Molecular and Cellular Endocrinology, 13 (1990) Rl-R6 Elsevier Scientific Publishers Ireland, Ltd.

MOLCEL 02380

Rapid Paper

Expression of the growth hormone-binding protein messenger RNA in the liver and extrahepatic tissues in the rat: co-expression with the growth hormone receptor Bjiirn Carlsson I, H&an Billig ‘, Lars Rymo 2 and Olle G.P. Isaksson ’ Departments

of I Physiology and 2 Medical Biochemistry,

University of Giiteborg, S-400 33 Giiteborg Sweden

(Received 13 August 1990; accepted 22 August 1990)

Key worak: Growth

hormone-binding protein mRNA; Growth hormone-receptor mRNA; (Rat)

A cDNA encoding a growth hormone-binding protein (GH-BP) was recently cloned from mouse and rat liver. The GH-BP in these species is identical to the extracellular part of the GH receptor (GH-R) with the transmembrane and intracellular domain substituted for a hydrophilic tail. In the present study the expression of the GH-BP and GH-R was studied in rat liver and extrahepatic tissues. Specific transcripts with estimated sizes of 1.2 kb (GH-BP) and 4.0 kb (GH-R) were found in the liver from both sexes. The expression of GH-BP increased with age up to puberty suggesting that it is developmentally regulated in a similar manner as GH-R. GH-BP mRNA was found in all extrahepatic tissues examined that contained GH-R mRNA. The ratio between the 1.2 kb and 4.0 kb transcripts varied between tissues indicating that GH-R and GH-BP transcripts may be separately regulated. The co-expression of GH-BP and GH-R suggests a functional role for the GH-BP in the local regulation of GH action.

Introduction

Growth hormone (GH) is an important regulator of postnatal growth and intermediary metabolism in many species, including man and rodent (Isaksson et al., 1988). The effects of GH are evoked by the interaction of the ligand with its cellular receptor. The response is not only dependent on the concentration of GH but also on the manner in which it is presented to its target cells (Jansson et al., 1985). Recent studies suggest that the tissue response is also dependent on the ex-

Address for correspondence: Dr. Bjam Carlsson, Department of Physiology, University of Gsteborg, P.O. Box 33031, S-400 33 Gateborg, Sweden. 0303-7207/90/$03.50

pression of the GH receptor (GH-R) (Maes et al., 1983; Mathews et al., 1989; Nilsson et al., 1990). In addition to growth hormone receptors on the target cells, a circulating growth hormone-binding protein (GH-BP) has been characterized in several species (Peeters and Frisen, 1977; Barnard and Waters, 1986; Baumann et al., 1986; Herington et al., 1986; Haldosen and Gustavsson, 1990). The GH-BP has been shown to decrease the clearance of GH, modify its distribution (Baumann et al., 1987) and decrease the binding of GH to its receptor (Waters et al., 1990). It has been suggested that the GH-BP is produced by a limited proteolytic cleavage of the receptor and the release of the extracellular domain (Trivedi and Doughaday, 1988). The primary sequence of the binding protein and the extracellular part of the receptor

0 1990 Elsevier Scientific Publishers Ireland, Ltd.

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in the rabbit are identical (Leung et al., 1987). In mouse and in rat the GH-R and the GH-BP are probably synthesized from separate mRNAs generated by alternative splicing (Baumbach et al., 1989; Smith et al., 1989). This GH-BP is identical to the GH-R but with a hydrophilic tail replacing the hydrophobic transmembrane domain and the intracellular domain. The GH-BP (Baumann et al., 1986) and the corresponding mRNA (Baumbath et al., 1989; Smith et al., 1989) are found in high concentrations in the liver indicating that this is a major site of production. However, there are indications that the GH-BP is present in the cytosol of extrahepatic tissues (Herington et al., 1986). The objective of the present study was to investigate the pattern of expression of the GH-BP and the GH-R in the liver and extrahepatic tissues of the rat. We demonstrate that the GH-BP mRNA is expressed in both the liver and in extrahepatic GH-target tissues. The ratio between the levels of GH-R and GH-BP mRNAs varies in different tissues suggesting separate regulation of the transcripts.

system (LKB, Stockholm, Sweden). The membranes were baked at 80 o C for 3 h, prehybridized at 60” C for 12 h in 50% formamide, 25 mM H,NaPO,, 25 mM HNa,PO,, 5 X SSC, 0.1% SDS, 1 mM EDTA, 0.05% BSA, 0.05% Ficoll, 0.05% PVP, 200 pg/rnl calf liver RNA, and 20 pg/ml salmon sperm DNA, and hybridized in the prehybridization buffer with the addition of 32P-labeled RNA probe. Antisense [ 32P]UTP-labeled RNA was synthesized using the EcoRI linearized plasmid pT7T3 18U (Mathews et al., 1989) as template. The pT7T3 18U plasmid contains a 560 bp BamHI fragment of the rat GH-R cDNA which encodes part of the extracellular domain of the GH-R identical to a part of the GH-BP. The membranes were washed in 0.1 X SSC, 0.1% SDS at 65 o C. When indicated the membrane was further treated with RNase A (1 pg/ml) at 37 o C, 30 min, and washed in 0.1 X SSC, 0.1% SDS at 37 o C. A 0.24-9.5 kb RNA ladder (BRL) was used to estimate transcript sizes.

Materials and methods rats (Alab, Animals. Female Sprague-Dawley Stockholm, Sweden) were housed under standardized environmental conditions with constant temperature (24-26 o C), humidity (50-608) and artificial 14 h light: 10 h dark cycle. The animals had free access to water and pelleted food. The rats were delivered at least 2 days before the start of the experiment. The animals were killed by cervical dislocation and the tissues were immediately removed, trimmed with micro-scissors and rapidly frozen in liquid nitrogen. All tissues were stored at -70°C until analysis. Northern blot analysis. Total RNA was prepared essentially according to Chomczynski and Sac&i (1987) with minor modifications (Nilsson et al., 1990). Poly(A)+ RNA was prepared with oIigo(dT)-spin column chromatography (Pharmacia, Uppsala, Sweden). 20 pg of total RNA, or when indicated, 2 pg of poly(A)+ RNA was electrophoresed in an agarose (l%)/formaldehyde (2.2 M) gel with ethidiumbromide. The RNA was transferred to Hybond-N membranes (Amersham, Buckinghamshire, U.K.) with a vacuum transfer

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Liver Fig. 1. Analysis of GH-R/GH-BP gene expression in livers from male (160 g) and virgin female (160 g) rats. Total RNA was electrophoresed, transferred and hybridized with a 32Plabeled RNA probe encoding part of the extracellular domain of the GH-R under conditions described in Materials and Methods.

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Northern blot analysis of hepatic total RsyA extracted from male and female rats using a Plabeled RNA probe recognizing both GH-R and GH-BP mRNA revealed the presence of two RNA species, 4.0 kb and 1.2 kb, respectively, in male and female liver (Fig. 1). These transcripts presumably encode GH-R and GH-BP, respectively, as indicated by previous investigations (Baumbach et al., 1989; Smith et al., 1989). When poly(A)+ RNA was analyzed with the same probe an additional transcript was found with an estimated size of 2.6 kb (Fig. 2). The band was resistant to high stringency washes and RNase treatment. It is well established that the expression of the hepatic GH-R is developmentally regulated. The amount of receptor increases from birth to puberty measured as the number of binding sites (Maes et al., 1983) or as the steady-state level of GH-R mRNA (Mathews et al., 1989). To investigate whether the GH-BP follows a similar pattern total RNA was extracted from the liver of male rats of various ages and analyzed for the presence of GH-BP and GH-R mRNA. As can be seen in Fig. 3 both the 4.0 kb and the 1.2 kb transcript were

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Liver Fig. 3. Analysis of GH-R/GH-BP gene expression in livers from male rats of various ages. Total RNA was electrophoresed, transferred and hybridized with a 32P-labeled RNA probe encoding part of the extracellular domain of the GH-R under conditions described in Materials and Methods.

present at all ages analyzed and the concentration of both RNAs increased during the first 6 weeks after birth. Although the liver is supposed to be the most important target tissue for GH, the GH-R gene is expressed in most tissues (Mathews et al., 1989) and direct effects of GH have been demonstrated in extrahepatic tissues (Isaksson et al., 1987). To investigate if the GH-BP is expressed in extrahepatic tissues total RNA was extracted from vari-

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Liver Fig. 2. Analysis of GH-R/GH-BP gene expression in livers from male (160 g) rats. Poly(A)+ RNA (2 pg) was electrophoresed, transferred and hybridized with a 32P-labeled RNA probe encoding part of the extracellular domain of the GH-R under conditions described in Materials and Methods.

Female Fig. 4. Analysis of GH-R/GH-BP gene expression in the liver and extrahepatic tissues from adult female rats (160 g). Total RNA was electrophoresed, transferred and hybridized with a 32P-labeled RNA probe encoding part of the extracellular domain of the GH-R under conditions described in Materials and Methods.

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Discussion

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1.2 Kb Male Fig. 5. Analysis of GH-R/GH-BP gene expression in the liver and extrahepatic tissues from adult male rats (160 g). Total RNA was electrophoresed, transferred and hybridized with a “P-labeled RNA probe encoding part of the extracellular domain of the GH-R under conditions described in Materials and Methods.

ous rat tissues and analyzed for the presence of GH-BP specific mRNA. All tissues examined in female rats that expressed the GH-R encoding 4.0 kb RNA species also contained the 1.2 kb transcript that codes for the GH-BP in the liver (Fig. 4). The ratio between the 4.0 and 1.2 kb transcripts varied between the tissues examined, indicating that they are separately regulated. In male rats the GH-R and the GH-BP mRNAs were detected in all tissues but the spleen (Fig. 5). GH-R or GH-BP transcripts could not be identified in the spleen of neither young nor adult rats (Fig. 6).

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Fig. 6. Analysis of GH-R/GH-BP gene expression in the spleen from male rats of different ages. Total RNA was electrophoresed, transferred and hybridized with a 32P-labeled RNA probe encoding part of the extra&h&u domain of the GH-R under conditions described in Materials and Methods.

In the present study we demonstrate that the GH-BP gene is transcribed in all tissues where GH-R mRNA could be detected. The spleen is the only tissue in which GH-BP and GH-R mRNA could not be found. The wide tissue distribution of GH-BP mRNA is consistent with extrahepatic production of GH-BP and this strongly indicates that GH-BP found in cytosolic preparations of extrahepatic tissues (Herington et al., 1986) is locally synthesized. Furthermore, GH-BP mRNA was recently found in adipose tissue (Frick et al., 1990). Fetal and early postnatal somatic growth of rodents is independent of GH in contrast to later growth (Palmiter et al., 1982). It has been shown that the expression of hepatic GH-R is developmentally regulated with increasing number of GH binding sites from birth to puberty (Maes et al., 1983). We demonstrate here that the expression of the GH-BP gene is developmentally regulated in a similar way as the GH-R gene (Mathews et al., 1989). We have identified two major transcripts (4.0 kb and 1.2 kb) in liver and in extrahepatic tissues showing that the GH-BP transcript is present in all tissues where the GH-R gene is active. An additional transcript with an estimated size of 2.6 kb was evident when Northern blot autoradiograms were exposed for longer times. Three RNAs, of similar sizes as found in the present study, hybridize to a GH-R cDNA probe in rabbit liver (Tiong et al., 1989). At present it is not known what the low abundant 2.6 kb transcript represents. The GH-R was recently recognized to be a member of a receptor super-family (Bazan et al., 1989). Other members include the prolactin receptor (Prl-R) (Boutin et al., 1988), the erythropoietin receptor (D’Andrea et al., 1989), the interleukin-2 receptor (IL-2-R) (Hatakeyama et al., 1989) the interleukin-4 receptor (IL-4-R) (Mosley et al., 1989), the interleukind receptor (IL-6-R) (Yamasaki et al., 1988), interleukin-7 receptor (IL-7-R) (Goodwin et al., 1990). Recently, cDNAs that encode soluble forms (receptor homologous binding proteins) of GH-R (Smith et al., 1989; Baumbath et al., 1989) Prl-R (Davis et al., 1989), IL-4-R (Mosley et al., 1989), and IL-7-R (Good-

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win et al., 1990) have been cloned. In addition, soluble receptors have been demonstrated at the protein level for IL-2-R (Rubin et al., 1985) and IL-6-R (Novick et al., 1989). However, soluble receptors as a phenomenon are not restricted to this receptor family. Instead, several non-related membrane anchored proteins have soluble counterparts. Examples of such are the LH-hCG receptor (Loosefelt et al., 1989), neural adhesion molecules (N-CAMS) (Gower et al, 1988), the major histocompatibility complex (MHC) antigens (Gussow and Ploegh, 1987) and the ~~o~ob~ molecules (Gough, 1987). The physiological role for soluble receptors is largely unknown. However, as discussed above there is increasing evidence that soluble forms of receptors are common for membrane bound receptors. Furthermore, many of the soluble receptors are expressed at high levels. This may indicate that the soluble forms of receptors are of importance for hormone action. Intravenous injection of GH-BP to rats decreases GH clearance and changes the pattern of GH ~st~bution in the rat (Baumann et al., 1987). However, locally expressed and regulated GH-BP may interact directly with GH at the cellular level and affect the balance between GH and GH-R. In vitro experiments indicate that GH-BP decreases the binding of GH to its membrane anchored receptor (Waters et al., 1990). Another interesting possibility is that the GH-BP represents an intracellular receptor for GH that has been internalized by cell surface receptors (Herington et al., 1986). It seems clear that the physiological effects of GH are regulated by the level of circulating hormone, the secretory pattern of the hormone and the number of receptor molecules available on the surface of the target cell. GH-BP might represent an additional control mechanism. It is likely the liver that is the major source of GH-BP but extrahepatic pr~uction of GH-BP may be a mechanism, not previously considered for the local regulation of GH action by modulating the availability of the hormone to its receptor.

We thank Lawrence Mathews for the rat GH-R cDNA. We also thank Harriet Thelander, Jane

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