Identification of novel CHD1-associated collaborative alterations of genomic structure and functional assessment of CHD1 in prostate cancer.

A clearer definition of the molecular determinants that drive the development and progression of prostate cancer (PCa) is urgently needed. Efforts to map recurrent somatic deletions in the tumor genome, especially homozygous deletions (HODs), have provided important positional information in the search for cancer-causing genes. Analyzing HODs in the tumors of 244 patients from two independent cohorts and 22 PCa xenografts using high-resolution single-nucleotide polymorphism arrays, herein we report the identification of CHD1, a chromatin remodeler, as one of the most frequently homozygously deleted genes in PCa, second only to PTEN in this regard. The HODs observed in CHD1, including deletions affecting only internal exons of CHD1, were found to completely extinguish the expression of mRNA of this gene in PCa xenografts. Loss of this chromatin remodeler in clinical specimens is significantly associated with an increased number of additional chromosomal deletions, both hemi- and homozygous, especially on 2q, 5q and 6q. Together with the deletions observed in HEK293 cells stably transfected with CHD1 small hairpin RNA, these data suggest a causal relationship. Downregulation of Chd1 in mouse prostate epithelial cells caused dramatic morphological changes indicative of increased invasiveness, but did not result in transformation. Indicating a new role of CHD1, these findings collectively suggest that distinct CHD1-associated alterations of genomic structure evolve during and are required for the development of PCa.

Yin Yang 1 regulates the transcriptional activity of androgen receptor.

The multifunctional protein Yin Yang 1 (YY1) has an important role in epigenetic regulation of gene expression. YY1 is highly expressed in various types of cancers, including prostate cancer. Currently, the mechanism underlying the functional role of YY1 in prostate tumorigenesis remains unclear. In this report, we investigated the functional interplay between YY1 and androgen receptor (AR), and the effect of YY1 on AR-mediated transcription. We found that YY1 physically interacts with AR both in a cell-free system and in cultured cells. YY1 is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate-specific antigen (PSA) promoter. However, ectopic YY1 expression in LNCaP cells did not further enhance the reporter driven by the PSA promoter, suggesting that an optimal level of YY1 is already established in prostate tumor cells. Consistently, YY1 depletion in LNCaP cells reduced endogenous PSA levels, but overexpressed YY1 did not significantly increase PSA expression. We also observed that YY1-AR interaction is essential to YY1-mediated transcription activity of AR and YY1 is a necessary component in the complex binding to the androgen response element. Thus, our study demonstrates that YY1 interacts with AR and regulates its transcriptional activity.

Preferential production of latent transforming growth factor beta-2 by primary prostatic epithelial cells and its activation by prostate-specific antigen.

Three mammalian isoforms of transforming growth factor-beta (TGFbeta) are known, TGFbeta1, 2, and 3, that have non-overlapping functions during development. However, their specific roles in cancers such as prostate cancer are less clear. Here we show that primary cultures of prostatic epithelial cells preferentially produce and activate the latent TGFbeta2 isoform. Paired cultures of normal and malignant prostate cells from prostate cancer patients produced predominantly the TGFbeta2 isoform, with 30- to 70-fold less TGFbeta1. By mono-Q ion exchange chromatography, three major peaks of latent TGFbeta2 activity were observed corresponding to the known small latent TGFbeta2 complex, the known large latent TGFbeta2 complex and a novel eluting peak of latent TGFbeta2. Although prostate cells are known to activate latent TGFbeta, the mechanism for activation is currently unclear. We investigated whether prostate specific antigen (PSA), a serine protease used as a clinical marker for prostate cancer, could play a role in the activation of latent TGFbeta. Unlike plasmin, a known activator of both latent TGFbeta1 and 2, PSA specifically activated the recombinant small latent form of TGFbeta2, but not TGFbeta1. Prostate epithelial cells, therefore, preferentially produce the TGFbeta2 isoform and PSA, a protease produced by the prostate, specifically targets the activation of this TGFbeta isoform. PSA-mediated activation of latent TGFbeta2 may be an important mechanism for autocrine TGFbeta regulation in the prostate and may potentially contribute to the formation of osteoblastic lesions in bone metastatic prostate cancer.

1alpha,25-dihydroxyvitamin D(3) (calcitriol) and its analogue, 19-nor-1alpha,25(OH)(2)D(2), potentiate the effects of ionising radiation on human prostate cancer cells.

Radiotherapy with external beam radiation or brachytherapy is an established therapeutic modality for prostate cancer. Approximately 30% of patients with localised prostate cancer relapse at the irradiated site. Secondary effects of ionising radiation (IR), for example, bowel and bladder complications, are common. Thus, the search for biological response modifiers that could potentiate the therapeutic effects of radiation and limit the occurrence of serious side effects is an important task in prostate cancer therapy. 1alpha,25-Dihydroxyvitamin D(3) (calcitriol), the active metabolite of vitamin D, and its analogues are under investigation for the treatment of several malignancies including prostate cancer. Here, we report that 1alpha,25-dihydroxyvitamin D(3) and its less calcaemic analogue 19-nor-1alpha,25-(OH)(2)D(2) (Zemplar) act synergistically with IR to inhibit the growth of the human prostate cancer cells in vitro. 1alpha,25-dihydroxyvitamin D(3) potentiated IR-induced apoptosis of LNCaP cells, and nanomolar doses of 1alpha,25-dihydroxyvitamin D(3) and 19-nor-1alpha,25-(OH)(2)D(2) showed synergistic inhibition of growth of LNCaP cells at radiobiologically relevant doses of IR (1-2 Gy). At higher doses of IR, the combination of 1alpha,25-dihydroxyvitamin D(3) and IR or 19-nor-1alpha,25-(OH)(2)D(2) and IR resulted in moderate antagonism. The synergistic effect at radiobiologically relevant doses of radiation suggests that a combination of 1alpha,25-dihydroxyvitamin D(3) or 19-nor-1alpha,25-(OH)(2)D(2) with IR could permit a reduction in the dose of radiation given clinically and thus potentially reduce treatment-related morbidity.

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2).

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.

25-Hydroxyvitamin D3, the prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic epithelial cells.

The hormonal metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is known to inhibit the proliferation of prostatic epithelial cells. This has stimulated interest in vitamin D compounds as therapeutic agents for prostate cancer. However, the therapeutic use of 1,25(OH)2D3 is limited because elevations in serum 1,25(OH)2D3 can cause dangerous elevations in serum calcium levels. We wondered whether the prohormone of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25-OH-D3), which is much less calcemic, could also achieve antiproliferative effects in prostatic cells. 25-OH-D3 is converted to 1,25(OH)2D3 by the mitochondrial enzyme 1-alpha-hydroxylase. We have recently shown that human prostatic cells also possess significant 1-alpha-hydroxylase activity (Schwartz et al., Cancer Epidemiol. Biomark. Prev., 7: 391-395, 1998). We studied 1-alpha-hydroxylase gene expression in four strains of primary human prostatic epithelial cells by reverse transcription PCR amplification (RT-PCR) of 1-alpha-hydroxylase. Human prostatic stromal cells were negative for 1-alpha-hydroxylase by RT-PCR. This led us to hypothesize that 25-OH-D3 would inhibit the proliferation of prostatic epithelial cells because 25-OH-D3 would be converted to 1,25(OH)2D3 intracellularly. We studied the effects of 25-OH-D3 and 1,25(OH)2D3 on the proliferation of prostatic epithelial cells using high density growth and clonal growth assays on two different primary cell strains derived from normal human prostatic peripheral zone. 25-OH-D3 and 1,25(OH)2D3 each inhibited growth in a dose- and time-dependent manner. Growth inhibition was evident at 1 nM, and maximal inhibition was observed at 100 nM within 10-12 days of exposure. The potencies of 25-OH-D3 and 1,25(OH)2D3 were not significantly different. These data demonstrate that 25-OH-D3, which previously was thought to have little biological activity, can become a potent antiproliferative hormone for prostatic cells that express 1-alpha-hydroxylase. Because 25-OH-D3 exhibits similar potency to 1,25(OH)2D3 but is less calcemic, 25-OH-D3 may offer a safer option than 1,25(OH)2D3 for prostate cancer therapy. Moreover, because 25-OH-D3 is produced endogenously from vitamin D, these findings support a potential role for vitamin D in the chemoprevention of prostate cancer.

Genetic basis of primary hyperoxaluria type II.

Primary hyperoxaluria Type II (PH2) is a rare monogenic disease characterized by excessive urinary oxalate and L-glycerate excretion. The severity of clinical complications in PH2 patients can range from none to end-stage renal failure secondary to massive deposits of calcium oxalate crystals in the kidney. The disease is a result of the absence of an enzyme with glyoxylate reductase and hydroxypyruvate reductase activities (GRHPR). Recent breakthroughs have occurred in our understanding of the molecular basis of PH2. In this article, we briefly review the literature concerning the clinical and biochemical characteristics of the disease and the enzyme associated with it. We describe the identification of the cDNA for the GRHPR enzyme using the expressed sequence tag database, the characterization of the human GRHPR gene, and the identification of mutations in patients with PH2. Insights gained from the molecular biology underlying this disease as they relate to relevant clinical issues such as potential therapeutic strategies are discussed.

The gene encoding hydroxypyruvate reductase (GRHPR) is mutated in patients with primary hyperoxaluria type II.

Primary hyperoxaluria type II (PH2) is a rare monogenic disorder that is characterized by a lack of the enzyme that catalyzes the reduction of hydroxypyruvate to D-glycerate, the reduction of glyoxylate to glycolate and the oxidation of D-glycerate to hydroxypyruvate. The disease is characterized by an elevated urinary excretion of oxalate and L-glycerate. The increased oxalate excretion can cause nephrolithiasis and nephrocalci-nosis and can, in some cases, result in renal failure and systemic oxalate deposition. We identified a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) cDNA clone from a human liver expressed sequence tag (EST) library. Nucleotide sequence analysis identified a 1198 nucleotide clone that encoded a 984 nucleotide open reading frame. The open reading frame encodes a predicted 328 amino acid protein with a mass of 35 563 Da. Transient transfection of the cDNA clone into COS cells verified that it encoded an enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Database analysis of human ESTs reveals widespread tissue expression, indicating that the enzyme may have a previously unrecognized role in metabolism. The genomic structure of the human GRHPR gene was determined and contains nine exons and eight introns and spans approximately 9 kb pericentromeric on chromosome 9. Four PH2 patients representing two pairs of siblings from two unrelated families were analyzed for mutations in GRHPR by single strand conformation polymorphism analysis. All four patients were homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45. The cDNA that we have identified represents the first characterization of an animal GRHPR sequence. The data we present will facilitate future genetic testing to confirm the clinical diagnosis of PH2. These data will also facilitate heterozygote testing and prenatal testing in families affected with PH2 to aid in genetic counseling.

Parathyroid hormone-related protein is not an autocrine growth factor for normal prostatic epithelial cells.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is the primary factor responsible for humoral hypercalcemia of malignancy. The hypercalcemic actions of PTHrP occur via stimulation of renal distal tubular calcium reabsorption and increased osteoclastic bone resorption. These effects of PTHrP are thought to be mediated through a common parathyroid hormone (PTH)/PTHrP receptor. In addition to the well-established role of PTHrP in bone remodeling, PTHrP is believed to be an important mediator of cellular growth and differentiation in a number of nonbony tissues. We recently demonstrated abundant expression of PTHrP in normal and malignant human prostatic tissues, and in cultured prostatic epithelial cells. METHODS: In vitro assays were used to test growth-regulatory activity of synthetic and endogenous PTHrP peptides on normal prostatic epithelial cells. RESULTS: No growth-regulatory activity could be demonstrated. CONCLUSIONS: PTHrP is not an autocrine growth factor for normal prostatic epithelial cells.

Prostate specific antigen cleaves parathyroid hormone-related protein in the PTH-like domain: inactivation of PTHrP-stimulated cAMP accumulation in mouse osteoblasts.

PURPOSE: To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA. MATERIALS AND METHODS: Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production. RESULTS: Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production. CONCLUSIONS: Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.

Parathyroid hormone--related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells.

Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways.

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.

Suramin, hydrocortisone, and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D(3) on prostatic epithelial cells.

The proliferation of prostatic epithelial cells is regulated by the complex interplay of numerous growth-stimulatory and growth-inhibitory factors. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been identified as a potent inhibitor of the growth of prostatic epithelial cells. Epidemiologic studies indicate that vitamin D deficiency may be a risk factor for the development of clinical prostate cancer, possibly due to increased growth and reduced differentiation of prostatic cells in an environment with decreased 1,25(OH)(2)D(3). The application of vitamin D or analogs in chemotherapy against prostate and other cancers is being explored by several investigators. In order to use vitamin D most efficaciously in a clinical setting, it may be beneficial to learn more about the interaction of 1,25(OH)(2)D(3) with other factors that regulate prostatic epithelial cellular growth. In this study, we examined the effect of the proliferative status of cultured cells on their ability to respond to 1,25(OH)(2)D(3), and found that minimally proliferative cells were equally as responsive to 1,25(OH)2D3 as actively dividing cells. We noted no apparent interaction of 1,25(OH)(2)D(3) with epidermal growth factor, insulin-like growth factor, cholera toxin, or transforming growth factor-ß, but we did find synergistic inhibitory effects of 1,25(OH)(2)D(3) with suramin and retinoic acid. Perhaps most noteworthy was the dramatic increase in potency of 1,25(OH)(2)D(3) that occurred upon deletion of hydrocortisone from the culture medium. Our in vitro studies indicate that combination therapy of vitamin D analogs with suramin, vitamin A analogs, or anti-glucocorticoids might be considered for prostate cancer.

Expression of growth hormone-binding protein with a hydrophilic carboxyl terminus by the mouse placenta: studies in vivo and in vitro.

GH-binding protein (GHBP) or GH receptor is present in numerous extrahepatic tissues in the rodent. From mid- to late gestation in the mouse, the maternal serum concentration of GHBP increases 30- to 50-fold. We have investigated whether the placenta might synthesize GHBP and potentially contribute to this increase. RNA was isolated from placentas and subjected to Northern analysis using a cDNA probe to the shared region of GHBP and GH receptor-encoding mRNAs. From day 8 to day 18 of gestation, the GHBP-encoding mRNA (1.4 kb) increased 45-fold in quantity. The GH receptor-encoding mRNA (4.2 kb) increased sixfold by day 14 and then remained steady until day 18. These changes which were not co-ordinated parallel reported changes in the steady-state concentrations of 1.4 and 4.2 kb mRNAs in maternal liver, suggesting shared regulatory factors. Extracts of freshly isolated trophoblasts were assayed for GHBP with a radioimmunoassay specific for GHBP with a hydrophilic carboxyl terminus. The cytosolic content of immunoreactive GHBP increased fourfold from mid- to late gestation. Trophoblasts were isolated from placentas and cultured for 2 days on collagen gels in defined medium. Cultured cells were at least 90% viable and secreted mouse placental lactogen-II (mPL-II). Immunocytochemistry was carried out simultaneously on cells cultured from day 7 to day 17 of gestation using a monoclonal antibody (MAb 4.3), which recognizes the hydrophilic C-terminus of GHBP. Cell-localized GHBP was present in trophoblasts cultured for 2 days, but GHBP was not detectable by radioimmunoassay or by immunoprecipitation in concentrated culture media from cultures treated with 100 ng mouse GH/ml or 100 ng mPL-II/ml or from untreated cultures. RNA was isolated from cells cultured in an identical manner to those analysed by immunocytochemistry. Three GH receptor/GHBP mRNA species of 8, 4.2 and 1.4 kb were observed. The quantity of 4.2 and 1.4 kb mRNAs did not change significantly in cultures from day 7 to day 15 of gestation but, in cultures from day 17 of gestation, the amount of 1.4 kb mRNA dropped significantly, while that of the 4.2 kb mRNA remained unchanged. GHBP- and GH receptor-encoding mRNAs are not co-ordinately regulated in vivo or in vitro. Although mPL-II was secreted into the medium by cultured trophoblasts, secretion of GHBP could not be detected. The culture medium may not contain the specific factors required for secretion of placental GHBP, or placental GHBP may not be destined for secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the hepatic growth hormone receptor and serum growth hormone-binding protein during pregnancy in the mouse: effects of litter size.

The regulation of hepatic GH receptor (GHR) and serum GH-binding protein (GHBP) during pregnancy in the mouse was investigated by manipulating the number of conceptuses carried by the dam. Animals carrying 1-4 conceptuses had significantly lower amounts of hepatic GHR (GH-binding activity) than mice carrying 10-13 conceptuses on days 9 and 13 of pregnancy. There was no significant difference in hepatic GHR on day 17 of pregnancy between animals carrying 1-4 and 10-13 conceptuses. Animals carrying 1-4 conceptuses had significantly lower concentrations of GHBP in serum than animals carrying 10-13 conceptuses on days 9, 13, and 17 of pregnancy. The relative amounts of liver GHR- and GHBP-encoding messages in animals with low and high conceptus numbers were investigated by Northern analysis. There were higher levels of both messages in animals carrying 10-13 conceptuses than in mice carrying 1-4 conceptuses. On day 13 of pregnancy, animals carrying 10-13 conceptuses had significantly higher levels of GHBP-encoding message than animals carrying 1-4 conceptuses. Total hepatic mass was not significantly different between animals with low and high conceptus numbers. No significant difference was found in GHBP concentration between blood from the uterine vein and the trunk in 17-day pregnant animals. Mouse placental lactogen-I (mPL-I), mPL-II, GH, and corticosterone concentrations were measured by RIA and related to hepatic GHR activity and serum GHBP concentration. Hepatic GHR activity and serum GHBP concentration were significantly correlated with each other on days 9, 13, and 17 of pregnancy. Hepatic GHR activity was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy. GHBP concentration was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy and with mPL-II and GH on day 13 of pregnancy. Data are consistent with the hypothesis that mPL-I, mPL-II, and GH may affect hepatic expression of the GHR/GHBP gene during pregnancy in the mouse.

Expression of the growth hormone receptor and growth hormone-binding protein during pregnancy in the mouse.

A 20-fold increase in the relative expression of the hepatic GH-binding protein (GHBP)-encoding message between nonpregnant and 17-day pregnant mice was found. The hepatic GH receptor (GHR)-encoding message increased 8-fold between nonpregnant and pregnant mice. The increase in both messages began on day 9 of pregnancy. The steady state level of the GHBP-encoding message continued to increase steadily until day 17 of pregnancy; however, by day 13 of pregnancy, the steady state level of the GHR-encoding message reached a plateau that continued to day 17. The ratio between the GHBP- and GHR-encoding messages gradually increased during the second half of pregnancy, reaching a maximum on day 17. There was a 10- to 16-fold increase in GH-binding capacity in liver microsomes and a 30- to 50-fold increase in serum GH-binding capacity between nonpregnant and late pregnant mice. The increase in hepatic GH-binding capacity began on day 9 of pregnancy and reached a plateau on day 11, which was maintained until the end of gestation. The increase in serum GH-binding capacity began on day 9 of pregnancy and continued to increase until day 17. No significant change in mouse (m) GHR (mGHR) or mGHBP affinity constants were observed between nonpregnant and pregnant mice; however, the mGHR had a 20-fold greater affinity for mGH than did the mGHBP. The serum GH concentration increased in the second half of pregnancy. The GHBP-bound and the free fractions of GH during pregnancy were predicted. While the bound fraction of GH is predicted to parallel the total GH concentration measured by RIA, the concentration of free mouse GH remains unchanged during pregnancy.

A mouse growth hormone-binding protein RIA: concentrations in maternal serum during pregnancy.

A RIA for mouse growth hormone-binding protein (mGHBP) was developed. A 28-amino acid synthetic peptide corresponding to the carboxyl-terminal 27 amino acids of mGHBP plus an additional cysteine residue at the amino-terminus was coupled to keyhole limpet hemocyanin and used as the antigen for antiserum production. The ability of the antiserum to recognize mGHBP was demonstrated by incubating mouse serum with 125I-iodomouse GH (mGH) in the presence of increasing concentrations of antiserum and subsequent immunoprecipitation. The antiserum precipitated mGHBP in a dose-dependent manner. The uncoupled synthetic peptide was used as the standard and radioligand in the RIA. Serial dilutions of sera from non-pregnant or 17-days-pregnant mice yielded displacement curves parallel to the synthetic peptide, with serum from 17-days-pregnant mice being 32 times more effective than serum from non-pregnant mice for a given dilution. The relative concentration of mGHBP in maternal serum on days 5, 11, and 15 of pregnancy was 1, 17, and 39, respectively.