Co-regulation of primary mouse hepatocyte viability and function by oxygen and matrix

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound.National Institutes of Health (U.S.) (Grant P50-GM068762-08)National Institutes of Health (U.S.) (Grant R01-EB010246-04)National Institutes of Health (U.S.) (Grant R01-ES015241)National Institutes of Health (U.S.) (Grant P30-ES002109

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

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.National Institutes of Health (U.S.) (Grant HL052212)National Institutes of Health (U.S.) (Grant HL066105)National Institutes of Health (U.S.) (Grant ES015241)National Institutes of Health (U.S.) (Grant GM068762

Quantitative analysis of cell decision processes in response to inflammatory cues and their role in mediating genotoxicity in hepatocytes

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (pages 189-211).While a link between chronic inflammation and cancer has been established, the mechanisms of genotoxicity in inflammatory environments remain poorly understood. We hypothesized that inflammation may provide cues that allow cells to survive in the face of significant DNA damage as well as cues that foster cell proliferation. This study sought to elucidate changes that influence hepatocyte decision processes under conditions of chronic inflammation by using a set of extracellular signals, both soluble and matrix-related, which can be varied systematically to create a diverse range of intracellular signaling states and phenotypic outcomes. We developed an easily translatable model system that can maintain primary mouse hepatocytes in a differentiated state and study the independent extracellular cues which regulate hepatocyte behavior during chronic inflammation. This model system allowed systematic variation of oxygen concentration as well as matrix composition and stiffness. Collagen, polyacrylamide, and RADA gels were used to create extracellular environments resembling the various stages of normal and fibrotic liver. Through careful control of medium depth and incubator oxygen levels, we determined that oxygen tension and extracellular matrix affected hepatocyte differentiation independently, and that both high oxygen and a compliant matrix environment are necessary for prolonged maintenance of primary mouse hepatocytes in vitro. Optimization of a quantitative imaging solution enabled the capture of rare events occurring in individual cells within a larger cell population. Using TNF-alpha, Fas ligand and, IL-6 cytokines in conjunction with oxygen and matrix cues, we investigated how extracellular stimuli influence cell fate in a pseudo-inflammatory environment and demonstrated that partial execution of apoptosis is a possible mechanism for genotoxicity in hepatocytes during chronic inflammation. The results of this study improve our understanding of how cues in the extracellular environment combine to influence the behavior primary mouse hepatocytes. The system we developed can be used as a platform for a multitude of in vitro applications including studies regarding drug toxicity and inflammation.by Lorenna Dianne Buck.Ph. D

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

<p>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 <i>t</i> test at 95% confidence intervals (*: p<0.05, **: p<0.005).</p

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

<p>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. <b>A</b> Effects of varying amounts of SR-BI expressing plasmid in the transfection together with either 0% (−) or 50% (+) PDZK1 expressing plasmid. <b>B</b> Effects of varying amounts of PDZK1 expressing plasmid transfected together with 1% SR-BI expressing plasmid.</p

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

<p>Hepatocytes were isolated from the livers of wild-type mice as previously described in Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069725#pone.0069725-Guo1" target="_blank">[58]</a>. 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). <b>A</b> 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>B</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.</p

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

<p>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. <b>A</b> 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>B</b> Effects of varying amounts of PDZK1 expressing plasmid transfected together with 1% SR-BI Δ509 expressing plasmid.</p