We examined the effects of angiotensin II (Ang II) on the differentiation of rat calvarial osteoblastic cells and on the formation of bone by these cells. Northern blotting analysis revealed that Ang II inhibited the expression of mRNA for osteocalcin, which is a protein that is specifically expressed during maturation of osteoblastic cells. Ang II decreased the activity of alkaline phosphatase, a marker of osteoblastic differentiation, in the cells, acting via the type 1 (AT1) receptor. We used von Kossa staining to examine the formation of mineralized nodules by osteoblastic cells. Both the number and the total area of mineralized nodules were quantified and shown to be decreased by 10(-7) M Ang II. The accumulation of calcium in cells and the matrix layer was also decreased by Ang II. Binding analysis with subtype-specific antagonists revealed the presence of AT1 receptors for Ang II in this culture system. Ang II caused a marked increase in the rate of production of intracellular cAMP in this system. Our data suggest that Ang II might be intimately involved in osteoblastic metabolism through its interaction with the AT1 receptor.

Abstract

The effects of interleukin (IL)-1 and granulocytemacrophage colony stimulating factor (GM-CSF), which are present in the mouse placenta, on the secretion of mouse placental lactogen (mPL)-1 and mPL-II by placental cells were tested in vitro. IL-lα and IL-1β, 2·5 nmol/l each, significantly inhibited mPL-II secretion by cells from days 9 and 12 of pregnancy, but did not affect mPL-II secretion by cells from day 7 of pregnancy or mPL-I secretion by cells from days 7, 9 or 12 of pregnancy. GM-CSF had no effect on mPL-I and mPL-II secretion by cells from days 7, 9 or 12 of pregnancy. The inhibitory effects of IL-1α and IL-1β on mPL-II secretion were completely eliminated by the addition of antibodies to IL-1α and IL-1β respectively. Western blot analysis for mPL-II indicated that IL-1α significantly reduced the intensity of the mPL-II band. Steady-state levels of mPL-II mRNA, assessed by Northern blot analysis, were reduced by incubation of placental cells from day 12 of pregnancy with 2·5 nmol/l IL-1α for 5 days. Co-incubation of 0·25 pmol/l IL-1α, 25 pmol/l IL-6, and 25 pmol/l tumor necrosis factor-α, each of which did not significantly inhibit mPL-II secretion by itself, together inhibited mPL-II secretion. These results suggest that IL-1, but not GM-CSF, is a potent inhibitor of mPL-II secretion after mid-pregnancy, and that the combined action of cytokines can inhibit mPL-II secretion.

Journal of Endocrinology (1995) 147, 423–429

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

Abstract

The regulation of mouse placental lactogen (mPL)-I and mPL-II secretion by activin and inhibin and the expression of activin and inhibin subunit mRNAs in the mouse decidua were examined. Activin-A at a concentration of 10 nm/l significantly inhibited mPL-II secretion by placental cells from days 9 and 12 of pregnancy. However, activin-A did not affect mPL-I secretion by cells from days 7 and 9 of pregnancy nor mPL-II secretion by cells from day 7 of pregnancy. By contrast, 10 nm/l inhibin activated mPL-II secretion by cells from day 12 of pregnancy. These effects of activin and inhibin on mPL-II secretion were dose-dependent. Follistatin, which binds to activin and blocks its bioactivity, completely eliminated the inhibitory effect of activin on mPL-II secretion. Incubation of placental cells from day 12 of pregnancy with activin-A resulted in a significant reduction of the mPL-II mRNA level assessed by Northern blot analysis. Northern blot analysis using poly(A)⁺ RNA extracted from the decidua indicated that mouse decidua, as well as the placenta, express all activin and inhibin subunits and that their gene expressions increased during gestation. The expression of these mRNAs in the decidua was much higher than those in the placenta. These findings suggest that activin and inhibin regulate mPL-II secretion and suggest the presence of an autocrine or paracrine regulation of mPL-II secretion in mouse placenta by activin and inhibin after mid-pregnancy in vivo.

Journal of Endocrinology (1995) 146, 469–474

Abstract

The placental members of the prolactin-GH-placental lactogen (PL) gene family of the mouse include mPL-I, mPL-II, proliferin (PLF) and proliferin-related protein (PRP). The aim of the present study was to assess the effects of tumour necrosis factor-α (TNF-α) on the secretion of these proteins in primary cultures of placental cells from days 7, 9 and 12 of pregnancy. The effects of epidermal growth factor (EGF) on the secretion of PLF and PRP were also determined. EGF has previously been shown to stimulate mPL-I and inhibit mPL-II secretion. Incubation of placental cells from day 7 of pregnancy for 5 days with 10 nmol human (h)TNF-α/1 did not affect the mPL-II concentration of the medium, but similar treatment of cells from days 9 or 12 of pregnancy resulted in a significant reduction in the mPL-II concentration of the medium by the second or third day of culture. The intracellular concentration of mPL-II, the number of cells that released mPL-II as assessed by reverse haemolytic plaque assay, and steady-state levels of mPL-II mRNA as assessed by Northern analysis were also reduced by hTNF-α treatment. The lowest concentration of hTNF-α that significantly inhibited mPL-II secretion by cells from day 12 of pregnancy was 0·01 nmol/l. hTNF-α treatment did not affect the secretion of mPL-I, PLF or PRP, as assessed by the concentrations of these proteins in the medium during a 5-day incubation. Incubation of the cells with 20 ng EGF/ml also did not affect the PLF or PRP concentration of the medium during 5 days of culture. To determine whether the effect of hTNF-α on mPL-II secretion was mediated by interleukin-6 (IL-6), the IL-6 concentration of the medium of control and hTNF-α-treated cells was determined. Bioactive and immuno-reactive IL-6 could not be detected in medium from either treatment group. The presence of binding sites for hTNF-α was assessed in cells from day 12 of pregnancy. Scatchard analysis detected a single class of binding sites having a K_d of 1·61±0·34 nmol/l, with about 1350 sites per cell. The results of this study demonstrate that hTNF-α inhibits the secretion of mPL-II by placental cells from days 9 and 12 of pregnancy, suggesting that TNF-α may be one of the factors that regulate the production of this hormone in vivo.

Journal of Endocrinology (1994) 143, 95–105

Insulin-like growth factor binding proteins (IGFBPs) are important local factors in the development of proliferative diabetic retinopathy. We investigated the effects of IGF-I and increased glucose concentrations on the release of IGFBPs and the growth of human retinal endothelial cells (HRECs). HRECs secrete IGFBPs-2 to -5. IGF-I stimulated thymidine incorporation and modified the pattern of IGFBPs, decreasing the inhibitory IGFBP-4 through down-regulation of its mRNA, and increasing IGFBP-5 which, per se, was able to modulate HREC growth, exerting post-transcriptional control. Studies using an antibody (alpha IR3) against the IGF-I receptor, and compounds with low affinity for IGFBPs, such as insulin and des(1-3)IGF-I, showed that an interaction between IGF-I and IGFBP-5 was necessary to detach this IGFBP from its binding sites. The dose of IGF-I that significantly decreased the IGFBP-4/IGFBP-5 ratio was the same that stimulated HREC growth. Chronic exposure to high concentrations of glucose was able to reduce HREC mitogenesis, interacting with the IGF system through a decrease in the stimulatory IGFBPs-2, -3 and -5, leaving the concentration of the inhibitory IGFBP-4 constant. These results extend our previous observations in endothelial cells and suggest that the IGFBP-4/IGFBP-5 ratio regulates IGF-I-induced growth of HRECs, whereas a general decrease in IGFBPs (except for IGFBP-4) was the anti-proliferative effect of chronic exposure to high glucose concentrations.

ABSTRACT

Adenylate cyclase and cyclic AMP phosphodiesterase activities in the thyroid gland were significantly reduced after hypophysectomy, followed by a gradual restoration of the enzyme activities to the levels seen in sham-operated rats whereas a slight and persistent reduction was evident in guanylate cyclase and cyclic GMP phosphodiesterase activities in the same tissue. These changes in enzyme activities were restored by TSH administration but not by ACTH. The recovery of activity produced by TSH administration was inhibited by cycloheximide. Hypophysectomy, or TSH and cycloheximide administration, did not produce any significant changes in the concentrations of calmodulin, suggesting that the alteration of these enzyme activities is not induced by a decrease in the concentration of calmodulin. Since forskolin activation of adenylate cyclase did not restore the reduced activity in the hypophysectomized rat thyroid to the level found in the sham-operated control rat thyroid, we conclude that there is a reduction of the amount of enzyme after hypophysectomy rather than a change of the active site on adenylate cyclase. The spontaneous restoration of adenylate cyclase and cyclic AMP phosphodiesterase activities after hypophysectomy implies that cyclic AMP-metabolizing enzymes are responsive to an autoregulatory mechanism in thyroid follicular cells.

J. Endocr. (1985) 105, 363–369

Abstract

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.

The results show that GHBP (as distinct from GH receptor) is expressed by the placenta in vivo and trophoblasts in vitro. From mid-gestation onwards, GHBP mRNA increases dramatically in vivo and the cytosolic content of GHBP in freshly isolated trophoblasts increases. This suggests an important local regulatory role for placental GHBP during gestation.

Journal of Endocrinology (1994) 140, 125–135