Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1

Abstract

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms.

Methodology/principal findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo.

Conclusions/significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

Figure 1. Time course of expression of SR-BI and PDZK1 in cultured primary mouse hepatocytes.

Hepatocytes were isolated from the livers of wild-type mice as previously described in Materials and Methods and . Immediately after isolation some of the cells were lysed (time  = 0) and others were plated at 80,000 cells/cm² onto collagen gels with 3% Matrigel and a 1.3 mm medium depth, as described in Materials and Methods. These cells were subsequently maintained in culture for the indicated times, lysed and lysates were analyzed for protein expression using immunoblotting (A) and mRNA expression using qRT-PCR (B). A For protein analysis lysates (20 µg protein) were subjected to SDS-PAGE and immunoblotting with polyclonal anti-SR-BI (mSR-BI⁴⁹⁵), polyclonal anti-PDZK1 and polyclonal anti-ε-COP (loading control) antibodies, and the proteins subsequently visualized using enhanced chemiluminescence detection. B For mRNA analysis Trizol lysates were subjected to qRT-PCR analysis and the approximate number of mRNA copies/cell was calculated by normalization to 18S rRNA abundance and expressed as percent of the number of copies at time 0. The average 100% of control copy numbers/cell at time 0 were: SR-BI, 19.3; PDZK1, 6.4.

Figure 2. Effects of PDZK1 co-transfection on SR-BI protein levels in COS cells.

COS cells were plated into the wells of 6 well plates on day 0 and transiently transfected with a total of 4 µg DNA (100%)/well on day 1 using the indicated plasmids encoding SR-BI, PDZK1 and an empty vector at the indicated relative concentrations (%). On day 3 the cells were harvested, lysed, and lysates (20 µg protein) were subjected to SDS-PAGE and immunoblotting with polyclonal anti-SR-BI (mSR-BI⁴⁹⁵), polyclonal anti-mouse PDZK1 and polyclonal anti-ε-COP (loading control) antibodies. A Effects of varying amounts of SR-BI expressing plasmid in the transfection together with either 0% (−) or 50% (+) PDZK1 expressing plasmid. B Effects of varying amounts of PDZK1 expressing plasmid transfected together with 1% SR-BI expressing plasmid.

Figure 3. Effects of PDZK1 co-transfection on SR-BI-mediated [³H]cholesteryl ester uptake from HDL in COS cells.

COS cells were plated into the wells of 6 well plates on day 0 and transiently transfected with a total of 4 µg DNA (100%)/well on day 1 using the indicated plasmids encoding SR-BI, PDZK1 and an empty vector at the indicated relative concentrations (%). On day 1, the cells were harvested, counted and plated into the wells of 24 well plates. On day 2, [³H]cholesteryl ester ([³H]CE) uptake from [³H]CE-HDL (10 mg of protein/mL, 2 hr, 37°C) was determined as described in Materials and Methods. All values represent receptor-specific activities calculated as the differences between activity in the absence (quadruplicate determinations) and presence (duplicate determinations) of a 40-fold excess of unlabeled HDL. Statistical analyses of data obtained with or without co transfection of the PDZK1 plasmid (50%) were performed using the unpaired two-tailed t test at 95% confidence intervals (*: p<0.05, **: p<0.005).

Figure 4. Effects of PDZK1 co-transfection on mutant SR-BI and SR-BII protein levels in COS cells.

COS cells were plated into the wells of 6 well plates on day 0 and transiently transfected with a total of 4 µg DNA (100%)/well on day 1 using at the indicated relative concentrations (%) of the indicated plasmids encoding PDZK1, a control empty vector and vectors encoding variants of SR-BI. These variants include SR-BI Δ509 (a mutant lacking a single amino acid at the C- terminus), SR-BI ΔC-term (a mutant lacking essentially the entire C- terminal cytoplasmic domain of SR-BI), and SR-BII (a splicing variant whose entire C-terminal cytoplasmic domain differs from that of SR-BI). On day 3 the cells were harvested, lysed, and lysates (20 µg protein) were subjected to SDS-PAGE and immunoblotting with polyclonal anti-SR-BI (KKB-1) and polyclonal anti-ε-COP (loading control) antibodies. A Effects of varying amounts of mutant SR-BI and SR-BII expressing plasmids in the transfection together with either 0% (−) or 50% (+) PDZK1 expressing plasmid. B Effects of varying amounts of PDZK1 expressing plasmid transfected together with 1% SR-BI Δ509 expressing plasmid.