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 gener ated 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 confir med by solution hybridisation methods. Thereafter, the cells were trea ted with rhGH at physiological (12.5, 25, 50 ng/ml) and supra-physiolo gical (150, 500 ng/ml) concentrations over the period of 48 h. At inte rvals, RNase protection assays were performed to determine GH-receptor /GH-BP mRNA levels, nuclear run-on assays were carried out to determin e 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 en coding the extracellular domain of human GH-receptor/GH-BP were identi cal to those previously detected using a probe for the sequences encod ing the transmembrane/intracellular domain of the human GH-receptor. I n 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-re ceptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulte d in a decrease of GH-BP released into the medium implying that enzyme s may be involved in the releasing mechanism. These data support the i dea 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 accura te reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP product ion is subject to r-haH-dependent post-transcriptional regulation, per haps 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 wit h caution.