Position-specific Gene Expression in the Liver Lobule Is Directed by the Microenvironment and Not by the Previous Cell Differentiation State

Abstract

Mechanisms directing position-specific liver gene regulation are incompletely understood. To establish whether this aspect of hepatic gene expression is an inveterate phenomenon, we used transplanted hepatocytes as reporters in dipeptidyl peptidase IV-deficient F344 rats. After integration in liver parenchyma, the position of transplanted cells was shifted from periportal to perivenous areas by targeted hepatic ablations with carbon tetrachloride. In controls, transplanted cells showed greater glucose-6-phosphatase and lesser glycogen content in periportal areas. This pattern was reversed when transplanted cells shifted from periportal to perivenous areas. Transplanted hepatocytes in perivenous areas exhibited inducible cytochrome P450 activity, which was deficient in periportal hepatocytes. Moreover, cytochrome P450 activity was rapidly extinguished in activated hepatocytes when these cells were transplanted into the nonpermissive liver of suckling rat pups. In cells isolated from the normal F344 rat liver, cytochrome P450 inducibility was originally greater in perivenous hepatocytes; however, periportal cells rapidly acquired this facility in culture conditions. These findings indicate that the liver microenvironment exerts supremacy over prior differentiation state of cells in directing position-specific gene expression. Therefore, persistence of specialized hepatocellular function will require interactions with regulatory signals and substrate availability, which bears upon further analysis of liver gene regulation, including in progenitor and/or stem cells.

Ischemic Preconditioning Affects Long-Term Cell Fate through DNA Damage–Related Molecular Signaling and Altered Proliferation

“…13,14 Cell viability was determined by trypan blue dye exclusion immediately after isolation and after suspension culture in RPMI 1640 medium with 10% fetal bovine serum and antibiotics for 6 or 12 hours at 4 C. For oxidative stress, 1 Â 10 4 hepatocytes/cm 2 were cultured overnight in 24-well collagen-coated dishes in Dulbecco's minimal Eagle's medium, antibiotics, and fetal bovine serum, followed by addition for 1 hour of 50 to 100 mmol/L tert-butylperoxide. Reduction of 0.5 mg/mL thiazolyl blue dye (MTT) over 90 minutes at 37 C was analyzed by lysing intracellular formazan in acid isopropanol with quantitation as described previously.…”

“…For DPPIV and g-glutamyltranspeptidase (GGT), sections were fixed in chloroforme acetone and were stained as described previously. 14,15 For heme oxygenase 1 (HMOX1), sections were stained with anti-rat HMOX1 antibody (Dr. Irving Listowsky, Albert Einstein College of Medicine). For apoptosis, terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) was used (ApopTag peroxidase in situ apoptosis kit; EMD Millipore, Billerica, MA).…”

“…For apoptosis, terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) was used (ApopTag peroxidase in situ apoptosis kit; EMD Millipore, Billerica, MA). 14 In negative controls, terminal deoxynucleotidyl transferase was omitted. Color was developed with diaminobenzidine (K3467; Dako North America, Carpinteria, CA).…”

“…14,15,18,19 For liver repopulation, DPPIV À rats were preconditioned with 30 mg/kg retrorsine at 6 and 8 weeks of age, and two-thirds PH was performed 4 weeks later. 22 At 1 week after PH, 5 Â 10 6 hepatocytes were injected into spleen in 0.25 mL serum-free RPMI 1640 medium.…”

“…To examine these possibilities, we used rats deficient in dipeptidyl peptidase IV (DPPIV À ) as a model for studies of liver injury induced by IP, carbon tetrachloride (CCl 4 ), or oxidative stress, along with assays of hepatocyte engraftment and proliferation, 14,15,18,19 followed by dissection of relevant cell signaling pathways.…”