A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.

Growth hormone (GH), brain development and neural stem cells.

A range of observations support a role for GH in development and function of the brain. These include altered brain structure in GH receptor null mice, and impaired cognition in GH deficient rodents and in a subgroup of GH receptor defective patients (Laron dwarfs). GH has been shown to alter neurogenesis, myelin synthesis and dendritic branching, and both the GH receptor and GH itself are expressed widely in the brain. We have found a population of neural stem cells which are activated by GH infusion, and which give rise to neurons in mice. These stem cells are activated by voluntary exercise in a GH-dependent manner. Given the findings that local synthesis of GH occurs in the hippocampus in response to a memory task, and that GH replacement improves memory and cognition in rodents and humans, these new observations warrant a reappraisal of the clinical importance of GH replacement in GH deficient states.

New insights into growth hormone receptor function and clinical implications.

Although used as a therapeutic for 50 years, it is only recently that the application of molecular techniques has provided a basis for understanding growth hormone's (GH) clinical actions. This article reviews progress in our current knowledge of the molecular mechanism of growth hormone (GH) receptor activation based on a number of physicochemical techniques, and documents insights gained into the means used by the activated GH receptor to control the expression of genes regulating growth and metabolism. These findings are related to disorders of short stature, and the therapeutic consequences are summarized.

New insights into growth hormone action.

It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma.

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Viral hepatitis? Which test should I order?

BACKGROUND: There are a multitude of viruses that may cause hepatitis. The laboratory diagnosis of viral hepatitis is important in order to plan immediate patient management, determine treatment choices and provide patient education in order to limit transmission of infections to others. OBJECTIVE: This article outlines laboratory investigations that may be routinely ordered to assist in determining the etiology of viral hepatitis and summarises some preventive and treatment strategies that may be adopted. DISCUSSION: Investigations to determine exposure to infection are routinely performed and include simple serological tests, while tests to follow the course of infection or response to treatment may involve newer molecular techniques, including polymerase chain reaction (PCR), genotyping and viral quantification.

Alterations in ciliary neurotrophic factor signaling in rapsyn deficient mice.

Rapsyn is a key molecule involved in the formation of postsynaptic specializations at the neuromuscular junction, in its absence there are both pre- and post-synaptic deficits including failure to cluster acetylcholine receptors. Recently we have documented increases in both nerve-muscle branching and numbers of motoneurons, suggesting alterations in skeletal muscle derived trophic support for motoneurons. The aim of the present study was to evaluate the contribution of target derived trophic factors to increases in motoneuron branching and number, in rapsyn deficient mice that had their postsynaptic specializations disrupted. We have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn deficient muscles compared with litter mate controls although those for NGF, BDNF, NT-3 and TGF-beta2 did not differ. We found that both the mRNA and the protein expression for suppressor of cytokine signaling 3 (SOCS3) decreased although janus kinase 2 (JAK2) did not change in the rapsyn deficient muscles compared with litter mate controls. These results suggest that failure to form postsynaptic specializations in rapsyn deficient mice has altered the CNTF cytokine signaling pathway within skeletal muscle, the target for motoneurons. This alteration may in part, account for the increased muscle nerve branching and motoneuron survival seen in rapsyn deficient mice.

Growth hormone (GH)-independent dimerization of GH receptor by a leucine zipper results in constitutive activation.

Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the growth hormone receptor (GHR) signaling domain. The entire extracellular domain of the GHR was replaced by the hemagglutinin-tagged zipper sequence of either the c-Fos or c-Jun transcription factor (termed Fos-GHR and Jun-GHR, respectively). Transient transfection of Fos-GHR or Jun-GHR resulted in activation of the serine protease inhibitor 2.1 promoter in Chinese hamster ovary-K1 cells to a level equal to that achieved by fully activated wild type GHR. Furthermore, stable expression of Jun-GHR alone or Fos-GHR and Jun-GHR together in the interleukin 3-dependent BaF-B03 cell line resulted in cell proliferation after interleukin 3 withdrawal at a rate equal to maximally stimulated wild type GHR-expressing cells. Activation of STAT 5b was also observed in Fos-Jun-GHR-expressing cells at a level equal to that in chronically GH-treated GHR-expressing cells. Thus, forced dimerization of the transmembrane and cytoplasmic domains of the GHR in the absence of the extracellular domain can lead to the constitutive activation of known GH signaling end points, supporting the view that proximity of Janus kinase 2 (JAK2) kinases is the essential element in signaling. Such constitutively active GH receptors may have particular utility for transgenic livestock applications.

Growth hormone and colorectal carcinoma: localization of receptors.

Growth hormone (GH) plays a crucial role in stimulating and controlling the growth, metabolism and differentiation of many mammalian cell types by modulating the synthesis of multiple mRNA species and a paracrine or autocrine mechanism of action has been proposed. These effects are mediated by the binding of GH to its membrane-bound receptor and involve a phosphorylation cascade that results in the modulation of numerous signaling pathways. To address the side/mode of action through which GH exerts its effects, a panel of well characterized monoclonal antibodies, directed against the hormone binding side of the receptor, was applied to immunohistochemically determine growth hormone receptor (GH-receptor) expression in poorly- moderate- to well differentiated col.orectal adenocarcinomas (n = 40) from the rectum, transverse-, ascending-, descending and sigmoid colons. Of five anti-growth hormone receptor monoclonal antibodies used, human GH- receptor specific Mab 263 consistently resulted in strong receptor expression in colorectal carcinoma tumour cells. Heterogeneity of immunoreactivity was found in primary and secondary tumour lesions with a variable range of positive cells. Staining was mainly intracellular, showing either a monotonous or granular pattern, with some nuclei also reactive. The presence of intracellular GH-receptors has been previously documented and is a result of endoplasmic reticulum and Golgi localization. Immunoreactivity in surface columnar cells, independent from pathological tissue, was weak to moderate. Epithelial cells from normal tissue, adjacent to tumour lesions, were of variable intensity. Goblet and mucous cells located at the crypt base immunostained faintly or were negative for the GH-receptors. Crypt base columnar cells strongly expressed the GH-receptor, but oligomucous cells were less reactive. In conclusion, this study indicates that receptor expression may be associated with malignancy of colorectal carcinoma and supports the hypothesis that GH may act locally in colorectal tissue. The demonstration of the presence of receptors for GH will be useful for site-specific studies of the evolution of gastrointestinal tract tumours, providing valuable information concerning cellular growth kinetics and tumour prognosis. It also raises questions regarding the administration of GH to cancer-induced cachexia patients and the possible oncogenic potential of the GH-receptor.

Insulin regulation of human hepatic growth hormone receptors: divergent effects on biosynthesis and surface translocation.

Insulin modulates the biological actions of GH, but little is known about its effect on human hepatic GH receptors (GHRs). Using the human hepatoma cell line HuH7 as a model, we investigated insulin regulation of total, intracellular, and cell surface GHRs and receptor biosynthesis and turnover. Insulin up-regulated total and intracellular GHRs in a concentration-dependent manner. It increased surface GHRs in a biphasic manner, with a peak response at 10 nmol/L, and modulated GH-induced Janus kinase-2 phosphorylation in parallel with expression of surface GHRs. The abundance of GHR messenger ribonucleic acid and protein, as assessed by RT-PCR and Western analysis, respectively, markedly increased with insulin treatment. To examine whether insulin regulates GHRs at the posttranslational level, its effects on receptor surface translocation and internalization were investigated. Insulin suppressed surface translocation in a concentration-dependent manner, whereas internalization was unaffected. Moreover, insulin actions on total GHRs and surface translocation were inhibited by PD98059 and wortmannin, respectively. In conclusion, insulin regulates hepatic GHR biosynthesis and surface translocation in a reciprocal manner, with surface receptor availability the net result of the divergent effects. The divergent actions of insulin appear to be mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, respectively.

Insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) immunoreactivity in normal and osteopetrotic (toothless, tl/tl) rat tibia.

Insulin-like growth factor-I (IGF-I) plays a major role in regulating cell growth. This study examined the immunohistochemical distribution of IGF-I and IGF-I receptor (IGF-IR) in tibias from normal and osteopetrotic (toothless, tl/tl) rats, following treatment with colony stimulating factor-1 (CSF-1). In normal rats, immunoreactivity for IGF-I and IGF-IR was detected in cells of the articular and epiphyseal cartilage, secondary ossification centres, zones of resting and proliferating chondrocytes and bone marrow. Bone marrow cells immunoreactive for IGF-I and IGF-IR were significantly reduced in the tl/tl rat (p < 0.001) compared with normal animals. Treatment of tl/tl rats with CSF-1 increased immunoreactivity for IGF-I and IGF-IR in bone marrow cells as well as the number of TRAP positive osteoclasts. This increase was the result of recruitment of a range of hematopoietic cell types, including eosinophils, polymorphs and a substantial number of monocyte-like cells demonstrating strong immunoreactivity to IGF-I/IGF-IR. The differences in relative immunoreactivity for IGF-I/IGF-IR by bone marrow cells in untreated and CSF-1-treated tl/tl rats indicate a CSF-1-dependent recruitment of cells bearing surface IGF-IRs which may be mediated by an increase in local or systemic IGF-I.

Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced transcription of egr-1 (early growth response factor-1) in 3T3-F442A preadipocytes.

In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis. Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (-624 to -250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.

A novel mutation affecting the interdomain link region of the growth hormone receptor in a Vietnamese girl, and response to long-term treatment with recombinant human insulin-like growth factor-I and luteinizing hormone-releasing hormone analogue.

A Vietnamese girl with Laron syndrome has been treated with recombinant human insulin-like growth factor-I for 4 yr from age 11.28 yr. Her height SD score increased from -6.3 to -4.7 without acceleration of bone age. Isolated breast development progressed despite pubertal suppression with luteinizing hormone-releasing hormone analogue, which was stopped after 3 yr because of growth deceleration. Facial coarsening was documented with serial photographs. Sequencing and in vitro analysis identified a homozygous base pair substitution in exon 6 of the proband's GH receptor (GHR), which changed amino acid 131 from proline to glutamine (P131Q) and disrupted GH binding. Both the P131Q-mutated human GHR and wildtype (wt) hGHR were transiently expressed in COS-1 cells, as demonstrated by Western blotting, but the P131Q-transfected cells did not bind 125I-hGH. Similarly, FDC-P1 cells transfected with wthGHR bound 125I-hGH with high affinity and proliferated in response to GH, whereas the P131Q hGHR cells did neither. In CHO-K1 cells cotransfected with wthGHR and the Egr-1 promotor linked to a luciferase reporter gene, GH evoked a 2.14 +/- 0.21-fold increase in luciferase activity, but there was no response in the cells carrying the P131Q hGHR mutation. From examination of the crystal structure of the GHR, we suggest that the P131Q mutation disrupts the interdomain link between the extracellular domains of the GHR, causing a conformational change that results in disruption of the GH binding site.

Cellular expression of growth hormone and prolactin receptors in human breast disorders.

Growth hormone (GH) and prolactin (PRL) exert their regulatory functions in the mammary gland by acting on specific receptors. Using isotopic in situ hybridization and immunohistochemistry, we have localized the expression of hGH receptor (hGHR) and hPRL receptor (hPRLR) in a panel of human breast disorders. Surgical specimens from adult females included normal breast, inflammatory lesions (mastitis) benign proliferative breast disease (fibroadenoma, papilloma, adenosis, epitheliosis), intraductal carcinoma or lobular carcinoma in situ, and invasive ductal, lobular or medullary carcinoma. Cases of male breast enlargement (gynecomastia) were also studied. In situ hybridization analysis demonstrated the co-expression of hGHR and hPRLR mRNA in all samples tested. Epithelial cells of both normal and tumor tissues were labelled. Quantitative estimation of receptor mRNA levels was regionally measured in areas corresponding to tumor cells and adipose cells from the same section. It demonstrated large individual variation and no correlation emerged according to the histological type of lesion. Receptor immunoreactivity was detected both in the cytoplasm and nuclei or in the cytoplasm alone. Scattered stromal cells were found positive in some cases, but the labeling intensity was always weaker than for neoplastic epithelial cells. Our results demonstrate the expression of the hGHR and hPRLR genes and their translation in epithelial cells of normal, proliferative and neoplastic lesions of the breast. They also demonstrate that stromal components express GHR and PRLR genes. Thus the putative role of hGH or hPRL in the progression of proliferative mammary disorders is not due to grossly altered levels of receptor expression.

Three cases of Anaerobiospirillum succiniciproducens bacteremia confirmed by 16S rRNA gene sequencing.

We describe three cases of Anaerobiospirillum succiniciproducens bacteremia from Australia. We believe one of these cases represents the first report of A. succiniciproducens bacteremia in a human immunodeficiency virus (HIV)-infected individual. The other two patients had an underlying disorder (one patient had bleeding esophageal varices complicating alcohol liver disease and one patient had non-Hodgkin's lymphoma). A motile, gram-negative, spiral anaerobe was isolated by culturing blood from all patients. Electron microscopy showed a curved bacterium with bipolar tufts of flagella resembling Anaerobiospirillum spp. Sequencing of the 16S rRNA genes of the isolates revealed no close relatives (organisms likely to be in the same genus) in the sequence databases, nor were any sequence data available forA. succiniciproducens. This report presents for the first time the 16S rRNA gene sequence of the type strain of A. succiniciproducens, strain ATCC 29305. Two of the three clinical isolates have sequences identical to that of the type strain, while the sequence of the other strain differs from that of the type strain at 4 nucleotides.

Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signaling.

Signal transduction by the growth hormone receptor (GHR) occurs through growth hormone (GH)-induced dimerization of two GHRs to form a trimeric complex. It is thought that dimerization alone is sufficient for signaling, since monoclonal antibodies (mAbs) against the extracellular domain of the GHR elicit proliferation of FDC-P1 cells transfected with a chimeric receptor comprising the extracellular domain of the GHR and the fibronectin and cytoplasmic domains of the murine granulocyte colony-stimulating factor receptor. We have screened 14 GHR mAbs for proliferative activity against characterized FDC-P1 and BaF-B03 cell lines stably expressing the full-length human, rabbit, or rat GHR, or the chimeric human GHR/granulocyte colony-stimulating factor receptor, and for transactivation of the c-fos promoter and STAT activation. With the chimeric receptor, eight mAbs were able to elicit proliferation, although there was no correlation between inhibition of hormone binding and agonist activity. In contrast, no mAbs were able to act as agonists with the full-length GHR FDC-P1 cell lines, although nine competed with GH for binding. A weak proliferative response was observed in the BaF-B03 cell lines with two of the mAbs (263 and 1C9), and the addition of anti-mouse F(ab)2 resulted in increased signaling in the hGHR BaF-B03 cell line to a plateau of 28 +/- 4% of the GH maximum for mAb 263. These data could indicate considerable stringency in the ability of mAbs to correctly dimerize the full-length GHR. However, the ability of mAb 263 to stimulate a mutant hGHR altered in the F'-G' loop of domain 2 was nearly abolished, concurrent with an increased affinity of this mAb for the receptor. Since the F'-G' loop undergoes a conformational change on GH binding and is necessary for full proliferative signaling, we propose that in addition to promoting receptor dimerization, mAb 263 may induce specific changes in receptor conformation similar to GH, which are required for the biological response.

Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells.

Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance.

Subpopulations of stromal cells from long-term human bone marrow cultures: ontogeny of progenitor cells and expression of growth hormone receptors.

Long-term culture of bone marrow derived stromal colony forming cells (S-CFC) in matrix and nutrient defined agar medium resulted in stromal cell colonies that pass sequentially through three distinct morphological stages: firstly, aggregated loose syncytium of round to avoid cells (stage I), a second developmental stage of large branching colonies in which the cells become enlarged, elongated with cytoplasmic projections forming a loosely anastomized network with adjacent cells (stage II), and finally cells become dissociated, loosing their long, thin cytoplasmic filaments and breaking their contacts with one another, but remain large and retain a bi-polar nature (stage III). Cells were also grown in liquid medium in a culture microenvironment closely resembling conditions of haemopoiesis in vitro. Using a panel of well defined monoclonal antibodies reactive against the rat, rabbit and human growth hormone receptors, this study found immunochemical evidence of the presence and localization of binding sites of growth hormone (GH) in the cell membrane and extra-nuclear Golgi area of long-term bone marrow derived human stromal cells in liquid and semi-solid nutrient agar mediums. GH-receptor immunoreactivity was present in small proliferating progenitor cells, myofibroblast-like cells, large reticular fibroblast cells, adipocytes and endothelial cells. Only MAb known to be reactive against human tissue resulted in strong immunoreactivity. The expression of GH-receptors not only on small proliferating, but also on the well differentiated cells, indicates a role for growth hormone on non-progenitor cells. GH-receptor immunoreactivity on differentiating and/or differentiated cells suggests that GH is also necessary for, or has a trophic function in differentiation. We propose that direct GH action is necessary not only for differentiation of progenitor cells as implied by the dual effector hypothesis, but also their subsequent clonal expansion, differentiation and maintenance.

The role of receptor dimerization domain residues in growth hormone signaling.

While there is a considerable amount of evidence that signal transduction by the growth hormone (GH) receptor requires receptor homodimerization, there has been no systematic study of the role of receptor dimerization domain residues in this process. In conjunction with the distances derived from the crystal structure of the hGH-hGH receptor (extracellular domain) complex, we have used a luciferase-based c-fos promoter reporter assay in transiently transfected Chinese hamster ovary (CHO) cells, and stable receptor expressing CHO cell populations to define the dimerization domain residues needed for effective signaling. In addition to alanine substitution, we have used both aspartate and lysine substitutions to allow us to provide evidence for proximity relations through charge complementation. Introduced cysteine substitutions were also used, but unlike the erythropoietin receptor, these were unable to generate constitutively active receptor. We conclude that serine 145, histidine 150, aspartate 152, tyrosine 200, and serine 201, but not leucine 146 or threonine 147 are required for effective signal transduction through the dimerization domain. This information may be valuable in designing small molecule antagonists of GH and other cytokines that block dimerization by binding to the dimerization domain.

Decreased growth hormone receptor expression in long bones from toothless (osteopetrotic) rats and restoration by treatment with colony-stimulating factor-1.

Growth hormone (GH) is known to regulate growth and development of skeletal tissues. This study examined the distribution of growth hormone receptor (GHR) expression in tibias from normal and osteopetrotic tl/tl rats. For normal 2 week-old rats, GHR expression was detected immunocytochemically in cells of the articular and epiphyseal cartilage, primary and secondary ossification centres, zone of resting cartilage and bone marrow. Within the marrow, GHR immunopositive cells were concentrated in the central cone and largely excluded from the zone of immature progenitors at the periphery. For the marrow haemopoietic compartment, GHR expression was almost restricted to the nucleus in large mononuclear cells, adipocytes and megakaryocytes. A population of small lymphocytelike cells in the marrow periphery expressed GHR on the plasma membrane. GHR was not detected in mature erythroid cells, macrophages, granulocytes, or osteoclasts. The expression of GHR was significantly reduced in bone marrow cells of the tl/tl rat (p < 0.001) compared with normal animals. Injection of recombinant CSF-1 into tl/tl rats every 48 hours for 2 weeks from birth restored GHR-positive cells to the central core of the marrow space. The most striking change was the appearance of substantial numbers of mononuclear cells expressing abundant GHR on the cell surface. We infer that these cells are a novel subset of CSF-1 responsive cells involved in bone resorption. The differences in relative expression of GHR by bone marrow cells in untreated and CSF-1-treated tl/tl rats suggests a CSF-1-dependent recruitment of cells bearing surface GHRs.