ToxSci Advance Access published online on February 27, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm034
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Gene Expression Profiling of Extracellular Matrix as an Effector of Human Hepatocyte Phenotype in Primary Cell Culture
1 Center for Molecular Toxicology and Carcinogenesis, 101 Life Sciences Bldg, The Pennsylvania State University, University Park, PA 16802, USA 2 Department of Pathology, University of Pittsburgh, 200 Lothrop St, 450 BST, Pittsburgh PA 15261, USA 3 Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C1-015, P.O. Box 19024, Seattle WA, 98109-1024
* Corresponding author: Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 101 Life Sciences Building, University Park, PA 16802. Phone: 814 863-1625; Fax: 814-863-1696; Email: cjo10{at}psu.edu
Received January 5, 2007; revision received February 14, 2007; accepted February 22, 2007
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Abstract |
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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 (ECM; MatrigelTM). In this investigation, we used DNA microarrays, quantitative RT-PCR, immunoblotting, and cell morphology analyses to evaluate the biological responses imparted by MatrigelTM overlays on primary cultures of human hepatocytes from five independent donors. Although inter-individual variability in responses was evident, our results demonstrated that MatrigelTM additions typically improved hepatocyte morphology and differentiation character. Results from RNA profiling experiments indicated that MatrigelTM 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, MatrigelTM 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 MatrigelTM -treated cultures. These data support the concept that by mediating a reduction in cellular stress and stress-signaling pathways, together with and enhancement of gap junctional cell-cell communication, ECM functions mechanistically to facilitate the differentiation character of primary human hepatocytes in culture.
Key Words: Extracellular matrix; primary hepatocytes; human; cell culture; microarray; phenobarbital; integrins; connexins.
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