0013-7227/91/1283-1238$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
Elevation of Growth Hormone (GH) and Prolactin Receptors in Transgenic Mice Expressing Ovine GH JACQUELINE M. ORIAN, KENNETH SNIBSON, JANET L. STEVENSON, MALCOLM R. BRANDON, AND ADRIAN C. HERINGTON* Department of Veterinary Preclinical Sciences (J.M.O., K.S., M.R.B.), The University of Melbourne, Parkville, Victoria 3052, Australia; and Prince Henry's Institute of Medical Research (J.L.S., A.C.R.), Monash Medical Centre, Melbourne, Victoria 3004, Australia
a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors. {Endocrinology 128: 1238-1246, 1991)
ABSTRACT. The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein l(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in
G
ROWTH HORMONE belongs to a family of polypeptide hormones which includes PRL and placental lactogen and whose members are related in their structure and function. Despite their relatedness, the different physiological effects of these hormones are mediated via distinct specific cell surface receptors (1). One aspect of elucidating the overall mechanism of GH action requires an understanding of the role GH itself plays in the regulation of its own target tissue receptors. Earlier studies have approached this issue by the daily administration of large doses of GH for relatively short periods of time (2) or by the sc implantation of rat pituitary cell lines (MTW or GH3) which secrete high levels of GH (3, 4). Such studies however have produced somewhat conflicting results. Human or rat GH has been reported to induce both GH receptors (GHR) and PRL receptors (PRL-R) in hypophysectomized and normal female and male rat liver, with PRL itself playing no role in the induction of PRL-R (5-8). In contrast, other reports have indicated that PRL induces Received Ocrober 1,1990. Address all correspondence and requests for reprints to: Dr. Jacqueline M. Orian, Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3052, Australia. * Present address: Department of Biochemistry, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
its own receptors in rat lung (9), liver (9, 10), kidney (11), Snell-dwarf mice (12), and cultured rat hepatocytes (13). These apparent discrepancies may result from the observations 1) that rat pituitary tumors secrete significant quantities of PRL as well as GH (14); 2) that semipurified preparations of GH and PRL have often been used; and 3) that the injection of heterologous PRL and GH is known to induce antibodies which can associate with tissues and mimic receptor up-regulation (15). The ability to generate transgenic mice provides an alternative approach to analyze physiological and metabolic effects of GH in vivo (16, 17). This technique has been used successfully to produce mice carrying GH genes from other species (18-21) and offers several advantages over other methods. First, the use of cloned genes ensures that the experimental animal is exposed to a single foreign protein. Also, since GH is produced in these animals from the fetal stage, it is likely to be recognized by their immune system as self, thus making this technique a more efficient delivery system while at the same time providing the means to study the effects of GH during early development. Furthermore, it allows measurement of several parameters over the whole lifespan of the animal, permitting a study of the relationship between the short term actions of the hormone (i.e. its
1238
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE effects on metabolism) and its long term effects (i.e. growth promotion). In the present study we have used transgenic mice expressing ovine GH (oGH) to investigate the induction of GH and PRL liver receptors. The mice carry a construct (MToGHl) consisting of the structural gene for oGH under the regulation of the mouse MT promoter (21). This promoter is from a housekeeping gene expressed in most tissues and is inducible by heavy metals and corticosteroids (22-24). It has previously been used in mice to direct high levels of expression of the GH gene from a number of species (16, 21). In a previous communication we have described transgenic mice expressing the MToGHl fusion gene (21). The major site of expression of oGH was the liver, and a number of transgenic mice expressed very high levels of oGH. Approximately 70% of founder generation (Fo) mice grew to be giant mice, being up to twice normal size, and three were selected for breeding to obtain second (Fi) and third (F2) generation mice. We have analyzed levels of GH and PRL receptor activity in these mice and now present the first evidence that both GH-R and PRL-R are induced in transgenic mice which express high levels of oGH.
Materials and Methods Materials The following were obtained from the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD): human GH (NIAMDD-hGH-I-1) and rat PRL (rPRL) (NIADKKrPRL-I-5) for iodination, ovine PRL (oPRL) (NIAMDDoPRL-15), bovine GH (bGH) (NIH-GH-B-18) and rPRL (NIADKK-rPRL-P-3) for unlabeled standards and rPRL antiserum (NIHDKK anti-rPRL-5-9). hGH for unlabeled standards was obtained from the Commonwealth Serum Laboratories, Melbourne, Australia. Probes for Northern analysis were obtained as follows: pG23 which contains a bGH cDNA was provided by F. Rottman (Case Western Reserve University, Cleveland, OH) (25), pUC.RGH.R.l which contains a 638 base pair fragment from the rabbit GH-R complementary DNA was provided by Genentech (San Francisco, CA) (26) and the plasmid containing the complete cDNA for the rat 18S ribosomal RNA (rRNA) (27) was provided by T. Adams (University of Melbourne, Australia). Oligonucleotide probes were synthesized using an Applied Biosystems synthesizer. [32P]ATP and nylon membrane were purchased from Amersham International (Amersham, UK); restriction enzymes were purchased from Pharmacia (Uppsala, Sweden) and New England Biolabs (Beverley, MA); phenyl methyl sulfonyl fluoride and aprotinin were purchased from Sigma Chemical Co. (St Louis, MO); Ultrogel AcA54 was purchased from LKB Produckter (Bromma, Sweden) and Sephadex G-50 from Pharmacia; mol wt markers for gel chromatography and polyacrylamide gel
1239
electrophoresis were obtained as previously described (28, 29); disuccinimidyl suberate (DSS) and IODO-GEN were purchased from Pierce Chemical Co. (Rockford, IL,); HEPES buffer was from Boehringer-Mannheim (Mannheim, West Germany). Generation of transgenic mice and DNA analysis Plasmid pMToGHl, a construct containing the mouse metallothionein-1 promoter fused to the oGH gene, has been described and is shown in Fig. 1 (21). A 2.5 kilobase pair JBstEIINdel fragment containing the mouse MT promoter (30) fused to the oGH gene was isolated from plasmid pMToGHl (Fig. 1), purified and microinjected into eggs taken from C57bl/6 x SJL/J hybrid mice as described previously (21). Fi and F2 mice were obtained by mating transgenic mice with normal C57bl/ 6 mice. For the identification of transgenic pups, DNA was isolated from tail biopsies as described (21) and analyzed by Southern blotting (32) using the radiolabeled 338 and 446 internal Pstl insert fragments of the bGH cDNA clone pG23 as hybridization probes. Surveillance of transgenic mice Mice were given a supplement of 25 mM ZnSO4 in their drinking water as described previously (21). A control group of mice which never received ZnSO4 supplementation was also maintained; these mice are identified in the footnote to Table 1. Mice were weighed weekly from weaning until 10 weeks of age. Thereafter they were weighed at two weekly intervals until 18 weeks of age at which time their weight had stabilized. Quantification of oGH and PRL in mouse serum oGH concentration in mouse sera was assayed as described previously (21). PRL was quantified in mouse serum, using a rPRL RIA. [125I]PRL was prepared as described below and used at 15,000 cpm/tube. The antiserum was used at a final dilution BstEII
EcoRI
BamHI
Kpnl BamHI Ndel
FIG. 1. Plasmid pMToGHl. A 1.9 kb BamHI-EcoRI DNA fragment containing the oGH gene was isolated from plasmid pGH3-12 (21) and modified at the 3' end by the addition of an adaptor to contain BamHI restriction sites at both ends. The modified 1.9 kb BamHI DNA fragment was then substituted for the hGH gene of the plasmid MThGHlll (21) to produce a 6.05 kb plasmid designated pMToGHl. The linear fragment indicated by the internal arrow was isolated and purified (31) and used for microinjection into mouse eggs. , pBr322 sequences; • 5'-sequence of the mouse MT gene; W3, oGH gene.
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
Endo.iwi Vo!128«No3
TABLE 1. Features of transgenic mice used in the study Mouse I.D. Generation Number"
Sex
Serum oGH (ng/ml)*
Relative growth (ratio)c
Age when killed (weeks)
Line 1
43 43-27-174
Fo F2
F M
13,600 4,000
1.9 1.2
25 53
Line 2
56 56-1 56-9 56-11 56-44 56-46 56-1-224 56-9-193 56-9-203 56-1-216 56-1-219
Fo
1,880 10,900 17,100 13,200 551 12,900 100 3,950 3,500
1.5 1.4 1.5 1.1 1.5 1.2 1.0 1.1
54 60 37 26 57 20
F2 F2 F2
M F F M F M M F F
1.0
F2
M
9
1.0
F2
M
11
1.0
Fo
F
304
2.0
F, F2 F,
M M M
1,200 500 28
1.3 1,5 1.0
Line 3
127 127-66 127-63-213 127-63-178
Fi
Fx Fi
F, Fx
18 19 29 30 43 63 30 54
" Mice 56, 56-1-224, and 127-63-213 were maintained on a zinc-free diet. 6 The average serum GH content in normal adult mice is 8.2 ± 2.6 ng/ml (62). c Weight of transgenic mice compared with sex-matched litter-mates at 8 weeks of age. Statistical analysis (Student's t test) shows that mice greater than 1.18 times normal mice are significantly larger than control mice.
of 1:10,000 in a final volume of 0.4 ml. The antiserum crossreactivity with oGH was less than 0.001%, and the sensitivity of the assay was 0.09 ng/tube. Sera used in both assays were from mice aged between 2 and 10 months. Microsome preparation Livers collected from transgenic mice, and from 16- to 19day pregnant mice, were immediately frozen in liquid nitrogen and stored at -70 C until required. Approximately 0.5 g portions were thawed, diced, and then homogenized in a Sorvall omnimixer in 5 vol of a solution containing 0.3 M sucrose, 1 mM phenyl methyl sulfonyl fluoride, and 1000 kallikrein inactivator units aprotinin/ml at 4 C. The homogenate was centrifuged at 1,500 X g for 20 min. The supernatant was collected and centrifuged at 15,000 X g for a further 20 min. The 15,000 X g supernatant was then centrifuged at 100,000 x g for 90 min. The pellet from the 100,000 X g centrifugation step was resuspended in 1 vol of a solution containing 25 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM phenyl methyl sulfonyl fluoride, and 1000 kallikrein inactivator units aprotinin/ml (HM buffer), and the protein concentration was determined by the method of Lowry et al. (33). Membrane preparations were frozen and stored at -20 C until used for binding studies. In some experiments, membranes were washed with MgCl2 to strip off any endogenous bound oGH hormone (34). This step was important due to the very high levels of GH circulating in some mice. Briefly, membranes were resuspended at a final concentration of 4 M MgCl2 and incubated at room temperature for 30 min. After the addition of 10 ml HM buffer membranes were collected by centrifugation at 15,000 x g for 20 min and washed once more in HM buffer. Protein concentration was assessed by the method of Lowry et al. (33).
Iodination hGH, bGH, and rPRL were iodinated as previously described (35) by using the IODO-GEN method (36) with a hormone to 125 I ratio of 2:1.125I-labeled hormone was separated on a Sephadex G-50 column (0.8 x 19 cm) and subsequently purified on Ultrogel AcA54 (1 X 55 cm). Specific radioactivities of 30-50 were obtained. Competitive binding assays Microsomal membranes (100 ng protein/tube) were incubated with 20,000 cpm [125I]hGH and unlabeled hGH, bGH, or oPRL at concentrations ranging from 5 to 10,000 ng/ml in a total volume of 250 n\ HM buffer containing 0.1% wt/vol BSA (HMB buffer). All assays were carried out in duplicate. After a 16 h incubation at room temperature, membrane-bound hormone was separated from free hormone by centrifugation at 1,500 X g for 20 min after the addition of 1 ml HMB buffer at 4 C. Membrane pellets were then counted for radioactivity. Specifically bound radioactivity was obtained after subtracting from each value the radioactivity remaining in the membrane pellets incubated in the presence of 10 ng unlabeled hormone. Coualent cross-linking of P2bIJhGH to membrane preparations All steps were carried out using silicone-treated glass tubes. [125I]hGH (40,000 cpm) was incubated in the absence of BSA with liver membranes in the presence or absence of excess unlabeled hGH. After binding equilibria were established, the entire reaction mixture was treated at 21 C for 15 min with DSS in dimethyl sulfoxide to give a final concentration of 0.05 mM DSS (37). The efficiency of covalent cross-linking was relatively low (5-10% of specifically bound hormone) but was similar in each tissue preparation examined. The cross-linking
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE reaction was stopped by boiling (3 min) 100 pi aliquots in the presence of 2% (wt/vol) sodium dodecyl sulfate (SDS) with or without dithiothreitol (100 mM). Each sample was then applied to a 7.5% vertical slab SDS-polyacrylamide gel, and electrophoresis was performed according to the method of Laemmli (38). Autoradiography was carried out as described previously (28, 37, 39). RNA isolation RNA was isolated from 100 mg liver obtained from transgenic mice or 19 day pregnant mice using the LiCl precipitation method described by Auffray and Rougeon (40). RNA concentration was measured by absorbance at 260 nm. Northern blot analyses Samples (20 /ug) of total liver RNA were run on formaldehyde/agarose gels and transferred to Nylon membranes as described (41). PRL receptor. For the detection of PRL receptor mRNA, blots were prehybridized in 5x SSPE (1 x SSPE = 0.18 M NaCl, 0.01 M sodium phosphate, pH 7.7, 1 mM EDTA), 0.1% SDS, 2x Denhardt's reagent (lx = 0.02% BSA, 0.02% polyvinyl pirolidone, 0.02% Ficoll) and 100 /xg/ml denatured salmon sperm DNA, at 50 C. Hybridizations were performed in the buffer solution described above at 50 C using an end-labeled (42) oligonucleotide probe (30-mer) corresponding to the Cterminal 10 amino acids of the cloned rat liver PRL-R (43). GH receptor and 18S rRNA. Blots were prehybridized in 5x SSPE, 0.25% sodium iV-laurylsarcosine, and 0.2 mg/ml denatured salmon sperm DNA. Hybridizations were performed in the same buffer solution. For the analysis of GH receptor transcripts, Northern blots were incubated with a radiolabeled 638 base pair cDNA fragment (44) from the rabbit GH-R at 58 C (26). For the analysis of 18S rRNA a radiolabeled cDNA probe (44), specific for the 18S rRNA was used (27). Incubations were performed at 63 C. Densitometric analysis Blots were analyzed on a Zeineh Scanning Densitometer, model SLR-TRFF (Biomed Instruments Inc.). The degree of hybridization of a specific band was quantified relative to the hybridization of the 1.9 kilobase (kb) band observed with the 18S rRNA probe.
Results Fourteen transgenic mice, generated from three giant F o generation mice (nos. 43, 56 and 127) were used in this study and are described in Table 1. Table 1 also shows three important features of these transgenic mice: 1) there is a high degree of variation in serum oGH levels within any pedigree, although variations in copy number of the transgene within any pedigree were a rare feature in these transgenic mice (data not shown); 2) a number of transgenic offspring of giant mice failed to express significantly high levels of serum oGH {e.g. mice 56-1-
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216, 56-1-219, and 127-63-178); and 3) a number of transgenic mice which had high levels of serum oGH did not respond to the hormone and were of normal size (e.g. 56-11, 56-1-224, and 56-9-203). These observations have been made previously on transgenic mice expressing oGH (21) as well as other GHs (18) and still remain unexplained. The levels of PRL in these transgenic mice were also assayed. The concentrations for nontransgenic control mice were between 1.8 and 16.0 ng/ml, and the concentrations for normal-size and giant transgenic mice, (both male and female) were within the normal range. The only notable difference between the groups was an apparent lowering of PRL concentration in the giant oGH expressing mice 6.0 ± 0.74 (mean ± SEM; n = 8) vs. 11.0 ± 2.0 (n = 6) in the normal size mice (P < 0.05) (data not shown). Receptor activity in microsomal preparations from livers of transgenic mice In initial assays, [125I]hGH was used as a tracer and unlabeled hGH was used as a competitor. Microsomal preparations from livers of normal and late pregnant mice were used as controls. Microsomal preparations from late pregnant mice usually show an 8- to 10-fold increase in specific binding of GH and PRL and have provided a useful model for the characterization of lactogenic receptors (45). All the transgenic mice with high serum oGH levels had elevated levels of receptor activity relative to normal mice (Fig. 2a). The levels of receptor activity in these transgenic mice were comparable to that observed in a late pregnant mouse. High levels of receptor activity were found in the high serum oGH mice irrespective of their sex, final size, or age at death. By contrast, transgenic mice which produced only low levels of serum oGH (56-1-216, 56-1-219 and 127-63-178) had low levels of receptor activity, comparable to that of the normal mouse. hGH is known to have both somatotrophic and lactogenic activities and can bind to both GH-R and PRL-R (46). Therefore to further investigate the nature of the receptors present in microsomal preparations from transgenic mice, competitive binding assays were performed using unlabeled bGH and oPRL, which are known to bind more specifically to each receptor class (45, 47, 48). Results in Fig. 2b show that both unlabeled bGH and oPRL displaced [125I]hGH from microsomal preparations from transgenic mice expressing elevated serum oGH levels. The percent specific binding in the presence of unlabeled oPRL was similar to that seen with unlabeled hGH. However, oPRL at high doses is known to cross-react with somatotrophic (GH) receptors (49) and hence the apparent abundance of PRL-R is
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
1242 a.
I?
_
_
^5 £ 8. 8- 40
j—
—
- °
r—n |—i
SPECIFIC COMPLEX
PROTEIN
(IOOK)
r—i
r—|
/GH-BINDING \ '
(
r—]
70K
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(48 K)
20 -
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122 K 92 K 82 K 7OK
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rinn n
Endo • 1991 Voll28>No3
_ 116K - 98 -
68
_ 45
b?
_ 30
FiG. 3. Cross-linking of [125I]hGH with microsomal membranes. [125I] hGH was chemically cross-linked to microsomal membranes in the
FIG. 2. Receptor activity in microsomal preparations from livers of transgenic mice, a, Displacement of [125I]hGH by unlabeled hGH from mouse liver microsomes. Microsomal membranes (100 fig protein) were incubated with 20,000 cpm [125I]hGH and either 0 or 10,000 ng unlabeled hGH. b, Displacement of [125I]hGH by unlabeled oGH, bGH, and oPRL. Microsomal membranes (100 ng protein) were incubated with 20,000 cpm [125I]hGH and either 0 or 10,000 ng unlabeled hGH (•), oPRL (M) bGH (•). In both panels a and b the data are plotted as the percent of total counts added that were specifically bound. Nonspecific binding was less than 10% of total counts added.
probably overestimated. The percent specific binding observed with the addition of unlabeled bGH was lower than that with unlabeled hGH and varied between samples but was still significant in most cases and was comparable to that observed in the pregnant mouse. These data indicate that both GH-R and PRL-R are present in transgenic mice expressing oGH and imply that both receptor types are elevated. To test the possibility that the apparently lower level of GH-R was due to endogenous, bound oGH interfering with bGH in the competitive assays, this experiment was repeated using microsomal preparations washed with 4 M MgCl2 to remove any membrane-bound oGH molecules (34). Similar results were obtained (data not shown). The above data therefore suggest that microsomal preparations isolated from transgenic mice expressing high oGH levels may contain elevated levels of more than one type of receptor, i.e. both GH-R and PRL-R. Cross-linking studies The receptors to which the various ligands in the previous experiments bound were further investigated by analysis on polyacrylamide gels of microsomal preparations cross-linked to [125I]hGH. Under reducing conditions (Fig. 3) the most prominent band had an Mr of
absence (lanes a, c, e, and g) or presence (lanes b, d, f, and h) of excess unlabeled hGH as described. After stopping the reaction by boiling in the presence of 2% (wt/vol) SDS, reduced samples were applied to a 7.5% SDS-polyacrylamide gel. Electrophoresis was performed by the method of Laemmli (38) and autoradiography performed as described (28, 37, 39). Molecular wts of protein markers are shown on the right; those of the [125I]hGH binding protein complexes or the binding proteins themselves (in parentheses) are shown on the left.
70,000 and was observed in preparations from three transgenic mice expressing high levels of oGH. Three other very minor bands (Mr 82,000, 92,000, and 122,000) were also observed. In the preparation obtained from pregnant mouse liver the same four bands were visualized. In all samples the appearance of these bands was completely inhibited by the addition of 10 /xg unlabeled hGH in the incubation mixture. The apparent mol wts of these labeled polypeptides minus that of hGH (22,000) are 48,000,60,000, 70,000, and 100,000 in transgenic mice and in the pregnant mouse. Cross-linking studies on microsomal preparations from rabbit, rat, and mouse livers have revealed that GH-R in rabbit and mouse liver consists primarily of polypeptides of Mr 100,000 and 60,000-70,000, while in the rat it consists of a single species of Mr 100,000 (50). In the case of PRL-R, the reported mol wts range between 35,000-45,000 (51-53). The above mol wts are very similar to those observed in cross-linking experiments using microsomal preparations from transgenic mice. It would appear therefore that the species binding predominantly to [125I]hGH in transgenic mice (the Mr 48,000 polypeptide) is the mouse PRL-R with a smaller amount of binding to GH-R (the Mr 60,000, 70,000, and 100,000 polypeptides). Northern blot analysis of RNA from livers of transgenic mice Total cellular liver RNA from transgenic mice was analyzed using a GH-R probe. Two RNA species of 3.9
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
and 1.2 kb were observed in all transgenic mice expressing high serum oGH levels (Fig. 4). These mRNA species were present at very low levels in transgenic mice expressing low levels of oGH. The sizes of these RNA species were similar to those observed for the pregnant mouse and are consistent with those previously described for the mouse GH-R by Smith et al. (26). Total cellular liver RNA was also analyzed with a probe specific for rat liver PRL-R. A major band corresponding to an mRNA species of 1.9 kb was observed in all preparations obtained from transgenic mice as well as from the pregnant mouse (Fig. 4). The low oGH expressing transgenic mice and the normal mouse had very low levels of this PRL-R transcript, which corresponds reasonably well with the primary transcript of 2.2 kb reported in rat tissues by Boutin et al. (43). An additional band corresponding to an RNA species of 2.7 kb was also observed in all high expressers and in the pregnant mouse. This band, which is clearly inducible by oGH, has not previously been identified as a major PRLR mRNA species (43). However, a very recent report (54) has described the presence of a long form of PRL receptor in ovary and liver with mRNA between 2.5 and 3 kb.
GH-R
1243
Densitometric analysis of autoradiograms was performed to obtain an estimate of the relative amounts of mRNA species observed. The data are shown in Fig. 5 and are expressed as a ratio of the intensity of each band with respect to that of the 18S rRNA on the same autoradiogram. Results for the two mRNA species observed on autoradiograms analyzed with the GH-R probe were as follows. The 1.2 kb species was elevated in most mice with elevated serum oGH with respect to the normal mouse, the only exception being mouse 56. The increase in intensity of the 1.2 kb species varied greatly between the mice and ranged from 1.6 times in mouse 56-9-193 to 8.0 times in mouse 56-46. In mice with low serum oGH, on the other hand, the amount of the 1.2 kb mRNA species was comparable to that of the normal mouse. In the case of the 3.9 kb species, elevated levels of mRNA were observed in most transgenic mice expressing high levels of serum oGH. The increase in mRNA levels ranged from 1.6 times in mouse 56 to 8.5 times in mouse 43-27-174. However it was also observed that mRNA levels for the 3.9 kb species were slightly elevated in the two transgenic mice expressing low levels of serum oGH and were approximately 1.4 times the levels observed in the normal mouse. Densitometric analysis of autoradiograms of Northern blots probed with the PRL-R probe showed that the relative levels of the 1.9 kb mRNA species were elevated in all transgenic mice expressing high serum oGH levels. The increase ranged from 1.9 times in mouse 127 to 11.9 times in mouse 56-46. Transgenic mice expressing low levels of serum oGH on the other hand showed no
RATIO GH-R 3.9kb 18SRNA PRL-R
MMHl > al -27 -1-9
18S
GH-R 1.2kb 18SRNA
PRL-R 2.7kb 18SRNA
PRL-R 1.9kb 18SRNA FlG. 4. Northern blot analysis of RNA from livers of transgenic mice. Total cellular liver RNA (20 ng) from each transgenic mouse, a 19 day pregnant mouse, and a normal mouse were electrophoresed on formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with the following probes under the conditions described. Top, a rabbit GH-R cDNA probe (44), 5-day exposure; Middle, an oligomer probe directed against PRL-R, 2-day exposure; Bottom, a rat 18S cDNA probe (27), 1-day exposure. The three probes were used sequentially on exactly the same filter.
Y7A V7"
^
FlG. 5. Densitometric analysis of Northern blots. Blots shown in Fig. 4 were analyzed as described in Materials and Methods. The degree of hybridization of each mRNA species was quantified relative to that of the 1.9 kb 18S rRNA for the same mouse sample.
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
increase in the 1.9 kb mRNA species. The expression of the 2.7 kb band closely resembled that of the 1.9 kb band. The increase ranged from 2.3 times in mouse 56 to 11 times in mouse 56-46. Two further observations were made from the data shown in Figs. 4 and 5. First, the relative increase in the mRNA species for GH-R and for PRL-R were very similar in each of the transgenic mice. Second, for both GH-R and PRL-R, the increases in mRNA levels occurred irrespective of the sex of the animals, their final size, and their age when killed.
Discussion These studies have shown that transgenic mice carrying an oGH fusion gene and with high serum levels of oGH have elevated levels of both GH-R and PRL-R on liver membrane preparations. Transgenic mice which did not express high levels of oGH did not have increased receptor expression. The increase in receptor activity was not related to the absolute level of oGH, the final size of the mice, or their sex. The observation that high levels of receptor activity were observed in transgenic mice expressing elevated serum oGH levels irrespective of the age at which they were killed strongly suggests that the receptor response to oGH is maintained throughout the life of these transgenic mice. Since the levels of PRL in these mice were similar to those of normal mice, the current data unequivocally support the proposition put forward by Baxter et al. (8) that GH is capable of inducing hepatic GH-R and PRL-R in vivo, and that PRL is not required for the induction of its own receptor. These data also show that the transgenic model represents a less complex and more reliable system than other in vivo models used previously for studies on GH and/or PRL regulation of receptor status. The receptors for GH as observed on autoradiographs of cross-linking experiments consisted of species of Mr 100,000, 70,000, and 60,000 for both the pregnant mouse and the transgenic mice. These Mr are similar to those reported in the literature (Mr 100,000 and Mr 60,00070,000) (50); however it is not clear why two species should be present within the Mr 60,000-70,000 range. This may reflect different glycosylation states of the single receptor polypeptide reported from cloning studies (55), and is perhaps related to the chronically elevated GH concentrations observed in pregnant and in transgenic mice. The Mr for PRL-R (48,000) was slightly above the range (Mr 35,000-45,000) commonly reported (51-53). However, in terms of binding characteristics and mRNA sizes it would appear that the receptors present in liver microsomal preparations from transgenic mice are bona fide GH and PRL receptors. In their analysis of GH-R mRNA from pregnant and
Endo • 1991 Voll28«No3
nonpregnant mice, Smith et al. (26) observed that both the 3.9 kb and the 1.2 kb species were enriched in pregnant mice compared with nonpregnant mice, although the relevant changes in each mRNA species were not quantified. Elevation of these two species was also observed in the pregnant mouse in our study; however densitometric analysis of Northern blots showed that these mRNA species were not enriched to the same degree in all the mice studied (pregnant or transgenic). There is strong evidence to support the suggestion that the 1.2 kb species encodes the serum GH binding protein while the 3.9 kb species encodes the membrane bound form of the receptor. Our densitometric data may indicate that in pregnancy, as well as in transgenic mice, the secreted form of GH-R is preferentially induced. There was no clear difference in receptor abundance between high expressers of oGH which responded (with increased growth) to oGH and high expressers which did not respond to oGH. Indeed almost all high expressers had high concentrations of GH and PRL receptor activity and elevated levels of mRNA for both GH-R and PRL-R. The only exception was mouse 56 which had elevated levels of mRNA for PRL-R but not for GH-R. However, this particular mouse had an abundance of hepatic tumors present at death (56), and it is possible that this may have selectively influenced expression of the GH-R gene. These observations suggest that the response to elevated serum GH is not directly related to the numbers of receptors present in liver microsomal preparations. However, it must be noted that these measurements were performed on preparations obtained from adult mice, and it is possible that the levels of receptor activity in these mice are different from those of juvenile mice when maximum growth rates are present. To further investigate whether receptor numbers are directly related to a positive response to serum oGH concentrations, these experiments should perhaps be repeated using juvenile mice. The data reported here on GH-R and PRL-R levels in oGH transgenic mice provide important insights into the physiological consequences of chronically elevated levels of GH on GH action at the cellular level. GH is well known to have a variety of actions in target tissues: metabolic actions, both acute insulin-like effects and longer term antiinsulin (diabetogenic) effects, as well as growth-promoting actions, many of which may be mediated via the insulin-like growth factor (IGF) family (57). The biological effects of GH have been difficult to analyze due to the lack of a biologically responsive in vitro system. The only useful system has been the isolated adipocyte in which a direct correlation has been made between receptor occupancy and metabolic function (58). Studies using this system (59) have shown that the acute metabolic effects of GH, e.g. glucose uptake
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
and oxidation, amino acid uptake and lipogenesis, are very sensitive to refractoriness in the presence of continued or prolonged exposure to GH. In transgenic mice where oGH levels are constitutively high it is most likely that metabolic tissues will also be refractory to GH and will not express the acute effects of GH. In contrast., the growth-related effects of GH are not subject to refractoriness, and this is evidenced in the current study by the increased growth of several of the mice expressing high levels of oGH and by increased levels of immunoreactive IGF-I (irIGF-I) and hepatic IGF-I mRNA in some of these mice (our unpublished observations). Both parameters were generally increased in those mice with elevated oGH levels, compared to transgenic mice which were not actively expressing the gene, although there was some variation in absolute levels. In some mice with marked hepatic pathology (56) the serum IGF-I levels were low despite increased hepatic IGF-I mRNA. The rise in IGFI mRNA and irIGF-I levels in oGH-expressing mice was generally consistent with the previous reports by Mathews et al. (60, 61) in much younger (
Vol. 128, No. 3 Printed in U.S.A.
Elevation of Growth Hormone (GH) and Prolactin Receptors in Transgenic Mice Expressing Ovine GH JACQUELINE M. ORIAN, KENNETH SNIBSON, JANET L. STEVENSON, MALCOLM R. BRANDON, AND ADRIAN C. HERINGTON* Department of Veterinary Preclinical Sciences (J.M.O., K.S., M.R.B.), The University of Melbourne, Parkville, Victoria 3052, Australia; and Prince Henry's Institute of Medical Research (J.L.S., A.C.R.), Monash Medical Centre, Melbourne, Victoria 3004, Australia
a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors. {Endocrinology 128: 1238-1246, 1991)
ABSTRACT. The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein l(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in
G
ROWTH HORMONE belongs to a family of polypeptide hormones which includes PRL and placental lactogen and whose members are related in their structure and function. Despite their relatedness, the different physiological effects of these hormones are mediated via distinct specific cell surface receptors (1). One aspect of elucidating the overall mechanism of GH action requires an understanding of the role GH itself plays in the regulation of its own target tissue receptors. Earlier studies have approached this issue by the daily administration of large doses of GH for relatively short periods of time (2) or by the sc implantation of rat pituitary cell lines (MTW or GH3) which secrete high levels of GH (3, 4). Such studies however have produced somewhat conflicting results. Human or rat GH has been reported to induce both GH receptors (GHR) and PRL receptors (PRL-R) in hypophysectomized and normal female and male rat liver, with PRL itself playing no role in the induction of PRL-R (5-8). In contrast, other reports have indicated that PRL induces Received Ocrober 1,1990. Address all correspondence and requests for reprints to: Dr. Jacqueline M. Orian, Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3052, Australia. * Present address: Department of Biochemistry, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
its own receptors in rat lung (9), liver (9, 10), kidney (11), Snell-dwarf mice (12), and cultured rat hepatocytes (13). These apparent discrepancies may result from the observations 1) that rat pituitary tumors secrete significant quantities of PRL as well as GH (14); 2) that semipurified preparations of GH and PRL have often been used; and 3) that the injection of heterologous PRL and GH is known to induce antibodies which can associate with tissues and mimic receptor up-regulation (15). The ability to generate transgenic mice provides an alternative approach to analyze physiological and metabolic effects of GH in vivo (16, 17). This technique has been used successfully to produce mice carrying GH genes from other species (18-21) and offers several advantages over other methods. First, the use of cloned genes ensures that the experimental animal is exposed to a single foreign protein. Also, since GH is produced in these animals from the fetal stage, it is likely to be recognized by their immune system as self, thus making this technique a more efficient delivery system while at the same time providing the means to study the effects of GH during early development. Furthermore, it allows measurement of several parameters over the whole lifespan of the animal, permitting a study of the relationship between the short term actions of the hormone (i.e. its
1238
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE effects on metabolism) and its long term effects (i.e. growth promotion). In the present study we have used transgenic mice expressing ovine GH (oGH) to investigate the induction of GH and PRL liver receptors. The mice carry a construct (MToGHl) consisting of the structural gene for oGH under the regulation of the mouse MT promoter (21). This promoter is from a housekeeping gene expressed in most tissues and is inducible by heavy metals and corticosteroids (22-24). It has previously been used in mice to direct high levels of expression of the GH gene from a number of species (16, 21). In a previous communication we have described transgenic mice expressing the MToGHl fusion gene (21). The major site of expression of oGH was the liver, and a number of transgenic mice expressed very high levels of oGH. Approximately 70% of founder generation (Fo) mice grew to be giant mice, being up to twice normal size, and three were selected for breeding to obtain second (Fi) and third (F2) generation mice. We have analyzed levels of GH and PRL receptor activity in these mice and now present the first evidence that both GH-R and PRL-R are induced in transgenic mice which express high levels of oGH.
Materials and Methods Materials The following were obtained from the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD): human GH (NIAMDD-hGH-I-1) and rat PRL (rPRL) (NIADKKrPRL-I-5) for iodination, ovine PRL (oPRL) (NIAMDDoPRL-15), bovine GH (bGH) (NIH-GH-B-18) and rPRL (NIADKK-rPRL-P-3) for unlabeled standards and rPRL antiserum (NIHDKK anti-rPRL-5-9). hGH for unlabeled standards was obtained from the Commonwealth Serum Laboratories, Melbourne, Australia. Probes for Northern analysis were obtained as follows: pG23 which contains a bGH cDNA was provided by F. Rottman (Case Western Reserve University, Cleveland, OH) (25), pUC.RGH.R.l which contains a 638 base pair fragment from the rabbit GH-R complementary DNA was provided by Genentech (San Francisco, CA) (26) and the plasmid containing the complete cDNA for the rat 18S ribosomal RNA (rRNA) (27) was provided by T. Adams (University of Melbourne, Australia). Oligonucleotide probes were synthesized using an Applied Biosystems synthesizer. [32P]ATP and nylon membrane were purchased from Amersham International (Amersham, UK); restriction enzymes were purchased from Pharmacia (Uppsala, Sweden) and New England Biolabs (Beverley, MA); phenyl methyl sulfonyl fluoride and aprotinin were purchased from Sigma Chemical Co. (St Louis, MO); Ultrogel AcA54 was purchased from LKB Produckter (Bromma, Sweden) and Sephadex G-50 from Pharmacia; mol wt markers for gel chromatography and polyacrylamide gel
1239
electrophoresis were obtained as previously described (28, 29); disuccinimidyl suberate (DSS) and IODO-GEN were purchased from Pierce Chemical Co. (Rockford, IL,); HEPES buffer was from Boehringer-Mannheim (Mannheim, West Germany). Generation of transgenic mice and DNA analysis Plasmid pMToGHl, a construct containing the mouse metallothionein-1 promoter fused to the oGH gene, has been described and is shown in Fig. 1 (21). A 2.5 kilobase pair JBstEIINdel fragment containing the mouse MT promoter (30) fused to the oGH gene was isolated from plasmid pMToGHl (Fig. 1), purified and microinjected into eggs taken from C57bl/6 x SJL/J hybrid mice as described previously (21). Fi and F2 mice were obtained by mating transgenic mice with normal C57bl/ 6 mice. For the identification of transgenic pups, DNA was isolated from tail biopsies as described (21) and analyzed by Southern blotting (32) using the radiolabeled 338 and 446 internal Pstl insert fragments of the bGH cDNA clone pG23 as hybridization probes. Surveillance of transgenic mice Mice were given a supplement of 25 mM ZnSO4 in their drinking water as described previously (21). A control group of mice which never received ZnSO4 supplementation was also maintained; these mice are identified in the footnote to Table 1. Mice were weighed weekly from weaning until 10 weeks of age. Thereafter they were weighed at two weekly intervals until 18 weeks of age at which time their weight had stabilized. Quantification of oGH and PRL in mouse serum oGH concentration in mouse sera was assayed as described previously (21). PRL was quantified in mouse serum, using a rPRL RIA. [125I]PRL was prepared as described below and used at 15,000 cpm/tube. The antiserum was used at a final dilution BstEII
EcoRI
BamHI
Kpnl BamHI Ndel
FIG. 1. Plasmid pMToGHl. A 1.9 kb BamHI-EcoRI DNA fragment containing the oGH gene was isolated from plasmid pGH3-12 (21) and modified at the 3' end by the addition of an adaptor to contain BamHI restriction sites at both ends. The modified 1.9 kb BamHI DNA fragment was then substituted for the hGH gene of the plasmid MThGHlll (21) to produce a 6.05 kb plasmid designated pMToGHl. The linear fragment indicated by the internal arrow was isolated and purified (31) and used for microinjection into mouse eggs. , pBr322 sequences; • 5'-sequence of the mouse MT gene; W3, oGH gene.
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
Endo.iwi Vo!128«No3
TABLE 1. Features of transgenic mice used in the study Mouse I.D. Generation Number"
Sex
Serum oGH (ng/ml)*
Relative growth (ratio)c
Age when killed (weeks)
Line 1
43 43-27-174
Fo F2
F M
13,600 4,000
1.9 1.2
25 53
Line 2
56 56-1 56-9 56-11 56-44 56-46 56-1-224 56-9-193 56-9-203 56-1-216 56-1-219
Fo
1,880 10,900 17,100 13,200 551 12,900 100 3,950 3,500
1.5 1.4 1.5 1.1 1.5 1.2 1.0 1.1
54 60 37 26 57 20
F2 F2 F2
M F F M F M M F F
1.0
F2
M
9
1.0
F2
M
11
1.0
Fo
F
304
2.0
F, F2 F,
M M M
1,200 500 28
1.3 1,5 1.0
Line 3
127 127-66 127-63-213 127-63-178
Fi
Fx Fi
F, Fx
18 19 29 30 43 63 30 54
" Mice 56, 56-1-224, and 127-63-213 were maintained on a zinc-free diet. 6 The average serum GH content in normal adult mice is 8.2 ± 2.6 ng/ml (62). c Weight of transgenic mice compared with sex-matched litter-mates at 8 weeks of age. Statistical analysis (Student's t test) shows that mice greater than 1.18 times normal mice are significantly larger than control mice.
of 1:10,000 in a final volume of 0.4 ml. The antiserum crossreactivity with oGH was less than 0.001%, and the sensitivity of the assay was 0.09 ng/tube. Sera used in both assays were from mice aged between 2 and 10 months. Microsome preparation Livers collected from transgenic mice, and from 16- to 19day pregnant mice, were immediately frozen in liquid nitrogen and stored at -70 C until required. Approximately 0.5 g portions were thawed, diced, and then homogenized in a Sorvall omnimixer in 5 vol of a solution containing 0.3 M sucrose, 1 mM phenyl methyl sulfonyl fluoride, and 1000 kallikrein inactivator units aprotinin/ml at 4 C. The homogenate was centrifuged at 1,500 X g for 20 min. The supernatant was collected and centrifuged at 15,000 X g for a further 20 min. The 15,000 X g supernatant was then centrifuged at 100,000 x g for 90 min. The pellet from the 100,000 X g centrifugation step was resuspended in 1 vol of a solution containing 25 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM phenyl methyl sulfonyl fluoride, and 1000 kallikrein inactivator units aprotinin/ml (HM buffer), and the protein concentration was determined by the method of Lowry et al. (33). Membrane preparations were frozen and stored at -20 C until used for binding studies. In some experiments, membranes were washed with MgCl2 to strip off any endogenous bound oGH hormone (34). This step was important due to the very high levels of GH circulating in some mice. Briefly, membranes were resuspended at a final concentration of 4 M MgCl2 and incubated at room temperature for 30 min. After the addition of 10 ml HM buffer membranes were collected by centrifugation at 15,000 x g for 20 min and washed once more in HM buffer. Protein concentration was assessed by the method of Lowry et al. (33).
Iodination hGH, bGH, and rPRL were iodinated as previously described (35) by using the IODO-GEN method (36) with a hormone to 125 I ratio of 2:1.125I-labeled hormone was separated on a Sephadex G-50 column (0.8 x 19 cm) and subsequently purified on Ultrogel AcA54 (1 X 55 cm). Specific radioactivities of 30-50 were obtained. Competitive binding assays Microsomal membranes (100 ng protein/tube) were incubated with 20,000 cpm [125I]hGH and unlabeled hGH, bGH, or oPRL at concentrations ranging from 5 to 10,000 ng/ml in a total volume of 250 n\ HM buffer containing 0.1% wt/vol BSA (HMB buffer). All assays were carried out in duplicate. After a 16 h incubation at room temperature, membrane-bound hormone was separated from free hormone by centrifugation at 1,500 X g for 20 min after the addition of 1 ml HMB buffer at 4 C. Membrane pellets were then counted for radioactivity. Specifically bound radioactivity was obtained after subtracting from each value the radioactivity remaining in the membrane pellets incubated in the presence of 10 ng unlabeled hormone. Coualent cross-linking of P2bIJhGH to membrane preparations All steps were carried out using silicone-treated glass tubes. [125I]hGH (40,000 cpm) was incubated in the absence of BSA with liver membranes in the presence or absence of excess unlabeled hGH. After binding equilibria were established, the entire reaction mixture was treated at 21 C for 15 min with DSS in dimethyl sulfoxide to give a final concentration of 0.05 mM DSS (37). The efficiency of covalent cross-linking was relatively low (5-10% of specifically bound hormone) but was similar in each tissue preparation examined. The cross-linking
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE reaction was stopped by boiling (3 min) 100 pi aliquots in the presence of 2% (wt/vol) sodium dodecyl sulfate (SDS) with or without dithiothreitol (100 mM). Each sample was then applied to a 7.5% vertical slab SDS-polyacrylamide gel, and electrophoresis was performed according to the method of Laemmli (38). Autoradiography was carried out as described previously (28, 37, 39). RNA isolation RNA was isolated from 100 mg liver obtained from transgenic mice or 19 day pregnant mice using the LiCl precipitation method described by Auffray and Rougeon (40). RNA concentration was measured by absorbance at 260 nm. Northern blot analyses Samples (20 /ug) of total liver RNA were run on formaldehyde/agarose gels and transferred to Nylon membranes as described (41). PRL receptor. For the detection of PRL receptor mRNA, blots were prehybridized in 5x SSPE (1 x SSPE = 0.18 M NaCl, 0.01 M sodium phosphate, pH 7.7, 1 mM EDTA), 0.1% SDS, 2x Denhardt's reagent (lx = 0.02% BSA, 0.02% polyvinyl pirolidone, 0.02% Ficoll) and 100 /xg/ml denatured salmon sperm DNA, at 50 C. Hybridizations were performed in the buffer solution described above at 50 C using an end-labeled (42) oligonucleotide probe (30-mer) corresponding to the Cterminal 10 amino acids of the cloned rat liver PRL-R (43). GH receptor and 18S rRNA. Blots were prehybridized in 5x SSPE, 0.25% sodium iV-laurylsarcosine, and 0.2 mg/ml denatured salmon sperm DNA. Hybridizations were performed in the same buffer solution. For the analysis of GH receptor transcripts, Northern blots were incubated with a radiolabeled 638 base pair cDNA fragment (44) from the rabbit GH-R at 58 C (26). For the analysis of 18S rRNA a radiolabeled cDNA probe (44), specific for the 18S rRNA was used (27). Incubations were performed at 63 C. Densitometric analysis Blots were analyzed on a Zeineh Scanning Densitometer, model SLR-TRFF (Biomed Instruments Inc.). The degree of hybridization of a specific band was quantified relative to the hybridization of the 1.9 kilobase (kb) band observed with the 18S rRNA probe.
Results Fourteen transgenic mice, generated from three giant F o generation mice (nos. 43, 56 and 127) were used in this study and are described in Table 1. Table 1 also shows three important features of these transgenic mice: 1) there is a high degree of variation in serum oGH levels within any pedigree, although variations in copy number of the transgene within any pedigree were a rare feature in these transgenic mice (data not shown); 2) a number of transgenic offspring of giant mice failed to express significantly high levels of serum oGH {e.g. mice 56-1-
1241
216, 56-1-219, and 127-63-178); and 3) a number of transgenic mice which had high levels of serum oGH did not respond to the hormone and were of normal size (e.g. 56-11, 56-1-224, and 56-9-203). These observations have been made previously on transgenic mice expressing oGH (21) as well as other GHs (18) and still remain unexplained. The levels of PRL in these transgenic mice were also assayed. The concentrations for nontransgenic control mice were between 1.8 and 16.0 ng/ml, and the concentrations for normal-size and giant transgenic mice, (both male and female) were within the normal range. The only notable difference between the groups was an apparent lowering of PRL concentration in the giant oGH expressing mice 6.0 ± 0.74 (mean ± SEM; n = 8) vs. 11.0 ± 2.0 (n = 6) in the normal size mice (P < 0.05) (data not shown). Receptor activity in microsomal preparations from livers of transgenic mice In initial assays, [125I]hGH was used as a tracer and unlabeled hGH was used as a competitor. Microsomal preparations from livers of normal and late pregnant mice were used as controls. Microsomal preparations from late pregnant mice usually show an 8- to 10-fold increase in specific binding of GH and PRL and have provided a useful model for the characterization of lactogenic receptors (45). All the transgenic mice with high serum oGH levels had elevated levels of receptor activity relative to normal mice (Fig. 2a). The levels of receptor activity in these transgenic mice were comparable to that observed in a late pregnant mouse. High levels of receptor activity were found in the high serum oGH mice irrespective of their sex, final size, or age at death. By contrast, transgenic mice which produced only low levels of serum oGH (56-1-216, 56-1-219 and 127-63-178) had low levels of receptor activity, comparable to that of the normal mouse. hGH is known to have both somatotrophic and lactogenic activities and can bind to both GH-R and PRL-R (46). Therefore to further investigate the nature of the receptors present in microsomal preparations from transgenic mice, competitive binding assays were performed using unlabeled bGH and oPRL, which are known to bind more specifically to each receptor class (45, 47, 48). Results in Fig. 2b show that both unlabeled bGH and oPRL displaced [125I]hGH from microsomal preparations from transgenic mice expressing elevated serum oGH levels. The percent specific binding in the presence of unlabeled oPRL was similar to that seen with unlabeled hGH. However, oPRL at high doses is known to cross-react with somatotrophic (GH) receptors (49) and hence the apparent abundance of PRL-R is
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
1242 a.
I?
_
_
^5 £ 8. 8- 40
j—
—
- °
r—n |—i
SPECIFIC COMPLEX
PROTEIN
(IOOK)
r—i
r—|
/GH-BINDING \ '
(
r—]
70K
1
(48 K)
20 -
'
122 K 92 K 82 K 7OK
—
rinn n
Endo • 1991 Voll28>No3
_ 116K - 98 -
68
_ 45
b?
_ 30
FiG. 3. Cross-linking of [125I]hGH with microsomal membranes. [125I] hGH was chemically cross-linked to microsomal membranes in the
FIG. 2. Receptor activity in microsomal preparations from livers of transgenic mice, a, Displacement of [125I]hGH by unlabeled hGH from mouse liver microsomes. Microsomal membranes (100 fig protein) were incubated with 20,000 cpm [125I]hGH and either 0 or 10,000 ng unlabeled hGH. b, Displacement of [125I]hGH by unlabeled oGH, bGH, and oPRL. Microsomal membranes (100 ng protein) were incubated with 20,000 cpm [125I]hGH and either 0 or 10,000 ng unlabeled hGH (•), oPRL (M) bGH (•). In both panels a and b the data are plotted as the percent of total counts added that were specifically bound. Nonspecific binding was less than 10% of total counts added.
probably overestimated. The percent specific binding observed with the addition of unlabeled bGH was lower than that with unlabeled hGH and varied between samples but was still significant in most cases and was comparable to that observed in the pregnant mouse. These data indicate that both GH-R and PRL-R are present in transgenic mice expressing oGH and imply that both receptor types are elevated. To test the possibility that the apparently lower level of GH-R was due to endogenous, bound oGH interfering with bGH in the competitive assays, this experiment was repeated using microsomal preparations washed with 4 M MgCl2 to remove any membrane-bound oGH molecules (34). Similar results were obtained (data not shown). The above data therefore suggest that microsomal preparations isolated from transgenic mice expressing high oGH levels may contain elevated levels of more than one type of receptor, i.e. both GH-R and PRL-R. Cross-linking studies The receptors to which the various ligands in the previous experiments bound were further investigated by analysis on polyacrylamide gels of microsomal preparations cross-linked to [125I]hGH. Under reducing conditions (Fig. 3) the most prominent band had an Mr of
absence (lanes a, c, e, and g) or presence (lanes b, d, f, and h) of excess unlabeled hGH as described. After stopping the reaction by boiling in the presence of 2% (wt/vol) SDS, reduced samples were applied to a 7.5% SDS-polyacrylamide gel. Electrophoresis was performed by the method of Laemmli (38) and autoradiography performed as described (28, 37, 39). Molecular wts of protein markers are shown on the right; those of the [125I]hGH binding protein complexes or the binding proteins themselves (in parentheses) are shown on the left.
70,000 and was observed in preparations from three transgenic mice expressing high levels of oGH. Three other very minor bands (Mr 82,000, 92,000, and 122,000) were also observed. In the preparation obtained from pregnant mouse liver the same four bands were visualized. In all samples the appearance of these bands was completely inhibited by the addition of 10 /xg unlabeled hGH in the incubation mixture. The apparent mol wts of these labeled polypeptides minus that of hGH (22,000) are 48,000,60,000, 70,000, and 100,000 in transgenic mice and in the pregnant mouse. Cross-linking studies on microsomal preparations from rabbit, rat, and mouse livers have revealed that GH-R in rabbit and mouse liver consists primarily of polypeptides of Mr 100,000 and 60,000-70,000, while in the rat it consists of a single species of Mr 100,000 (50). In the case of PRL-R, the reported mol wts range between 35,000-45,000 (51-53). The above mol wts are very similar to those observed in cross-linking experiments using microsomal preparations from transgenic mice. It would appear therefore that the species binding predominantly to [125I]hGH in transgenic mice (the Mr 48,000 polypeptide) is the mouse PRL-R with a smaller amount of binding to GH-R (the Mr 60,000, 70,000, and 100,000 polypeptides). Northern blot analysis of RNA from livers of transgenic mice Total cellular liver RNA from transgenic mice was analyzed using a GH-R probe. Two RNA species of 3.9
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
and 1.2 kb were observed in all transgenic mice expressing high serum oGH levels (Fig. 4). These mRNA species were present at very low levels in transgenic mice expressing low levels of oGH. The sizes of these RNA species were similar to those observed for the pregnant mouse and are consistent with those previously described for the mouse GH-R by Smith et al. (26). Total cellular liver RNA was also analyzed with a probe specific for rat liver PRL-R. A major band corresponding to an mRNA species of 1.9 kb was observed in all preparations obtained from transgenic mice as well as from the pregnant mouse (Fig. 4). The low oGH expressing transgenic mice and the normal mouse had very low levels of this PRL-R transcript, which corresponds reasonably well with the primary transcript of 2.2 kb reported in rat tissues by Boutin et al. (43). An additional band corresponding to an RNA species of 2.7 kb was also observed in all high expressers and in the pregnant mouse. This band, which is clearly inducible by oGH, has not previously been identified as a major PRLR mRNA species (43). However, a very recent report (54) has described the presence of a long form of PRL receptor in ovary and liver with mRNA between 2.5 and 3 kb.
GH-R
1243
Densitometric analysis of autoradiograms was performed to obtain an estimate of the relative amounts of mRNA species observed. The data are shown in Fig. 5 and are expressed as a ratio of the intensity of each band with respect to that of the 18S rRNA on the same autoradiogram. Results for the two mRNA species observed on autoradiograms analyzed with the GH-R probe were as follows. The 1.2 kb species was elevated in most mice with elevated serum oGH with respect to the normal mouse, the only exception being mouse 56. The increase in intensity of the 1.2 kb species varied greatly between the mice and ranged from 1.6 times in mouse 56-9-193 to 8.0 times in mouse 56-46. In mice with low serum oGH, on the other hand, the amount of the 1.2 kb mRNA species was comparable to that of the normal mouse. In the case of the 3.9 kb species, elevated levels of mRNA were observed in most transgenic mice expressing high levels of serum oGH. The increase in mRNA levels ranged from 1.6 times in mouse 56 to 8.5 times in mouse 43-27-174. However it was also observed that mRNA levels for the 3.9 kb species were slightly elevated in the two transgenic mice expressing low levels of serum oGH and were approximately 1.4 times the levels observed in the normal mouse. Densitometric analysis of autoradiograms of Northern blots probed with the PRL-R probe showed that the relative levels of the 1.9 kb mRNA species were elevated in all transgenic mice expressing high serum oGH levels. The increase ranged from 1.9 times in mouse 127 to 11.9 times in mouse 56-46. Transgenic mice expressing low levels of serum oGH on the other hand showed no
RATIO GH-R 3.9kb 18SRNA PRL-R
MMHl > al -27 -1-9
18S
GH-R 1.2kb 18SRNA
PRL-R 2.7kb 18SRNA
PRL-R 1.9kb 18SRNA FlG. 4. Northern blot analysis of RNA from livers of transgenic mice. Total cellular liver RNA (20 ng) from each transgenic mouse, a 19 day pregnant mouse, and a normal mouse were electrophoresed on formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with the following probes under the conditions described. Top, a rabbit GH-R cDNA probe (44), 5-day exposure; Middle, an oligomer probe directed against PRL-R, 2-day exposure; Bottom, a rat 18S cDNA probe (27), 1-day exposure. The three probes were used sequentially on exactly the same filter.
Y7A V7"
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FlG. 5. Densitometric analysis of Northern blots. Blots shown in Fig. 4 were analyzed as described in Materials and Methods. The degree of hybridization of each mRNA species was quantified relative to that of the 1.9 kb 18S rRNA for the same mouse sample.
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
increase in the 1.9 kb mRNA species. The expression of the 2.7 kb band closely resembled that of the 1.9 kb band. The increase ranged from 2.3 times in mouse 56 to 11 times in mouse 56-46. Two further observations were made from the data shown in Figs. 4 and 5. First, the relative increase in the mRNA species for GH-R and for PRL-R were very similar in each of the transgenic mice. Second, for both GH-R and PRL-R, the increases in mRNA levels occurred irrespective of the sex of the animals, their final size, and their age when killed.
Discussion These studies have shown that transgenic mice carrying an oGH fusion gene and with high serum levels of oGH have elevated levels of both GH-R and PRL-R on liver membrane preparations. Transgenic mice which did not express high levels of oGH did not have increased receptor expression. The increase in receptor activity was not related to the absolute level of oGH, the final size of the mice, or their sex. The observation that high levels of receptor activity were observed in transgenic mice expressing elevated serum oGH levels irrespective of the age at which they were killed strongly suggests that the receptor response to oGH is maintained throughout the life of these transgenic mice. Since the levels of PRL in these mice were similar to those of normal mice, the current data unequivocally support the proposition put forward by Baxter et al. (8) that GH is capable of inducing hepatic GH-R and PRL-R in vivo, and that PRL is not required for the induction of its own receptor. These data also show that the transgenic model represents a less complex and more reliable system than other in vivo models used previously for studies on GH and/or PRL regulation of receptor status. The receptors for GH as observed on autoradiographs of cross-linking experiments consisted of species of Mr 100,000, 70,000, and 60,000 for both the pregnant mouse and the transgenic mice. These Mr are similar to those reported in the literature (Mr 100,000 and Mr 60,00070,000) (50); however it is not clear why two species should be present within the Mr 60,000-70,000 range. This may reflect different glycosylation states of the single receptor polypeptide reported from cloning studies (55), and is perhaps related to the chronically elevated GH concentrations observed in pregnant and in transgenic mice. The Mr for PRL-R (48,000) was slightly above the range (Mr 35,000-45,000) commonly reported (51-53). However, in terms of binding characteristics and mRNA sizes it would appear that the receptors present in liver microsomal preparations from transgenic mice are bona fide GH and PRL receptors. In their analysis of GH-R mRNA from pregnant and
Endo • 1991 Voll28«No3
nonpregnant mice, Smith et al. (26) observed that both the 3.9 kb and the 1.2 kb species were enriched in pregnant mice compared with nonpregnant mice, although the relevant changes in each mRNA species were not quantified. Elevation of these two species was also observed in the pregnant mouse in our study; however densitometric analysis of Northern blots showed that these mRNA species were not enriched to the same degree in all the mice studied (pregnant or transgenic). There is strong evidence to support the suggestion that the 1.2 kb species encodes the serum GH binding protein while the 3.9 kb species encodes the membrane bound form of the receptor. Our densitometric data may indicate that in pregnancy, as well as in transgenic mice, the secreted form of GH-R is preferentially induced. There was no clear difference in receptor abundance between high expressers of oGH which responded (with increased growth) to oGH and high expressers which did not respond to oGH. Indeed almost all high expressers had high concentrations of GH and PRL receptor activity and elevated levels of mRNA for both GH-R and PRL-R. The only exception was mouse 56 which had elevated levels of mRNA for PRL-R but not for GH-R. However, this particular mouse had an abundance of hepatic tumors present at death (56), and it is possible that this may have selectively influenced expression of the GH-R gene. These observations suggest that the response to elevated serum GH is not directly related to the numbers of receptors present in liver microsomal preparations. However, it must be noted that these measurements were performed on preparations obtained from adult mice, and it is possible that the levels of receptor activity in these mice are different from those of juvenile mice when maximum growth rates are present. To further investigate whether receptor numbers are directly related to a positive response to serum oGH concentrations, these experiments should perhaps be repeated using juvenile mice. The data reported here on GH-R and PRL-R levels in oGH transgenic mice provide important insights into the physiological consequences of chronically elevated levels of GH on GH action at the cellular level. GH is well known to have a variety of actions in target tissues: metabolic actions, both acute insulin-like effects and longer term antiinsulin (diabetogenic) effects, as well as growth-promoting actions, many of which may be mediated via the insulin-like growth factor (IGF) family (57). The biological effects of GH have been difficult to analyze due to the lack of a biologically responsive in vitro system. The only useful system has been the isolated adipocyte in which a direct correlation has been made between receptor occupancy and metabolic function (58). Studies using this system (59) have shown that the acute metabolic effects of GH, e.g. glucose uptake
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GH AND PRL RECEPTORS IN oGH TRANSGENIC MICE
and oxidation, amino acid uptake and lipogenesis, are very sensitive to refractoriness in the presence of continued or prolonged exposure to GH. In transgenic mice where oGH levels are constitutively high it is most likely that metabolic tissues will also be refractory to GH and will not express the acute effects of GH. In contrast., the growth-related effects of GH are not subject to refractoriness, and this is evidenced in the current study by the increased growth of several of the mice expressing high levels of oGH and by increased levels of immunoreactive IGF-I (irIGF-I) and hepatic IGF-I mRNA in some of these mice (our unpublished observations). Both parameters were generally increased in those mice with elevated oGH levels, compared to transgenic mice which were not actively expressing the gene, although there was some variation in absolute levels. In some mice with marked hepatic pathology (56) the serum IGF-I levels were low despite increased hepatic IGF-I mRNA. The rise in IGFI mRNA and irIGF-I levels in oGH-expressing mice was generally consistent with the previous reports by Mathews et al. (60, 61) in much younger (
