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S. Johansson, B. Husman, G. Norstedt, and G. Andersson


Recent studies have implicated the involvement of pituitary factor(s) in the regulation of hepatic epidermal growth factor receptor (EGF-R) levels. In the present study the possible role of GH as a regulator of EGF-R has been examined by measuring hepatic EGF-R mRNA and EGF binding in intact and GH-deficient rats and mice before and after administration of GH. Using a human EGF-R probe, 10·5 and 6·0 kb transcripts were detected in mouse and rat liver by Northern gel analysis. EGF-R mRNA was quantified by solution hybridization, and EGF binding determined by incubation of 125I-labelled EGF with a low-density membrane fraction.

Levels of hepatic EGF-R mRNA and binding of EGF in female rats were about two-thirds of those in male rats. GH-deficient (lit/lit) male and female mice had approximately 10 and 25% respectively of the levels of EGF-R mRNA and EGF binding of intact male and female mice. Furthermore, hypophysectomized rats exhibited a reduced level of EGF-R mRNA and EGF binding, corresponding to about 20% of the levels detected in intact male rats. When hypophysectomized male and female rats received recombinant human GH (hGH) either as intermittent injections or by continuous infusion using osmotic minipumps, the EGF-R mRNA and EGF binding levels increased to about half those of the intact male animals. No differences between intermittent or continuous administration of hGH on the induction of EGF-R mRNA or EGF binding could be seen. The correlation between mRNA and binding levels suggests regulation at a pretranslational level.

There was a reduction of EGF-R mRNA to female levels when intact male rats were given hGH. An additional reduction of EGF binding levels was seen in both intact male and female rats exposed continuously to GH. This may indicate additional translational and/or post-translational regulatory mechanisms.

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C. Möller, P. Arner, T. Sonnenfeld, and G. Norstedt


In this study a solution hybridization assay was evaluated for its application to the measurement of levels of specific mRNAs. The evaluation included parameters such as incubation time, hybridization stringency and probe concentration/structure. Both short (50 bases derived from synthetic oligonucleotides) and long (125–147 bases) RNA probes, derived from cloned sequences, could be used to obtain quantitative information on specific mRNA species.

The solution hybridization assay was used to compare the levels of insulin-like growth factor-I (IGF-I) and IGF-II mRNAs in various rat and human tissues. In the rat the liver was the main source of IGF-I mRNA (approximately 400 molecules/cell), but significant levels were also found in extrahepatic tissues such as fat and muscle (3–50 molecules/cell). Human liver contained approximately 100-fold less IGF-I mRNA than rat liver. Human fat, muscle and placenta contained levels of IGF-I mRNA (2–8 molecules/cell) similar to those in the liver. Levels of IGF-II mRNA in rat and human tissues were similar, in that the expression was greatest in the placenta (approximately 200 molecules/cell). Species differences were evident, however, since human liver and fat contained significant amounts of IGF-II mRNA (15–20 molecules/cell), while the rat counterparts had almost undetectable levels. Young and old rats were used to examine the influence of age on the expression of IGF-I and GH receptor mRNAs in the liver. Levels of both IGF-I mRNA and GH receptor mRNA were found to decrease with age (2.8-fold and 1.7-fold respectively).

It is concluded that low levels of IGF mRNAs can be detected using the solution hybridization assay and that there are considerable species differences within and between tissues with regard to steady-state levels of IGF-I and IGF-II mRNAs.

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B Enberg, A Hulthén, C Möller, G Norstedt, and S M Francis


The mechanism by which GH transmits a signal to the nucleus via its membrane-bound receptor is unknown. To study this process, Buffalo rat liver (BRL), rat hepatoma (FAO), human hepatoma (HepG2) and Chinese hamster ovary (CHO) cell lines were transfected with GH receptor cDNA, and stable clones expressing GH receptor mRNA and protein were selected. From previous in vivo studies it is known that GH regulates the expression of the rat hepatic serine protease inhibitor (SPI) 2.1 gene at the transcriptional level. However, in all the cell lines tested, SPI gene expression was less than 0·2% of that measured in rat liver, and GH did not affect the expression of the endogenous SPI gene in GH receptor-expressing cells.

A 45 bp GH-responsive element (GHRE) has previously been defined in the SPI 2.1 gene. A construct containing six repeats of this GHRE was assembled with the thymidine kinase promoter and a chloramphenicol acetyl transferase (CAT) reporter gene. Transient transfection of this reporter gene resulted in GH stimulation of CAT activity in all GH receptor-transfected cell lines. A 33-fold induction was measured in the GH receptor-expressing BRL cells. Induction of CAT activity was observed after 8 h of GH treatment in the BRL-GHR638 cell line. Stable BRL cell lines expressing GH receptors with carboxy-terminal truncations (GHR380 and GHR454) did not show increased CAT activity on GH stimulation. This suggests that more than half of the intracellular domain of the GH receptor is required to activate transcription of the SPI 2.1 gene.

It is concluded that the use of GH receptor-expressing cell lines in combination with the GH-regulated reporter system described here provides a good model for studying intracellular signalling after GH stimulation.