Transcriptional and translational regulation of the splicing isoforms of the growth hormone receptor by glucocorticoids.
Glucocorticoids exert multiple effects on the growth hormone IGF-I axis. The GH receptor is expressed as an active, full-sequence molecule and a truncated, inactive one that lacks the intracellular signaling domain. We used the HuH7 human hepatoma cell line to investigate the effect of glucocorticoids on growth hormone receptor mRNA and protein expression. cDNA quantification was performed by Real Time Quantitative PCR. Growth hormone receptor expression at the protein level was studied by fluorescence-activated cell sorter analysis using specific polyclonal antibodies raised against the two isoform unique C-terminal sequences. Cells were treated with pharmacologic doses of dexamethasone (Dex 10 -7 - 10 -5 M) to assess its acute (1 hour or overnight) and chronic effects (7 days). Dex induced a dose-dependent increase of both the full (427 %) and truncated (180 %) mRNAs. At the protein level, Dex upregulated the full sequence more (183 %) than the truncated (126 %) protein. For a better understanding of this regulation system, cells were incubated with Dex 10 -6 M for 24 h in the absence or presence of a transcriptional inhibitor, actinomycin D, or a translational inhibitor, cycloheximide. Actinomycin D had no effect on Dex-induced upregulation, while cycloheximide blocked the truncated mRNA but not the full sequence mRNA upregulation, suggesting that this effect of glucocorticoids is a post-transcriptional event. After 7 days of chronic treatment, Dex induced a dose-dependent downregulation of the active receptor without any changes in the expression of the truncated isoform either at the mRNA or protein levels. We conclude that short-term glucocorticoid treatment results in an enhancement of the growth hormone receptor expression, while long-term treatment has a suppressive effect, through both transcriptional and translational mechanisms ultimately influencing both isoforms of the receptor.
Vottero
A
A
NICHD/PREB, NIH, Bethesda, MD, USA. vottero@nemmo.unipr.it
Kimchi-Sarfaty
C
C
Kratzsch
J
J
Chrousos
G P
GP
Hochberg
Z
Z
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007536:Isomerism; D008113:Liver Neoplasms / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D005938:Glucocorticoids; D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D003907:Dexamethasone