Phenobarbital responsiveness as a uniquely sensitive indicator of hepatocyte differentiation status: requirement of dexamethasone and extracellular matrix in establishing the functional integrity of cultured primary rat hepatocytes.
We used a serum-free, highly defined primary hepatocyte culture model to investigate the mechanisms whereby dexamethasone (Dex) and extracellular matrix (ECM) coordinate cell differentiation and transcriptional responsiveness to the inducer, phenobarbital (PB). Low nanomolar levels of Dex and dilute concentrations of ECM overlay were essential in the maintenance of normal hepatocyte physiology, as assessed by cell morphology, LDH release, expression of the hepatic nuclear factors C/EBPalpha, -beta, -gamma, HNF-1alpha, -1beta, -4alpha, and RXRalpha, expression of prototypical hepatic marker genes, including albumin and transferrin, and ultimately, cellular capacity to respond to PB. The loss of hepatocyte integrity produced by deficiency of these components correlated with the activation of several stress signaling pathways including the MAPK, SAPK/JNK, and c-Jun signaling pathways, with resulting nuclear recruitment of the activated protein-1 (AP-1) complex. In Dex-deficient cultures, normal cellular function, including the PB induction response, was largely restored in a dose-dependent manner by reintroduction of nanomolar additions of the hormone, in the presence of ECM. Our results demonstrate critical and cooperative roles for Dex and ECM in establishing hepatocyte integrity and in the coordination of an array of liver-specific functions. These studies further establish the PB gene induction response as an exceptionally sensitive indicator of hepatocyte differentiation status.
Sidhu
Jaspreet S
JS
Department of Environmental Health, University of Washington, Seattle, WA 98105, USA.
Liu
Fei
F
Omiecinski
Curtis J
CJ
D000418:Albumins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003907:Dexamethasone / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins / Q000378:metabolism* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005809:Genes, Regulator / Q000187:drug effects / Q000502:physiology; D005819:Genetic Markers / Q000187:drug effects / Q000502:physiology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008954:Models, Biological; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000187:drug effects / Q000378:metabolism; D015533:Transcriptional Activation; D014168:Transferrin / Q000187:drug effects / Q000378:metabolism
D000418:Albumins; D016895:Culture Media, Serum-Free; D016326:Extracellular Matrix Proteins; D005819:Genetic Markers; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014168:Transferrin; D003907:Dexamethasone; D010634:Phenobarbital