Target Of Growth Hormone Action Research Articles

WIP1 is a novel specific target for growth hormone action

DNA damage repair (DDR) is mediated by phosphorylating effectors ATM kinase, CHK2, p53, and γH2AX. We showed earlier that GH suppresses DDR by suppressing pATM, resulting in DNA damage accumulation. Here, we show GH acting through GH receptor (GHR) inducing wild-type p53-inducible phosphatase 1 (WIP1), which dephosphorylated ATM and its effectors in normal human colon cells and three-dimensional human intestinal organoids. Mice bearing GH-secreting xenografts exhibited induced colon WIP1 with suppressed pATM and γH2AX. WIP1 was also induced in buffy coats derived from patients with elevated GH from somatotroph adenomas. In contrast, decreased colon WIP1 was observed in GHR-/- mice. WIP1 inhibition restored ATM phosphorylation and reversed GH-induced DNA damage. We elucidated a novel GH signaling pathway activating Src/AMPK to trigger HIPK2 nuclear-cytoplasmic relocation and suppressing WIP1 ubiquitination. Concordantly, blocking either AMPK or Src abolished GH-induced WIP1. We identify WIP1 as a specific target for GH-mediated epithelial DNA damage accumulation.

Growth Hormone Induces Transforming Growth Factor-Beta-Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria.

The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR-array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor-beta-induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH-dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN.

The Glomerular Podocyte as a Target of Growth Hormone Action: Implications for the Pathogenesis of Diabetic Nephropathy

Involvement of the growth hormone (GH) / insulin-like growth factor 1 (IGF-I) axis in the pathogenesis of diabetic nephropathy (DN) is strongly suggested by studies investigating the impact of GH excess and deficiency on renal structure and function. GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney. In the human a direct relationship has been noted between the activity of the GH/IGF-1 axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis. Conversely, states of GH deficiency or deficiency or inhibition of GH receptor (GHR) activity confer a protective effect against DN. The glomerular podocyte plays a central and critical role in the structural and functional integrity of the glomerular filtration barrier and maintenance of normal renal function. Recent studies have revealed that the glomerular podocyte is a target of GH action and that GH's actions on the podocyte could be detrimental to the structure and function of the podocyte. These results provide a novel mechanism for GH's role in the pathogenesis of DN and offer the possibility of targeting the GH/IGF-1 axis for the prevention and treatment of DN.

Growth Hormone (GH)-dependent Expression of a Natural Antisense Transcript Induces Zinc Finger E-box-binding Homeobox 2 (ZEB2) in the Glomerular Podocyte: A NOVEL ACTION OF GH WITH IMPLICATIONS FOR THE PATHOGENESIS OF DIABETIC NEPHROPATHY

Growth hormone (GH) excess results in structural and functional changes in the kidney and is implicated as a causative factor in the development of diabetic nephropathy (DN). Glomerular podocytes are the major barrier to the filtration of serum proteins, and altered podocyte function and/or reduced podocyte number is a key event in the pathogenesis of DN. We have previously shown that podocytes are a target for GH action. To elucidate the molecular basis for the effects of GH on the podocyte, we conducted microarray and RT-quantitative PCR analyses of immortalized human podocytes and identified zinc finger E-box-binding homeobox 2 (ZEB2) to be up-regulated in a GH dose- and time-dependent manner. We established that the GH-dependent increase in ZEB2 levels is associated with increased transcription of a ZEB2 natural antisense transcript required for efficient translation of the ZEB2 transcript. GH down-regulated expression of E- and P-cadherins, targets of ZEB2, and inhibited E-cadherin promoter activity. Mutation of ZEB2 binding sites on the E-cadherin promoter abolished this effect of GH on the E-cadherin promoter. Whereas GH increased podocyte permeability to albumin in a paracellular albumin influx assay, shRNA-mediated knockdown of ZEB2 expression abrogated this effect. We conclude that GH increases expression of ZEB2 in part by increasing expression of a ZEB2 natural antisense transcript. GH-dependent increase in ZEB2 expression results in loss of P- and E-cadherins in podocytes and increased podocyte permeability to albumin. Decreased expression of P- and E-cadherins is implicated in podocyte dysfunction and epithelial-mesenchymal transition observed in DN. We speculate that the actions of GH on ZEB2 and P- and E-cadherin expression play a role in the pathogenesis of microalbuminuria of DN.

IGF-I and GH: Potential use in gene doping

Gene doping is the term given to the potential misuse of gene therapy for the purposes of enhancing athletic performance. Insulin like growth factor-I (IGF-I), the prime target of growth hormone action, is one candidate gene for improving performance. In recent years a number of transgenic and somatic gene transfer studies on animals have shown that upregulation of IGF-I stimulates muscle growth and improves function. This increase in muscle IGF-I is not reflected in measurable increases in circulating IGF-I. Whilst the responses obtained in the animal studies would appear to give clear benefits for performance, the transfer of such techniques to humans still presents many technical challenges. Further challenges will also be faced by the anti doping authorities in detecting the endogenously produced products of enhanced gene expression.

Lack of an association between GHR exon 3 polymorphism and diabetic nephropathy in the Genetics of Kidneys in Diabetes (GoKinD) population

To the Editor: Growth hormone (GH) signalling via the GH receptor (GHR) forms the GH/GHR axis and plays an important role in metabolism. There is a genomic deletion of full-length exon 3 (d3 isoform) in the GHR gene. Previously, the GHRd3 isoform is found to be significantly associated with increased responsiveness to growth hormone [1]. Although the consensus is lacking in subsequent studies [2], GHRd3 has also been found to be associated with hypertension among stroke patients [3]. Recently, a report from our research group has demonstrated that the homozygosity for the GHRd3 allele may have the protective effect on the prevalence of type 2 diabetes [4]. Furthermore, evidence has suggested that the glomerular podocyte is a target for GH action, and the GH/GHR axis may play a role in the development of diabetic nephropathy (DN) [5, 6]. A recent study has shown that deficiency of GHR in mice (Ghr knockout) causes a reduction in systolic blood pressure and plasma renin levels, as well as an increase in aortic endothelial NO synthase (eNOS) levels [7]. We thus hypothesise that GHR exon 3 polymorphism may be involved in the pathogenesis of DN. To test this hypothesis, we have genotyped GHR exon 3 polymorphism in the participants selected from the Genetics of Kidneys in Diabetes (GoKinD) study [8]. This GoKinD cohort consists of 663 (351 male and 312 female) type 1 diabetes patients with DN (cases) and 622 (252 male and 370 female) patients without DN (controls). Among the patients, ~92% were of European descent, while ~8% were Americans of Black, Asian, Hispanic or Indian descent. All type 1 diabetes patients were diagnosed according to the World Health Organization criteria [9]. The patients with DN had persistent proteinuria or end-stage renal disease (not due to condition other than diabetes). The patients without DN had persistent normal albuminuria despite them having had type 1 diabetes for at least 15 years and never having been treated with ACE inhibitors. The detailed information of the GoKinD cohort is available at GoKinD database [10] and reference [8]. The study was approved by the local ethics committees and material transfer agreement was completed prior to the study. Genotyping of GHR exon 3 polymorphism was performed with a multiplex PCR protocol [1, 11] and the primers G1, G2 and G3 are recorded in GenBank with the accession number of AF155912. PCR variables were: initial denaturing at 94°C for 5 min; 35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 90 s; and a final extension at 72°C for 10 min. Genotypes with GHRfI and/or -d3 alleles were detected by 1% agarose gel electrophoresis. For genotyping quality control in the present study, the patients with and without DN were distributed randomly across PCR plates. Successful genotype calls were ≥95% and plates were randomly genotyped twice for duplication accuracy, which was calculated to be 98%. Furthermore, the sample sizes in both cases (type 1 diabetes with DN, n=633) and controls (type 1 diabetes without DN, n=622), which produced 1,266 case alleles and 1,244 control alleles, were sufficiently Diabetologia (2008) 51:2333–2334 DOI 10.1007/s00125-008-1145-2

Identification of the Glomerular Podocyte as a Target for Growth Hormone Action

GH excess in both the human and transgenic animal models is characterized by significant changes in blood pressure and renal function. The GH/GH receptor (GHR) axis is also implicated in the development of diabetic nephropathy. However, it is not clear whether GH's actions on renal function are due to indirect actions mediated via changes in blood pressure and vascular tone or due to direct action of GH on the kidney. We hypothesized that functional GHRs are expressed on the glomerular podocyte enabling direct actions of GH on glomerular function. Real-time PCR, immunohistochemistry, and Western blot analysis of murine podocyte cells (MPC-5) and kidney glomeruli demonstrated expression of GHR mRNA and protein. Exposure of both murine and human podocytes to GH (50-500 ng/ml) resulted in an increase in abundance of phosphorylated signal transducer and activator of transcription-5, Janus kinase-2, and ERK1/2 proteins. Exposure of podocytes to GH also caused changes in the intracellular distribution of the Janus kinase-2 adapter protein Src homology 2-Bbeta, stimulation of focal adhesion kinase, increase in reactive oxygen species, and GH-dependent changes in the actin cytoskeleton. We conclude that glomerular podocytes express functional GHRs and that GH increases levels of reactive oxygen species and induces reorganization of the actin cytoskeleton in these cells. These results provide a novel mechanistic link between GH's actions and glomerular dysfunction in disorders such as acromegaly and diabetic glomerulosclerosis.

HUMAN GROWTH HORMONE: RESEARCH AND CLINICAL PRACTICE

Overview of Human Growth Hormone: Research and Clinical Practice, Roy G. Smith. Part I Research. GHRP Historical Perspective: Basic and Clinical, C. Y. Bowers. The Design of Peptidomimetic Growth Hormone Secretagogues, Arthur A. Patchett and Matthew J. Wyvratt. Molecular Characterization of Growth Hormone Secretagogue Receptors, Andrew D. Howard, Scott D. Feighner, Roy G. Smith, and Lex H. T. Van der Ploeg. Central Actions of Peptide and Nonpeptide Growth Hormone Secretagogues, Suzanne L. Dickson and Gareth Leng. Pharmacology of Growth Hormone-Releasing Hormone and Its Peptide Analogs, David H. Coy. Somostatin Receptor Subtypes and Regulation of GH Secretion, Gurkeerat Singh, George Liapakis, and Terry Reisine. Mathematical Modeling of the GH Release Axis, David Brown, Elinor A. Stephens, Gareth Leng, and Roy G. Smith. Activation of the Human Growth Hormone Receptor: Structure and Function of the Ligand-Receptor Complex, Kenneth H. Pearce, and James A. Wells. The Central Nervous System as a Direct Target for Growth Hormone Action, Pamela A. Bennett and Iain C. A. F. Robinson. Part II Clinical Practice. Molecular Defects of the Growth Hormone Axis, Linda Anne DiMeglio, Paul L. Hofman, and Ora Hirsch Pescovitz. Growth Hormone: Relevance to Pediatrics, Barry B. Bercu and Howard J. Heinze. Growth Hormone Deficiency in Adults: The Rationale for Growth Hormone Replacement, Paul V. Carroll, Emanuel R. Christ, and Peter H. Soenksen. Growth Hormone and Osteoporosis, Howard B. A. Baum and Anne Klibanski. Growth Hormone and Syndrome X, Gudmundur Johannsson, Per Marin, Jan-Ove Johansson, and Bengt-Ake Bengtsson. Interactions Between Growth Hormone Secretion and Sleep, Eve Van Cauter and Georges Copinschi. Growth Hormone in AIDS, Kathleen Mulligan and Morris Schambelan. Clinical and Physiological Studies With Growth Hormone-Releasing Hormone, George R. Merriam and Fernando Cassorla. Clinical Use of Growth Hormone Secretagogues, Mark A. Bach and Glenn J. Gormley. Index.

Expression of growth hormone receptor by peripheral blood lymphocytes in children: evaluation in clinical conditions of impaired growth.

It is widely accepted that the haematopoietic system is a target of growth hormone action and that GH may act as a lymphokine. The expression of GH receptors (GHR) on human peripheral blood lymphocytes (PBL) has been reported previously in adult donors by dual fluorochrome flow cytometry. The aim of this study was to apply the cytofluorimetric method to the analysis of GHR expression on PBL in various human conditions characterized by different patterns of growth due to age or physiopathological conditions. PBL from 38 normal (control) subjects (7 newborns, 18 prepubertal children, 13 adults) were studied in order to provide age-related physiological data. Twenty-two short children (18 with idiopathic short stature, 4 with Ullrich-Turner syndrome) were studied to determine the expression of GHR in conditions of impaired longitudinal growth which may or may not require GH treatment. Analysis was performed using a fluorescein isothiocyanate (FITC)-conjugated antibody specific for the GHR (mAb263) and phycoerythrin (PE)-anti CD2 (T and natural killer cells) or PE-anti CD2 (B cells) in dual fluorochrome flow cytometric assays. Results were expressed as mean fluorescent intensity (MFI). Adult CD2+ coils exhibited a significantly higher GHR expression (MFI 347 +/- 40) than that expressed in children and newborns (MFI 285 +/- 36 and 299 +/- 41, respectively, P < 0.001). A significantly increased expression of GHR on CD2+ cells was also found in short children (MFI 330 +/- 42 vs 285 42- 36, respectively; P < 0.002), whereas Ullrich-Turner syndrome patients did not show any difference from their age and gender matched controls (254 +/- 52 and 288 +/- 40, respectively). A negative relationship was found between GHR expression on CD2+ cells and height-SDS (r - 0.54, P < 0.0001) or BMI (r - 0.4, P < 0.015) in controls and short children, independent of their GH secretory status. Expression of GHR and CD20+ cells was higher than that expressed on CD2+ cells in all subjects. No appreciable differences were found in the MFI levels of GHR expression on CD20+ cells either among the different age group controls or between short children or Ullrich-Turner syndrome patients. A significant downregulation of expression was shown in CD20+ (P < 0.008) but not CD2+ cells after 6 months of GH treatment in 6 short children who had a poor response to GH provocative tests. GH receptor expression on immune cells in non-syndromic short children appears to be inversely related to the linear growth expression and BMI of the subjects, contrary to findings with hepatic derived serum GHBP. This finding may reflect alternate exon usage in lymphoid cells, and indicates that GH has a distinctive role in the immune system.

Growth hormone and reproduction

It has become apparent that the reproductive and somatotrophic axes may often be interlinked, and that the ovary may be a target for growth hormone action. Within the ovary, there exists a complex regulating system involving growth factors, which promote gonadotrophin action, and their binding proteins, which modulate these actions. The realization of the existence of this fine-tuning system has prompted clinical trials of cotreatment with growth hormone and gonadotrophins, mainly directed at the poor responder to gonadotrophin stimulation. The effect of this treatment is dependent on the hormonal status and 'ovarian age' of the patient. Studies have delineated a possible role for additional growth hormone in those with a lack of growth hormone but it has little place in a normal system. Although an improvement in pregnancy rate has not yet emerged, more research should be forthcoming on the effect of growth hormone and growth factors on the follicle, oocyte and endometrium and their possible role in polycystic ovary syndrome.

Sex differences in binding of human growth hormone to isolated rat hepatocytes.

Since liver is a target for growth hormone action, binding of 125I-labeled human growth hormone to enzymatically isolated rat hepatocytes was studied. Specific binding was shown with hepatocytes from both male and female animals. There was a single class of receptors for human growth hormone on cells from males (affinity constant, Ka = 1.16 x 10(9) liters/mole; sites per cell, q = 6200). In males, bovine growth hormone was almost as potent as human growth hormone in displacing bound 125I-labeled human growth hormone, while ovine prolactin was about 1000 times less potent. Cells from female rats bound more 125I-labeled human growth hormone than cells from males. The cells from females contained at least two classes of receptors for human growth hormone. The receptor of highest affinity had the same affinity for human growth hormone as the single receptor found in males (Ka = 0.96 x 10(9) liters/mole). However, there were three to four times as many of these receptors per cell in females (q = 21,000). In females, bovine growth hormone and ovine prolactin were both about 20 times less potent than human growth hormone. Treatment of male rats with estrone produced cells that show the same binding characteristics as females. These results indicate that human growth hormone binds to a somatogenic receptor in hepatocytes from male rats. In females and estrogen-treated males, the receptors that bind human growth hormone recognize lactogenic as well as somatogenic properties. This suggests that the lactogenic and growth-promoting effects of human growth hormone in the rat are mediated by different receptors.