The growth hormone receptor exon 3-deleted/full-length polymorphism and response to growth hormone therapy in prepubertal idiopathic short children.

OBJECTIVE: The primary aim of the study was to evaluate d3-GHR as a possible cause of increased GH sensitivity in children with delayed infancy-childhood transition (DICT). The secondary aim was to investigate the impact of the GHR exon 3 deleted/full-length (d3/fl) polymorphism on GH treatment response in prepubertal children classified as having idiopathic short stature (ISS). DESIGN: Study subjects included 167 prepubescent longitudinally followed children classified as having ISS. Children were randomized to standard-dose GH treatment (33 µg kg(-1) day(-1)), to double-dose treatment (67 µg kg(-1) day(-1)), or to an untreated control group. Growth and metabolic outcome were evaluated at birth (n = 166), after one year of treatment (n = 59) and at adult height (n = 145). Genotyping of the GHR d3/fl polymorphism was performed using TaqMan SNP genotyping of tagSNP rs6873545. RESULTS: Birth and early growth data did not reach the predetermined level of statistical significance for difference between genotypes. Growth and IGF-1 response after one year of GH treatment did not differ between genotypes. IGFBP-3SDS was higher in untreated d3-GHR carriers than in untreated fl/fl individuals, whereas there was insufficient evidence for higher IGFBP-3SDS in treated d3-GHR carriers. Genotype did not explain the growth response to treatment, and no differences in heightSDS, height gain, or difference in height to midparental heightSDS between genotype groups were found at adult height. CONCLUSION: The common GHR d3/fl polymorphism is probably not a cause of DICT in children with ISS, and our results do not suggest that the d3-GHR genotype is associated with increased sensitivity to GH in children with ISS.

Construction of a soluble human GH-receptor/EGF-receptor hybrid and its activation by GH.

To develop a cell-free system that can be used to measure cytokine bioactivity we have designed a soluble hybrid molecule consisting of the extracellular domain of the GH-receptor (GHR) and the intracellular domain of the epidermal growth factor receptor (EGFR). A DNA construct encoding this hybrid-receptor was inserted into a baculoviral expression vector and expressed in Sf9-cells. Activation of the hybrid-receptor by ligand-induced dimerization can be measured as the incorporation of radiolabeled phosphate into a biotinylated tyrosine kinase peptide substrate. The kinase activity in samples stimulated with GH (10 ng/ml) increased 5-fold compared to samples without addition of GH. This is the first example of a functional hybrid-receptor where the transmembrane domain has been deleted. Our results suggest that such hybrid-receptors may be used for detection of GH and other cytokine-receptor activating substances in biological fluids.

Selective introduction of antisense oligonucleotides into single adult CNS progenitor cells using electroporation demonstrates the requirement of STAT3 activation for CNTF-induced gliogenesis.

We have developed a novel method in which antisense DNA is selectively electroporated into individual adult neural progenitor cells. By electroporation of antisense oligonucleotides against signal transducer and activator of transcription 3 (STAT3) we demonstrate that ciliary neurotrophic factor (CNTF) is an instructive signal for astroglial type 2 cell fate specifically mediated via activation of STAT3. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway induced only a transient increase in glial fibrillary acidic protein (GFAP) expression, and inhibition of this signaling pathway did not block the induction by CNTF of glial differentiation in progenitor cells. In addition we show that microelectroporation is a new powerful method for introducing antisense agents into single cells in complex cellular networks.

Growth hormone receptor interaction with Jak proteins differs between tissues.

Janus kinases (Jak) play an important role in the initial steps of cytokine receptor signaling. The specificity of the four members of the Jak family (Jak1, Jak2, Jak3, and Tyk2) for different cytokine receptors is not fully understood. Recent studies have indicated that a specific cytokine receptor can activate several Jak and that this may differ between tissues. The growth hormone receptor (GHR) is believed to interact predominantly with Jak2, but studies on cell lines have shown that it may also induce phosphorylation of Jak1 and Jak3. Little is known about the interaction between the GHR and Jak in tissues. Our aim, therefore, was to elucidate which Jak interact with the GHR in two target tissues for GH, liver and adipose tissue. Western blot analysis showed that all four members of the Jak family are present in both rat liver and adipose tissue. However, coprecipitation using an anti-GHR antibody revealed that only Jak1 and Jak2 were associated with the GHR in these tissues. The relative amount of Jak1 and Jak2 that coprecipitated with the GHR differed markedly between tissues. In the liver, Jak2 dominated, and only a small amount of Jak1 was detected. In adipose tissue, at least one third of the coprecipitated Jak was Jak1. This is the first study to show that both Jak1 and Jak2 are associated with the GHR in rat tissues. The difference in the ratio between GHR-associated Jak1 and Jak2 in liver and adipose tissue may indicate that GHR signaling in different tissues could differ in terms of Jak specificity.

Prolactin (PRL) receptor gene expression in mouse adipose tissue: increases during lactation and in PRL-transgenic mice.

There are indications that PRL may exert important metabolic actions on adipose tissue in different species. However, with the exception of birds, the receptor has not been identified in white adipose tissue. The present study was designed to examine the possible expression and regulation of the PRL receptor (PRLR) in mouse adipose tissue. The long PRLR messenger RNA (mRNA) splice form (L-PRLR) and two short splice forms (S2- and S3-PRLR) were detected in mouse adipose tissue by RT-PCR. Furthermore, L-PRLR mRNA was detected by ribonuclease protection assay. Immunoreactive PRLR with a relative molecular mass of 95,000 was revealed by immunoblotting. Furthermore, L-PRLR mRNA expression was demonstrated in primary isolated adipocytes. In mouse adipose tissue, the level of L-PRLR mRNA expression increased 2.3-fold during lactation compared with those in virgin and pregnant mice. In contrast, in the liver the expression of L-PRLR increased 3.4-fold during pregnancy compared with those in virgin and lactating mice. When comparing the levels of L-PRLR expression in virgin female and male mice, no difference was detected in adipose tissue. However, in virgin female liver the expression was 4.5-fold higher than that in male liver. As PRL up-regulates its own receptor in some tissues, we analyzed L-PRLR expression in PRL-transgenic female and male mice. In PRL-transgenic mice L-PRLR expression was significantly increased in both adipose tissue (1.4-fold in females and 2.4-fold in males) and liver (1.9-fold in females and 2.7-fold in males) compared with that in control mice. Furthermore, in female PRL-transgenic mice retroperitoneal adipose tissue was decreased in weight compared with that in control mice. However, no difference was detected when comparing the masses of parametrial adipose tissue. Our results suggest a direct role for PRL, mediated by PRLR, in modulating physiological events in adipose tissue.

The growth hormone receptor associates with Jak1, Jak2 and Tyk2 in human liver.

Studies in cell lines have shown that Jak2 is the primary tyrosine kinase involved in signal transduction by the growth hormone receptor (GHR). In addition, growth hormone (GH) stimulates tyrosine phosphorylation of Jak1 and Jak3 in certain cell lines, while the effect on Tyk2 has not been analysed. We have investigated the expression of Jak proteins in human liver and analysed their interactions with the GHR. Using Western blot analysis and immunohistochemistry, we demonstrate that Jak1, Jak2, Jak3 and Tyk2 are present in human liver. Immunoprecipitation by an antibody against the GHR (Mab 263) followed by immunoblotting with specific antibodies against Jak proteins showed that Jak1, Jak2 and Tyk2 were associated with the GHR in this tissue. We conclude that the GHR associates with Jak1, Jak2 and Tyk2 in human liver. Although experiments in vitro indicate that Jak2 mediates GH signalling, our results open the possibility that other Jak proteins may influence GHR signalling in human liver.