Materials and Methods Cell culture

1. Early molecular events involved in sterol efflux induced by micellar sterols.A study with a polarized Caco-2 intestinal cell model Roberto Rivabene 1, Ida Blotta 1, Sabrina Mirante 1, Francesca Prestinaci 1, Livia Pisciotta 2, Stefano Bertolini 2, and Alfredo Cantafora1 1Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy 2Department of Internal Medicine, University of Genoa, Italy Materials and Methods Cell culture The human colon carcinoma cell lineCaco-2 was routinely subcultured on 25 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4g/l glucose, 0.2 mM L-glutamine, 1% non-essential amino acids, 100U/ml penicillin, 50 mg streptomycin and 10% (v/v) fetal bovine serum, and maintained at 37°C in a atmosphere of 5% CO2, 95% air. For the experiments, once the flasks reached 80% confluence, the cells were split and seeded as reported in Figure 1. All experiments were performed 20-22 days of culture. Experimental design 24 hours before the start of each experiment, the maintenance medium was removed. Then, 2 ml of serum-free DMEM containing cholesterol or cholesterol plus 10% b-sitosterol enriched micelles were added to the apical chamber, whether 2 ml of serum-free DMEM alone was added to the basolateral chamber. In untreated cells, 2 ml of serum-free DMEM alone were added in both the compartments.An additional control in which cells were treated with all micellar constituents but cholesterol and/or b-sitosterol has also considered. Analysis of gene expression by Real Time Quantitative PCR The cell suspension corresponding to about 2x106 cells was centrifuged at low speed for 5 min. The cell pellet was immediately submitted to total RNA extraction by silica membrane spin columns with the NucleoSpin RNAII extraction kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s protocol. Purified RNA was reverse transcipted (M-MuLV reverse transcriptase) according to the protocols supplied by the manufacturer. Quantitative gene expression was performed by real-time PCR using the MX3000P quantitative PCR system (Stratagene, La Jolla, CA).PCR primers (Tab.1) were designed using a commercial software (DNASIS, Ver. 2.5 – Hitachi Software Engineering Co., France) using the mRNA sequences reported in a public database. ELISA for ApoA-I and ApoB The basolateral medium collected at the end of incubation (24 h) was immediately diluted 1:1 with a PBS buffer containing 0.1 % Tween-20, 1 % BSA, 0.05 M glycine and protease inhibitors cocktail. ApoA-I and ApoB were estimated by sandwich enzyme-linked immunosorbent assays, by using monoclonal antibodies against the two apolipoproteins. Background • Cholesterol homeostasis within the cell is maintained by balancing cholesterol absorption and endogenous cholesterol synthesis with mechanisms able to favour the export of cholesterol excess in order to prevent its accumulation. • Nuclear receptors of the Retinoid-X-receptor (RXR) heterodimer family, such as the Liver–X-receptors (LXR) and the Farnesoid-X-receptor (FXR), were recently indicated as regulators of key genes involved cholesterol efflux, such as different members of the ATP-binding cassette (ABC) transporters family, e.g. ABCA1, ABCG5 and ABCG8. • AIM • To study the early time-related expression of a wide panel of genes known to be involved in cholesterol efflux, following the exposition of polarized Caco-2 intestinal cells to micellar cholesterol or cholesterol/b-sitosterol mixtures. In addition, the basolateral secretion of the major intestinal apolipoproteins (ApoA-I and ApoB) was also evaluated. Results Figure. 1 Figure. 3 Table. 1 Figure 1 - characterization of Caco-2 cell monolayer. Cells were seeded onto Millicell tissue culture plate inserts (30 mm diameter, 0.4 mm pore size), and left to differentiate in 20-22 days of culture. The degree of biochemical differentiation of Caco-2 cell monolayer was checked over the period of culture and the integrity of monolayers was assessed by the development of a significant trans-epithelial electrical resistance (TEER). Before treatments and in order to ascertain if Caco-2 cells have reached the typical fully differentiated enterocyte morphology, cells were processed for scanning electron microscopy (SEM). As can be seen, cells show morphological characteristics resembling those of small intestinal enterocytes, with a well polarized columnar body with typical brush border microvilli projecting perpendicular to the cell surface. Figure. 2 • Figure 2 - Time-related expression of PS1 (panel A) and PS2 (panel B) genes in SK-N-BE cells incubated from 6 to 24 hrs with THG. • Table 1- Primer pairs for the amplification of the different messenger analyzed by real-time PCR. The sizes of amplification products from cDNA or DNA templates and their distances from poly A tails are also indicated. • Figure 3 -Effect of 24 h treatment with THG on GSH content and GSH/GSSG ratio in SK-N-BE cells Controls: serum-free DMEM cultivated cells; FA: cells incubated with all micellar constituents but Mean ± SD of six experiments, each performed in triplicate, are shown in figure. *P<0.001 Conclusion • Micellar cholesterol and more consistently b-sitosterol influx within Caco-2 cells induced and early increased time-related expression of FXR, ABCA1 and APOA1, but only for b-sitosterol a consistent (P<0.001) increased ApoA-I secretion into the basolateral compartment was appreciated. • Conversely, and differently from what has been previously reported, the genetic expression of LXRa, ABCG5, ABCG8, PPARg and APOB were unmodified. • These events suggest that micellar sterols induce a rapid mechanism of basolateral efflux that is initiated by FXR/RXR heterodimer formation, supported by ABCA1 and APOA1 gene activation, and sustained by the higher amount of intestinal lipid-free ApoA-I basolateral secretion, which function as extracellular cholesterol acceptor (Fig. 4). • The FXR pathway, rather the LXRa one (as reported by others), may be critical for cholesterol homeostasis at intestinal level and possibly represent a useful drug target for regulation of intestinal cholesterol metabolism. Figure. 4 Figure 4 Acknowledgments This work was partially supported by the research project “Studio clinico e sperimentale di alterazioni biochimiche causa di dislipidemie e loro sequele (C3BO)” from the Ministry of Health. We gratefully acknowledge the skillful collaboration of Dr. Rocco Careri