V. K. M. Han

Ligand-Dependent Nuclear Receptor Corepressor LCoR Functions by Histone Deacetylase-Dependent and -Independent Mechanisms

LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.

Characteristic's of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

Brain growth retardation due to the expression of human insulin like growth factor binding protein-1 in transgenic mice: an in vivo model for the analysis of igf function in the brain

Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation.

Dual Functional Roles for the X-linked Lymphoproliferative Syndrome Gene Product SAP/SH2D1A in Signaling through the Signaling Lymphocyte Activation Molecule (SLAM) Family of Immune Receptors

The X-linked lymphoproliferative (XLP) syndrome gene encodes a protein named SAP or SH2D1A that is composed of a single Src homology 2 (SH2) domain. Two models have been proposed for its function in lymphocyte signaling. One postulates that it acts as an inhibitor of interactions between the phosphatase SHP-2 and the immune receptor SLAM. The other suggests that it functions as an adaptor to promote the recruitment of a kinase, FynT, to SLAM. Here, we provide evidence in support of both roles for SAP. Using an array of peptides derived from the SLAM family of receptors, we demonstrate that SAP binds with comparable affinities to the same sites in these receptors as do the SH2 domains of SHP-2 and SHIP, suggesting that these three proteins may compete against one another in binding to a given SLAM family receptor. Furthermore, in vitro and in vivo binding studies indicate that SAP is capable of binding directly to FynT, an interaction mediated by the FynT SH3 domain. In cells, FynT was shown to be indispensable for SLAM tyrosine phosphorylation, which, in turn, was drastically enhanced by SAP. Because SAP also blocked the recruitment of SHP-2 to SLAM in these cells, we propose a dual functional role for SAP in SLAM signaling by acting both as an adaptor for FynT and an inhibitor to SHP-2 binding. The physiological relevance of the dual functional role for SAP is underscored by the observation that disease-causing SAP mutants exhibited significantly reduced affinities to both FynT and SLAM.

Decline in Number of Elevated Blood CD3+ CD56+ NKT Cells in Response to Intravenous Immunoglobulin Treatment Correlates with Successful Pregnancy

Patients with elevated blood natural killer (NK) cells may be offered intravenous immunoglobulin (IVIG) treatment, but there is controversy about the utility of blood NK cell testing. Human CD56+ NK cells include several subpopulations that include the putatively cytotoxic CD56+ CD16+ subset. In mouse models of pregnant failure, NKT cells appear to be important. However, a mouse model may only be pertinent to a subset of patients, as recurrent pregnancy failure is a heterogenous group.

Determinants of small for gestational age birth at term.

BACKGROUND   Being born small for gestational age (SGA) is an indicator of intrauterine growth restriction (IUGR) and later health risks. This study investigated determinants of severe and moderate SGA (respectively, birthweight <3rd percentile and 3rd to <10th percentile for gestational age and sex). METHODS   A total of 2195 term pregnancies from a prospective cohort were studied. Prenatal data arose from maternal interview at 10-22 weeks of gestation and perinatal data were collected from hospital charts. Severe and moderate SGA were classified by Canadian population standards. Risk factors for SGA were identified from fitting multivariable logistic regression models. RESULTS   Multivariable associations with severe SGA were: maternal age ≥ 35 [odds ratio (OR) 3.2 [95% confidence interval (CI) 1.4, 6.9]], maternal smoking during pregnancy (OR 5.3 [95% CI 2.4, 11.7]), preeclampsia (OR 4.6 [95% CI 1.6, 13.2]) and threatened preterm labour (OR 3.9 [95% CI 1.3, 11.4]). Primiparity was associated with both severe and moderate SGA with OR 2.4 [95% CI 1.1, 5.1] and OR 1.9 [95% CI 1.3, 2.9] respectively. Underweight pre-pregnancy body mass index was associated with moderate SGA (OR 2.4 [95% CI 1.2, 5.0]). Inclusion of placental weight, in the final model attenuated the associations. CONCLUSIONS   This study demonstrated different determinants for severe and moderate SGA. We speculate that the majority of severe SGA infants are IUGR while moderate SGA infants may be a mixture of IUGR and constitutionally small newborns. This study has also contributed evidence linking preterm labour and SGA as two, potentially related, outcomes of overlapping causal mechanisms reflective of ischaemic placental disease.

Effects of Maternal Global Nutrient Restriction on Fetal Baboon Hepatic Insulin-Like Growth Factor System Genes and Gene Products

Knowledge of altered maternal nutrition effects on growth-regulating systems is critical to understanding normal and abnormal fetal development. There are many reports of hepatic fetal IGF system responses to maternal nutrient restriction (MNR) during pregnancy in rodents and sheep but none in nonhuman primates. We determined effects of MNR on the fetal baboon hepatic IGF system. Social groups of female baboons were fed ad libitum, controls, or 70% controls (MNR) from 0.16 to 0.5 gestation and fetuses delivered by cesarean section. Fetal liver tissue was analyzed for IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 mRNA by in situ hybridization and quantitative RT-PCR and protein by immunohistochemistry (IHC); IGF-I receptor, IGF-II receptor by quantitative RT-PCR and IHC and IGFBP-1 by in situ hybridization and IHC. MNR did not alter fetal body or liver weight. Fetal hepatic glycogen staining increased with MNR. MNR reduced fetal hepatic IGF-I and IGF-II and increased IGFBP-1 mRNA and decreased IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor protein and increased protein for IGFBP-1 and IGFBP-3. MNR increased caspase-3, indicating apoptosis and decreased Akt staining, indicating decreased nutrient sensing. In conclusion, whereas fetal body and liver weights did not change in response to moderate MNR during the first half of baboon pregnancy, the major indices of function of the hepatic IGF system measured were all reduced.

Randomized controlled trial of very early continuous distending pressure in the management of preterm infants

Application of continuous distending pressure at birth (very early CDP) should stabilize the immature airways and reduce the severity of respiratory distress syndrome (RDS) in preterm infants. Eighty-two preterm infants of less than 32 weeks gestation were randomly assigned at birth to early treatment group (TG), in which CDP of 6 cm water pressure was applied at birth by the nasopharyngeal route (NP-CDP), or to control group (CG), in which CDP was applied when indicated for established criteria (pO2 less than 50 mmHg in FiO2 greater than 0.5). Characteristics of the infants in the two groups were comparable. No statistically significant difference between the two groups was found in the incidence of RDS. The course of RDS, and oxygen and ventilatory requirements also did not appear to be changed. In blood gas parameters of most of the time frames, no significant difference was found between the two groups when the results were analyzed according to the assigned group. When the results were analyzed separately for the infants who developed RDS, infants in TG appear to have fared worse from the therapy in terms of oxygenation, as indicated by significantly higher FiO2 (P less than 0.01) and lower a/A (P less than 0.01) values on the third day of the course of RDS, as compared to infants in CG. The incidence of complications was comparable in the two groups. Four infants from TG (9.3%) and one from CG (2.6%) died (P = NS). We conclude that VECDP by nasopharyngeal route does not reduce the incidence of RDS and does not appear to improve the outcome and may worsen the severity of RDS when compared to application of CDP for established criteria.

Apoptosis in the preterm and near term ovine fetal brain and the effect of intermittent umbilical cord occlusion

Programmed cell death or apoptosis plays a central role during the development of the brain, but can also be activated by hypoxic/ischemic insult. The purpose of the present study was to determine the regional distribution of apoptotic cells in the preterm and near term ovine fetal brain and thus in relation to the maturation of neurobehavioural activity, and the effect of intermittent umbilical cord occlusion (UCO), which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete inflation of an occluder cuff for 90 s every 30 min for 3 to 5 h each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and apoptosis staining using the TUNEL assay method. The number of TUNEL positive cells for both the preterm and near term control group animals was low but with a significant regional hierarchy whereby values were higher in the cerebellar peduncle and cortex and lower in the cortical grey and white matter, hippocampus, and pons. While the apoptotic indices (expressed as TUNEL positive cells/1000 cells or high powered field) for most brain regions were not significantly changed between the preterm and near term control group animals, that for the hippocampus and pons were increased approximately 5- and 4-fold, respectively, (both P<0.05), in the near term animals. Intermittent UCO with severe but limited hypoxemia and no cumulative acidosis to ensure longer term survival, had no significant effect on apoptotic indices in the brains of either the preterm or near term animals, although hippocampal values for both occlusion groups were increased approximately 2-3-fold. Levels of apoptosis noted for the ovine fetal brain at 0.75 to 0.90 of gestation are thus low and likely approaching the basal levels of later life, but there are regional differences and changes over this period although little change in response to intermittent cord occlusion as studied, with implications for behavioural state activity and antenatal hypoxic insults in the brains development.

Insulin-like growth factor binding protein-6 (IGFBP-6) interacts with DNA-end binding protein Ku80 to regulate cell fate.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a growth inhibitory protein that regulates the availability of insulin-like growth factors (IGFs). We recently reported that IGFBP-6 exerts intracellular actions via its translocation to the nucleus. We now show that IGFBP-6 co-purifies by tandem-affinity with nuclear proteins involved in DNA stability and repair such as Ku80, Ku70, histone H2B and importin-alpha. Furthermore, this report shows that IGFBP-6 and Ku80 interact specifically using two active binding sites for Ku80 in IGFBP-6. One of the binding sites [196RKR199], as part of the NLS-sequence in IGFBP-6 also binds importin-alpha which may selectively compete with Ku80 regulating its trafficking to the nucleus. Moreover, IGFBP-6 co-localized with Ku80 based on a cell cycle pattern. Overexpression of IGFBP-6 increased the nuclear Ku80 in mitotic cells and reduced it post-mitosis. It is known that if highly expressed IGFBP-6 induces apoptosis and in our model, the down-regulation of Ku80 by specific siRNAs enhanced the apoptotic effect caused by the IGFBP-6 overexpression. This study demonstrates that IGFBP-6 alters cell survival by potentially regulating the availability of Ku80 for the DNA-repair process. This action represents a novel mechanism by which growth inhibitory proteins such as IGFBP-6 regulate cell fate.