Mariko Horii

Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase.

Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells.

Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation.

We demonstrate here that neuronal nitric‐oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca2+/calmodulin (CaM)‐dependent protein kinase I (CaM‐K I1‐293). Substitution of Ser741 to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser741 by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM‐binding domain. CaM‐K I1‐293 gave phosphorylation of nNOS at Ser741 in transfected cells, resulting in 60–70% inhibition of nNOS activity. Wild‐type CaM‐K I also did phosphorylate nNOS at Ser741 in transfected cells, but either CaM‐K II or CaM‐K IV did not. These results raise the possibility of a novel cross‐talk between nNOS and CaM‐K I through the phosphorylation of Ser741 on nNOS.

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

Significance Human pluripotent stem cells (hPSCs) continue to be underappreciated as a model for studying trophoblast differentiation. In this study, we provide a reproducible, two-step protocol by which hPSCs can be differentiated into bipotential cytotrophoblast (CTB) stem-like cells and subsequently into functional, terminally differentiated trophoblasts. In addition, we provide evidence that the response of hPSC-derived CTBs to low oxygen is similar to that of primary CTBs. Finally, using trisomy 21-affected hPSCs, we show, for the first time to our knowledge, that hPSCs can model a trophoblast differentiation defect. We propose that hPSCs are superior to other currently available models for studying human trophoblast differentiation. Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6–8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2+/p63+ CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2+/p63+ CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G+ EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

Rare sugar d-allose enhances anti-tumor effect of 5-fluorouracil on the human hepatocellular carcinoma cell line HuH-7

d-Allose is a novel anti-tumor monosaccharide that causes cell growth inhibition, specifically of the cancer cells, by inducing the tumor suppressor gene thioredoxin interacting protein (TXNIP). The commonly used anti-tumor drug, 5-fluorouracil (5-FU), blocks the cell cycle by inhibiting thymidylate synthase, and is also known to induce TXNIP gene expression. In this study, we examined the synergistic effect of d-allose and 5-FU and the role of TXNIP on cancer cell growth. The treatment of HuH-7 cells with d-allose or 5-FU inhibited the cell growth in a dose-dependent manner (75.2+/-2.7% with 50 mM d-allose and 66.1+/-2.7% with 0.5 mug/ml 5-FU) and d-allose enhanced the anti-tumor effect of 5-FU (55.3+/-1.1 %). TUNEL analysis did not show any evidence of apoptosis with either d-allose or 5-FU treatment. 5-FU suppressed the expression of p27(kip1), p53, and cyclin E, whereas d-allose induced p53 and reduced cyclins D, A, and E. The expression of p27(kip1) remained unchanged by d-allose at transcriptional level, but increased at the protein level suggesting an increase in protein stability by TXNIP. d-Allose and to a lesser extent 5-FU induced TXNIP expression significantly (808.4+/-122.9% and 186.8+/-32.9%, respectively) and the combination of both further enhanced TXNIP expression. As d-allose has no known side effects on normal cells, the combination of d-allose and 5-FU might be a potent candidate for cancer therapy.

Nitric oxide prevents phosphorylation of neuronal nitric oxide synthase at serine1412 by inhibiting the Akt/PKB and CaM-K II signaling pathways.

Neuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the central nervous system. However, the mechanisms by which nNOS activity is regulated are not completely understood. In the present study, the effects of protein kinases on the phosphorylation of nNOS in GH3 rat pituitary tumor cells were evaluated. We show that phosphorylation of nNOS at Ser1412 could be induced by the phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) agonist insulin, the calcium/calmodulin-dependent protein kinase II (CaM-K II) agonist A23187 or the cAMP-dependent protein kinase A (PKA) agonist IBMX, respectively. The phosphorylation levels of nNOS at Ser1412, induced by activation of Akt/PKB or CaM-K II, but not by PKA signaling, were reduced by pre-treatment with the NO donor diethylamine-NONOate. This inhibitory effect could be reversed by addition of a reducing reagent, dithiothreitol. Furthermore, the levels of phosphorylation of nNOS at Ser1412, induced by Akt/PKB or CaM-K II but not by PKA signaling, were enhanced by inhibition of nNOS activity with 7-nitroindazole. These findings suggest that the activation of nNOS can be catalyzed by at least three protein kinases, Akt/PKB, CaM-K II or PKA. NO generated from nNOS feedback prevents the activation of nNOS by inhibiting either Akt/PKB or CaM-K II but not PKA signaling.

Hypoxia Directs Human Extravillous Trophoblast Differentiation in a Hypoxia-Inducible Factor–Dependent Manner

Villous cytotrophoblasts are epithelial stem cells of the early human placenta, able to differentiate either into syncytiotrophoblasts in floating chorionic villi or extravillous trophoblasts (EVTs) at the anchoring villi. The signaling pathways regulating differentiation into these two lineages are incompletely understood. The bulk of placental growth and development in the first trimester occurs under low oxygen tension. One major mechanism by which oxygen regulates cellular function is through the hypoxia-inducible factor (HIF), a transcription factor complex stabilized under low oxygen tension to mediate cellular responses, including cell fate decisions. HIF is known to play a role in trophoblast differentiation in rodents; however, its role in human trophoblast differentiation is poorly understood. Using RNA profiling of sorted populations of primary first-trimester trophoblasts, we evaluated the first stage of EVT differentiation, the transition from epidermal growth factor receptor+ villous cytotrophoblasts into human leukocyte antigen-G+ proximal column EVT (pcEVT) and identified hypoxia as a major pcEVT-associated pathway. Using primary cytotrophoblasts, we determined that culture in low oxygen directs differentiation preferentially toward human leukocyte antigen-G+ pcEVT, and that an intact HIF complex is required for this process. Finally, using global RNA profiling, we identified integrin-linked kinase and associated cytoskeletal remodeling and adhesion to be among HIF-dependent pcEVT-associated signaling pathways. Taken together, we propose that oxygen regulates EVT differentiation through HIF-dependent modulation of various cell adhesion and morphology-related pathways.

Hepatoblastoma in an infant with paternal uniparental disomy 14

A 29‐year‐old primigravida developed polyhydramnios at 24 weeks of gestation, requiring six serial amnioreductions. In addition, prenatal ultrasound examinations revealed a fetus with small stomach pouch, small thorax, slightly shortened limbs, and skin edema; paternal uniparental disomy 14(upd(14)pat) phenotype was suspected. At 37 weeks, the patient delivered a 2558 g female infant with characteristic facial features, webbed neck, thoracic deformity, abdominal wall defect, skin edema, overlapping fingers, placentomegaly, and small thorax with ‘coat‐hanger’ appearance of the ribs on chest X‐ray. A phenotype consistent with upd(14)pat was confirmed by DNA analysis. Although the infants condition was initially stable, hepatoblastoma was subsequently detected and right hepatectomy was performed on day 224. On day 382, the infant was discharged with in‐home respiratory management.

Female Genital Tract Development and Disorders of Childhood

Abstract Female reproductive tract development is a complex process intricately tied to the patterning of the male (Wolffian) reproductive tract and renal anlage. Development starts from undifferentiated mesoderm known as the genital ridge, with germ cells migrating to this location from the yolk sac. The mullerian duct begins as an invagination of the coelomic epithelium at the top of the genital ridge and elongates by active cell proliferation using the Wolffian duct as a guide. Many genes have been linked to female reproductive tract development, but only a few have been directly implicated by animal knockout models; this is because the simplex female reproductive tract pattern (i.e., one with a single cervix and uterus, with two separate fallopian tubes) is limited to humans and other primates. Most recently, genomic sequencing has identified more genes, mostly transcription factors and extracellular signaling molecules, and mutations in those genes, associated with malformations of the female reproductive tract. The most common congenital abnormality of the human female genital tract occurs when the paired mullerian ducts fail to fuse or the subsequent septum fails to resorb, yielding a spectrum of uterine anomalies, including uterus didelphys and bicornuate uterus. External female genital tract development requires both the absence of a key male determining factor (SRY) and presence of its antagonist (WNT4). Virilization of genetically female fetuses is due to excessive androgens from congenital adrenal hyperplasia or maternal blood.

Placental Correlates of Unanticipated Fetal Death

Abstract Examination of the placenta is a critical component in evaluating the etiology of intrauterine demise. This is particularly true as pregnancy progresses, as the cause of demise beyond the first trimester is less likely to be due to intrinsic fetal disease and more likely due to “errors of the intrauterine environment”; that is, as a result of causes extrinsic to the fetus. As a general principle, the longer intrauterine stressors that ultimately result in demise are present, the more likely that placental “footprints” of those stressors will (1) accrue and (2) be detectable by gross or microscopic examination. This chapter lists segregated specific mechanisms of demise based on the timing of evolution from onset to demise. Of relevance are the following concepts, the literature for which remains in evolution. These concepts pertain largely to subacute and chronic intrauterine pathologies, although superimposed acute manifestations of preexisting disorders can add to the overall burden of fetal stress and concomitant risk for untoward fetal/neonatal outcome. (1) As a general rule, the more advanced or extensive a pathologic process, the greater the potential risk for untoward fetal/neonatal outcome. (2) Threshold criteria are accruing for identifying the severity/extent of specific placental lesions that herald an increased risk of untoward outcome. (3) The coexistence of separate pathologic processes, each of which can have fetal/neonatal implications, compounds their cumulative effects in at least an additive, if not multiplicative manner. (4) The same processes that lead to unexpected fetal demise can also result in neonatal morbidity and mortality.

Placental Development and Complications of Previable Pregnancy

Abstract Evaluation of the products of conception in the previable period requires knowledge of fetal and placental development across gestation. The placenta is comprised of two types of trophoblast—cytotrophoblast and syncytiotrophoblast, which comprise the outer linings of chorionic villi, the villous compartment of the placenta, required for gas and nutrient exchange. The extravillous trophoblast compartment includes cells that invade the uterus and remodel maternal vessels to establish blood flow into the intervillous space of the placenta, chorionic trophoblast in the fetal membranes, and cells within the intraplacental trophoblast islands. Abnormalities of fetal and placental development can result in pregnancy complications. Abnormal fetal development, secondary to aneuploidy or environmental disruption (e.g., amnion band sequence), can lead to severe fetal malformations and pregnancy loss. Abnormalities of placental implantation can lead to ectopic pregnancy, with the fallopian tube being the most common site. To rule out an ectopic pregnancy, endometrial curettings should ideally be examined grossly and histologically. An intrauterine pregnancy should not be diagnosed on the basis of mature chorionic villi fragments, remote implantation site nodule, or sole presence of decidua and gestational endometrium. In early spontaneous pregnancy loss, histologic findings are usually not specific for a particular cause of pregnancy loss; karyotypic abnormalities are present in 50% to 75% of these specimens, particularly in the setting of recurrent pregnancy loss. In the setting of late pregnancy loss with a normal fetus, inflammatory abruption, the pathologic correlate of cervical incompetence, should be strongly considered.