Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigeltrade mark) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBPalpha, HNF4alpha, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.
Previously, we demonstrated that primary cultures of rat hepatocytes evidence higher levels of differentiated function when cultured in the presence of a dilute overlay of extracellular matrix (Matrigel). In this investigation, we used DNA microarrays, quantitative RT-PCR, immunoblotting, and cell morphology analyses to evaluate the biological responses imparted by Matrigel overlays on primary cultures of human hepatocytes from five independent donors. Although interindividual variability in responses was evident, our results demonstrated that Matrigel additions typically improved hepatocyte morphology and differentiation character. Results from RNA-profiling experiments indicated that Matrigel additions enhanced hepatocyte RNA expression levels associated with a battery of differentiated features, to levels comparable to those seen in vivo, for genes such as the cytochrome P450s, solute carrier family members, sulfotransferases, certain nuclear transcription factors, and other liver-specific markers, such as albumin, transferrin, and response to the inducer, phenobarbital. In contrast, Matrigel additions were generally associated with reduced RNA expression levels for several cytokeratins, integrins, and a number of stress-related pathways. Decreases in integrin protein expression were similarly detected, although enhanced levels of the gap junction-associated protein, connexin 32, were detected in Matrigel-treated cultures. These data support the concept that ECM functions mechanistically to augment the differentiation character of primary human hepatocytes in culture by mediating a reduction in cellular stress response signaling and by enhancing gap junctional cell-cell communication.
Activation of the Raf kinase signal transduction pathway in skeletal myoblasts causes a complete cessation of myofiber formation and muscle gene expression. The negative impacts of the signaling pathway are realized through downstream activation of mitogen and extracellular kinase (MEK) phosphorylation-dependent events and MEK-independent signal transmission. MEKK1, a kinase that can physically associate with Raf, may contribute to the MEKindependent signaling in response to elevated Raf activity. Myogenic cells overexpressing activated Raf and kinasedefective MEKK1 remain differentiation-defective, suggesting that MEKK1 does not contribute to the inhibitory actions of Raf. However, constitutive activation of MEKK1 dramatically inhibits biochemical and morphological measures of muscle formation. MEKK1 inhibits MyoD-directed transcriptional activity without altering the ability of the protein to form heterodimers with E2A proteins or bind DNA. By contrast, the transcriptional activity of E47, the preferred dimer partner of the myogenic regulatory factors, is severely compromised by MEKK1-initiated signaling. Inhibition of MEK1/2 and JNK1/2 function did not reinstate E47-directed transcription, indicating that these two downstream kinases likely are not involved in the MEKK1-controlled transcriptional block. Inhibition of p38 signaling overcame the negative effects exerted by MEKK1 on the amino terminus of E47. Closer examination indicates that E47 is phosphorylated in vitro by p38, and deletion analysis predicts that the critical amino acid(s) phosphorylated by p38 lie outside of the minimal transcriptional activation domains. Thus, modification of E47 by p38 likely disrupts higher order protein complex formation that is necessary for muscle gene transcription.
Cathepsin E (CTSE) is an aspartic protease that has been linked to antigen processing and innate immunity. Elevated levels of CTSE expression have also been associated with several forms of cancer, including carcinomas exhibiting highly invasive character. In this study, we performed DNA microarray experiments, together with quantitative reverse transcriptase PCR analyses and enzymatic activity determinations to identify human CTSE as a novel target gene for regulation by the constitutive androstane receptor (CAR), a nuclear receptor activated by the liver tumor promoting agent, phenobarbital. In particular, two motifs within the 5′-flanking region of the human CTSE gene were identified as direct sites of interaction with CAR/RXRα heterodimers, a direct repeat-3 site at position −766 and a direct repeat-4 site at position −1407. Thus, these studies demonstrate CAR-mediated regulation of CTSE within primary hepatocyte cultures from several individual donors and suggest that elevated CTSE activity may play a functional role in the etiology of hepatocarcinogenesis. KeywordsCathepsin E; Constitutive androstane receptor; Hepatocytes; Primary hepatocytes; Liver; Human; Hepatocytes Cathepsin E (CTSE) 1 is an intracellular aspartic protease that hydrolyzes various biologically active peptides [1]. Unlike related proteases, CTSE is localized outside of the lysosomal system and likely functions in extralysosomal proteolysis [2]. Although its exact biological functions remain elusive, several physiological roles of CTSE have been reported, including roles in immune defense and antigen processing [3][4][5]. In this respect, CTSEdeficient mice exhibit markedly increased susceptibility to both gram positive and gram negative bacterial infection, likely due to impaired regulation of bacterial elimination [5]. Given the apparent association of enhanced CTSE expression with hepatocellular carcinoma, we hypothesize that liver tumor promoting agents may function to enhance CTSE expression levels in liver and that increased proteolytic activity may underlie certain mechanistic aspects of the tumor promotion process. In the studies reported here, we identify CTSE expression in human hepatoctyes. In particular, microarray expression profiling analyses indicated that exposures to the classical liver tumor promoter, phenobarbital (PB), resulted in increased CTSE transcript levels within primary hepatocyte cultures from several individual donors, a result that was confirmed by quantitative RT-PCR experiments. Further, we demonstrate that the constitutive androstane receptor (CAR) acts as a transcriptional regulator of the human CTSE gene and that these events lead to PB-inducible increases in functional hepatic CTSE enzymatic activity. Materials and methods ChemicalsPhenobarbital sodium salt was obtained from UWMC Drug Services (Seattle, WA). Clotrimazole, meclizine and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). CITCO was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Cell cultureCO...
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