Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.

Examination of the role of CSF-1 independence in myc retrovirus induced monocyte tumorigenesis.

These studies were initiated as an attempt to estimate the number and nature of genetic changes that are required in addition to c-myc deregulation during monocyte tumorigenesis, and to determine whether the oncogenic changes that can be created in vitro resemble the actual changes that occur in vivo. We found that superinfecting myc-immortalized monocytes with a colony stimulating factor-1 (CSF-1) expressing retrovirus strongly promoted tumorigenesis, whereas granulocyte/macrophage-CSF (GM-CSF) and v-fms retroviruses, or the spontaneous acquisition of CSF-1 independence did so only moderately. In addition myc-infected monocytes isolated from mice at a stage prior to tumor formation are more tumorigenic than in vitro myc-immortalized monocytes, but they were still largely CSF-1 dependent, and were not as tumorigenic as reinnoculated tumor cells. In the simplest model only two oncogenic activations are required for monocyte/macrophage transformation, immortalization of the cells with c-myc and deregulation of the CSF-1 gene. However, not all mechanisms that result in loss of CSF-1 dependence lead to full tumorigenicity, suggesting that in vivo tumorigenesis may involve multiple secondary events including growth factor independence.

A functional polyadenylation signal is embedded in the coding region of chicken growth hormone receptor RNA.

A study of chicken GH receptor (cGHR) expression has revealed that the two major liver and skeletal muscle transcripts of the cGHR are developmentally expressed. Expression of the larger (4.7 kilobases) transcript increases with age. The smaller transcript (0.7 kilobases) is a truncation product, resulting from alternative usage of a functional polyadenylation [poly(A)] signal embedded in the coding sequence. The extent to which alternative cleavage and polyadenylation occur displays some tissue and sex specificity. Cleavage and polyadenylation occur down-stream of the AATAAA portion of the poly(A) signal (cGHR positions 304-309) and up-stream of a GT-rich sequence. The truncated transcript appears to be translated, based on its association in vivo with polyribosomes, although the physiological role of the putative protein product of this truncated transcript is as yet unknown. Three other avian species (quail, turkey, and duck) also show a polyadenylated truncation of the GHR message due to a poly(A) signal at the same location in the coding sequence. In cell culture expression, mutation of AATAAA to AACAAG prevents production of the truncated transcript. In a chimeric construct, the signal and neighboring sequence from the cGHR are sufficient to confer cleavage and polyadenylation upon the rat GHR, a gene that otherwise lacks the internal poly(A) signal. Alternative polyadenylation within the coding region of a structural gene is discussed as a heretofore unknown means of post-transcriptional regulation of a gene product.

Identification of the origin of the growth hormone-binding protein in rat serum.

GH specifically interacts with a soluble binding protein in serum. The GH-binding protein (GHBP) has been shown to contain the extracellular portion of the cell surface GH receptor (GHR). In rats and mice there is a unique mRNA that encodes the GHBP. This mRNA contains an alternatively spliced exon that replaces the transmembrane and intracellular domains of the receptor with a short hydrophilic carboxy-terminus of 17 and 25 amino acids, respectively, in rats and mice. In humans and other species no mRNAs encoding the GHBP have been identified, suggesting that the GHBP is in these cases a proteolytically processed GHR. In this study a monoclonal antibody (GHBP 4.3) was raised to the rat GHBP using as immunogen a synthetic peptide containing the unique C-terminal 17 amino acids that are not found in the rat GHR. As predicted, this antibody is specific to rat GHBP and does not cross-react with rat GHR. In combination with polyclonal and monoclonal antibodies that recognize both GHBP and GHR, this antibody was used to show that all, or most, of the GHBP in rat serum is indeed derived from the alternatively spliced GHBP mRNA and not from proteolytic processing of the GHR. In addition, endogenous rat serum GHBP was found to exist in two forms, with apparent mol wt of 52 and 44 kDa, arising from a single protein core of 32 kDa by extensive glycosylation. The concentrations of GHBP in male and female rat plasma were also estimated to be 300 and 575 ng/ml, respectively (measured in nonglycosylated GHBP equivalents).

One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR1, is sexually dimorphic and regulated by GH.

In the rat, alternatively spliced messenger RNA (mRNA) species encode GH receptor (GHR) and GH-binding protein (GHBP). Additionally, these mRNAs are alternatively spliced in the 5'-untranslated region, resulting in at least two classes of GHR and GHBP mRNA with distinct first exons and identical coding regions. These alternative first exons define two unique classes of GHR and GHBP mRNA (called GHR1 and GHR2). The GHR1 class of RNA is expressed only in the liver, is far more abundant in females than males, and is particularly abundant during pregnancy. GHR1 RNA is induced later in development than is GHR2. Additional classes of GHR and GHBP RNA may also exist. The genomic structure of the GHR1 first exon reveals a putative promotor region with no TATA box, CAAT box, or other sequence elements suggesting specific responses. An in vivo approach was used to investigate the regulation of GHR1 expression. In female rats, gonadectomy was found to reduce the percentage of steady state GHR1 RNA levels in the liver, whereas male castration resulted in an induction of GHR1 RNA. However, short-term treatment with estrogen or testosterone had little effect, suggesting that direct regulation of GHR1 expression may occur through effector(s) other than gonadal steroids. Hypophysectomy abolished GHR1 RNA in females. Treatment of hypophysectomized females and castrated males with GH by single injection did not significantly induce GHR1 RNA, but treatment by continuous infusion of GH did. Little change in non-GHR1 RNA levels was observed for each of these treatments. The results suggest that: 1) the sexual dimorphism observed in total GHR and GHBP RNA in rat liver is attributable to the sexually dimorphic expression of the GHR1 class of RNA; 2) the sexually dimorphic pattern of GH release in rats regulates the GHR1 class of RNA; 3) changes in GHR and GHBP expression observed on gonadectomy, hypophysectomy, GH treatment, and pregnancy are best attributed to GHR1 regulation; and 4) since GHR1 is liver specific, the observed increases in serum GHBP concentration in response to sex steroids, GH pattern, and pregnancy are likely to originate from the liver.

Ultrastructural colocalization of growth hormone binding protein and pituitary hormones in adenohypophyseal cells of the rat.

GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.

Cellular localization of the growth hormone binding protein in the rat.

In the rat a GH-binding protein (GHBP) exists that is derived from the GH receptor gene by an alternative messenger RNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxy terminus. Previous immunohistochemical studies detailing the localization of the GH receptor binding protein (BP) have used monoclonal antibodies that recognize extracellular region-specific epitopes common to both the GH receptor and GHBP. In this study we have used a monoclonal antibody (MAb 4.3) specific for the carboxy terminus of the rat GHBP to map its somatic distribution in the rat and have compared this distribution with that of a MAb recognizing both the BP and the GH receptor. A variety of tissues including the skeletal and muscular systems, the gastrointestinal tract and derivatives, the male and female reproductive systems, skin, central and peripheral nervous systems, and the 18 day gestation fetus were investigated. The distribution of GHBP immunoreactivity (MAb 4.3) was widespread and identical to that previously reported for the extracellular region of the GH receptor (MAbs 263 and 43). Immunoreactivity was both cytoplasmic and nuclear, indicating a possible role for the GHBP in intracellular function. GHBP immunoreactivity was predominantly associated with epithelial/endothelial cell subtypes and with mesenchymal elements such as muscle, chondrocytes, and osteoblasts, as previously described for the GH receptor extracellular region. We also report here the distribution of the GH receptor/GHBP in the kidney, cardiovascular, and respiratory systems. The most prominent immunoreactivity (MAbs 4.3 and 263) was associated with the distal convoluted tubules and collecting ducts of the kidney, with the epithelium and smooth muscle of the broncho-alveolar tree (including type I and II pneumocytes), with the Purkinje and myocardial fibers of the heart and with the endothelium and smooth muscle of blood vessels. Thus we have identified sites of direct GH action in the cardiovascular, renal, and respiratory systems. In conclusion, the extensive cellular distribution of the GHBP in the rat indicates physiological function(s) other than the binding of GH in plasma. Since GHBP mRNA has also been reported in a number of tissues, it may be that the GHBP is synthesized locally to mediate intracellular transport of GH and/or transcriptional regulation by GH in a variety of target tissues.

A rapid and sensitive binding assay for growth hormone releasing factor.

A binding assay for growth hormone releasing factor (GRF) has been developed using scintillation proximity assay (SPA) technology. Binding conditions were validated by several criteria. Equilibrium binding was attained within three hours at 22 degrees C in crude membrane fractions of HEK293 (293-P2) and GH4C1 (GH4-P1) cells transfected with the porcine GRF receptor. Saturation binding isotherms produced a KD of 296 pM and a Bmax of 4.7 pmols/mg membrane protein in 293-P2 cells. Cells not expressing the GRF receptor displayed no specific binding for the ligand. Competition binding curves produced the following rank order of potency for tested peptides: GRF analogs D-Ala2 = D-Arg2 (IC50 approximately 1 nM) >> PACAP > secretin, VIP (EC50 > 100 nM). Somatostatin (SRIF) binding was also adapted to the SPA format in a GH4C1 cell line transfected with the SRIF receptor subtype 2 (SSTR2) and in HEK293 cells transfected with the SRIF receptor subtype 5 (SSTR5). This assay represents a major improvement for binding measurements of these and potentially many other ligands for G-protein linked receptors, requiring no separation of bound from free hormone, allowing detailed pharmacological evaluations and enabling measurement of equilibrium binding in real time. In the 96-well format, it is suitable for high throughput screening.

Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues.

Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.

Tissue distribution and ontogeny of growth hormone receptor messenger ribonucleic acid and ligand binding to hepatic tissue in the midgestation sheep fetus.

While circulating GH concentrations are high in fetal life, skeletal growth is only slightly reduced by GH deficiency in utero. This has been explained by the relatively low binding of GH to fetal hepatic tissue, suggesting a lack of GH receptors (GHR). The GHR also recognizes ovine placental lactogen (oPL), which may have a specific role either as a fetal growth-promoting hormone or in regulating fetal metabolism. We investigated GHR expression and membrane binding of ovine (o) GH and oPL in various ovine fetal tissues and in maternal liver at different gestational stages. Singleton-bearing ewes at 51, 95, and 120 days gestation were killed. Liver, muscle, kidney, and brain samples were taken from the fetuses as well as placentas and livers from the ewes (n = 3/gestational age). GHR mRNA measured by Northern blot analysis was expressed at high levels in maternal liver at all gestational stages. A major band was observed at 4.4 kilobases (kb), and three minor bands were observed at 2.5, 1.7, and 8.1 kb. In fetal and placental tissue, only the 4.4-kb band was detected. This was present as early as day 51 of gestation in liver, kidney, lung, heart, and placenta and increased slightly with advancing gestation. On day 51, the expression of GHR mRNA in muscle was negligible, but by day 95, muscle expressed higher concentrations than fetal liver. Placental samples showed only a slight signal, with no change over the gestational range studied. In situ hybridization revealed the placental mRNA to be primarily associated with the decidua. Hepatic tissue showed specific binding to [125I]oGH and [125I]oPL from 51 days gestation. [125I]oPL showed a higher [51 days, 17.9 +/- 1.9% (mean +/- SEM); 95 days, 11.5 +/- 1.6%; 120 days, 16.3 +/- 0.9%] specific binding to the liver membranes than [125I]oGH (51 days, 2.1 +/- 0.7%; 95 days, 2.6 +/- 0.3%; 120 days, 3.5 +/- 0.4%). We conclude that oGHR are present as early as day 51 of gestation in various tissues, including liver. The message appears later in skeletal muscle than in liver. As the GH receptor binds oPL with higher potency than oGH, the parallel ontogenic changes in [125]oGH and [125]oPL binding in the liver do not support the presence of a PL receptor under independent developmental regulation.

Measurement of growth hormone-binding protein in the rat by a ligand immunofunctional assay.

We have developed a ligand immunofunctional assay (LIFA) for quantifying the circulating functional GH-binding protein (GHBP) in the rat. This two-site solid-phase assay uses a capture monoclonal antibody (4.3) specific to the hydrophilic C-terminal segment of rat GHBP (rGHBP), saturation of binding with human GH, and a detection system of rabbit antihuman GH polyclonal antibody and peroxidase-conjugated antirabbit immunoglobulin G antibody. Results were compared with Scatchard estimates derived by immuno-precipitation with monoclonal antibody 4.3. This assay was used to determine the GHBP levels in male and female rats and to investigate the diurnal properties and dynamics of GH and GHBP interaction in 15-min blood sampling over a 6-h period. The dynamic range of the rLIFA was 0.15-20.0 nM recombinant rGHBP, with intraassay and interassay coefficients of variation of 10.5% (n = 20) and 12.9% (n = 12), respectively. Serum GHBP levels determined by the rLIFA and those derived from Scatchard estimates were strongly correlated (n = 8; beta = 0.55; r2 = 0.89; P = 0.0005). Male rats had lower GHBP levels (6.5 +/- 0.7 nM; mean +/- SE; n = 14) than female rats (35.4 +/- 2.7 nM; n = 15; P = 0.0001). In the diurnal study, male rats had higher GH peaks (312.5 +/- 121.6 ng/ml; n = 7) than female rats (96.5 +/- 15.4 ng/ml; n = 9; P < 0.0001). In contrast to the pulsatile secretion of GH, GHBP levels in both sexes remained stable and showed no relationship to secretory pulses of GH. However, the GH bursts significantly altered the distribution of the GH-GHBP complex in male rats. By saturation and mass analysis, the greater GH pulsatile secretion in male rats resulted in occupancy of GHBP from less than 5% at nadir to about 80% at secretory peaks, in contrast to the less than 5-15% range of GHBP occupancy in female rats. In male rats, greater than 80% of GH at secretory peaks existed in the free form, whereas in female rats, 16-23% of GH existed in the free form during pulsatile secretion. In summary, the rLIFA shows good correlation to Scatchard analysis using an identical antibody. We conclude that this assay provides a rapid, sensitive, and accurate measurement of the circulating functional GHBP in the rat, and that it facilitates the study of GH and GHBP dynamics under a range of physiological conditions.

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Interaction of monoclonal antibodies with growth hormone-binding protein and its complex with growth hormone.

The properties of four independent lines of monoclonal antibodies (MAbs) specific to rat GH-binding protein (GHBP) were examined. Three MAbs, designated GHR-12, GHR-13 and GHR-16, were raised against the entire GHBP molecule. The fourth MAb, designated as GHBP4.3, was raised against the 17 amino acid residues at the C-terminal end of rat GHBP. The interaction of these antibodies with GHBP and their effect on GH binding to GHBP were analysed by conventional competition binding assays and surface plasmon resonance, i.e. with a Biospecific Interaction Analysis (BIAcore) instrument. The binding affinity of these MAbs to GHBP ranged from 29 nmol/l to 30.9 pmol/l. The pair-wise antibody binding to GHBP on BIAcore suggested that GHR-13 and GHR-16 recognized different antigenic determinants while part of the GHR-12 epitope might be shared with the other antibodies. The antibodies inhibited the interaction of GH with GHBP in the competition binding assay. However, in sequential binding on the BIAcore instrument, they were able to bind GHBP after its interaction with GH, indicating that the inhibition observed in the competition binding assay resulted from steric hindrance rather than direct interference with the GH-binding site of GHBP. The present findings, therefore, suggest that these antibodies are useful for investigating GHBP and its interaction with GH.

Thyroid glands: novel sites of growth hormone action.

Thyroid hormones inhibit the synthesis and release of GH in avian species. This may represent a feedback mechanism, since GH enhances the peripheral production of tri-iodothyronine (T3). The possibility that GH may also have direct effects on thyroidal function was therefore investigated. The basal and thyrotrophin-induced release of thyroxine (T4) from incubated chicken thyroid glands was not enhanced, however, in the presence of chicken GH. Contrarily, GH impaired T4 release in a dose-related way. These actions were probably mediated by specific receptors, since binding sites for radiolabelled GH were demonstrated on the plasma membranes of chicken thyroid glands. Expression of the GH receptor gene in these tissues was also demonstrated using a cRNA probe for the rabbit liver GH receptor, which specifically hybridized with RNA moieties of 4.4 kb, 2.7 kb and 1.0 kb. Moreover, reverse transcription of thyroidal RNA and its amplification in the presence of 3'- and 5'-oligonucleotide primers coding for the extracellular or intracellular domains of the GH receptor generated electrophoretically separable fragments of 500 bp and 800 bp respectively, as would be expected from analysis of the hepatic GH receptor cDNA sequence. Digestion of the 500 bp fragment with NcoI or EcoRI also produced moieties of expected size (350 bp and 150 bp or 325 bp and 175 bp respectively), as did BamHI or HaeIII digestion of the 800 bp fragment (yielding fragments of 550 bp and 275 bp or 469 bp and 337 bp respectively). Translation of the GH receptor mRNA was also indicated by the immunocytochemical demonstration of GH receptors in thyroid follicular and parafollicular cells, using a specific polyclonal antibody raised against the chicken GH-binding protein. These results therefore provide evidence, for the first time, of GH receptor gene expression in thyroid tissue and the translation of functional GH receptors in thyroid glands. These results also demonstrate differential effects of GH on the extracellular concentrations of T3 and T4, which may permit subtle regulation within the somatotroph-thyroid axis.

Hormonal regulation of the female enriched GH receptor/binding protein mRNA in rat liver.

At least two classes of mRNA for the GH receptor (GHR) and GH binding protein (GH BP) with different 5' untranslated first exons exist in the rat. One such class, the GHR1 is predominantly expressed in the liver of female rats. The hepatic expression of the GHR1 mRNA in normal and hypophsectomized rats of both sexes was studied by employing an RNase protection/solution hybridization assay. Normal females expressed 10-fold more GHR1 mRNA than males, hypophysectomy of female rats decreased the GHR1 level to that observed in male rats. Continuous GH treatment of hypophysectomized male and female rats for 6 days increased the expression of GHR1 mRNA to levels found in normal females, whereas intermittent GH treatment without effect. Bovine GH(bGH) induced the GHR1 expression in a time- and dose-dependent manner in primary cultures of adult rat hepatocytes as determined by solution hybridization. Maximal induction was achieved after 72 h of treatment with 50 ng bGH/ml medium. Female enriched expression of receptor and binding protein mRNAs raises the possibility that they participate in determining the ability of the liver to respond differently to the male and female GH secretory patterns. Our in vitro model utilizing cultures of primary adult rat hepatocytes could be used to address this issue as well as explore a hormonal interplay in regulation of GHR1 expression.

Generation of growth hormone binding protein by avian growth plate chondrocytes is dependent on cell differentiation.

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plates in situ and in cultured chondrocytes. In the epiphyseal growth-plate, chondrocytes at different stages of differentiation located at the proliferative and upper hypertrophic zones express the GH-R gene. In culture, addition of ascorbic acid facilitated chondrocyte differentiation as evaluated by decrease in collagen type II gene expression and increase in alkaline phosphatase activity and osteopontin gene expression. Both the ascorbic acid-treated and untreated chondrocytes expressed the gene coding for the chicken growth hormone receptor (cGH-R), but only the undifferentiated cells were capable of binding the hormone. This reduction in GH-binding resulted in alteration in GH-dependent regulation of the GH-R gene expression: only the undifferentiated chondrocytes responded to chicken GH (cGH) by down-regulation of the cGH-R gene expression. Chondrocyte differentiation induced by either ascorbic acid or retinoic acid was associated with the appearance of two growth hormone binding-proteins (GHBPs) in the culture medium with estimated MWs of 32 and 70 kDa, respectively. These GHBPs differ in their MW from the major GHBP found in chicken plasma. Chondrocyte GHBPs specifically bind [125I]cGH, which can be displaced by an excess of unlabeled cGH. The differentiation-dependent increase in the 70 kDa GHBP was observed also using specific chicken GHBP antiserum. Our data suggest that the reduction of the differentiated chondrocytes response to GH is due to differentiation-dependent loss of the extracellular domain of the GH-R, resulting in a lack of functional receptors on the cell surface and generation of GHBP.

Homogeneous pharmacologic and cell-based screens provide diverse strategies in drug discovery: somatostatin antagonists as a case study.

The development of high throughput, homogeneous pharmacologic and functional assays and their implementation in screening combinatorial libraries has increased the pace of stochastic drug discovery in recent years. New, noninvasive approaches involving radiometric proximity assays, an array of fluorescence-based technologies, and reporter gene constructs in mammalian and nonmammalian systems are providing more options for the selection of specific therapeutic targets. The increasing sophistication of homogeneous assay designs has also served as a springboard to better lead validation in drug discovery initiatives. This review examines these approaches in the context of new drug discovery strategies which combine a growing repertoire of high throughput screening techniques. The utility and importance of cell-based assays vis-a-vis pharmacologic (cell-free) assays is considered with specific reference given to yeast-based functional screens and homogeneous binding methodologies used to search for somatostatin antagonists and other potential growth hormone secretagogues. Also considered is the custom tailoring of specific chemical libraries which provide yet another level of target selectivity. The advantages and shortcomings of these various technologies are discussed in light of emerging trends toward higher throughput and nanoscale formats which are pushing the limits of measurable response at the cellular and molecular level.

Integration of the BALB/c ecotropic provirus into the colony-stimulating factor-1 growth factor locus in a myc retrovirus-induced murine monocyte tumor.

The development of tumors is thought to be a multistage process that requires an unknown number of genetic or epigenetic changes in a single cell. We previously described a murine monocyte tumor which was induced by a helper-free c-myc retrovirus and which also contained a DNA rearrangement at the colony-stimulating factor-1 (CSF-1) locus. The CSF-1 gene rearrangement gave rise to high levels of growth factor production and autocrine growth, implicating this secondary event in tumorigenesis. This CSF-1 gene rearrangement was found to be the result of integration of the BALB/c ecotropic retrovirus. Restriction enzyme mapping and DNA sequence analysis demonstrated that the novel provirus is identical to the BALB/c endogenous ecotropic provirus, indicating that infection was probably not due to the creation of a recombinant virus in vivo. The proviral integration site was mapped 3 kilobases 5' of the CSF-1 promoter and in an opposite transcriptional orientation, indicating that activation of CSF-1 expression was the result of the presence of the retroviral enhancer element.