Divergent regulation of the class II P-glycoprotein gene in primary cultures of hepatocytes versus H35 hepatoma by glucocorticoids.
We investigated whether the glucocorticoid-mediated mechanisms controlling P-glycoprotein (pgp2 or mdr1b) are similar in normal hepatocytes compared with the H35 hepatoma cell line. In primary rat hepatocytes, dexamethasone (DEX) caused a dose- and time-dependent decrease in the amount of the pgp2 mRNA, which correlated with functional pgp2 expression (intracellular accumulation of [3H]vincristine). The suppression of pgp2 mRNA was specific for glucocorticoids because a representative estrogen and progestin were without effect, and DEX suppression of pgp2 mRNA could be reversed by cotreatment with an anti-glucocorticoid. DEX suppression of pgp2 mRNA appears to be posttranscriptional because following actinomycin D inhibition of new RNA synthesis, the pgp2 transcript disappeared at a faster rate in DEX treated versus untreated hepatocytes. Moreover, transcriptional activity of chloramphenicol acetyltransferase plasmids containing the pgp2 promoter in primary rat hepatocytes was unaffected by DEX treatment. Thus, suppression of pgp2 mRNA by glucocorticoids in primary hepatocytes is due to a decrease in pgp2 mRNA stability. In contrast, in the H35 hepatoma cell line, DEX dose dependently increased pgp protein and pgp2 mRNA, effects which parallel transcriptional activation of the pgp2 promoter. Activation of the pgp2 promoter was specific for glucocorticoids since a representative estrogen had no significant effect on transcription of the pgp2 promoter and RU486 blocked DEX activation of pgp2 transcription. Transcriptional activation of the pgp2 promoter was not due to a global up-regulation of basal transcription factors because DEX treatment did not activate either a herpes simplex virus thymidine kinase promoter or the SV40 early gene promoter. Further studies with a panel of pgp2 5' sequence deletion plasmids revealed that the minimal promoter (-66 bp) was not activated by DEX. In contrast, inclusion of sequences up to -177 bp restored DEX-dependent transcriptional activation. These are the first studies to demonstrate that glucocorticoids regulate pgp2 by different mechanisms in normal rat hepatocytes compared to the H35 hepatoma cell line.
Schuetz
J D
JD
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA.
Silverman
J A
JA
Thottassery
J V
JV
Furuya
K N
KN
Schuetz
E G
EG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005938:Glucocorticoids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism*; D015735:Mifepristone / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014444:Tyrosine Transaminase / Q000378:metabolism; D014750:Vincristine / Q000096:biosynthesis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005938:Glucocorticoids; D012333:RNA, Messenger; D015735:Mifepristone; D004958:Estradiol; D014750:Vincristine; D003907:Dexamethasone; D014444:Tyrosine Transaminase