Differential distribution of binding sites for 125I-insulin-like growth factor II on trophoblast membranes of human term placenta.

The syncytiotrophoblast, which is delineated by two polar membranes (the microvillous and the basal plasma membranes), is the main placental structural element controlling maternal-fetal exchanges. These studies of the full-term placenta were undertaken in order to determine whether the microvillous membranes, which are bathed by the maternal intervillous circulation, and basal plasma membrane, which lines the fetal blood capillaries, have binding sites for insulin-like growth factor (IGF)-II. The microvillous and basal plasma membranes were purified and found to bind 125I-IGF-II with significantly different (p < 0.0001) Kd (0.51 and 1.02 nM, respectively). There were more available binding sites in the microvillous (4.4+/-0.3 pmol/mg protein) than in the basal (2.7+/-0.4 pmol/mg protein) plasma membranes (p < 0.0001). Both membranes contained three major (250, 135, and 130 kDa) 125I-IGF-II/binding-site protein complexes as determined by affinity cross-linking and PAGE. The 250-kDa band (type 2 IGF receptor) was the main band in the basal plasma membranes (46% total bound 125I-IGF-II). The 135-kDa band (insulin-receptor alpha subunit) was the main one in the microvillous membranes (48% total bound 125I-IGF-II). The amounts of 130-kDa band (type 1 IGF-receptor alpha subunit) in the two types of membranes were similar (30% total bound 125I-IGF-II). Only IGF-II displaced 125I-IGF-II from the 250-kDa band, while 125I-IGF-II bound to the 135-kDa band was displaced by insulin, and ligand bound to the 130-kDa band was displaced by IGF-I. Thus there are IGF receptors in both types of membranes of syncytiotrophoblast in the human full-term placenta, and the distributions of the IGF and insulin receptors are asymmetrical. This may reflect the fact that they face and interact with two independent, different media. Maternal IGF may influence the syncytiotrophoblast by binding to receptors on the microvillous membranes, while fetal IGF may also influence syncytiotrophoblast functions by activating receptors in the basal plasma membranes.

Identification and characterization of 125I-insulin-like growth factor-II binding sites on the muscular layer of stem villi vessels of human term placenta.

The primary function of the placenta is to ensure an optimal environment for fetal growth and development. In normal pregnancy, placental vascular tone regulation assures fetus well-being and normal development by maintaining adequate blood flow so as to ensure materno-fetal exchanges. In human placenta, synthesis of insulin-like growth factor (IGF)-II and specific binding sites have been previously characterized in the trophoblast; in contrast, no studies have dealt with this subject in the fetoplacental vascular system, particularly in stem villi vessels. We thus investigated whether membranes of the muscular layer of stem villi vessels contained 125I-IGF-II binding sites. Two complementary approaches were used: 125I-IGF-II binding and affinity cross-linking studies. 125I-IGF-II labeled, in a saturable and noncooperative manner, a single class of high-affinity binding sites characterized by a Kd of 1.24 +/- 0.26 nM (n = 6), a maximum binding capacity (Bmax) of 3.02 +/- 0.45 pmol/mg protein, and a Hill coefficient of 1.00 +/- 0.15. Competitors for 125I-IGF-II binding to membranes were in the order of potency IGF-II > IGF-I. Insulin was not a competitor. Affinity cross-linking of membranes with 125I-IGF-II, followed by SDS-PAGE and autoradiography, revealed two labeled bands: a protein complex of 250 kDa, which corresponds to the type II IGF receptor, and another of 135 kDa, corresponding to the type I IGF receptor. Only IGF-II could displace 125I-IGF-II binding from the major 250-kDa band, while 125I-IGF-II bound to the minor 135-kDa band was displaced by either IGF-I, IGF-II, or insulin. In conclusion, high levels of specific binding sites for 125I-IGF-II are present in the muscular layer of stem villi vessels, which are considered placenta resistance vessels. The involvement of both type I and type II IGF receptors in the growth-promoting action of IGF-II remains to be determined in the fetoplacental vascular system.

Inhibition of chondrocyte cathepsin B and L activities by insulin-like growth factor-II (IGF-II) and its Ser29 variant in vitro: possible role of the mannose 6-phosphate/IGF-II receptor.

Lysosomal enzymes and IGF-II both bind to the mannose 6-phosphate (M6P)/IGF-II receptor. This receptor targets newly synthesized lysosomal enzymes to lysosomes. The functional meaning of IGF-II binding to this receptor is not well known. We have postulated that IGF-II, the Ser29 IGF-II variant (vIGF-II) and IGF-I on lysosomal cathepsin B and L activities from post-natal rabbit chondrocytes in vitro. This effect was compared with the ability of each peptide to stimulate chondrocyte-sulfated proteoglycan synthesis. The sulfating dose-response relationship of the IGF peptides corresponded to their relative binding affinities for the type I-IGF receptor (IGF-I > IGF-II > vIGF-II). The intracellular cathepsin B and L activities were inhibited in a time- and dose-dependent manner by IGF-II or vIGF-II. Maximal inhibition of cathepsin B and L activities (40 and 30% below controls, respectively) was found after an 8 h treatment with 100 ng/ml IGF-II or vIGF-II. By contrast, IGF-I up to 1 micrograms/ml or insulin up to 2 micrograms/ml had no inhibitory effect. The relative potency pattern corresponded to the binding profile of each ligand for the M6P/IGF-II receptor. A treatment of chondrocytes with IGF-I or insulin transiently increased the binding of radiolabelled IGF-II at the cell surface to approximately 120% of controls, whereas IGF-II or vIGF-II had no effect. Thus, it is unlikely that the inhibition of lysosomal enzyme activities by IGF-II peptides could result from a redistribution of M6P/IGF-II receptors from intracellular compartments to the plasma membrane. We hypothesize that internalized IGF-II peptides could occupy the intracellular M6P/IGF-II binding sites required for targeting of cathepsins B and L to lysosomes.

Purification and characterization of insulin-like growth factor II (IGF II) and an IGF II variant from human placenta.

In order to purify variant IGF II peptides from human placenta, we have developed a purification procedure combining heparin affinity chromatography and cation-exchange, reversed-phase and size-exclusion HPLC. Two peptides were purified, both having apparent M(r) values of ca. 7300 Da as evaluated by SDS-PAGE. N-Terminal sequencing revealed IGF II and an IGF II variant in which Ser29 was replaced by the tetrapeptide Arg-Leu-Pro-Gly. The final yield of variant IGF II was about eight-fold lower than that of IGF II. Both pure peptides were functionally active as they bound to type I and type II IGF receptors from ovine and human placental membranes, as determined by crosslinking experiments and displacement curve studies.

[Effect of growth hormone on the differentiation of chondrocytes from prepuberal rabbits in serum-free culture and on the radioimmunologic activity of Sm-C/IGF1 measured in the culture medium]. / Effet de GH sur la différenciation de chondrocytes de lapin prépubère en culture sans sérum et sur l'activité radioimmunologique Sm-C/IGF1 measurable dans le milieu de culture.

This study concerns the immunoreactive somatomedin C secretion by prepubertal rabbit epiphyseal chondrocytes cultured in a defined serum-free medium. In such culture conditions, chondrocytes mainly synthesized Type II collagen (80% of total collagen) during 10 days. A small amount of Type I collagen was also found with a significant (p less than 0.05) higher level during the period of cell multiplication (6.4 +/- 1.5%) than when cells reached confluency (0.9 +/- 0.2%). During the 10 days of culture without serum and without hormone added, a Sm-C/IGF1 activity was measured by RIA at a mean level of 30 +/- 5 mU/ml/10 micrograms DNA. This value was significantly higher (p less than 0.001) than in the medium not incubated with the cells (1.7 +/- 0.9 mU/ml). When hGH was added to the culture medium during the period of cell division, the level of Sm-C/IGF1 activity was significantly elevated at 39 +/- 4 mU/ml/10 micrograms DNA (p less than 0.05) and at 55 +/- 3 mU/ml/10 micrograms DNA (p less than 0.001) with 50 ng/ml and 100 ng/ml hGH concentrations respectively. On the contrary, no difference was observed at confluency in treated and non treated cells.