Molecular and Cellular Endocrinology, 14 (1990) 213-219

Elsevier Scientific Publishers Ireland, Ltd.

213

MOLCEL 02399

Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone

Jiangming Luo and Liam J. Murphy Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg R3E 0 W3, Canada

(Received 13 August 1990; accepted 13 September 1990)

Key words: Glucocorticoid; Insulin-like growth factor; Growth retardation

Dexamethasone (DXM), a potent long acting glucocorticoid results in growth retardation when administered to children and experimental animals. We have used ligand blotting and RNA blotting techniques to examine the effects of DXM on serum insulin-like growth factor binding protein 3 (IGFBP-3) levels and hepatic IGFBP-3 mRNA abundance. A time- and dose-dependent increase in the 39-42 kDa serum IGF binding proteins and in IGFBP-3 mRNA abundance was observed in DXM-treated rats. A significant increase in serum IGF binding capacity was seen with as little as 0.1 pg/lOO g body weight. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h following DXM administration. IGFBP-3 mRNA levels reached a peak at 3 h (1.9 f 0.2 fold, p < 0.005) and declined to normal levels in 6-12 h after DXM administration. Since the IGF binding proteins may be able to inhibit the action of the IGFs, enhanced expression of IGFBP-3 may be one of the mechanisms involved in DXM-induced growth retardation.

Introduction

Glucocorticoid excess whether as a result of exogenous administration or as a result of pitu- itary or adrenal tumours is associated with growth retardation (Blodgett et al., 1956; Loeb, 1976). Several mechanisms have been proposed to ex- plain this unwanted effect of glucocorticoid ther- apy. These mechanisms include inhibition of pitu- itary growth hormone secretion (Smals et al., 1986; Kaufmann et al., 1899), inhibition of growth hormone signal transduction and impaired in- sulin-like growth factor-l (IGF-I) production

Address for correspondence: Liam J. Murphy, MB, PhD, University of Manitoba, Room 435, Basic Medical Science

Building, 110 Bannatyne Ave., Winnipeg R3E OW3, Canada.

(Asakawa et al., 1982; Luo and Murphy, 1989), inhibition of IGF-I action by circulating inhibitors (Uterman and Phillips, 1985), inhibition of IGF-I action at target tissues (Keret et al., 1976) and direct effects of glucocorticoids on skeletal tissue matrix production (Silbermann and Maor, 1978). While each of these mechanisms may be im- portant in glucocorticoid induced growth retarda- tion additional mechanisms may also be involved.

Circulating insulin-like growth factors (IGFs) measured by various techniques including bioas- says, receptor assays, protein-binding and im- munoassays, have been reported to be low, normal or high in patients with Cushing’s syndrome and individuals receiving glucocorticoid therapy (Elders et al., 1975; Furlanetto et al., 1977; Thoren et al., 1981). Furthermore, there appears to be no relationship between serum IGF-I concentrations

0303-7207/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland, Ltd.

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and growth velocity in children with gluco- corticoid excess (Gourmelen et al., 1982). This latter observation suggests steroid-induced inhibi- tors may be important in the impaired growth observed in children with hypercorticoidism. The presence of such an inhibitor has been demon- strated in a cartilage bioassay (Uterman and Phil- lips, 1985). However, the nature of this steroid-in- duced inhibitor has not been determined.

The IGFs are present in the circulation and tissue extracts in association with high affinity binding proteins. The function of these binding proteins has not been resolved. Most reports to date have focused on the ability of these binding proteins to inhibit IGF-I action in a variety of bioassays including the classical cartilage bioassay (Meuli et al., 1978; Drop et al., 1979; Knauer and Smith, 1980; Burch et al., 1990). However, at least under some circumstances, the binding proteins may be able to enhance IGF-I action (Elgin et al., 1987). We have previously demonstrated that dexamethasone (DXM) enhances the expression of insulin-like growth factor binding protein 1 (IGFBP-1) in the rat (Luo and Murphy, 1990). This binding protein, however, appears to con- stitute only a small proportion of the total 1251- IGF-I binding capacity in adult rat serum (Donovan et al., 1989) and the small DXM-in- duced increase in IGFBP-1 is likely to have little effect on total serum ‘251-IGF-I binding capacity. In both human and adult rat serum IGFBP-3, a glycosylated, growth hormone dependent protein with an apparent molecular weight of 39-42 kDa is the predominant ‘*‘I-IGF-I binding protein identified by ligand binding studies (Donovan et al., 1989). In this study we have used ligand and Northern blotting to examine the effects of DXM on IGFBP-3 expression in the rat.

Materials and methods

Animals Male Sprague-Dawley rats at 4 weeks of age

and 80-100 g body weight were obtained from the University of Manitoba breeding facility (Wirmi- peg, Canada). Groups of four or five rats were injected i.p. with DXM (Sigma, St. Louis, MO, U.S.A.), 6 pg/lOO g body weight and killed by decapitation at various times l-24 h after injec-

tion. In a separate study groups of four or five rats were killed 1 h after a single injection of varying doses of DXM, 0.1-60 pg/lOO g body weight. Control rats received an equivalent volume of vehicle; 15% ethanol, 85% phosphate-buffered saline (PBS). In the chronic study groups of five rats received daily injections for 6 days of vehicle containing 0, 1, 6, or 60 pg of DXM. These animals were killed 9 h after the final injection.

Ligand blotting Two types of assays were used. Firstly total

available serum IGF-I binding activity was quantitated by a dot-blot assay. Serial dilutions of sera from individual rats were immobilized on nitrocellulose paper using a filtration vacuum manifold (Schleicher and Schuell, Keene, NH, U.S.A.). The nitrocellulose paper was washed and incubated with ‘251-IGF-I (Amersham Canada, Oakville, Ontario, Canada) as described by Unter- man et al. (1989). Since no attempt is made to remove endogenous IGFs, this assay reflects avail- able ‘251-IGF-I sites present in the serum. In ad- dition serum samples, pooled from the various treatment groups were boiled, resolved on a 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel and transferred to nitrocellulose paper and processed as described by Hossenlopp et al. (1986). The individual serum IGF binding proteins were visualized after incubation with ‘251-IGF-I and autoradiography.

RNA extraction and RNA blot hybridization Total RNA was extracted from individual rat

liver using the guanidinium isothiocyanate-cesium chloride method (Chirgwin et al., 1979). The abundance of IGFBP-3 mRNA in individual rat samples was quantitated by Northern blot hy- bridization. Hybridization was performed at 42 o C in a solution containing 50% formamide. A oligonucleotide with a sequence corresponding to the complement of nucleotides 54-114 of the pub- lished sequences for rat IGFBP-3 (Shimasaki et al., 1989) was radiolabelled using T4 polynucleo- tide kinase and [ y- 32 P]ATP. Filters were also hy- bridized with a 28s ribosomal probe (Gonzalez et al., 1985) as a control for RNA loading. The ribosomal probe was radiolabelled by nick-trans- lation using [ 32P]dCTP with reagent obtained from

Amersham Canada (Oakville, Ontario, Canada). All autoradiographs were analyzed by densitome- try. Significant differences between treated and control rats were determined for various parame- ters using Student’s t-test.

Results

When sera from control rats were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and ligand blotting, six IGF binding proteins were identified. These proteins had apparent molecular weights of 42, 40, 39, 30, 29 and 24 kDa. The 42, 40 and 39 kDa binding proteins most probably represent IGFBP-3 which is known to be glyco- sylated and consequently poorly resolved by SDS-PAGE (Zapf et al., 1990). IGFBP-3 con- stituted the majority of the serum ‘251-IGF-I bind- ing capacity and accounted for 62.8 f 7.3% of the total densitometric signal when ligand blot autora- diograms were quantitated (Fig. 1). In contrast the 30 kDa binding protein which may represent IGFBP-1 represent less than 10% of the total serum ‘2SI-IGF-I binding capacity. Each of the six binding proteins detected in serum from control rats was also present in sera from DXM-treated rats.

MOLECULAR 1251-IGF-I BINDING WEIGHT CAPACITY

Fig. 1. Identification and abundance of insulin-like growth

factor binding proteins. Pooled serum samples (9 al) from

normal control rats were analyzed in a 12% SDS-PAGE and

transferred to nitrocellulose for ligand blotting with ‘251-IGF-I.

The autoradiographs were quantitated by densitometry. The

percentage of the total autoradiographic signal has been used

to determine the contribution of the various binding proteins

to the total ‘251-IGF-I binding capacity of the serum. Data

represents the mean f SEM, n = 3. The apparent molecular

weights were determined by comparison with protein molecu-

lar weight standards.

01 3 61224

Fig. 2. The time course of induction of serum IGF binding

proteins following DXM administration. Panel A depicts a

ligand blot of serum from rats at various times after a single

injection of 6 pg/lOO g body weight of DXM. Pooled serum

samples (9 ~1) were analyzed in a 12% SDS-PAGE and trans-

ferred to nitrocellulose for ligand blotting with lz51-IGF-I. The

apparent molecular weights were determined by comparison

with protein molecular weight standards. In panel E, the

abundance of the 42, 40 and 39 kDa proteins has been quanti-

tated by densitometry and expressed as a percentage of the

time 0 control rats.

The effect of a single injection of DXM, 6 pg/lOO g body weight, on serum IGFBP-3 is shown in Fig. 2. An increase in the 42, 40 and 39 kDa binding proteins was apparent 1 h after DXM administration. Using an antisense oligonucleotide probe, hepatic IGFBP-3 was detected as a single 2.6 kb band on Northern blots. A representative Northern blot of hepatic RNA from rats killed at

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various times after DXM treatment is shown in Fig. 3. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h after a single injection of DXM (Fig. 3B). IGFBP-3 mRNA levels reached a peak at 3 h (1.9 f 0.2 fold, p c 0.005) and declined to normal levels in 6-12 h after DXM administration.

The dose dependence of the DXM effect on IGFBP-3 was examined in Fig. 4. A dose-depen- dent increase in serum IGFBP-3 was observed with as little as 0.1 pg/lOO g body weight (Fig. 4A

28s

0 13 61224

12 16 24 TIME AFTER,DXM-hours

Fig. 3. The time course of induction of hepatic IGFBP-3

mRNA following DXM administration. Panel A depicts a

representative Northern blot of RNA extracted from rats killed

at various times after a single injection of DXM, 6 pg/lOO g

body weight. The pattern of hybridization obtained with an

antisense oligonucleotide probe is shown in the upper autora-

diographs. As a control for gel loading the pattern of hybrid-

ization obtained with a 28s ribosomal probe is depicted in the

lower autoradiogram. In panel B data from four or five rats

per time point have been quantitated by densitometry and

IGFBP-3 mRNA abundance has been expressed as a per-

centage of the time 0, control rats. *. * * p < 0.05 and P i

0.005, respectively, for the difference between treated and

control rats.

and B). The most dramatic increase was seen in the 39 kDa binding protein. Smaller increases were also consistently seen in the 42 and 40 kDa binding proteins. A similar dose-dependent in- crease was seen when total IGF binding activity was quantitated in individual rat serum samples by the dot-blot assay (Fig. 4C). When IGFBP-3 mRNA was quantitated in rats which received varying doses of DXM a significant increase was observed only with 6 pg/lOO g body weight (Fig. 40).

Varying amounts of DXM were administered chronically to rats over a period of 6 days. A previously reported by us, DXM at 6 and 60 pg/lOO g body weight resulted in significant growth retardation (Luo and Murphy, 1989). In sera from these animals there was a dose-depen- dent increase in IGFBP-3, as determined by ligand blotting of pooled sera and in total IGF binding capacity quantitated by the dot-blot analysis of individual rat sera (Fig. 5A and B respectively). An increase in IGFBP-3 mRNA abundance was also observed in hepatic tissue from rats treated chronically with DXM (Fig. 5C).

Discussion

Glucocorticoid excess is associated with marked skeletal growth retardation in both children and experimental animals (Blodgett et al., 1956; Loeb, 1976). In a previous report from this laboratory we have demonstrated that DXM inhibits growth hormone (GH) induction of hepatic and skeletal IGF-I expression in hypophysectomized rats and reduced steady state IGF-I mRNA levels in pitui- tary-intact rats (Luo and Murphy, 1989). We now report that DXM enhances hepatic expression of the GH-dependent IGF binding protein, IGFBP-3. The rat IGFBP-3 is a protein of 265 amino acids with four potential Asn-linked glycosylation sites (Shimasaki et al., 1989). The 39-42 kDa binding proteins identified in our ligand blotting experi- ments probably represent IGFBP-3. All three of these binding proteins are reduced in sera from hypophysectomized animals and increase in re- sponse to GH treatment (Zapf et al., 1990). How- ever, since antisera directed against rat IGFBP-3 are not available, it is not possible to determine whether the 42, 40 and 39 kDa proteins are post-

217

translational modified products of the single IGFBP-3 gene or rather represent one or more separate and distinct binding proteins. If analo- gous to human IGFBP-3, all three of binding proteins are likely to be the product of a single rat IGFBP-3 gene, differing only in the degree of glycosylation. The difficulty in resolving these three proteins on SDS-PAGE argues in favour of this latter hypothesis.

Administration of DXM caused a time- and dose-dependent increase in IGFBP-3 mRNA and in the serum IGF binding capacity. This was unexpected, since the GH dependence of IGFBP-3 appears to be mediated by IGF-I (Zapf et al.,

A.

24*

1990) and under the same conditions used here, we have previously reported that DXM decreases hepatic IGF-I mRNA steady state levels (Luo and Murphy, 1989). Indeed, when the nitrocellulose filters from this study were probed with IGF-I cDNA similar findings to those previously re- ported by us were observed. Thus, it would seem unlikely that the effects of DXM on IGFBP-3 expression are mediated via IGF-I. The effects of DXM on IGFBP-3 expression appear to involve a different mechanism since inhibition of GH-signal transduction in the liver should result in a de- crease rather than an increase in hepatic IGFBP-3 expression.

8 0 0.1 1 6 80 v)

Fig. 4. The dose-dependent induction of IGFBP-3 expression by DXM. In panel A sera (9 ~1) pooled from rats killed 1 h after

injection of varying amounts of DXM were analyzed by SDS-PAGE and ligand blotting. The apparent molecular weights of the

major binding proteins were determined by comparison with protein molecular weight standards. In panel 3, the abundance of the

42, 40 and 39 kDa proteins have been quantitated by densitometry. In panel C, r2%GF binding capacity of sera from individual

rats, four or five rats per group has been quantitated using a dot-blot assay. In panel D, the relative abundance of IGFBP-3 mRNA

has been determined in hepatic RNA extracted from individual rats, four or five rats per group. In each case data have been

expressed as a percentage of the untreated control rats. *‘* * p < 0.05 and P < 0.01, respectively, for the difference between treated

and control rats.

218

B. 6 = 300- s 8 **

-

Fig. 5. The effects of chronic administration of DXM on IGFBP-3 expression. In panel A, pooled serum samples were analyzed by SDS-PAGE and ligand blotting. The 42,4O and 39 kDa binding proteins corresponding to IGFBP-3 were quanti- tated by densitometry. In panel B, “‘1-1GF binding capacity of sera from individual rats, four or five rats per group has been quantitated using a dot-blot assay. In pane1 C, the relative abundance of hepatic IGFBP-3 mRNA was de- termined In each case data have been expressed as a per- centage of the untreated control rats. *** * p < 0.05 and P -c 0.005, respectively, for the difference between treated and

control rats.

We have previously reported that hepatic ex- pression of IGFBP-1 is also up-regulated by DXM

(Luo and Murphy, 1990). The small increase in the 30 kDa binding protein following DXM ad- ministration, identified in this study by ligand

blotting, confirms our previous observations using specific antibodies to IGFBP-1. However, the ef- fect of DXM on IGFBP-1 is quite small compared to the increase in the 39-40 kDa binding proteins. Furthermore, if IGFBP-1 accounts for all of the 29 kDa binding protein identified by ligand blot- ting, IGFBP-1 appears to constitute less than 10% of the total ‘251-IGF-I binding capacity.

The physiological role of the IGF binding pro- teins remains controversial. The simplest view is that they serve to prolong the half-life of IGFs

and protect against the hypoglycemic effects of the large amounts of IGF present in the circula-

tion. In a number of different systems, IGF bind- ing proteins appear to be able to inhibit the ac- tions of IGF-I (Meuli et al., 1978; Drop et al., 1979; Knauer and Smith, 1980; Burch et al., 1990). However, there is at least one report that human IGFBP-1 is able to enhance IGF-I action (Elgin et al., 1987). Most studies to date which address the physiological role of the binding proteins have used human IGFBP-1 purified from amniotic fluid. The action of IGFBP-3 in these assays has not been investigated.

In this study, DXM at doses which retarded growth and inhibited IGF-I expression, enhanced IGFBP-3 expression. If the function of IGFBP-3

is to limit the action of the IGFs then enhanced expression of this binding protein together with IGFBP-1 may reduce bioavailability of IGF-I.

This together with the DXM-induced inhibition of IGF-I expression may be responsible in part for the pronounced growth retardation which is char- acteristic of glucocorticoid excess.

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