Gene expression profiling of extracellular matrix as an effector of human hepatocyte phenotype in primary cell culture.
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.
Page
Jeanine L
JL
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, 101 Life Sciences Building, The Pennsylvania State University, University Park, PA 16802, USA.
Johnson
Mary C
MC
Olsavsky
Katy M
KM
Strom
Steven C
SC
Zarbl
Helmut
H
Omiecinski
Curtis J
CJ
D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016000:Cluster Analysis; D003094:Collagen / Q000494:pharmacology*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004338:Drug Combinations; D004777:Environment; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000502:physiology; D007797:Laminin / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011509:Proteoglycans / Q000494:pharmacology*; D012313:RNA / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D018076:DNA, Complementary; D004338:Drug Combinations; D016023:Integrins; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D012313:RNA; D003094:Collagen