Naoya Emoto

Basic Fibroblast Growth Factor (FGF) in the Central Nervous System: Identification of Specific Loci of Basic FGF Expression in the Rat Brain

The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be constitutively expressed in the central nervous system. As such, while it is difficult to detect any mRNA encoding basic FGF in the heart, lung, kidneys, ovaries, liver, or pituitary of rats, the levels are easily detected in brain. A regional analysis of the expression of basic FGF mRNA in brain reveals that it is widely distributed in the cortex (frontal, parietal, and occipital), the hippocampus, hypothalamus, and pons. Only a few loci of basic FGF synthesis are detected by in situ hybridization and include layers 2 and 6 of the medial (cingulate) cortex, the indusium griseum, fasciola cinereum, and field CA2 of the hippocampus. The identification of specific cell populations in the brain, and particularly in the hippocampus, that synthesize basic FGF supports the notion that this potent neurotrophic factor is involved in normal CNS function and that the presence (or absence) of its expression may be linked to the pathogenesis of the neurogenerative diseases characterizing these various loci. The significance of these findings with respect to the regulation of basic FGF expression in peripheral tissue and the central nervous system is discussed.

The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells.

We investigated the effects of tumor necrosis factor (TNF)/cachectin on follicle-stimulating hormone (FSH)-induced aromatase activity in cultured rat granulosa cells using the stereospecific transfer of 3H from [1 beta-3H] androstenedione into 3H2O. TNF (10 pg/ml-10 ng/ml) inhibited FSH (250 ng/ml)-induced aromatase activity in a concentration-dependent manner, and 10 ng/ml of TNF completely abolished the FSH-induced aromatase activity. A time course analysis of the effects of TNF showed that TNF had no effect on induced aromatase activity, but inhibited the further induction of the enzyme by FSH. TNF (10 ng/ml) also inhibited the ability of TGF beta (1 ng/ml) to enhance aromatase activity and increase progesterone synthesis. Thus, TNF is a component of the complex array of proteins that modulate ovarian function and, as such, may play a physiological role in the regulation of the granulosa cell. In view of its association with cachexia, it may also play a pathophysiological role in the suppression of reproductive function during chronic illness.

Autocrine role of insulin-like growth factor (IGF)-I in a human thyroid cancer cell line

An established cell line (TC-cell, clone 78) derived from human thyroid papillary cancer cells was investigated for production of peptide growth factors. The cells had specific binding sites for insulin-like growth factor-I (IGF-I) and responded to this growth factor with increased proliferation. Culture medium conditioned by TC cells was found to contain insulin-like growth factor (IGF)-I and IGF-binding protein(s). Furthermore, reverse transcription-polymerase chain reaction revealed expression of IGF-I mRNA. When monoclonal antibody to IGF-I receptors (alpha IR3) was added, the growth of TC cells cultured in serum-free medium was significantly reduced. The growth rate of the cells was restored when the antibody was removed from the medium. These results strongly suggest that TC cells produce IGF-I, which is involved in the regulation of their own growth.

Basic fibroblast growth factor (FGF-2) in renal cell carcinoma, which is indistinguishable from that in normal kidney, is involved in renal cell carcinoma growth.

To investigate the role of basic fibroblast growth factor (FGF) in renal cell carcinoma growth, we have analyzed the expression of mRNA of basic FGF. In 7 of 15 cases, basic FGF mRNA level in renal cell carcinoma tissues was higher than that in corresponding normal tissues. However, the tumor-to-normal ratios of expression levels are chiefly less than 2.0 and, in 5 cases, are even less than 1.0. Furthermore, there was no correlation between the ratio and the clinical stage. In protein analysis, we could not find any difference between basic FGF extracted from renal cell carcinomas and that from normal kidney tissues in bioactivity, immunoreactivity, molecular weight and affinity to heparin. On the other hand, anti-basic FGF monoclonal antibody inhibited the growth of a renal cell carcinoma cell line, VMRC-RCW, and this inhibition was reversed by an extraphysiological amount of exogenous basic FGF (100 ng./ml.). These results suggest that basic FGF itself may have no pivotal role in renal cell carcinoma etiology but is involved in the growth of renal cell carcinomas in an autocrine manner.

Effect of insulin-like growth factor-I on growth hormone-releasing factor receptor expression in primary rat anterior pituitary cell culture

We examined the effect of insulin-like growth factor-I (IGF-I) on GH-releasing factor (GRF) receptor expression in the primary rat anterior pituitary cell culture. The levels of GRF receptor mRNA were dose-dependently reduced by IGF-I treatment for 24 h. To clarify whether altered levels of GRF receptor mRNA contribute to GRF receptor concentrations, we examined the GH response to GRF in vitro. There was no difference in basal GH secretion between control and IGF-I pretreated cells, while GRF-stimulated GH secretion in cells pretreated with IGF-I for 24 h was significantly lower than that in control cells. Moreover, specific [125I] Tyr10-human GRF binding to pituitary cells was reduced significantly by IGF-I treatment. These results suggest that IGF-I acts directly on the pituitary and participates in the regulation of GRF receptor expression.

Growth factors increase pericellular proteoglycans independently of their mitogenic effects on A10 rat vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells with the accumulation of proteoglycans in the extracellular matrix is one of the significant changes found in atherosclerotic lesions. In order to clarify the relationship between pericellular proteoglycan and cell growth, we established a simple method for quantitatively estimating the amount of pericellular proteoglycans and investigated the effects of various growth factors on the synthesis of pericellular proteoglycans by cultured A10 rat smooth muscle cells. Analysis of trypsin accessible [35SO4]-labeled material in the pericellular area of the A10 cell culture by Q-sepharose anion-exchange chromatography showed two peaks. One peak, eluted at 0.55 M NaCl, disappeared after treatment with 2 mU/ml of heparitinase, indicating that heparan sulfates (HS) were present. The other peak, which eluted at 0.65 M NaCl, disappeared with 20 mU/ml of chondroitinase ABC, indicating the presence of chondroitin sulfates and dermatan sulfates (CS/DS). We estimated the effects of several growth factors on the synthesis of the pericellular proteoglycans by measuring heparitinase- and chondroitinase-ABC-sensitive radioactivities. Although PDGF-AB significantly stimulated cell proliferation and the synthesis of pericellular CS/DS, its dose-dependent effect on the cell growth did not coincide with that on the proteoglycan synthesis. IGF-I (1 nM) increased pericellular CS/DS but not the cell number, while basic FGF (1 nM) and EGF (1 nM) increased the cell number but not pericellular CS/DS. All the growth factors we examined had no effect on the synthesis of pericellular HS. These results indicate that growth factors increase pericellular proteoglycans independently of their mitogenic effects.

Increased pituitary growth hormone-releasing factor (GRF) receptor messenger ribonucleic acid expression in food-deprived rats

In prolonged food-deprived rats, growth hormone (GH) secretion in response to exogenous GH-releasing factor (GRF) is enhanced both in vivo and in vitro. We tested the hypothesis that, in fasted rats, GRF receptors in the pituitary may be up-regulated. The expression of mRNA for the GRF receptor in the pituitary and GRF binding to the pituitary membrane were examined in adult male Wistar rats deprived of food for 72 h. The level of GRF receptor mRNA in the pituitary was significantly increased after 48 h food deprivation and was more than 3 times the level in control rats after 72 h food deprivation. GRF binding to the pituitary was significantly increased after 72 h food deprivation. The results of the present study provide evidence regarding changes in the GH axis in fasted rats, involving increased pituitary responsiveness to GRF and an increase in the pituitary membrane GRF receptor concentration.

Phorbol ester, not growth hormone releasing factor, consistently stimulates growth hormone release from somatotroph adenomas in culture

In order to study the mechanism of GH secretion from somatotroph adenoma cells, we have compared the effect of 12–O‐tetradecanoyl phorboi‐13‐acetate (TPA) with that of growth hormone releasing factor (GRF) on GH secretion from human somatotroph adenoma cells cultured in monolayer. Pituitary adenoma cells were obtained from 13 patients with acromegaly undergoing surgery. On the 7th day of culture, the cells were exposed for 2 h to secretagogues. All 13 adenoma cell cultures (100%) responded to TPA (1·6–16·0 nmol/I) with a two‐ to six‐fold increase in GH release (240·37% Increase of control: mean±SE). The response was detectable within 10 min, and was maximal at 2 h. Phosphollpase C (7·7 mmol/I) also stimulated a two‐to ten‐fold Increase In GH release in all four adenomas examined (100%). GH release was stimulated by GRF (2·0 nmol/I) in eight out of 12 adenoma cells (67%), but the magnitude of the responses to GRF (60·18% Increase of control: mean ± SE) were much smaller than that of TPA. Five out of 13 adenomas secreted detectable amount of PRL Into the medium and these five adenomas (100%) responded to TPA (16·0 nmol/I) with a two‐ to six‐fold Increase. These observations indicate that the activation of protein kinase C is the consistent stimulator in GH and PRL secretion In human somatotroph adenoma cells. However, It is not determined whether the protein kinase C

Increased activity of insulin-like growth factor-binding protein in human thyroid papillary cancer tissue.

It has been shown that both insulin‐like growth factor‐I (IGF‐I) and IGF‐binding proteins (IGFBPs) are produced by thyroid cells in culture and that the cells respond to IGF‐I with increased DNA synthesis, suggesting an autocrine/paracrine role of IGF‐I in the regulation of thyroid cell growth. We investigated the tissue contents of immunoreactive IGF‐I (irIGF‐I) and IGFBPs in human papillary carcinoma and compared them with those of normal thyroid tissue. When irIGF‐I was measured after separation of the IGFBPs by gel‐filtration, its content in carcinoma tissue was not different from that in adjacent normal tissue (566±58 vs. 424±75 pg/mg protein, N = 10). Nor was there any difference in the abundance of IGF‐I mRNA expression determined by slot blot analysis. On the other hand, IGFBP activity measured in terms of 125I‐IGF‐I binding was significantly higher in cancer extracts. Western ligand blot analysis of IGFBPs revealed several species (24–42 kDa) of IGFBPs. The IGF‐I‐binding activity of 38–41 kDa species (corresponding to IGFBP‐3) was not different between extracts of cancer tissue and those of normal tissue, whereas that of 28–32 kDa species was significantly higher in cancer tissue extracts. Since IGFBPs have been reported to modulate cellular responses to IGF‐I, the present data suggest that higher IGFBP activity in cancer tissue is involved in regulating growth of thyroid papillary carcinoma cells.

Mechanism of inhibitory actions of minocycline and doxycycline on ascitic fluid production induced by mouse fibrosarcoma cells

Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5-4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HCl) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC50s of 5 microM and 8 microM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 microM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.