Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer.

BACKGROUND: Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. METHODS: An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. RESULTS: At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. CONCLUSION: These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Inhibiting Eph kinase activity may not be "Eph"ective for cancer treatment.

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by "leaky" or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

Identification and characterization of specific binding proteins for growth hormone in normal human sera.

The well-recognized "big" forms (45,000-100,000 mol wt) of immunoreactive human growth hormone (hGH) in human serum have been reported to be random aggregates or formal polymers. However, we have now investigated the possibility that they are protein-bound forms. After incubation of monomeric 125I-hGH with normal serum, gel chromatography indicated a peak of bound 125I-hGH (at approximately 120,000 mol wt), which was completely displaced by excess unlabeled hGH. When serum alone was chromatographed two peaks of specific binding were subsequently detected, the major peak, eluting between 74,000 and 85,000 mol wt corresponded to the 125I-hGH-binding protein complex observed at approximately 120,000 mol wt. Using a mini-gel filtration system for separating bound from free hormone, binding of 125I-hGH by normal human serum was dependent on time (equilibrium was reached in 2 h at 21 degrees C), temperature (21 degrees C greater than 37 degrees C), Ca2+ and serum concentrations. Binding was reversible and highly specific for hGH, not being displayed by GH or prolactins from several species. Scatchard analysis revealed linear plots with an affinity (KA) of 0.32 +/- 0.06 X 10(9) M-1 (n = 7). Human serum with low endogenous hGH levels, when added to rabbit liver membranes, decreased the binding of 125I-hGH in this tissue in a dose-dependent manner. These data indicate that human sera contain a specific, high affinity binding protein for hGH and that this may account, at least in part, for the known size heterogeneity of GH in serum. Its effect on GH binding to target tissues may indicate a role for the binding protein in the regulation of GH action.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells.

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines.

While oestrogen, progesterone and growth factors, including growth hormone (GH), are clearly implicated in the pathogenesis of breast cancer, there is now evidence that the newly described ghrelin axis is also involved. The aims of this study were to investigate the expression of the ghrelin axis in breast cancer tissues and cell lines and to examine the effect of ghrelin on breast cancer cell proliferation in vitro. Ghrelin and its functional receptor, the growth hormone secretagogue receptor (GHSR) type 1a, were expressed in normal breast tissue and breast cancer specimens and cell lines. In contrast, the truncated GHSR type 1b isoform was exclusively expressed in breast carcinoma, suggesting that it has potential as a diagnostic marker. Ghrelin treatment significantly increases the proliferation of the MDA-MB-435 and MDA-MB-231 breast cancer cell lines in vitro. In addition, we have described the expression of a human preproghrelin isoform, exon 3-deleted preproghrelin, which encodes mature ghrelin plus a novel C-terminal peptide. Quantitative RT-PCR was used to demonstrate that this mRNA isoform is highly expressed in the MDA-MB-435 metastatic breast cancer cell line relative to the benign MCF-10A breast epithelial cell line. The unique C-terminal peptide of exon 3-deleted preproghrelin is expressed in the glandular epithelium of breast cancer tissues, with high-grade carcinoma exhibiting the strongest immunoreactivity. The data presented here suggest that components of the ghrelin axis may represent novel markers for breast cancer and potential therapeutic targets.

Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

Expression of the ghrelin axis in the mouse: an exon 4-deleted mouse proghrelin variant encodes a novel C terminal peptide.

Ghrelin, an n-octanoylated 28-amino-acid peptide capable of inducing GH secretion and food intake in humans and rats, is the endogenous ligand for the GH secretagogue receptor (GHS-R). Here we describe the expression and tissue distribution of the ghrelin/GHS-R axis in the mouse. We also report for the first time the identification of a novel mouse ghrelin mRNA variant in which there is a complete deletion of exon 4. Translation of this variant mRNA yields a protein containing ghrelin and an alternative C-terminal domain with a unique C-terminal peptide sequence. RT-PCR with primers specific for mouse ghrelin was used to demonstrate the mRNA expression of the full preproghrelin transcript and the exon 4-deleted variant in multiple mouse tissues. Real-time PCR was also employed to quantitate mRNA expression of ghrelin, the novel isoform and a previously reported ghrelin gene variant, ghrelin gene-derived transcript. We also demonstrated the tissue expression of the functional GHS-R in the mouse. Immunohistochemistry, employing antibodies raised against the mature human n-octanoylated ghrelin peptide and the putative C-terminal peptide encoded by the exon 4-deleted proghrelin variant, was used to demonstrate protein expression of ghrelin and the variant in multiple mouse tissues including stomach, kidney, and reproductive tissues. The coexpression of ghrelin and its receptor in a wide range of murine tissues suggests varied autocrine/paracrine roles for these peptides. Exon 4-deleted proghrelin, a novel mouse proghrelin isoform with a unique C-terminal peptide sequence, is also widely expressed in the mouse and thus may possess biological activity in these tissues.

Expression of growth hormone secretagogue receptor type 1a, the functional ghrelin receptor, in human ovarian surface epithelium, mullerian duct derivatives, and ovarian tumors.

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R), is a newly identified, ubiquitously expressed molecule that has been involved in a wide array of endocrine and nonendocrine functions, including cell proliferation. In this context, our group recently reported the expression of ghrelin and its functional receptor, the GHS-R type 1a, in the human ovary and testis as well as several testicular tumors. Ovarian malignancies, however, remain unexplored. Notably, a vast majority of ovarian tumors derive from the surface epithelium, which originates from the celomic epithelium. Considering the proven expression of ghrelin in the human ovary, and its reported effects in the proliferative activity of different cancer cell lines, we aimed at evaluating whether the ovarian surface epithelium as well as related reproductive structures and tumors are potential targets of ghrelin. To this end, expression of GHS-R1a was analyzed by immunohistochemistry in a panel of normal, metaplastic, and neoplastic tissues. Uniform GHS-R1a immunostaining was detected throughout the ovarian surface epithelium. Likewise, ciliated cells within the fallopian tube epithelium showed strong GHS-R1a expression. In contrast, other celomic derivatives, such as endometrium and endocervix, were negative for GHS-R1a immunoreactivity. In keeping with data from normal tissues, inclusion cysts from the surface epithelium expressed GHS-R1a. Similarly, benign serous tumors resembling fallopian tube epithelium were also positive, whereas serous cystadenocarcinomas showed GHS-R1a expression only in highly differentiated specimens. In contrast, other neoplasms, such as mucinous cystadenomas and cystadenocarcinomas, endometrioid tumors, clear cell carcinomas, and Brenner tumors, did not express GHS-R1a. In conclusion, our results demonstrate that the ovarian surface epithelium and related tumors are potential targets for systemic or locally produced ghrelin because they express the functional type 1a GHS-R. Considering the relevant role of the ovarian surface epithelium in key physiological events (such as ovulation) and neoplastic transformation of the ovary, the potential actions of ghrelin in those phenomena merit further investigation.

Focal induction of IGF binding proteins in proximal tubules of diabetic rat kidney.

Diabetes-associated kidney enlargement is associated with increased kidney insulinlike growth factor I (IGF-I) binding. IGF-I binds to the type I IGF receptor, which mediates most of its actions, and to specific binding proteins (IGFBPs), which modulate its actions. To explore the nature and extent of IGF-I binding in the kidney, in vitro autoradiography was used to map the distribution of IGF binding in control and diabetic rat kidney. Specificity studies were performed with increasing concentrations of unlabeled IGF-I, IGF-II, des(1-3)IGF-I (an IGF-I derivative that binds to receptors normally but with decreased affinity to binding proteins), and insulin. In control rats, diffuse binding was found throughout the kidney with increased density in the papilla. Binding specificity in the cortex and outer medulla was typical of the type I IGF receptor (IGF-I = des[1-3]IGF-I greater than IGF-II much greater than insulin). Binding in the outer medulla of diabetic kidney was typical of the type I IGF receptor. A marked focal increase in proximal tubular binding occurred in 13 of 22 postpubertal diabetic rats. Binding specificity of the proximal tubular binding was consistent with the predominance of an IGF binding protein (IGF-I = IGF-II greater than des[1-3]IGF-I with minimal displacement by insulin). Northern-blot analysis revealed increased IGFBP-1 and IGFBP-3 mRNA in cortical tissue from diabetic rats displaying increased proximal tubular binding but not from diabetic rats not displaying this phenomenon. As cell surface association of IGFBPs is linked to potentiation of IGF activity, a possible mechanism for potentiation of local IGF-I action may be provided.

Postreceptor signaling mechanisms for Growth Hormone.

Recent data have shed significant new light on the mechanisms involved in the transmission of a biologic signal by GH. Following ligand-induced dimerization of the GH receptor, multiple cascades are involved in GH signaling. These include activation of nonreceptor tyrosine kinases, in particular JAK2, which is a mechanism shared by the newly described cytokine receptor superfamily. Furthermore, several classic pathways (for example, guanine-nucleotide-binding proteins and protein kinase C), shared by numerous hormones, growth factors, and neurotransmitters, are also involved in many of the actions of GH. The interrelationships between the various signaling pathways for GH have not yet been fully defined. This review briefly summarizes the current state of knowledge with respect to the processes involved in the effects of GH in target cells.

Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors.

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization of a growth hormone-specific receptor from rabbit liver.

Membrane preparations from rabbit liver, known to possess GH-specific binding sites, have been solubilized with Triton X-100 and the binding characteristics of [125I]-human GH (hGH) and [125I]-bovine GH (bGH) subsequently studied. Specific binding of the hGH and bGH by the solubilized preparation was demonstrated of bound and free hormone by either polyethylene glycol precipitation or by Sephadex G-100 chromatography. Binding of hGH was both rapid and reversible and was displaced only by other growth hormones (bovine and ovine) and not by lactogenic hormones (ovine and human prolactins, human placental lactogen). As shown by Scatchard analysis specific binding of [125I]-bGH exhibited a lower binding affinity and capacity than did [125I]-hGH. Overall, the characteristics of the binding reaction for hGH were not significantly different from those reported for the particulate membrane preparation. The solubilization process did not appear to alter the binding protein(s) therefore, and permits a further study of the isolation, purification and properties of the binding protein(s) itself.

Identification of a specific inhibitor of nonsuppressible insulin-like activity in a partially purified human serum fraction.

During purification of low molecular weight (MW) nonsuppressible insulin-like activity (NSILA) from Cohn fraction IV-I of human serum, a fraction from Sephadex G-75 chromatography was gel filtered on Biogel P-30 in 1% formic acid. NSILA activity was all eluted in "fraction III" (Kav 0.4-0.9) with a recovery (compared to applied activity of 216 +/- 21% (mean +/- SE, n = 6). To test the possibility that this increase was due to removal of an inhibitor, a series of mixing experiments was performed. Total inhibition of fraction III occurred on mixing with fraction II (Kav 0.1-0.4), which had no intrinsic activity of its own. Fraction I (Kav 0.01) had no effects. Inhibition by fraction II was dose dependent, nondialysable, partially heat sensitive (boiling, 15 min) and totally destroyed by trypsin. Estimations of the MW of the inhibitor are 16,000-18,000. The inhibitor was shown to be specific for low MW NSILA by inhibiting the stimulatory effects of an acid-ethanol extract of human serum but not insulin or the acid-stable high MW form of nonsuppressible insulin-like activity. Inhibition of NSILA was observed in both rat adipocyte (insulin-like) and costal cartilage (sulfation) bioassays. Lineweaver-Burk analysis suggested the inhibitor acted in a competitive fashion. These studies have demonstrated the presence of a specific inhibitor of NSILA in Cohn fraction UV-I of normal human serum. The identity and physiological role of the inhibitor are as yet unknown.

Tissue distribution, characterization, and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat.

The distribution of GH receptor (GHR) and GH-binding protein (GHBP) mRNAs in multiple rat tissues was examined by Northern blotting using a cDNA fragment encoding the common extracellular domain of the GHR and the serum GHBP. Both GHR and GHBP mRNAs [4.5 and 1.2 kilobases (kb), respectively] were present in liver, kidney, adrenal, heart, muscle, ovary, mammary gland, gastrointestinal tract, and adipose tissue, but were barely or not detectable in testis, thymus, or brain. These observations suggest that GH exerts direct effects across a broad spectrum of rat tissues. Nuclease protection analysis also confirmed the presence in extrahepatic tissues of a GHR mRNA with up to 50% of the cytoplasmic domain being identical in sequence to that of the hepatic GHR mRNA. This suggests, but does not prove, that different receptor classes with differing intracellular signalling mechanisms may not exist. It is also clear from our studies that liver was the most abundant source of the truncated (1.2 kb) mRNA and is, therefore, believed to be the primary site of GHBP synthesis. Also, more importantly, at least in the rat, it was only GHBP mRNA that was up-regulated to any extent during pregnancy (female vs. pregnant, P less than 0.001). No significant changes were observed in the abundance of the full-length (4.5 kb) GHR mRNA. This pregnancy-related change in GHBP mRNA was also accompanied by a comparable increase in the actual level of measurable serum GHBP (female vs. pregnant, P = 0.005). Little change was seen in hepatic membrane binding. These data suggest that the GHBP and GHR are both widely coexpressed, but that the expression is not always coordinately regulated. This raises the possibility that the GHBP and GHR may have distinct roles in the regulation of GH delivery and action.

Water-soluble hepatic growth hormone receptors: structural studies using gel chromatography and chemical cross-linking.

Gel filtration of a water-solubilized human GH (hGH)-binding protein from rabbit liver has indicated that the binding species as determined by polyethylene glycol precipitation, was of mol wt greater than or equal to 300,000. In contrast, gel filtration of 125I-hGH-binding protein complex revealed in addition a major binding species of mol wt approximately 100,000. This species was shown not to be detectable by polyethylene glycol precipitation. Further, this same binding protein was observed after incubation with 125I-bovine GH but not 125I-ovine PRL, suggesting that it was specific for the somatotrophic but not lactogenic receptor of pregnant rabbit liver. Covalent cross-linking studies using disuccinimidyl suberate and sodium dodecyl sulfate gel electrophoresis, under reducing and nonreducing conditions, revealed a predominant binding subunit of mol wt 57,000, as well as smaller amounts of a mol wt 124,000 species. These data indicate that the structure of the binding protein involves no intersubunit disulfide bonds and suggest that the primary hGH binding subunit of pregnant rabbit liver has a mol wt of 57,000 and exists naturally as a dimer (mol wt, 100-124,000) and perhaps larger oligomers of mol wt greater than or equal to 300,000.

Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture.

At 37 degrees C, cultured rat adipocytes bound [125I]human GH ([125I]hGH) rapidly, with binding being detectable within 1 min of incubation. The bound [125I]hGH was then internalized (within 10 min) and accumulated in the cell interior until a steady state was reached (by 60 min). At this time, where the rates of GH internalization, processing, and release are equivalent, 55% of total cell-associated [125I]hGH was intracellular. Internalization of [125I]hGH by acutely isolated (noncultured) adipocytes was preceded by a 20-min lag phase indicative of a temporary postbinding defect. The lag phase was not seen with cultured adipocytes. After preloading of [125I]hGH into the cell interior, cultured cells rapidly released [125I]hGH (t1/2 = 20-30 min) into the extracellular medium as both intact (25%) and degraded (75%) GH. The release of intact vs. degraded GH was distinguishable on the basis of kinetics and temperature dependence. In order to determine when internalized [125I]hGH entered a catabolic compartment, cultured adipocytes were incubated with [125I]hGH and the composition of intracellular GH was determined as a function of time. All [125I]hGH internalized during the first 20 min was intact. Between 20 and 30 min some of the internalized [125I]hGH entered a catabolic compartment and degradation products began accumulating within the adipocytes. Release of degraded [125I]hGH from cultured adipocytes began at 60 min. The processing of GH through the complete degradative pathway (binding, internalization, degradation, release) required a period of 1 h at 37 degrees C.

Processing of growth hormone by rat adipocytes in primary culture: differentiation between release of intact hormone and degradative processing.

Rat adipocytes in primary culture have been used to study the intracellular processing of GH. These classic target cells for GH have been shown to process GH through two pathways: a nondegradative pathway which resulted in the rapid release of intact GH, and a slower, degradative pathway which involved the degradation of GH and release of degraded ligand. Differentiation between the two pathways was on the basis of differences in their kinetics and temperature dependence. The present study has investigated the relative characteristics of the two pathways further. Incubation of [125I]human GH ([125I]hGH)-preloaded adipocytes with extracellular unlabeled hGH (400 ng/ml) resulted in an increase in the absolute amount of [125I]hGH released. The increased amount of [125I]hGH released was all intact. Extracellular, unlabeled hGH had no effect on the rate or amount of degraded [125I]hGH released. This suggests that the nondegradative pathway is sensitive to the number of internalized hormone-receptor complexes and that GH which is not immediately degraded or stored in the degradative pathway, is redirected and processed via the faster non-degradative pathway. Ammonium chloride (known to inhibit the lysosomal degradation of many polypeptide hormones) markedly inhibited the absolute amount of [125I]hGH released from preloaded adipocytes. This inhibition was due to an effect on the release of degraded [125I]hGH. NH4Cl had no effect on the rate or amount of intact [125I]hGH released. Finally, it was found that dinitrophenol and sodium fluoride (agents known to deplete cellular energy) inhibited the release of degraded GH but not intact GH suggesting that the degradative pathway involves an energy-dependent step, most likely the fusion of hormone-containing vesicles with the lysosomal membrane. The mechanism of release of intact hormone by energy-independent means is not yet known. These data indicate that the processing of GH by cultured rat adipocytes is complex and involves at least two independently regulated pathways, a predominant degradative route and a nondegradative route. Further studies are required to assess the possible roles of these pathways in the metabolic actions of GH in adipocytes.

Tissue distribution and regulation of insulin-like growth factor (IGF)-binding protein-3 messenger ribonucleic acid (mRNA) in the rat: comparison with IGF-I mRNA expression.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is the most abundant IGFBP in rat and human sera. The present study demonstrates the expression of the rat IGFBP-3 gene in a large number of tissues and coexpression, but not necessarily equal expression, with IGF-I mRNA. Tissues with a major abundance of IGFBP-3 were kidney, antrum of stomach, placenta, uterus, and liver. Changes in hepatic and renal levels of IGFBP-3 mRNA were analyzed after hypophysectomy (with and without GH treatment) and in the developing postnatal rat. These results were compared to changes in IGF-I mRNA levels under the same physiological conditions. Using S1 nuclease analysis, IGFBP-3 mRNA was present in the kidney and liver of 1-day-old rats and rose significantly in both organs by week 1. Thereafter, levels remained relatively constant, particularly in the liver. This is in marked contrast to the hepatic IGF-I pattern, which showed a continual rise up to 8 weeks. Hepatic IGFBP-3 gene expression was partially GH dependent, with IGFBP-3 mRNA levels falling (approximately 50%) after hypophysectomy and rising slightly after GH treatment. These changes were much less dramatic than those in IGF-I mRNA. In contrast, the renal levels of IGFBP-3 mRNA increased after hypophysectomy, (approximately 100%), but did not decrease with GH treatment. These data suggest that IGFBP-3 mRNA abundance is regulated differently in different tissues, and in at least some tissues is less sensitive to regulation than is IGF-I mRNA.

Cloning and characterization of the promoter for the rat insulin-like growth factor-binding protein-3 gene.

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the adult circulation, is produced by a wide range of cell and tissue types. IGFBP-3 appears to be regulated by transcriptional and/or posttranslational mechanisms in a species-, cell-, and development-specific manner. In vitro and in vivo studies suggest that a number of factors (e.g. cAMP, GH, insulin-like growth factor-I, epidermal growth factor, TSH, and FSH) can act as transcriptional regulators of IGFBP-3 in particular cell types. To address the mechanistic basis for these observations, we isolated the rat IGFBP-3 gene and began characterization and analysis of the hormonal regulation of its promoter. The rat IGFBP-3 gene is located within 2 adjacent EcoRI fragments spanning about 10 kilobases. Southern analysis indicated a single copy gene. A 1.18-kilobase fragment 5' to the translation initiation codon has been sequenced and showed 65% homology with the corresponding human IGFBP-3 sequence. The region between -100 and -1 bp relative to the transcription start site showed 85% homology. The transcription start site was 118 basepairs (bp) up-stream of the initiation codon, and a TATA box consensus was located 27 bp 5' to this CAP site. No CAAT box was present, but a CpG island was identified. Consensus sequences for a number of putative response elements (e.g. activating protein-2, insulin, TSH/insulin-like growth factor, and GH) were present within -700 bp of the CAP site. A series of 5'-truncated chloramphenicol acetyltransferase reporter constructs has been transfected into both COS-1 cells and the rat thyroid cell line FRTL-5. Both basal and hormonally responsive (TSH and phorbol ester) promoter activities have been localized within the first 472 bases of the promoter region. These data indicate that suitable transfected cell systems can be established in which additional investigations can be undertaken into the mechanisms of cell- and species-specific hormonal regulation of IGFBP-3 gene expression.

Hepatic binding of human and bovine growth hormones and ovine prolactin in the dwarf "little" mouse.

Binding of human (hGH) and bovine (bGH) GH and ovine PRL (oPRL) has been compared in liver membranes from GH-deficient dwarf "little" mice (lit/lit) and their normal-sized littermates (lit/+). Binding of [125I]hGH to lit/lit membranes was dependent on time, temperature, and membrane concentration and was reversible. Scatchard plots of the binding of [125I]hGH to male and female lit/lit and lit/+ membranes were linear, with no significant differences between binding affinities (overall mean +/- SE, 1.42 +/- 0.27 X 10(9) M-1; n = 24). The hormonal specificity of binding was complex, with hGH being displaced by both somatotropic (bGH) and lactogenic (oPRL) competitors, indicating the presence of a mixed population of receptors. This conclusion was supported by the specific binding of both [125I]bGH and [125I]oPRL to membranes from male and female lit/lit and lit/+ mice. No differences in the specific binding of [125I]bGH to any membrane type was observed, indicating that GH receptors were at normal levels in lit/lit mice despite their deficiency of pituitary and serum GH. A sex difference in hGH and oPRL binding was seen only in normal (lit/+) mice. Male and female lit/lit mice exhibited the same degree of binding as normal female mice. These studies have demonstrated that dwarf little mice have normal levels of hepatic GH and PRL receptors, with binding characteristics not different from those of normal mice. Thus, it would appear that the mechanism of regulation of GH receptors by GH itself is different in this animal model of GH deficiency than in the Snell dwarf mouse and the hypophysectomized rat, where GH receptor levels are very low or absent. The failure of lit/lit mice to grow normally despite normal levels of GH receptor raises questions regarding the site and mechanism of the growth defect in the little mouse.