Transcriptional Regulation of Rat Insulin-Like Growth Factor-Binding Protein-1 Expression by Growth Hormone

Charita Seneviratne, Jiangming Luo, and Liam J. Murphy4 Departments of Internal Medicine and Physiology University of Manitoba Winnipeg, Canada R3E 0W3

The low mol wt insulin-like growth factor-binding protein (IGFBP-1) originally isolated from amniotic fluid has been considered to be GH independent. In this report we have examined the effect of GH on hepatic IGFBP-1 expression in the hypophysectomized rat. Using a rat IGFBP-1 cDNA probe we now demonstrated that hepatic IGFBP-1 expression is up-regulated in the GH-deficient rat. In addition, using ligand blotting, an increase in the abundance of a 30-kDa [125I]IGF-I-BP was detected in both hepatic extracts and serum from hypophysectomized rats. After a single ip injection of GH, the IGFBP-1 transcription rate was reduced within 30 min to the levels seen in the sham-operated control rats. Similarly, hepatic IGFBP-1 mRNA abundance was reduced after both acute GH injection and chronic GH treatment for 8 days. These data demonstrate that IGFBP-1 expression is inversely regulated by GH. (Molecular Endocrinology 4: 1199-1204, 1990)

INTRODUCTION

Insulin-like growth factors (IGFs) are present in the circulation in association with proteins (1, 2). These binding proteins (IGFBP), which have high affinity for both IGF-I and IGF-II, have been reported to inhibit the action of IGFs in various bioassays (3, 4). There is, however, one as yet unconfirmed report that human IGFBP-1 may facilitate IGF-I action (5). Since the BPs may regulate the availability of circulating and locally synthesized IGF-I, they may serve to modulate the biological actions of the IGFs in vivo. Clearly, factors that regulate expression of the BP are important to our understanding of the regulation of the biological actions of the IGFs. At least three immunologically distinct IGFBPs exist in tissue fluids and plasma from rat and man (1, 2). In plasma from adult rats and human subjects, the major-

ity of IGF-I immunoreactivity is associated with a GHdependent, 150- to 200-kDa protein complex. This complex dissociates under acid conditions into a IGFBP with an apparent molecular mass of 43 kDa on reduced sodium dodecyl sulfate (SDS)-gels and an 100-kDa nonIGF-binding subunit (6). The 43-kDa subunit has been recently cloned (7). Since the predicted molecular mass of the encoded protein is only 28.7 kDa, it appears to be posttranslationally modified, mostly likely via N- and O-linked glycosylation (7). In current terminology this protein has been designated IGFBP-3. A second BP with an apparent molecular mass between 25-40 kDa is also present in plasma and appears to be identical to the IGFBP present in amniotic fluid and to placental protein-12 (8, 9). This BP, IGFBP-1, in contrast to IGFBP-3, has been considered to be GH independent by many investigators (10-12), although at least two studies have suggested that the serum concentrations of IGFBP-1 are increased in GH-deficient individuals (13, 14). There is general agreement that in human subjects, plasma levels of IGFBP-1 correlate inversely with plasma insulin concentrations (11, 12). Furthermore, in vitro experiments with human fetal liver explants have demonstrated that secretion of this protein is inhibited by insulin (15). A third type of BP, IGFBP-2, is present in fetal, but not adult, rat serum and in conditioned medium from buffalo rat liver cells (16). A cDNA that encodes the rat homolog of human IGFBP-1 has been isolated from a rat decidual cDNA library in this laboratory (17). Here, we have examined the expression of IGFBP-1 in the liver of hypophysectomized (hypox) and pituitary-intact rats. In this report we demonstrate that expression of IGFBP-1 is transcriptionally down-regulated by GH in hypox rats.

RESULTS AND DISCUSSION

We have recently isolated a cDNA from a rat decidual library which encodes the rat homolog of the human low mol wt IGFBP extracted from amniotic fluid. There is 66% sequence similarity between the predicted rat

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and human IGFBP-1 proteins. This BP has been considered to be GH independent (11,18). An unexpected finding in our initial experiments was the observation that the abundance of the 1.6-kilobase (kb) IGFBP-1 mRNA was increased in hepatic RNA from hypox rats (Fig. 1). A significant increase in IGFBP-1 mRNA abundance was demonstrated in hypox rats compared to that in pituitary-intact rats (6.5 ± 1.7-fold; n = 9; compared to sham-operated controls, P < 0.05). Since we have previously shown that the abundance of IGFBP-1 mRNA is higher in neonatal rat liver than in mature rats (17), the possibility that hypophysectomy arrested an age-related decline in hepatic IGFBP-1 abundance was examined. In rats hypophysectomized at 200-250 g, a significant increase in hepatic IGFBP-1 mRNA was also seen (17.9 ± 3.7-fold; n = 4; compared to shamoperated controls, P < 0.05). We next examined the effects of GH on hepatic IGFBP-1 transcription and IGFBP-1 mRNA abundance. In hypox rats, transcription of IGFBP-1 was significantly increased (~4-fold) compared to that in pituitary-intact sham-operated rats (Fig. 2A). The increase in IGFBP-1 transcription in hypox rats was of sufficient magnitude to account for the increased IGFBP-1 mRNA abundance. GH administration rapidly reduced IGFBP-1 transcription. Thirty minutes after GH administration, IGFBP-1 transcription was not significantly different from that in pituitary-intact rats. There was a gradual increase in IGFBP-1 transcription over the subsequent 5 h after GH injection. Similarly, IGFBP-1 mRNA abundance decreased after GH administration; however, the pattern of decline and recovery was slightly delayed compared to the data obtained in the in vitro transcription assay (Fig. 2B). In addition to the major transcript of 1.6 kb, additional transcripts of 6.0, 4.8, 4.1, 3.5, and 2.5 kb were apparent in hepatic RNA from the untreated hypox rats.

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