinases, including Erk1/2, can alter PPAR␣ and PPAR␣ co-activator activity in a liganddependent and -independent manner (17-25). Recent findings link Erk1/2 kinases with nuclear receptors and lipid export via ABCA1. First, enhanced Erk1/2 signaling increases ABCA1 expression and ABCA1-mediated phos...

Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1

“…ERK1/2 regulates SR-BI protein stability [29] and its expression was induced in SNTA-B−/− mice liver (Fig. 6A, B).…”

“…Hepatic overexpression of sphingomyelin synthases 1 and 2 induces liver SR-BI expression [48] while treatment of hepatoma cells with sphingomyelinase does not affect SR-BI protein (own unpublished results). Recently it has been shown that ERK1/2 regulates SR-BI protein stability in HuH7 hepatocytes by a mechanism involving ligand-independent activation of PPARα [29]. ERK1/2 and phosphorylated ERK2 are increased in SNTA/B2−/− liver.…”

Sub-Nanomolar Sensitivity of Nitric Oxide Mediated Regulation of cGMP and Vasomotor Reactivity in Vascular Smooth Muscle

“…The velocity and rate constants for the inactive and active NO and cGMP receptors (sGC, PDE5, P-PDE5, and PDE1, respectively), were determined assuming G(0) = 4.16 nM, PDE5a(0) = 0.8, PDE5b(0) = 0, PDE5c(0) = 0, and PDE5d(0) = 0.2 at time zero. Values from previously studies ( Vp 1 , Kp 1 , Kp 2 , Vp 3 , Kp 3 , Vp x , and Kp x ; see Table 3) and as determined (GC max and Vp 2 ) were calculated by the Levenberg-Marquardt method of least squares curve fitting of the proposed model to actual data using the MinErr function of MathCad, as shown in the Supplementary Material and previously described (Wood et al, 2011). The resulting values were then combined to determine the velocity of cGMP hydrolysis ( Vd ) by solving:…”

“…The short biological half-life of NO (Griffith et al, 1984; Cocks et al, 1985; Hakim et al, 1996; Wood et al, 2011) has previously served to explain the transient in nature of NO-induced vasorelaxation (Ignarro et al, 1987, 1988; Palmer et al, 1987; Amezcua et al, 1988; Moncada et al, 1991b). However, the kinetics of the NO/cGMP/PKG signaling mechanism has not yet been described in VSM.…”

“…This approach allowed for the direct vascular response to NO to be determined without interference of NO decay. Combination of this NO delivery method with the single wavelength cGMP biosensor, FlincG (Nausch et al, 2008), has recently been described as an exceedingly sensitive NO detector in intact cells, using cGMP as a gauge of sGC activity (Batchelor et al, 2010; Wood et al, 2011). Sub-nanomolar concentrations of NO were detected, albeit dependent on sGC expression level, providing insight into the mechanism for the biological effects of NO at very low concentrations (Batchelor et al, 2010).…”