Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line.
The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH-receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH-receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH-receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/intracellular domain of the human GH-receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH-BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution.
Mullis
P E
PE
Department of Paediatric Endocrinology, Children's Hospital, University of Bern, Inselspital, Switzerland.
Holl
R W
RW
Lund
T
T
Eblé
A
A
Brickell
P M
PM
D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D017077:Culture Media, Conditioned; D005786:Gene Expression Regulation / Q000187:drug effects*; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012260:Ribonucleases; D013696:Temperature; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013006:Growth Hormone; D012260:Ribonucleases; C051815:somatotropin-binding protein