Karen Nygard

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.

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.

Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.

Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix‐associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type‐I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three‐dimensional type‐I collagen lattices. Inhibition of Rho‐associated protein kinase activity significantly attenuated the effects of matrix‐associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix‐associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho‐associated protein kinase‐dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.

Synaptic development and neuronal myelination are altered with growth restriction in fetal guinea pigs.

This study examines aberrant synaptogenesis and myelination of neuronal connections as possible links to neurological sequelae in growth-restricted fetuses. Pregnant guinea pig sows were subjected to uterine blood flow restriction or sham surgeries at midgestation. The animals underwent necropsy at term with fetuses grouped according to body weight and brain-to-liver weight ratios as follows: appropriate for gestational age (n = 12); asymmetrically fetal growth restricted (aFGR; n = 8); symmetrically fetal growth restricted (sFGR; n = 8), and large for gestational age (n = 8). Fetal brains were perfusion fixed and paraffin embedded to determine immunoreactivity for synaptophysin and synaptopodin as markers of synaptic development and maturation, respectively, and for myelin basic protein as a marker for myelination, which was further assessed using Luxol fast blue staining. The most pertinent findings were that growth-restricted guinea pig fetuses exhibited reduced synaptogenesis and synaptic maturation as well as reduced myelination, which were primarily seen in subareas of the hippocampus and associated efferent tracts. These neurodevelopmental changes were more pronounced in the sFGR compared to the aFGR animals. Accordingly, altered hippocampal development involving synaptogenesis and myelination may represent a mechanism by which cognitive deficits manifest in human growth-restricted offspring in later life.

Maternal nutrient restriction in guinea pigs leads to fetal growth restriction with evidence for chronic hypoxia

BackgroundWe determined whether maternal nutrient restriction (MNR) in guinea pigs leading to fetal growth restriction (FGR) impacts markers for tissue hypoxia, implicating a mechanistic role for chronic hypoxia.MethodsGuinea pigs were fed ad libitum (Control) or 70% of the control diet before pregnancy, switching to 90% at mid-pregnancy (MNR). Near term, hypoxyprobe-1 (HP-1), a marker of tissue hypoxia, was injected into pregnant sows. Fetuses were then necropsied and liver, kidney, and placental tissues were processed for erythropoietin (EPO), EPO-receptor (EPOR), and vascular endothelial growth factor (VEGF) protein levels, and for HP-1 immunoreactivity (IR).ResultsFGR–MNR fetuses were 36% smaller with asymmetrical growth restriction compared to controls. EPO and VEGF protein levels were increased in the female FGR–MNR fetuses, providing support for hypoxic stimulus and linkage to increased erythropoiesis, but not in the male FGR–MNR fetuses, possibly reflecting a weaker link between oxygenation and erythropoiesis. HP-1 IR was increased in the liver and kidneys of both male and female FGR–MNR fetuses as an index of local tissue hypoxia, but with no changes in the placenta.ConclusionChronic hypoxia is likely to be an important signaling mechanism for the decreased fetal growth seen with maternal undernutrition and appears to be post-placental in nature.

BDNF and TrkB in the Preterm and Near-term Ovine Fetal Brain and the Effect of Intermittent Umbilical Cord Occlusion

We have determined the developmental change in immunoreactivity for brain-derived neurotrophic factor and its high-affinity tyrosine kinase receptor, TrkB, in the ovine fetal brain with advancing gestation and in response to intermittent umbilical cord occlusion, 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 umbilical cord occlusions performed in the experimental group animals by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brains perfusion fixed and prepared for subsequent histology with the distribution of brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity determined by immunohistochemistry. In the control group animals brain-derived neurotrophic factor immunoreactivity decreased in the gray matter, thalamus, and hippocampus but increased in the white matter, while tyrosine kinase receptor immunoreactivity decreased in all regions (most P < .01), with advancing gestation consistent with the developmental change from neurogenesis/gliagenesis to myelination over this time period. Intermittent umbilical cord occlusion as studied with severe but limited hypoxemia resulted in a variable decrease in brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity for all brain regions in the preterm animals (most P < .01) when protein turnover is higher, but a selective increase in brain-derived neurotrophic factor immunoreactivity in the hippocampus of the near-term animals consistent with a heightened vulnerability for necrotic/apoptotic injury at this time. As such, brain-derived neurotrophic factor—tyrosine kinase receptor in the ovine fetal brain may be altered with intermittent hypoxic insults over the latter part of pregnancy with potential for longer term neurologic consequences.

Exposure of decidualized HIESC to low oxygen tension and leucine deprivation results in increased IGFBP-1 phosphorylation and reduced IGF-I bioactivity

Phosphorylation of decidual IGFBP-1 enhances binding of IGF-I, limiting the bioavailability of this growth factor which may contribute to reduced placental and fetal growth. The mechanisms regulating decidual IGFBP-1 phosphorylation are incompletely understood. Using decidualized human immortalized endometrial stromal cells we tested the hypothesis that low oxygen tension or reduced leucine availability, believed to be common in placental insufficiency, increase the phosphorylation of decidual IGFBP-1. Multiple reaction monitoring-MS (MRM-MS) was used to quantify IGFBP-1 phosphorylation. MRM-MS validated the novel phosphorylation of IGFBP-1 at Ser58, however this site was unaffected by low oxygen tension/leucine deprivation. In contrast, significantly elevated phosphorylation was detected for pSer119, pSer98/pSer101 and pSer169/pSer174 sites. Immunoblotting and dual-immunofluorescence using phosphosite-specific IGFBP-1 antibodies further demonstrated increased IGFBP-1 phosphorylation in HIESC under both treatments which concomitantly reduced IGF-I bioactivity. These data support the hypothesis that down regulation of IGF-I signaling links decidual IGFBP-1 hyperphosphorylation to restricted fetal growth in placental insufficiency.

Location, Location, Location: Appraising the Pleiotropic Function of HMGB1 in Fetal Brain

We read with interest the paper by Zhang et al entitled “HMGB1 translocation after ischemia in the ovine fetal brain” (1). High-mobility group box 1 protein (HMGB1) is a fascinating protein with “many faces,” yet we are only beginning to uncover its roles in the developing brain (2). The findings of Zhang et al add to the much-needed body of literature in the field and validate our work in the same animal model and at the same near-term gestational age. Under intermittent umbilical cord occlusions (UCOs) insult with 3–4 hours of worsening acidemia and pH<7.00, we found an intracellular translocation of HMGB1 in neurons (3, 4), astrocytes (4), and microglia (5, 6) measured 24 hours post-insult. Interestingly, under control conditions, we found the dominant localization of HMGB1 to cytosol in neurons and astrocytes but not in microglia. Acidemic insult due to UCOs caused subtle and brain region-specific reverse shifts in neuronal HMGB1 patterns of cortical gray matter (3). Our findings seem to stand in contrast to those by Zhang et al. A number of methodological differences in quantification of HMGB1 translocation distinguish the present paper by Zhang et al from our approach and may explain the contrasting findings in the dominant neuronal HMGB1 localization. A stronger scenario of 30 min cerebral ischemia was modeled by Zhang et al with immunohistochemical (IHC) analyses performed at 48 and 72 hours post-insult whereas we attempted to mimic the generic human labor process with multiple repetitive, intermittent 1min lasting global ischemia episodes and IHC at 24 hours post-insult. It is possible that with the more severe brain ischemia apoptosis pathways have been triggered and the nuclear HMGB1 translocated to the cytosol en route to act as the extracellular cytokine for microglia (7). With the DAB chromogen stain used, the delineation between nucleus and cytosol without a counterstain is less clear than with fluorescence or dual/triple stains. It is not clear how the authors determined where to draw these lines except by visually recognizing the translocation. Rather than contouring around specific brain regions, the authors drew around where they thought they saw positive translocation, and then measured the percentage size of that outline relative to the whole brain contour. Next, the authors deliberately selected only cells showing evidence of HMGB1 translocation. An additional selection bias may have been difficult to avoid as the authors chose the already larger pyramidal cortical gray matter cells as “translocated” because they have large nuclei, in which they could see the translocation better. We suggest that an additional systematic approach of all layers needs to be undertaken with quantification of cytosol and nuclear HMGB1 signals. This may reveal additional translocated cells being the granular cells, which may seem as non-translocated because of their tight cytosol and dark nuclei. Lastly, formalin immersion fixation only penetrates tissue at a rate of approximately 1.6 mm/hour and after penetration it still needs to create crosslinks to fix proteins. We and others perfuse and then immerse. The fixation is then coming from two directions at once (i.e. inside vessels and outside brain) and reaches the tissues faster to stabilize them. The areas the authors outlined as having heightened HMGB1 cytosolic signal seem close to a ventricle where formalin would be entering from, and their high signal may be reflective of good perfusion there vs. deep within the tissue. In light of the strong focus on the inflammatory roles of HMGB1 in the extracellular space in the last decade of research it is worth remembering that HGMB1 is also essential for life per se and for tissue regeneration processes (8). Perhaps in line with the many faces of HMGB1 during development and in various cell types in health and disease, HMGB1 nucleo-cytosolic distribution patterns are remarkably tissueand time-specific (8–10). A predominantly cytosolic location of HMGB1 has been reported in developing brain, promoting neurite outgrowth and implicated in early neuronal development in vitro and in vivo (8,9,11). Thus, alterations in nucleo-cytosolic predominance of HMGB1 in developing brain may not only result in pro-inflammatory and neurodestructive effects due to its extracellular functions but also in disruption of physiological neurodevelopmental programs due to its intracellular, notably also cytosolic, functions, which are yet to be well defined in mammals.

Impact of ventilation-induced lung injury on the structure and function of lamellar bodies

Alterations to the pulmonary surfactant system have been observed consistently in ventilation-induced lung injury (VILI) including composition changes and impairments in the surface tension reducing ability of the isolated extracellular surfactant. However, there is limited information about the effects of VILI on the intracellular form of surfactant, the lamellar body. It is hypothesized that VILI leads to alterations of lamellar body numbers and function. To test this hypothesis, rats were randomized to one of three groups, nonventilated controls, control ventilation, and high tidal volume ventilation (VILI). Following physiological assessment to confirm lung injury, isolated lamellar bodies were tested for surfactant function on a constrained sessile drop surfactometer. A separate cohort of animals was used to fix the lungs followed by examination of lamellar body numbers and morphology using transmission electron microscopy. The results showed an impaired ability of reducing surface tension for the lamellar bodies isolated from the VILI group as compared with the two other groups. The morphological assessment revealed that the number, and the relative area covered by, lamellar bodies were significantly decreased in animals with VILI animals as compared with the other groups. It is concluded that VILI causes significant alterations to lamellar bodies. It is speculated that increased secretion causes a depletion of lamellar bodies that cannot be compensated by de novo synthesis of surfactant in these injured lungs.

IUGR is associated with marked hyperphosphorylation of decidual and maternal plasma IGFBP-1

Context The mechanisms underpinning intrauterine growth restriction (IUGR), as a result of placental insufficiency, remain poorly understood, no specific treatment is available, and clinically useful biomarkers for early detection are lacking. Objective We hypothesized that human IUGR is associated with inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling, increased protein kinase casein kinase-2 (CK2) activity, and increased insulin-like growth factor-binding protein 1 (IGFBP-1) expression and phosphorylation in decidua and that maternal plasma IGFBP-1 hyperphosphorylation in the first trimester predicts later development of IUGR. Design, Setting, and Participants Decidua [n = 16 appropriate-for-gestational age (AGA); n = 16 IUGR] and maternal plasma (n = 13 AGA; n = 13 IUGR) were collected at delivery from two different cohorts. In addition, maternal plasma was obtained in the late first trimester from a third cohort of women (n = 7) who later delivered an AGA or IUGR infant. Main Outcome Measures Total IGFBP-1 expression and phosphorylation (Ser101/Ser119/Ser169), mTOR, AAR, and CK2 activity in decidua and IGFBP-1 concentration and phosphorylation in maternal plasma. Results We show that decidual IGFBP-1 expression and phosphorylation are increased, mTOR is markedly inhibited, and AAR and CK2 are activated in IUGR. Moreover, IGFBP-1 hyperphosphorylation in first-trimester maternal plasma is associated with the development of IUGR. Conclusions These data are consistent with the possibility that the decidua functions as a nutrient sensor linking limited oxygen and nutrient availability to increased IGFBP-1 phosphorylation, possibly mediated by mTOR and AAR signaling. IGFBP-1 hyperphosphorylation in first-trimester maternal plasma may serve as a predictive IUGR biomarker, allowing early intervention.