Modulators of Protein Kinase C Affect SR-BI-Dependent HDL Lipid Uptake in Transfected HepG2 Cells

Abstract

SR-BI is a cell surface HDL receptor that mediates selective uptake of the lipid cargo of HDL, an important process in hepatocytes, driving reverse cholesterol transport from cells in the artery wall. To facilitate examination of factors that modulate SR-BI activity in hepatocytes, we have generated fluorescent protein-tagged versions of SR-BI that allow for facile monitoring of SR-BI protein levels and distribution in transfected cells. We show that deletion of the C-terminal cytosolic tail does not affect the distribution of SR-BI in HepG2 cells, nor is the C-terminal cytosolic tail required for SR-BI-mediated uptake of HDL lipids. We also demonstrate that the phorbol ester, PMA, increased, while protein kinase C inhibitors reduced SR-BI-mediated HDL lipid uptake in HepG2 cells. These data suggest that protein kinase C may modulate selective uptake of HDL lipids including cholesterol in hepatocytes, thereby influencing hepatic HDL cholesterol clearance and reverse cholesterol transport.

Figure 1

Diagrams of the eGFP or mRPF-tagged SR-BI and SR-BI-ΔC transgenes. The constructs consist of either SR-BI or SR-BI-ΔC tagged with either eGFP or mRFP on their amino termini. The locations of putative transmembrane domains (TMDs) are also shown. Cytosolic regions are shown in yellow and extracellular regions are shown in blue.

Figure 2

Expression of eGFP-SR-BI or eGFP-SR-BI-ΔC in HepG2 cell lines. Cell lysates prepared from HepG2[eGFP-SR-BI] (lane 1), HepG2[eGFP-SR-BI-ΔC] (lane 2), HepG2 (untransfected) (lane 3), and HepG2[eGFP] cells (lane 4) were subjected to SDS-PAGE and immunoblotting for eGFP (upper panel) or β-actin (lower panel).

Figure 3

Localization of fluorescent protein-tagged SR-BI containing or lacking the C-terminal tail in transfected HepG2 cells. HepG2 cells transfected with eGFP-SR-BI ((a)–(c)), eGFP-SR-BI-ΔC ((d)–(f)), mRFP-SR-BI ((g)–(i)), or mRFP-SR-BI-ΔC ((j)–(l)) were fixed, stained with DAPI and imaged by wide field fluorescence microscopy in the blue ((a), (d), (g), and (j)), green ((b) and (e)), or red ((h) and (k)) channels. Merged images of the blue and green ((c) and (f)) or the blue and red ((i) and (l)) channels are also shown. All images are set to the same scale. Scale bar in (a) = 10 μm.

Figure 4

Colocalization of fluorescent protein-tagged SR-BI and SR-BI-ΔC in transfected HepG2 cells. HepG2 cells transfected with both eGFP-SR-BI and mRFP-SR-BI-ΔC ((a)–(d)) or with both mRPF-SR-BI and eGFP-SR-BI-ΔC ((e)–(h)) expression plasmids were fixed, stained with DAPI, and imaged by wide-field fluorescence microscopy in the blue ((a) and (e)), green ((b) and (f)), and red ((c) and (g)) channels for DAPI, eGFP, and mRFP, respectively. Merged images are shown in panels (d) and (h). All images are set to the same scale. Scale bar in (a) = 10 μm.

Figure 5

SR-BI-ΔC retains DiI-HDL lipid uptake activity. HepG2 cells and stably transfected HepG2[eGFP-SR-BI] and HepG2[eGFP-SR-BI-ΔC] cells were incubated with DiI-HDL and analyzed by flow cytometry for DiI fluorescence (a) or eGFP fluorescence (b). The specific uptake of DiI lipid is shown in panel (a). The geometric mean eGFP fluorescence for HepG2[eGFP-SR-BI] and HepG2[eGFP-SR-BI-ΔC] cells is shown in panel (b). The experiment was performed in triplicate, and error bars represent standard deviation. *P < .05 by the Student's t-test.

Figure 6

The protein kinase C inhibitor calphostin C reduces SR-BI-mediated DiI-HDL lipid uptake in HepG2 cells independently of SR-BI's C-terminal tail. DiI-HDL lipid uptake (panels (a)-(c)) and eGFP fluorescence (panels (d) and (e)) were measured by flow cytometry. HepG2[eGFP-SR-BI] cells untreated or treated with 0.5 μM PMA, 7.5 μM chelerythrine chloride, 1 μM calphostin C, 300 nM BLT-1, individually or in combination as indicated panels (a) and (d). HepG2[eGFP-SR-BI-ΔC] cells untreated or treated with PMA, calphostin C, or BLT-1 either individually or in combination as indicated panels (b) and (e). Panel (c): untransfected HepG2 cells treated without or with PMA or calphostin C individually as above. The data represent average ± standard deviations of triplicate samples. *P < .05 compared to control by Student's t-test.

Figure 7

Modulators of PKC activity do not affect SR-BI distribution in HepG2 cells. HepG2[eGFP-SR-BI] cells were serum starved for 4 hrs and then refed medium containing 5% FBS and either vehicle (0.1% DMSO), 0.5 μM PMA, or 7.5 μM chelerythrine chloride and incubated for 1 hr. Cells were fixed with 2.5% paraformaldehyde and imaged by confocal microscopy.