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Regulation of the level of epidermal growth factor by oestrogen in the submandibular gland of female mice

H. Kurachi and T. Oka

ABSTRACT

The concentration of epidermal growth factor (EGF) in the submandibular gland of immature female mice (less than 6 weeks old) was low, ranging from 0·07 to 0·17 pmol (0·4 to 1·0 ng)/mg wet wt, but increasing to 7·61 pmol/mg wet wt by 8 weeks of age. It remained relatively constant up to 24 weeks of age, but thereafter the glandular EGF concentration increased again during the post-reproductive period to reach 62·9 pmol/mg wet wt. In contrast, the glandular EGF content in male mice increased greatly during the first 17 weeks of age to about 310 pmol/mg wet wt, and thereafter remained relatively constant up to 48 weeks of age. Ovariectomy of mature virgin mice markedly increased the glandular concentration of EGF to about 74·4 pmol/mg wet wt 4 weeks after the operation. This increase was suppressed by oestradiol-17β administered to ovariectomized mice at a dose of 1 μg/mouse per day but not by 1 mg progesterone. Histological studies indicated that granular convoluted tubular cells that produced EGF in the submandibular gland were much less abundant in 12-week-old female and in oestrogen-treated ovariectomized mature mice than in 40-week-old female and ovariectomized mature mice. We conclude that oestrogen suppresses the concentration of EGF in the submandibular gland of female mice.

J. Endocr. (1986) 109, 221–225

DOI:
https://doi.org/10.1677/joe.0.1090221
Volume 109: Issue 2,
Pages:
221–225 (5 total)
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Changes in epidermal growth factor concentrations of submandibular gland, plasma and urine of normal and sialoadenectomized female mice during various reproductive stages

H. Kurachi and T. Oka

ABSTRACT

The concentrations of epidermal growth factor (EGF) were measured by radioimmunoassay in the submandibular gland, plasma and urine of adult female C3H/HeN mice whilst virgin and during pregnancy, lactation and after lactation. During gestation there was a significant increase in the submandibular EGF concentration which was five to seven times higher than that found in virgin mice. The level of EGF in the gland remained high during the period of lactation and even several weeks after lactation. Plasma EGF levels were also increased during the periods of pregnancy, lactation and after lactation when compared with those of virgin mice. These increases were, however, apparent only between 24.00 and 08.00 h, because of circadian variations in circulating EGF. The level of plasma EGF was significantly (P<0·05) higher during the 24.00–08.00 h period than during the 12.00–20.00 h period in all stages examined. Concentrations of EGF in the urine of virgin, pregnant, lactating and primiparous mice remained relatively constant, and the levels were much higher than those in the plasma. Similar studies using sialoadenectomized pregnant and lactating mice indicated that the plasma levels of EGF were below the level of sensitivity of the assay (<16·5 pmol/l (<0·1 ng/ml)) even during the 24.00–08.00 h period, whereas urinary EGF remained at high levels which were similar to those of normal pregnant and lactating mice. These results suggest that submandibular EGF contributes to the increase in plasma EGF which occurs after gestation, but is not the major source of the urinary EGF.

J. Endocr. (1985) 106, 197–202

DOI:
https://doi.org/10.1677/joe.0.1060197
Volume 106: Issue 2,
Pages:
197–202 (6 total)
Free access

High concentration of glucose decreases glucose transporter-1 expression in mouse placenta in vitro and in vivo

K Ogura, M Sakata, M Yamaguchi, H Kurachi, and Y Murata

Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1.

DOI:
https://doi.org/10.1677/joe.0.1600443
Volume 160: Issue 3,
Pages:
443–452 (10 total)
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Involvement of epidermal growth factor in inducing adiposity of aged female mice

H Adachi, H Kurachi, H Homma, K Adachi, T Imai, M Sakata, Y Matsuzawa, and A Miyake

Abstract

Aged mice exhibit an increase in their body weight (BW), which is associated with fat deposit increase. Epidermal growth factor (EGF) concentration in the submandibular gland also increases with aging. We examined the effects of elevated EGF on the adiposity of aged female mice. Studies were started in two groups of animals consisting of sham-operated (n=10) and sialoadenectomized (n=10, Sx; surgical removal of the submandibular glands) mice at 8 weeks of age. Body weight gain and food intake were measured throughout 78 weeks of age in these two groups. Body weight was significantly less in the Sx group throughout 78 weeks, while food intake was not changed by Sx after 12 weeks of age. To examine further if EGF plays a role in the induction of adiposity in aged female mice, sham-operated animals were given 100 μl anti-EGF rabbit antiserum (anti-EGF group, n=5) or normal rabbit serum (control group, n=5) every 3 days, and Sx animals were given 5 μg/day EGF (Sx+EGF group, n=5) or saline (Sx group, n=5) from 78 weeks of age for 3 weeks. At 81 weeks of age, all animals of these four groups were killed, and carcass fat deposition and fat cell sizes were measured. Although the relative weights (weight ratio to BW) of the liver and kidney were not changed by Sx and anti-EGF treatment, the relative weights of mesenteric and subcutaneous fat tissues and adipocyte weights were significantly decreased in Sx and anti-EGF groups compared with the control group. Moreover, both acyl-CoA synthetase (ACS) and lipoprotein lipase (LPL) mRNA levels were significantly decreased by Sx or anti-EGF administration in mesenteric and subcutaneous fat tissues. On the other hand, EGF administration to Sx animals had no effect on BW, fat tissues and adipocyte weights, and ACS and LPL mRNA levels. The results, however, were consistent with the fact that adipose tissue EGF receptors were down regulated in Sx mice. These findings suggest that EGF may play a role in the induction of adiposity in aged female mice.

Journal of Endocrinology (1995) 146, 381–393

DOI:
https://doi.org/10.1677/joe.0.1460381
Volume 146: Issue 3,
Pages:
381–393 (13 total)
Restricted access

Involvement of epidermal growth factor (EGF)/EGF receptor autocrine and paracrine mechanism in human trophoblast cells: functional differentiation in vitro

K Amemiya, H Kurachi, H Adachi, K-I Morishige, K Adachi, T Imai, and A Miyake

Abstract

We have studied the expression of epidermal growth factor (EGF) and EGF receptors (EGF-R) in isolated human trophoblast cells at various stages of differentiation and also the biological significance of the EGF/EGF-R autocrine and paracrine mechanism. Cytotrophoblast cells were isolated from human placental tissues of 6–9 weeks of gestation. Trophoblast cells underwent morphological and functional differentiation during in vitro culture. The expression of EGF and EGF-R protein and mRNA was studied in trophoblast cells cultured for 0–5 days, using immunocytochemical staining, and reverse transcription and polymerase chain reaction. Monoclonal antibodies (mAbs) against EGF and EGF-R showed specific staining in trophoblast cells at all stages of differentiation. Both EGF and EGF-R gene transcripts were detected in RNA samples isolated from trophoblast cells at all stages. These data suggest the presence of an EGF/EGF-R autocrine and paracrine mechanism in human trophoblast cells. Next, we examined the biological significance of this mechanism on trophoblast cell differentiation in vitro. EGF added to the culture medium significantly increased human chorionic gonadotrophin-β (hCG-β) secretion and, more importantly, anti-EGF neutralizing mAbs significantly reduced both hCG-β and human placental lactogen secretion from trophoblast cells in culture. All these results suggest that human trophoblast cells express both EGF and EGF-R, and that EGF may play an important role in the functional differentiation of human trophoblast cells.

Journal of Endocrinology (1994) 143, 291–301

DOI:
https://doi.org/10.1677/joe.0.1430291
Volume 143: Issue 2,
Pages:
291–301 (11 total)
Free access

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

M Yoneda, T Nishizaki, K Tasaka, H Kurachi, A Miyake, and Y Murata

Using digitonin-permeabilized GH3 cells, we investigated both the release of prolactin (PRL) and changes in the cytoskeleton. We determined that permeabilized GH3 cells released PRL in a dose-dependent manner upon addition of micromolar Ca(2+). Phalloidin, a filamentous actin (F-actin) stabilizing agent, inhibited both Ca(2+)-dependent and -independent PRL release, whereas cytochalasin B, a destabilizing agent, had almost no effect on the release. Observation with a confocal laser scanning microscope revealed that F-actin existed mainly in the cortical region in the quiescent state. Increased cytosolic Ca(2+) induced a change in F-actin distribution: F-actin in the cortical region decreased, whereas F-actin inside the cells increased. This change in F-actin distribution was not observed when phalloidin was added. Addition of cytochalasin B induced patchy F-actin spots, but the pattern of the changes of F-actin distribution did not change. The time course of change in F-actin distribution showed that the F-actin network in the cortical region was reduced within 1 min, and Ca(2+)-dependent release of PRL continued for up to 20 min. These results suggest that the F-actin network near the membrane acts as a barrier to exocytosis and that Ca(2+) directly controls the cytoskeletal changes.

DOI:
https://doi.org/10.1677/joe.0.1660677
Volume 166: Issue 3,
Pages:
677–687 (11 total)
Free access

Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid

T Takeda, H Kurachi, T Yamamoto, Y Nishio, Y Nakatsuji, K Morishige, A Miyake, and Y Murata

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

DOI:
https://doi.org/10.1677/joe.0.1590323
Volume 159: Issue 2,
Pages:
323–330 (8 total)
Restricted access

8-Bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture

M Sakata, M Yamaguchi, T Imai, C Tadokoro, Y Yoshimoto, Y Oka, H Kurachi, and A Miyake

Abstract

Glucose plays an important role in fetal development and energy metabolism. Facilitative glucose transporter-1 (GLUT1) has been found in placenta. However, little is known about GLUT1 modulation in placental cells. To examine changes in mouse placental GLUT1 levels caused by 8-bromo-cAMP, we performed 2-deoxyglucose uptake experiments, Northern blot analysis and immunoblot analysis using a primary mouse placental cell culture. Immunohistochemical analysis showed that GLUT1 was localized to the ectoplacental cone and the labyrinth zone of mouse placentas on days 7 and 11 of pregnancy respectively. Treatment of mouse placental cells with 250 μmol/l 8-bromo-cAMP resulted in a significant (P<0·01) decrease in glucose uptake on days 2–5 of culture. The inhibitory effect of 8-bromo-cAMP on glucose uptake was concentration-dependent. Glucose uptake was also inhibited by 100 μg/l cholera toxin and by 0·1 mmol/l forskolin. Northern blot and immunoblot analysis revealed that both GLUT1 mRNA and protein levels were also decreased by 8-bromo-cAMP. These findings suggest that 8-bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture.

Journal of Endocrinology (1996) 150, 319–327

DOI:
https://doi.org/10.1677/joe.0.1500319
Volume 150: Issue 2,
Pages:
319–327 (9 total)
Free access

Rab3B is essential for GnRH-induced gonadotrophin release from anterior pituitary cells

K Tasaka, N Masumoto, J Mizuki, Y Ikebuchi, M Ohmichi, H Kurachi, A Miyake, and Y Murata

Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca2+ concentration ([Ca2+]). Rab3B, a member of the small GTP-binding protein Rab family, is known to be involved in Ca(2+)-regulated exocytosis in pituitary cells. However, it is not known whether Rab3B functions in the physiological process regulated by GnRH in gonadotrophs. In this study using antisense oligonucleotide against Rab3B (AS-Rab3B) we determined that Rab3B is involved in GnRH-induced gonadotrophin release. Rab3B immunopositive cells were reduced in 24% of pituitary cells by AS-Rab3B. This treatment did not affect the population of gonadotrophs or the intracellular contents of gonadotrophins. However, AS-Rab3B significantly inhibited the total amount of basal and GnRH-induced gonadotrophin released from pituitary cells. These results show that Rab3B is involved in basal and GnRH-induced gonadotrophins release but not the storage of gonadotrophins. Next, the changes in [Ca2+] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca2+] was not different in the two groups, but exocytosis was significantly inhibited in Rab3B-negative cells. These results suggest that Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs.

DOI:
https://doi.org/10.1677/joe.0.1570267
Volume 157: Issue 2,
Pages:
267–274 (8 total)
Restricted access

Menstrual cycle specific expression of epidermal growth factor receptors in human fallopian tube epithelium

K Adachi, H Kurachi, H Adachi, T Imai, M Sakata, H Homma, O Higashiguchi, T Yamamoto, and A Miyake

Abstract

We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n=3), late follicular (n=3) and luteal (n=3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. 125I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (P<0·01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain M r 170 000 EGF receptor protein. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n=3 for each stage) from the tubal ampullary portion. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor a) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n=3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen.

Journal of Endocrinology (1995) 147, 553–563

DOI:
https://doi.org/10.1677/joe.0.1470553
Volume 147: Issue 3,
Pages:
553–563 (11 total)