Marie-Catherine Postel-Vinay

Growth hormone can act as a cytokine controlling survival and proliferation of immune cells: new insights into signaling pathways

While growth hormone (GH) is classically defined as a peptide hormone, recent evidence supports a role for GH acting as a cytokine in the immune system under conditions of stress, counteracting immunosuppression by glucocorticoids. Lymphoid cells express the GH receptor, which belongs to the cytokine receptor superfamily, and GH can be produced by immune tissues, suggesting an autocrine/paracrine mode of action of GH. GH can act as a cytokine, promoting cell cycle progression of lymphoid cells and preventing apoptosis. These effects of GH were shown to be mainly mediated by the PI-3 kinase/Akt pathway and the transcription factor NF-kappaB. Expression of several cell cycle mediators, as well as Bcl-2, c-Myc and cyclin proteins were found to be regulated by GH. Survival of immune cells under conditions of stress was promoted by NF-kappaB. Thus, GH acts not only as a hormone but also as a cytokine, playing a potentially important role in immune system cells. Lastly, in this mini-review, we will discuss whether the discovery of these molecules in GH signaling pathways offers new insights into additional mechanisms of action whereby GH regulates apoptosis, proliferation and neoplastic transformation of cells of the immune system.

Improvement of diagnostic criteria in growth hormone insensitivity syndrome: solutions and pitfalls

A survey to identify children and adolescents with primary growth hormone insensitivity syndrome (GHIS) yielded 38 patients who were positively identified using a scoring system that included five criteria: height, basal growth hormone (GH), GH binding protein, basal insulin‐like growth factor 1 (1GF‐I) and the increase of IGF‐I after 4 days of GH administration (IGF generation test). Because of an overlap of the accepted and excluded groups with respect to points scored, an attempt was made to improve the scoring system. The new criteria were: height below –3 SDS, basal GH 4 mU/I or above, GH binding below 10%, basal IGF‐I and basal IGF binding protein‐3 (IGFBP‐3) below the 0.1 centile for age, an increase of IGF‐I in the IGF generation test less than 15 μg/1, and the increase of IGFBP‐3 less than 0.4 mg/1. With this scoring system, a clear separation between the accepted and the excluded groups was obtained. IGFBP‐3 was included to give the GH‐dependent parameters of the IGF system more weight and because the accuracy of IGFBP‐3 in the IGF generation tests was greater than the accuracy of IGF‐I, when the group of patients with GHIS was compared with a group of patients with GH deficiency. Unexpectedly, the IGF generation test was unable to segregate both cohorts completely. In the GHIS‐positive group, a significant correlation was found between basal IGF‐I or IGFBP‐3 levels corrected for age (SDS) and height SDS (r= 0.49, p < 0.002 and r= 0.61, p < 0.0001, respectively). There was also a significant correlation between the changes of IGF‐I or IGFBP‐3 in the IGF generation test and height SDS. That is, the patients with a slight response to GH were those with the least growth retardation, suggesting the existence of partial GH insensitivity.

Binding of human growth hormone to rat liver membranes: Lactogenic and somatotropic sites

Binding sites for human growth hormone have recently been identified in various tissues from various species [l-6] . In certain cases, such as with cultured human lymphocytes [5,6] and rabbit liver membranes [l-3] , these sites bind exclusively or predominantly growth hormones; in other cases, such as with rat liver membranes, they also bind hormones possessing lactogenic activity [3,4] . On the basis of these findings, the existence of two distinct types of hGH binding sites, ‘somatotropic’ and ‘lactogenic’, has been postulated. Earlier studies [2-41 have shown that, in liver membranes of female rats, the hGH binding sites have almost exclusively ‘lactogenic’ specificity; it was also demonstrated that the hGH binding capacity increases in female rats after puberty. With liver membranes of male rats, there was much less binding than with those of female animals, and the specificity of the binding sites was not assessed. In the present studies we demonstrate the existence, in rat liver membranes, of ‘somatotropic’ sites in addition to the ‘lactogenic’ sites. In membranes of male rats the somatotropic sites account for virtually all the binding sites for hGH, whereas in those of female animals, they represent only a small fraction of the hGH binding sites. The variation, with sex and age, of the somatotropic sites has been investigated

Growth Hormone Exerts Antiapoptotic and Proliferative Effects through Two Different Pathways Involving Nuclear Factor-κB and Phosphatidylinositol 3-Kinase1

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kB (NF-kB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21 and repressed cyclin-dependent kinase inhibitor p27. The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G1/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kB activation by GH, suggesting a possible link between PI 3-kinase and NF-kB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kB-dependent and -independent manner. (Endocrinology 142: 147–156, 2001) G INITIATES its wide variety of biological effects by binding to the GH receptor (GHR). After GHR dimerization, Jak-2 recruitment and activation promote its autophosphorylation and phosphorylation of GHR on tyrosine residues. In addition, a large number of other cellular proteins become tyrosyl phosphorylated in response to GH (1). Two signaling pathways have been identified to mediate the GH proliferative action: the mitogen-activated protein (MAP) kinases, designated extracellular regulated kinase-1 (Erk-1) and Erk-2, and signal transducers and activators of transcription. Molecules implicated in MAP kinase activation by GH were identified in 293 cells (2), and their involvement was confirmed in 3T3 F442A preadipocytes (3). The Shc/Grb2/SOS/Ras/Raf/MEK (MAP/Erk kinase)/MAP kinase pathway activated by GH led to the activation of several transcription factors, such as Elk-1, a member of the ternary complex factor family. GH induces phosphorylation of Elk-1 via Erk-1 and -2 and promotes the transcription of early response genes such as c-fos, egr-1, and junB (4). Furthermore, a correlation was demonstrated between insulin receptor substrate-1 (IRS-1) expression and GH-induced MAP kinase activation and proliferation in GHR-expressing 32D cells (5). Together, these data strongly suggest an important role for the MAP kinase pathway in the GH prolif-

Monkey Growth Hormone (GH) Receptor Gene Expression EVIDENCE FOR TWO MECHANISMS FOR THE GENERATION OF THE GH BINDING PROTEIN

The growth hormone receptor (GHR) cDNA was cloned from the liver of Rhesus macaque using polymerase chain reaction. As deduced from the nucleotide sequence, the mature GHR is a protein of 620 amino acids which presents 94.1% identity with the human receptor. The monkey GHR (mkGHR) expressed in 293 cells presented the expected specificity for a primate GHR and was able to transduce a transcriptional effect of GH. Human GH was able to activate tyrosine phosphorylation of both the tyrosine kinase JAK2 and the receptor in 293 cells co-transfected with mkGHR and JAK2 cDNAs. The GH binding protein (GHBP), the soluble short form of the GHR, was also present in monkey serum. Expression of the GHR cDNA in eucaryotic cells indicated that the GHBP can be produced by proteolytic cleavage of the membrane receptor. Northern blot analysis of GHR gene expression in different tissues allowed us to identify three different transcripts of 5.0 and 2.8 kilobase pairs and a smaller one of 1.7 kilobase pairs which could encode a GHBP. Rapid amplification of cDNA extremities (3′-RACE-polymerase chain reaction) was used to identify a cDNA encoding a protein in which the transmembrane and cytoplasmic domains of the receptor are substituted by a short sequence of 9 amino acids. This transcript was present in various tissues and could encode a GHBP as well, suggesting for the first time that two different mechanisms can coexist for the generation of the GHBP: proteolytic cleavage of the membrane receptor and a specific mRNA produced by alternative splicing.

Nutritional Status and Growth Hormone-Binding Protein

To study the effects of nutrition on growth hormone (GH) receptor status, the plasma GH-binding protein was evaluated under conditions of poor nutrition, anorexia nervosa, celiac disease, and obesity. Nine patients, aged 12-30 years, presented anorexia nervosa and had a mean weight loss of -19% of their initial weight at the time of the study. Ten patients with celiac disease, aged 3-14 years, had a mean height at -4.2 SD, and normal body weight for height. Fourteen severely obese children, aged 3-10 years, had a mean body mass index (BMI) of 25.7 +/- 0.9. GH-binding protein was low in patients with anorexia nervosa (16.8 +/- 1.9% of radioactivity) and in patients with celiac disease (16.1 +/- 2.2%) whereas it was very high in obese children (57.2 +/- 3.3%). A strong correlation was found between GH-binding protein and BMI. GH-binding protein was also correlated with insulin-like growth factor-1 plasma levels. Nutrition is an important regulator of the GH receptor/binding protein. The growth failure presented by undernourished children is associated with partial GH resistance and low GH receptor level. On the contrary, children with obesity and normal growth have a high GH receptor level.

The Proliferative and Antiapoptotic Actions of Growth Hormone and Insulin-Like Growth Factor-1 Are Mediated through Distinct Signaling Pathways in the Pro-B Ba/F3 Cell Line1

Biological actions of GH can be direct or mediated through insulin-like growth factor I (IGF-I). In the interleukin-3 (IL-3)-dependent Ba/F3 cell line, IGF-I induces cell cycle entry and proliferation. Ba/F3 cells expressing the rat GH receptor (Ba/F3 GHR cells) have been shown to escape from apoptosis and to proliferate under GH stimulation. Using the Ba/F3 GHR cell model, we sought to dissect the signals elicited specifically by IGF-I or GH. In contrast to IGF-I or IL-3, GH is able to maintain cell cycle entry of Ba/F3 GHR cells cultured for 7 days in the absence of serum. The presence of IGF-I messenger RNA was not detected by RT-PCR, and by RIA, IGF-I was not found in culture medium of Ba/F3 GHR cells, unstimulated or stimulated by GH. Moreover, the addition of an anti-IGF-I antibody that blocks IGF-I effects suggests that the actions of GH are not mediated by IGF-I, but appear to be direct. GH or IGF-I stimulation increased expression of cyclins A and D(1) with comparable kinetics, whereas expression of p21(waf1/cip1) seemed delayed in IGF-I-stimulated cells compared with that in GH-stimulated cells. Contrary to GH or IL-3, IGF-I did not induce nuclear factor-kappaB DNA-binding activity in Ba/F3 cells. Inhibition of nuclear factor-kappaB through expression of the mutant IkappaBalpha (A32/36) abrogated the GH-mediated survival signal, but did not result in alterations of the cell cycle in Ba/F3 GHR cells treated with IGF-I. Phosphatidylinositol 3-kinase was required for both survival and proliferative responses to IGF-I. Transfection of a dominant negative form of AKT (AH-AKT) resulted in suppression of IGF-I-mediated cell survival, but not of the antiapoptotic effect of GH in Ba/F3 GHR cells. Thus, GH and IGF-I are able to promote cell survival and proliferation through independent and different pathways in Ba/F3 cells.

Role of prolactin and growth hormone on thymus physiology.

Intrathymic T-cell differentiation is under the control of the thymic microenvironment, which acts on maturing thymocytes via membrane as well as soluble products. Increasing data show that this process can be modulated by classical hormones, as exemplified herein by prolactin (PRL) and growth hormone (GH), largely secreted by the pituitary gland. Both PRL and GH stimulate the secretion of thymulin, a thymic hormone produced by thymic epithelial cells. Conversely, low levels of circulating thymulin parallel hypopituitary states. Interestingly, the enhancing effects of GH on thymulin seem to be mediated by insulinlike growth factor (IGF-1) since they can be abrogated with anti-IGF-1 or anti-IGF-l-receptor antibodies. The influence of PRL and GH on the thymic epithelium is pleiotropic: PRL enhances in vivo the expression of high-molecular-weight cytokeratins and stimulates in vitro TEC proliferation, an effect that is shared by GH and IGF-1. Differentiating T cells are also targets for the intrathymic action of PRL and GH. In vivo inoculation of a rat pituitary cell line into old rats results in restoration of the thymus, including differentiation of CD4-CD8- thymocytes into CD4+CD8+ cells. Furthermore, PRL may regulate the maintenance of thymocyte viability during the double-positive stage of thymocyte differentiation. Injections of GH into aging mice increase total thymocyte numbers and the percentage of CD3-bearing cells, as well as the Concanavalin-A mitogenic response and IL-6 production by thymocytes. Interestingly, similar findings are observed in animals treated with IGF-1. Lastly, the thymic hypoplasia observed in dwarf mice can be reversed with GH treatment. In keeping with the data summarized earlier is the detection of receptors for PRL and GH on both thymocytes and thymic epithelial cells. Importantly, recent studies indicate that both cell types can produce PRL and GH intrathymically. Similarly, production of IGF-1 and expression of a corresponding receptor has also been demonstrated. In conclusion, these data strongly indicate that the thymus is physiologically under control of pituitary hormones PRL and GH. In addition to the classical endocrine pathway, paracrine and autocrine circuits are probably implicated in such control.

The GH Receptor and Signal Transduction

The primary structure of the growth hormone (GH) receptor in rabbits and humans determined by complementary DNA cloning revealed a single membrane-spanning protein of approximately 620 amino acids. A binding protein (bp) specific for GH has been identified in the serum of a number of species. In rabbits and man, a single 4.5-kb transcript has been identified that encodes the full-length receptor. In rats and mice, however, a smaller transcript produced by alternative splicing has been reported which is specific for the GHbp. Recently, the X-ray crystallographic structure of GH and its receptor have clearly shown the formation of an unusual homodimer, consisting of one molecule of GH and two molecules of hGHbp. Formation of the GH dimer is a necessary prerequisite for biological activity. The transcriptional activity of wild-type and mutant forms of GH receptor has been determined by co-transfecting the promoter of a GH-responsive gene, coupled to CAT along with the receptor cDNA. A 25-amino acid region near the transmembrane domain has been shown to be important for functional activity, although 8 amino acids (known as Box 1), rich in prolines, is essential. Alanine scanning mutagenesis has revealed that individual substitution of each residue is without effect, while the replacement of the last 2 or all 4 of the prolines abolishes activity. Finally, GH has been shown to induce rapid tyrosine phosphorylation of several proteins in cells expressing the receptor, one of which has recently been identified as the kinase JAK2 and another as MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone receptor signalling

The growth hormone (GH) receptor belongs to the superfamily of transmembrane proteins that includes the prolactin (PRL) receptor and a number of cytokine receptors. Two forms exist for the GH receptor: the membrane-bound form is a protein of 620 amino acid residues with a unique transmembrane domain; the GH-binding protein (GHBP), which is a soluble short form, is identical to the extracellular domain of the membrane receptor. In man and many other species, GHBP is believed to result from proteolytic cleavage of the membrane receptor; in human tissues, only one mRNA form of 4.5 kb encoding the full-length receptor has been detected. In rodents, GHBP is encoded by a specific mRNA of 1.2kb. Binding of GH to its receptor results in dimerization of the receptor, phosphorylation of the tyrosine kinase JAK2 and of the receptor, followed by a cascade of protein phosphorylations. Transcription factors belonging to the signal transducers and activators of transcription (STAT) family are involved in the effects of GH on the transcription of genes such as c-fos, serine protease inhibitor Spi 2.1 and beta-casein. GH is able to activate several STAT proteins including STAT1, 3 and 5. The JAK-STAT pathway is a main pathway for GH effects on gene transcription. Other signalling molecules are involved in GH action through different pathways: GH is able to activate mitogen activated protein (MAP) kinases; the hormone can utilize insulin receptor substrate-1 (IRS-1) and induces the association of phosphatidylinositol 3-kinase with IRS-1. Two main functional regions have been defined in the cytoplasmic domain of the GH receptor by testing the activity of mutant forms of the receptor in several systems: Box 1, a proline-rich sequence in the membrane proximal part, is necessary for all GH effects and is probably the region of association with JAK2; the C-terminal region is required for the induction of specific genes. Other molecules involved in the mechanisms of action of GH remain to be identified. As the same signalling pathways are used by many ligands, explanations for the specificity of the cellular effects have to be determined.