Cloning and uterus/oviduct-specific expression of a novel estrogen-regulated gene (ERG1)

The steroid hormone estrogen profoundly influences growth and differentiation programs in the reproductive tract of cycling and pregnant mamals. It is thought that estrogen exerts its cellular effects by regulating the expression of specific target genes. We utilized a messenger RNA differential display method to identify the genes whose expression is modulated by estrogen in the preimplantation rat uterus. Here we report the cloning of a novel gene (ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that ERG1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length ERG1 cDNA indicates that it has an open reading frame of 1821 nuceotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. The extracellular part of the protein contains several distinct structural motifs. These include a zona pellucida binding domain, which is present in a number of proteins such as the zona pellucida sperm binding proteins, and uromodulin, In addition, there is a repeat of a motif called CUB domain, which exists in a number of genes involved in development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its unique pattern of expression in the uterus and oviduct and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy.

Estradiol increases the production of sex hormone-binding globulin but not insulin-like growth factor binding protein-1 in cultured human hepatoma cells.

OBJECTIVE: To study the effects of E2 on insulin-like growth factor binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production with cultured human HepG2 hepatoma cells. DESIGN: Experimental cell culture. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): Addition of E2 to cell culture medium. MAIN OUTCOME MEASURE(S): Intracellular and released concentrations of IGFBP-1 and SHBG. RESULT(S): Estradiol did not affect the intracellular or extracellular IGFBP-1 concentration, whereas the intracellular SHBG concentration increased significantly in response to 0.5-2.5 microM of E2. CONCLUSION(S): Whereas the two binding proteins share a number of regulatory factors, their regulation by E2 is dissimilar in human hepatoma cells. Estradiol does not affect the intracellular or secreted IGFBP-1 concentration, but it does increase the production of SHBG.

Prostaglandin F2 alpha stimulates release of insulin-like growth factor binding protein-3 from cultured human granulosa-luteal cells.

Human ovarian follicular fluid contains a number of insulin-like growth factor binding proteins (IGFBP) of which IGFBP-3 is the most abundant. IGFBP-3 synthesis is growth hormone-regulated. We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on IGFBP-3 secretion by cultured human granulosa-luteal cells from follicular aspirates of women participating in an in-vitro fertilization programme. The IGFBP-3 concentration was measured using a specific monoclonal immunofluorimetric assay. Contrary to a previous report on unstimulated follicles, this study demonstrated a positive correlation between follicular fluid IGFBP-3 concentration and follicular size. PGF2 alpha was found to stimulate in a dose-dependent fashion the secretion of IGFBP-3. Significant (P < 0.05) effects were found at PGF2 alpha concentrations of 10(-8), 10(-7) and 10(-6) M. Because IGFBP-3 inhibits progesterone production stimulated by insulin-like growth factor (IGF)-I, the PGF2 alpha-induced stimulation of IGFBP-3 production may be one of the mechanisms whereby PGF2 alpha exerts its luteolytic effect via the IGF system.

Phosphorylation of insulin-like growth factor-binding protein-1 increases in human amniotic fluid and decidua from early to late pregnancy.

Different fractions of insulin-like growth factor-binding protein-1 (IGFBP-1) from anion exchange chromatography represent differently phosphorylated forms as demonstrated by protein kinase and alkaline phosphatase treatments. The major fraction is non-phosphorylated. Three minor fractions are more phosphorylated and, in native polyacrylamide gel electrophoresis (PAGE), they migrate faster than the major fractions. We studied the changes in phosphorylation of decidual and amniotic fluid IGFBP-1 during pregnancy. Both in decidua and in amniotic fluid the degree of phosphorylation increased from early to late pregnancy, as indicated by faster mobility of IGFBP-1 in native PAGE and increased relative amount of the phosphorylated forms in anion exchange chromatography. The more phosphorylated forms had higher IGF-binding affinity than the less phosphorylated forms. As the degree of phosphorylation of IGFBP-1 is highest in full term decidua it is likely that the inhibitory role of IGFBP-1 is accentuated in the end of pregnancy.

Thyroxine withdrawal is accompanied by decreased circulating levels of insulin-like growth factor-binding protein-1 in thyroidectomized patients.

We carried out a study on the effect of T4 withdrawal on serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels in 10 thyroidectomized patients with papillary thyroid cancer receiving T4 replacement therapy. As controls we also measured serum sex hormone-binding globulin (SHBG) levels, known to be regulated by T4. Each patient acted as his/her own control, studied both before and 30 days after T4 withdrawal. On the average, the IGFBP-1 level decreased by 36% from 66 +/- 9 to 44 +/- 8 micrograms/L (mean +/- SE; P = 0.0007) during T4 withdrawal. The SHBG level decreased by 47% from 78 +/- 17 to 46 +/- 13 nmol/L (P = 0.0001). The fasting insulin levels were unaffected by T4 withdrawal. There also was an 18% decline in the serum IGF-I concentration after T4 withdrawal (P = 0.011). It is concluded that in athyreotic patients receiving T4, withdrawal of the drug decreases serum IGFBP-1 and SHBG concentrations and IGF-I levels. In view of the possible role of IGFBP-1 in glucose counterregulation, these results indicate a novel mechanism by which T4 may exert its metabolic action on liver carbohydrate metabolism.

Thyroxine treatment increases circulating levels of insulin-like growth factor binding protein-1: a placebo-controlled study.

OBJECTIVE: Thyroid hormones affect carbohydrate metabolism in the liver, and hepatic insulin-like growth factor binding protein-1 (IGFBP-1) participates in glucose counter-regulation, so we studied the effects of oral thyroxine on serum IGFBP-1. DESIGN: The study was performed on a placebo-controlled cross-over basis covering 3 months' thyroxine and 3 months' placebo administration. PATIENTS: Eight patients taking anticonvulsant medication and four patients with hypothalamic hypothyroidism were given thyroxine, 150-200 micrograms daily for 3 months, or placebo. MEASUREMENTS: Serum IGFBP-1, sex hormone binding globulin, free T3 and free T4, TSH and IGF-I levels were measured after an overnight fast before treatment, and at the end of each 3-month period. RESULTS: Anticonvulsant medication had no significant effect on serum IGFBP-1. After 3 months' thyroxine treatment the serum IGFBP-1 levels (69; 58-167 micrograms/l; median and interquartile range, respectively) were significantly higher than those after placebo treatment (44; 23-58 micrograms/l; P = 0.002), or the pretreatment levels (54; 19-81 micrograms/l, P = 0.005). The IGFBP-1 levels rose in all 12 patients after thyroxine treatment, the median rise being 2.1-fold compared to placebo levels. No change was found in serum IGF-I concentrations. CONCLUSIONS: Oral thyroxine produces a rise in serum IGFBP-1 levels without a change in serum IGF-I concentration.

Phosphorylation of insulin-like growth factor-binding protein-1 from different sources.

During purification, insulin-like growth factor binding protein-1 from amniotic fluid was separated into five different peaks by anion exchange chromatography. These peaks represent differently phosphorylated forms of IGFBP-1. The major peak (peak 1) is non-phosphorylated. Peaks 3, 4, and 5 are more phosphorylated and, in native polyacrylamide gel electrophoresis (PAGE), they migrate faster than peaks 1 and 2. The more phosphorylated forms have higher IGF-I-binding affinity. Both dephosphorylated and phosphorylated peaks enhanced IGF-I stimulated DNA-synthesis in fetal skin fibroblast cell culture. They, however, inhibited the binding of IGF-I to the same cells. The phosphorylation of IGFBP-1 was changed during pregnancy. In decidua and in amniotic fluid the degree of phosphorylation increased from early to late pregnancy, as indicated by faster mobility of IGFBP-1 in native PAGE and increased relative amount of the more phosphorylated peaks in anion exchange chromatography. Human ovarian follicular fluid, culture media from human granulosa cells and endometrial adenocarcinoma cells (HEC-1-B) consisted mostly of the non-phosphorylated form of IGFBP-1.

Tri-iodothyronine and cycloheximide enhance insulin-like growth factor-binding protein-1 gene expression in human hepatoma cells.

The growth-regulating actions of IGFs are modulated by their binding proteins (IGFBPs). The serum concentration of IGFBP-1 is down-regulated by insulin, and in-vitro studies have demonstrated that IGFBP-1 secretion from various tissues and cells can be stimulated by theophylline, forskolin, oestrogen and progesterone. We have studied the effects and mechanisms of thyroid hormone action on IGFBP-1 gene expression and secretion by human hepatoma cells in vitro. Tri-iodothyronine dose-dependently enhanced IGFBP-1 secretion in serum-free HepG2 cell cultures after 24-48 h of exposure, as measured by a specific immunofluorometric assay. This was accompanied by an increase (+ 50%) in the amount of IGFBP-1 mRNA, which could be prevented by cycloheximide, a protein synthesis inhibitor. Cycloheximide transiently enhanced (+ 200%) the accumulation of IGFBP-1 mRNA at 3-12 h of incubation, when no effect of tri-iodothyronine was observed. It is concluded that thyroid hormone stimulates IGFBP-1 secretion slowly by enhancing IGFBP-1 gene expression by a protein mediator. The acute stimulation of IGFBP-1 gene transcription by cycloheximide associates this gene with a number of growth-related genes encoding growth- and tumour-associated peptides.

Epidermal growth factor enhances insulin-like growth factor binding protein-1 synthesis in human hepatoma cells.

Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 mRNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile protein repressor protein is involved in the turnover IGFBP-1 mRNA.

Epidermal growth factor stimulates production of insulin-like growth factor-binding protein-1 in human granulosa-luteal cells.

Insulin-like growth factor-I (IGF-I) enhances and epidermal growth factor (EGF) inhibits gonadotrophin-induced aromatization in granulosa cells. Our previous studies have shown that human ovarian granulosa cells synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) which inhibits IGF-stimulated DNA synthesis. The present study addresses the effect of EGF and gonadotrophins in the regulation of IGFBP-1 release by human granulosa cells cultured in serum-free medium. At concentrations of 1-100 micrograms/l EGF was found to stimulate IGFBP-1 secretion. This was not due to cell proliferation, as the viable cell count remained unaffected. Growth hormone and gonadotrophins had no effect on IGFBP-1 secretion when added alone to culture medium. These results suggest that EGF regulates IGFBP-1 secretion in human granulosa-luteal cells.

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.