ToxSci Advance Access originally published online on February 27, 2007
Toxicological Sciences 2007 97(2):384-397; 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
* Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, 101 Life Sciences Building, The Pennsylvania State University, University Park, Pennsylvania 16802
Department of Pathology, University of Pittsburgh, 200 Lothrop Street, 450 BST, Pittsburgh, Pennsylvania 15261
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop C1-015, PO Box 19024, Seattle, Washington 98109-1024
1 To whom correspondence should be addressed. Fax: (814) 863-1696. E-mail: cjo10{at}psu.edu.
Received January 5, 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 (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.
Key Words: extracellular matrix; primary hepatocytes; human; cell culture; microarray; phenobarbital; integrins; connexins.
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