J. Anim. Sci. 2002. 80:1962-1969
© 2002 American Society of Animal Science
Inability of heterologous growth hormone (GH) to regulate GH binding protein in GH-transgenic swine1
D. Cifone*,
F. P. Dominici*,
V. G. Pursel
and
D. Turyn*,2
* Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (1113) Buenos Aires, Argentina, and
and
Gene Evaluation and Mapping Laboratory, USDA, ARS, Beltsville, MD 20705
2 Correspondence:
Departamento de Química Biológica (phone: 5411-4962-5506; fax: 5411-4962-5457; E-mail:
dturyn{at}qb.ffyb.uba.ar).
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Abstract
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Transgenic pigs expressing bovine, ovine, or human growth hormone (GH) structural genes fused to mouse metallothionein-I (mMT-bGH), ovine MT (oMT-oGH), or mouse transferrin (mTf-hGH) promoters were used to study the effects of GH on the regulation of serum GH-binding protein (GHBP). In the 14 transgenic pigs studied, circulating concentrations of heterologous GH ranged from 15 to 2,750 ng/mL. Using chromatographic methods, specific binding of GH was detected in serum from normal pigs but was undetectable in serum from all the transgenic pigs used, probably as a result of the high serum concentrations of heterologous GH present in these animals. Thus, to avoid interference of binding by high GH concentrations, serum samples were subjected to immunoblotting using a specific anti-GHBP antibody. A specific 54-kDa band was detected in normal pig serum as well as in sera from mMT-bGH, oMT-oGH, and mTf-hGH pigs. Additionally, sera from transgenic mMT-bGH pigs and their sibling controls were subjected to immunoprecipitation with an anti-GHBP antibody followed by immunoblotting with the same antibody. With this technique, we detected two specific bands of 53 and 45 kDa that could represent different degrees of glycosylation of GHBP. As determined by densitometric analysis the amount of GHBP in transgenic pig sera was similar to that detected in sera of the respective control animals. The amount of circulating GHBP remained unchanged even in oMT-oGH and mTf-hGH pigs that were exposed from birth to circulating concentrations of GH as high as 2,750 ng/mL. Thus, we conclude that heterologous GH do not act as modulators of the serum GHBP in pigs.
Key Words: Binding Proteins • Pigs • Somatotropin
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Introduction
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Growth hormone binding proteins (GHBP) have been identified in the blood of many species (Herington et al., 1986; Smith et al., 1989; Amit et al., 1992). The main GHBP activity in serum corresponds to the extracellular growth hormone (GH)-binding domain of the GH receptor (GHR; Leung et al., 1987; Smith et al., 1989), but there are differences in the generation of the GHBP among species. As in humans and rabbits, pig serum GHBP might be derived by enzymatic cleavage of the GHR (Leung et al., 1987; Baumann et al., 1994).
The physiological regulation of GHBP is partially understood. Serum concentration of GHBP increases during pregnancy in the mouse (Smith et al., 1988; Cramer et al., 1992; Ilkbahar et al., 1995). An increase of GH circulating concentration results in up-regulation of GHBP in several species (Amit et al., 1992; Sotelo et al., 1995; Gonzalez et al., 1999). The study of the effect of GH on GHBP in pigs has led to contradictory observations. The administration of homologous GH to pigs under different conditions resulted either in an increase (Mullins and Davis, 1992), a decrease (Davis et al., 1994), or in no significant changes of the circulating concentration of GHBP (Mullins and Davis, 1992; Evock-Clover et al., 1993; Combes et al., 1997). The present work was undertaken with the aim of clarifying this issue. We have worked with transgenic pigs that harbor three different genomic constructs, and as a consequence they express either bovine, ovine, or human GH. These animals appear to be useful in vivo models to study the effects of GH on GHBP concentration in peripheral circulation because they were exposed to supranormal levels of GH since birth. We have investigated the functional and molecular characteristics of the GHBP present in the serum of transgenic pigs overexpressing heterologous GH. In this report we present novel evidence that indicates GH does not regulate serum concentration of GHBP in pigs.
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Materials and Methods
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Chemicals
The reagents and apparatus for SDS-PAGE and immunoblotting were obtained from Bio-Rad (Richmond, CA). Tris, BSA fraction V, PMSF, aprotinin, Sephacryl S-200 HR, nitrocellulose membranes, and Kodak X-OMAT XAR 5 films were obtained from Sigma Chemical Co. (St. Louis, MO). Na125I was purchased from Dupont (Boston, MA). Enhanced chemiluminescence (ECL) reagents were from Amersham Corp. A polyclonal antibody raised in rabbits against recombinant bovine GHBP (anti-GHBP) which recognizes porcine GHBP (Davis et al., 1994), as well as recombinant bovine GHBP used as a positive control, were kindly provided by Monsanto Chemical Corp. (St. Louis, MO). Protein A-Sepharose 6 MB was purchased from Pharmacia (Uppsala, Sweden).
Animals
Transgenic pigs were derived from animals produced by microinjection of the bGH gene fused to the promoter sequence of the murine metallothionein-I (mMT) gene (mMT-bGH; Miller et al., 1989), the oGH gene fused to the promoter sequence of the ovine metallothionein (oMT) gene (oMT-oGH; Pursel et al., 1997), or the hGH gene fused to the promoter sequence of the murine transferrin (Tf) gene (mTf-hGH; Pursel et al., unpublished data) into the pronuclei of eggs from superovulated pigs. The presence of the transgene was detected by Southern blot analysis as described (Pursel et al., 1990). Normal siblings of transgenic pigs were used as controls. All animals used were female. The mMT-bGH pigs were all from the same line (first-generation offsprings) and were used at the age of 3, 4, and 5 mo (three transgenic and three control animals for each age group). The oMT-oGH pigs were first-generation offspring and were used at the age of 5 mo (three transgenic and three control animals). The mTf-hGH pigs were grand-progeny of a founder and were used at the age of 5 mo (two transgenic and two control animals). All animals used in this research were maintained under experimental protocols approved by the Beltsville Area Institutional Animal Care and Use Committee. Animals were provided diets (corn-soybean diet with 18% crude protein plus 0.25 lysine) on an ad libitum basis. Blood was collected after an overnight fast, and serum was separated and kept at -70°C.
Hormones
Bovine GH (bGH) and ovine GH (oGH) were obtained through the National Hormone and Pituitary Program, NIDDK, NIH, USA. Recombinant human GH (hGH) was kindly provided by BioSidus (Buenos Aires, Argentina).
Growth Hormone Radioimmunoassays
Circulating concentrations of bGH, hGH, and oGH were measured by specific RIA as described (Sotelo et al., 1993). All RIA measurements were performed in duplicate within the same assay. The intraassay coefficients of variations for each RIA ranged between 4 and 6% and the interassay coefficients of variations ranged between 7 and 9%. The sensitivity for these determinations was approximately 2 ng/mL for each hormone assayed (ovine, bovine, and human GH). The levels of porcine GH (pGH) in normal pigs were determined with a previously described RIA (Miller et al., 1989).
Determination of GHBP Activity in Serum
The general procedure for this determination has been adapted from previous research (Amit et al., 1992; Davis et al., 1992) and was described previously in detail (Sotelo et al., 1995). Human GH was iodinated using limiting amounts of chloramine T, as described previously (Aguilar et al., 1992) to a specific activity of 70 to 120 µCi/µg. This radioligand was used rather than pGH because it has been shown previously that specific binding to porcine GHBP is higher with 125I-labeled hGH than with 125I-labeled pGH (Amit et al., 1992). Pig sera (20 µL) were incubated overnight at 4°C with 10µL buffer (50 mM Tris [pH 7.4], 0.1% BSA) containing 40,000 cpm of [125I]hGH. A parallel incubation was carried out in the presence of an excess of unlabeled hGH (1 µg) to evaluate nonspecific binding. The entire incubation mixture was then subjected to exclusion chromatography on Sephacryl S-200 HR (1.5 x 12 cm). The elution buffer was 50 mM Tris, 0.2% bovine serum albumin, 150 mM NaCl (pH 7.5). Flow rates were 0.5 mL/min. The dissociation of the GH-GHBP complexes during elution was less than 8% (data not shown; Sotelo et al., 1995). For estimation of molecular weight, columns were calibrated with [125I]hGH (22 kDa) and [125I]BSA (66 kDa).
Immunoblotting
This determination was performed essentially as described (Dominici et al., 1999). Serum samples containing the same amount of protein (60 µg) were subjected to SDS-PAGE (10% bis-acrilamide) in a Bio-Rad miniature lab gel apparatus (Mini-Protean, Bio-Rad). Prestained molecular weight standards were myosin (209 kDa), ß-galactosidase (124 kDa), BSA (80 kDa), ovalbumin (49.5 kDa), carbonic anhydrase (34.8 kDa), and soybean trypsin inhibitor (28.9 kDa). Electrotransfer of proteins from the gel to nitrocellulose was performed for 1 h at 100 V in the Bio-Rad miniature transfer apparatus (Mini-Protean) as described by Towbin et al. (1979). Nonspecific binding sites were blocked by incubating the blots for 1 h at room temperature in blocking buffer (5% nonfat dried milk, 10 mM Tris, 150 mM NaCl, and 0.1% Tween). The nitrocellulose blots were incubated with the polyclonal anti-GHBP antibody (1:200) diluted in blocking buffer overnight at 4°C. After extensive washing, blots were incubated with horseradish peroxidase-linked second antibody followed by chemiluminiscence detection (ECL-Amersham). Band intensities were estimated by using Gel-Pro Analyzer software (Media Cybernetics). Bovine recombinant GHBP was used as a positive control. Serum samples from each animal used (14 transgenic and 14 control pigs) were analyzed individually.
Immunoblotting after Immunoprecipitation
Aliquots of serum from normal and transgenic pigs containing equal amounts of total protein (8 mg) were diluted to 500 µL with immunoprecipitation buffer (100 mM Tris (pH 7.4), 10 mM EDTA, 2 mM PMSF, and 0.1 mg/mL aprotinin) and incubated at 4°C overnight with the anti-GHBP antibody (4 µL). The ability of the antibody to precipitate GHBP was checked by measuring [125I]hGH binding to normal pig serum before and after being subjected to incubation with the antibody. Under these conditions, more than 90% of the GHBP activity present was precipitated. After incubation, 50 µL of protein A-Sepharose (50%, vol/vol) was added to the mixture. The preparation was further incubated with constant rocking for 2 h at 4°C and centrifuged at 3,000 x g for 1 min at 4°C. The supernate was discarded and the precipitate was washed three times with immunoprecipitation buffer. The final pellet was resuspended in 50 µL of reducing sample buffer, boiled for 5 min, and subjected to inmunoblotting using the anti-GHBP antibody as described above.
Statistics
The differences between the matched studies were analyzed by Student’s t-test, using the InStat statistical program by GraphPad software (San Diego, CA). The level of significance used was P < 0.05.
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Results
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Determination of GHBP activity by chromatographic analysis
Sera from normal and transgenic pigs were incubated with [125I]hGH for 24 h at 4°C and the resulting mixtures were purified on Sephacryl S-200 HR columns. The gel filtration profiles obtained are shown in Figure 1
. Two peaks of radioactivity were detected in normal pig serum, as was described already for pigs and other species (Amit et al., 1992; Sotelo et al., 1995). Peak I accounted for [125I]hGH bound to GHBP (or GHBP) and was almost completely abolished when an excess of unlabeled hGH was included in the incubation mixture (Figure 1: A, C, and E). Peak II corresponded to free [125I]hGH (Figure 1: A, C, and E). When sera from mMT-bGH, oMT-oGH, and mTf-hGH transgenic pigs were analyzed, only one peak of radioactivity corresponding to unbound [125I]hGH was detected (Figure 1: B, D, and F). Specific binding of [125I]hGH to serum GHBP expressed as a percentage of total [125I]hGH added to serum was 22 ± 6, 24 ± 7, and 20 ± 2% for 5-mo-old mMT-bGH, oMT-oGH, and mTf-hGH controls, respectively (Table 1). In contrast, when sera from 5-mo-old mMT-bGH, oMT-oGH, and mTf-hGH transgenic pigs were analyzed, the gel filtration profiles displayed only one peak of radioactivity that corresponded to the elution volume of [125I]hGH (Figure 1: B, D, and F). No differences were detected after incubation with an excess of unlabeled GH (Figure 1: B, D, and F), which indicated that no specifically bound radioactivity was present in the serum from any of the transgenic pigs lines analyzed.
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Figure 1. Growth hormone-binding protein (GHBP) activity in serum: representative profiles of gel filtration on Sephacryl S-200 HR (1.5 x 12 cm) of mixtures of serum from mMt-bGH (B), oMT-oGH (D), mTf-hGH (F) transgenic pigs or their respective controls (serum from matched normal pigs: A, C, and E) and [125I]hGH incubated for 24 h at 4°C in the absence ( ) or presence (•) of 1 µg unlabeled hGH. Peak I corresponds to [125I]hGH bound to GHBP and peak II corresponds to unbound [125I]-hGH.
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Table 1. Age, body weight, circulating concentrations of GH and [125I]hGH binding activity in serum of the 14 normal and 14 transgenic pigs used in the present study
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The absence of GH binding activity in transgenic pigs’ sera could be ascribed to a saturation of the serum GHBP by the heterologous GH present in high amounts in the blood of these animals. Thus, to further investigate the presence of GHBP activity in serum from transgenic pigs, serum samples from mMT-bGH, oMT-oGH, and mTf-hGH transgenic pigs were applied to Sephacryl S-200HR (1.5 x 12 cm). Fractions whose elution volume corresponded to the molecular weight of GH were discarded, and fractions whose elution volume corresponded to GHBP or GH-GHBP complexes (determined from the gel filtration profiles obtained with sera of normal pigs, Figure 1A
, C, and E) were collected, pooled, and applied to a longer Sephacryl S-200 HR column (1.5 x 50 cm). Growth hormone-binding activity was investigated in all of the fractions eluted from the second column, but the presence of GHBP activity was not detected in sera from any of the 14 transgenic pigs analyzed (data not shown).
Detection of GHBP by Immunoblotting
The presence of GHBP was investigated by immunoblotting analysis using an antibody against recombinant bovine GHBP that cross-reacts with pig GHBP (Davis et al., 1994). In contrast to the chromatographic analysis, this technique is not affected by the concentration of GH in serum. Results from these studies are shown in Figure 2A and demonstrated the presence of GHBP in the serum of normal and transgenic pigs. A specific band of about 54 kDa was detected in normal and in all transgenic pig serum (Figure 2A). Moreover, as determined by densitometric analysis, this GHBP was present in a concentration similar to that estimated for normal pig serum (Figure 2B). The relative abundance of GHBP in serum of mMT-bGH transgenic pigs was similar at 3, 4, or 5 mo of age (data not shown). Sera obtained from 5-mo-old mMT-bGH transgenic pigs and their corresponding controls were subjected to immunoprecipitation using the anti-GHBP antibody followed by immunoblotting with the same antibody. This procedure allowed the detection of two specific bands of 53 and 45 kDa in both normal and transgenic pig serum (Figure 3).
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Figure 2. Analysis of serum GHBP by immunoblotting: aliquots of serum containing equal amounts of protein were resolved on 10% SDS-PAGE, transferred to nitrocellulose, and detected with an anti-GHBP antibody as described in Materials and Methods. A: GHBP immunoreactivity in pig serum. Serum samples from 5-mo old mMT-bGH, oMT-oGH, and mTf-hGH transgenic pigs (T) were loaded along with samples obtained from their respective controls (normal pigs; N). Each assay was performed in duplicate. The arrows indicate the specific bands obtained whose molecular weight (54 kDa) was calculated by comparison with molecular weight standards (not shown) run in parallel. B: GHBP content in serum as determined by densitometric analysis. Data are the mean ± SEM of three different experiments performed in duplicate (n = 3/ group), except for mTf-hGH pigs and their corresponding controls, for which the average of two individual values is shown.
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Figure 3. Immunoblotting after immunoprecipitation: growth hormone-binding protein (GHBP) was immunoprecipitated from serum of 5-mo-old mMT-bGH transgenic (T) and control (N) pigs by incubation with an anti-GHBP antibody (anti-GHBP), transferred to nitrocellulose, and immunoblotted with the same antibody as described in Materials and Methods. The apparent molecular weights of the two specific bands obtained are indicated and were calculated by comparison with molecular weight standards run in parallel (data not shown).
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Discussion
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The regulation of GHBP and GHR is not fully elucidated (Baumann et al. 1994), and some controversial findings were reported in different animal studies (Turyn and Dellacha, 1978; Amit et al., 1992). We have demonstrated an important up-regulation of GHR in liver membranes and serum GHBP in transgenic mice that overexpress bGH, mouse (m) GH or hGH genes and exhibits different degrees of elevation of circulating GH concentrations (Aguilar et al., 1992; Sotelo et al., 1995; Gonzalez et al., 1999). In the pig, no correlation between serum GHBP and liver GHR has been found (Schnoebelen-Combes et al., 1996; Combes et al., 1997). However, the hypothesis that in this species, serum GHBP can be an indicator of total GHR status has not been excluded (Combes et al., 1997). It has been shown that membrane-associated GHBP is the principal component of GH binding in membranes prepared from liver of normal and pregnant mice (Camarillo et al., 1998; Frick et al. 1998; Gonzalez et al., 2001). This observation shows that the regulation of the GHBP-GHR system is complex and at the same time indicates that serum GHBP is a suitable target for the study of this system.
Growth hormone treatment is known to induce weight gain in pigs (Chung et al., 1985; Campbell et al., 1989), but it is not known whether this change is associated with alterations of plasma GHBP levels. Studies on the regulation of GHBP by GH in pigs have generated contradictory observations. Treatment of pigs with pig GH (pGH) for 7 wk resulted in an increase of serum GHBP levels, but in the same study it was found that when pigs were treated with GH for a shorter period (2 wk), no significant changes were found in GHBP levels (Mullins and Davis, 1992). However, Ambler et al. (1992) found that treatment of pigs daily with pGH for a similar period (12 d) resulted in an increase of GHBP levels. In another study in pigs, treatment with pGH using two different doses resulted in a reduction of serum GHBP (Davis et al., 1994), whereas other authors reported that serum GHBP concentration remained unaltered after pGH administration (Evock-Clover et al., 1993; Combes et al., 1997). Several variables could have influenced these results, among them the length of treatment, the dose of GH used, as well as the number of daily injections employed. The age or hormonal status of the animals studied might also be relevant (Ambler et al., 1992; Davis et al., 1994; Combes et al., 1997).
In the present study, chromatographic techniques have been used to measure functional (able to bind GH) GHBP. In transgenic pig serum, we could not detect functional GHBP activity, a fact that is probably a consequence of the high serum concentrations of GH present in these animals. Our results may reflect that in the serum from transgenic pigs with high circulating concentrations of GH, almost all the GHBP is bound to GH, and therefore totally saturated with the foreign GH.
The relative abundance of GHBP in transgenic pigs was analyzed by immunoblotting, a technique that proved adequate to characterize serum GHBP in other species (Hochberg et al., 1993; Gonzalez et al., 1999). Specific immunoreactivity with an anti-GHBP antibody was detected in the serum from every line of transgenic pigs investigated. We detected a single broad band that is present in an amount similar to that detected in normal pig serum. Its apparent molecular weight (54 kDa) correlates well with previous estimations (Davis et al., 1992). Moreover, the apparent molecular weight of the GHBP detected in the serum of the transgenic pigs was identical to the GHBP present in normal pig serum. To verify these results, sera from one of the lines of transgenic pigs we have used in this study (mMt-bGH) and their sibling controls were subjected to immunoprecipitation with the anti-GHBP antibody and the resulting precipitates were subjected to Western blotting analysis using the same antibody. The use of this technique allowed the detection of two specific bands of 53 and 45 kDa that could represent different degrees of glycosylation of GHBP, as was found previously in the rat (Frick et al., 1998).
In a previous report, we described a three- to four-fold increase in the circulating concentration of GHBP in transgenic mice expressing a bGH transgene, in which plasma bGH levels were elevated up to 40-fold (Sotelo et al., 1995). Similarly, in transgenic mice expressing high concentrations of mGH, we have also described an 8- to 12-fold increase in the concentration of serum GHBP (Gonzalez et al., 1999). Based on these findings, we speculated that continuous endogenous production of GH attained in GH-transgenic pigs would also stimulate high concentrations of GHBP in serum. Thus, the fact that GH failed to up-regulate serum GHBP in transgenic pigs, even under the same conditions that induced GHBP in mice, provides strong evidence to support the idea that such up-regulation is not present in this species.
In summary, in the present study we have demonstrated that in transgenic pigs the continuous presence high concentrations of heterologous growth hormones, such as bGH, oGH, and hGH, fails to induce a permanent up-regulation of serum GHBP in adult pigs. However, because we have investigated the activity and concentration of GHBP only in adult animals, the possibility that a transient up-regulation of GHBP may take place in these animals shortly after birth, at weaning, or at puberty cannot be excluded.
Our present results give further support to the conclusion that heterologous GH do not act as modulators of serum growth hormone-binding protein in pigs. Our data add to the previously raised hypothesis that the mechanism of GHBP up-regulation by GH is species-specific (Combes et al., 1997; Sotelo et al., 1995; González et al., 1999). The reason for this difference between species is unknown, but it could be related to the mechanism by which GHBP is generated: enzymatic proteolysis in pigs, rabbits, and humans vs alternate splicing of mRNA in rodents.
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Implications
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Previous studies in pigs that focused on the regulation of growth hormone binding protein levels by growth hormone provided conflicting results, and no clear relationship between them could be established. The present data indicate that the continuous presence from birth of high levels of heterologous growth hormones (bovine, ovine, or human) does not induce growth hormone-binding protein in pigs, results that are opposite to those found in mice under similar conditions. Previous research has demonstrated that homologous growth hormone is capable of inducing growth hormone-binding proteins in pigs; therefore, our data add to the previously raised hypothesis that the mechanism of growth hormone-binding protein up-regulation by growth hormone is species-specific.
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Footnotes
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1 The authors wish to thank A. F. Parlow, Pituitary Hormones and Antisera Center and NIDDKD, for providing bGH and reagents for bGH, hGH, and oGH radioimmunoassays. Daniel Turyn is Career Investigator from the National Research Council (CONICET) of Argentina. Support for these studies was provided by the University of Buenos Aires and CONICET (to DT). Fernando Dominici is a post-doctoral fellow from CONICET. 
Received for publication October 23, 2001.
Accepted for publication March 4, 2002.
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Abstract
Introduction
