Joanne Paquin

Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes

We recently discovered the existence of the oxytocin/oxytocin receptor (OT/OTR) system in the heart. Activation of cardiac OTR stimulates the release of atrial natriuretic peptide (ANP), which is involved in regulation of blood pressure and cell growth. Having observed elevated OT levels in the fetal and newborn heart at a stage of intense cardiomyocyte hyperplasia, we hypothesized a role for OT in cardiomyocyte differentiation. We used mouse P19 embryonic stem cells to substantiate this potential role. P19 cells give rise to the formation of cell derivatives of all germ layers. Treatment of P19 cell aggregates with dimethyl sulfoxide (DMSO) induces differentiation to cardiomyocytes. In this work, P19 cells were allowed to aggregate from day 0 to day 4 in the presence of 0.5% DMSO, 10−7 M OT and/or 10−7 M OT antagonist (OTA), and then cultured in the absence of these factors until day 14. OT alone stimulated the production of beating cell colonies in all 24 independently growing cultures by day 8 of the differentiation protocol, whereas the same result was obtained in cells induced by DMSO only after 12 days. Cells induced with OT exhibited increased ANP mRNA, had abundant mitochondria (i.e., they strongly absorbed rhodamine 123), and expressed sarcomeric myosin heavy chain and dihydropyridine receptor-α1, confirming a cardiomyocyte phenotype. In addition, OT as well as DMSO increased OTR protein and OTR mRNA, and OTA completely inhibited the formation of cardiomyocytes in OT- and DMSO-supplemented cultures. These results suggest that the OT/OTR system plays an important role in cardiogenesis by promoting cardiomyocyte differentiation.

Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system

Neural apoptosis‐regulated convertase‐1/proprotein convertase subtilisin‐kexin like‐9 (NARC‐1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low‐density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT–PCR analyses were performed following retinoic acid (RA)‐induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC‐1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element‐binding protein‐2, which normally up‐regulates NARC‐1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC‐1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole‐mount in situ hybridization revealed that the pattern of expression of zebrafish NARC‐1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC‐1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain–midbrain boundaries, leading to embryonic death at ∼ 96 h after fertilization. These data support a novel role for NARC‐1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.

Nitric Oxide Signaling in Oxytocin‐Mediated Cardiomyogenesis

Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain‐2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G‐nitro‐l‐arginine‐methyl‐ester (l‐NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO‐sensitive guanylyl cyclases. The NO generator S‐nitroso‐N‐acetylpenicillamine (SNAP) reversed the l‐NAME inhibition of cell beating and GFP expression. In OT‐induced cells, l‐NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, α‐myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT‐induced P19Cl6‐GFP cells revealed ventricular CM coexpressing sarcomeric α‐actinin and GFP, with some cells exclusively expressing α‐actinin, most likely of the SM phenotype. The OT‐mediated production of CM, but not SM, was diminished by l‐NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of l‐NAME. Surprisingly, l‐NAME alone decreased the expression of anti‐stage specific embryonic antigen‐1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6‐GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.

The NCI-N87 cell line as a gastric epithelial barrier model for drug permeability assay.

The objective of this study was to evaluate the human NCI-N87 cell line as a model for gastric permeability drug studies under pH conditions of the stomach. The optimal conditions that led NCI-N87 cells to form a typical differentiated gastric epithelial barrier were a seeding density of 2.5 × 10⁵ cells/cm² on porous inserts and growth in serum-complemented RPMI-1640 medium until 18-27 days post-confluency. The resulting cell monolayers showed moderately high transepithelial electrical resistance (TEER) values of about 500 Ω cm², cells of polygonal morphology expressing E-cadherin and ZO-1 proteins at their contact surfaces, and production of mucus clusters. The monolayers withstood apical pH of 7.4 down to 3.0 with the basal pH fixed at 7.4. The apparent permeability coefficients (P(app)) of model compounds were evaluated in the apical-to-basolateral and basolateral-to-apical directions under different pH gradients. The monolayers were impermeable to the integrity marker Lucifer Yellow (low P(app) of 0.3-1.1 × 10⁻⁶ cm/s). The furosemide P(app) (0.4-1.5 × 10⁻⁵ cm/s) were slightly dependent on pH but remained moderate. The caffeine P(app) (4.2-5.0 × 10⁻⁵ cm/s) were higher and insensitive to pH changes. The NCI-N87 cell line provides a useful in vitro tool to assess gastric drug permeability and absorption under physiologic conditions prevailing in the human stomach.

Structural and immunological homology of human and porcine pituitary and plasma IRCM-serine protease 1 to plasma kallikrein: marked selectivity for pairs of basic residues suggests a widespread role in pro-hormone and pro-enzyme processing

IRCM-serine protease 1 (SP1), originally isolated from porcine pituitaries and exhibiting preference for cleavage at pairs of basic residues has now been isolated in sufficient quantities to be structurally characterized from both porcine and human pituitaries and plasmas. Whereas the porcine protease shows a high degree of amino acid sequence homology to human plasma pre-kallikrein, the human homologue exhibits an identity of sequence in the first 25 residues of each chain (regulatory and catalytic chains). In addition, human plasma and pituitary IRCM-SP1 and human plasma pre-kallikrein show virtually identical immunological and molecular properties. These data strongly suggest that IRCM-SP1 and plasma pre-kallikrein originate from the same gene product. Purified extracts from perfused rat pituitaries show that 32% of the IRCM-SP1 activity found in normal rat pituitaries, still remain. These data together with the demonstrated association of IRCM-SP1 with particulate fractions of the pituitary suggest that IRCM-SP1 represents a tissue form of plasma pre-kallikrein. The characterization of the digestion products obtained upon reaction of IRCM-SP1 with pro-insulin, ACTH1-39, pro-dynorphin and pro-enkephalin-derived peptides, somatostatin-28, and a pro-renin-like peptide confirmed the high degree of cleavage selectivity of this enzyme for pairs of basic residues.

Deglycosylated ceruloplasmin maintains its enzymatic, antioxidant, cardioprotective, and neuronoprotective properties

Ceruloplasmin (CP), an important serum antioxidant, is a blue copper glycoprotein with ferroxidase and oxidase activities. Among other physiological actions, plasma CP was shown to protect isolated rat hearts and cultured P19 neurons exposed to oxidative stress conditions, raising the possibility of using this protein in the treatment of cardiac and neuronal diseases related to oxidative damage. However, since therapeutic applications of CP must be compatible with restrictions in the administration of blood derivatives to humans, there is a need to produce the protein by genetic engineering. To help in the choice of adequate expression systems, we undertook this study to determine if the carbohydrate moiety on the protein is essential for its functions. CP was completely deglycosylated using N-glycosidase F under nondenaturing conditions. Deglycosylated CP was found to retain most of the conformational, antioxidant, and enzymatic properties of the native protein in vitro. Moreover, both forms of the protein had similar cardioprotective and neuronoprotective effects against oxidative stress as evaluated with isolated rat hearts undergoing ischemia-reperfusion and with cultured P19 neurons exposed to xanthine-xanthine oxidase. The data thus indicate that the carbohydrate moiety of CP is not essential for its enzymatic and protective actions. Accordingly, even the use of expression systems that do not glycosylate mammalian proteins could provide a recombinant CP that retains its therapeutic potential.

Skeletal and cardiac myogenesis accompany adipogenesis in P19 embryonal stem cells.

P19 embryonic carcinoma cells resemble normal embryonic stem (ES) cells. They generate cardiac and skeletal myocytes in response to retinoic acid (RA) or oxytocin (OT). RA treatment followed by exposure to triiodothyronine (T3) and insulin induces ES cells differentiation into adipocytes and skeletomyocytes. On the other hand, OT (10(-7) M) was reported to inhibit 3T3 preadipocyte maturation. The present work was undertaken to determine whether P19 cells have an adipogenic potential that could be affected by OT. Cells were treated with RA (10(-6) M)/T3+insulin (adipogenic protocol) or 10(-7) M OT (cardiomyogenic protocol), and analyzed by polymerase chain reaction, immunotechniques, and cytochemistry. Oil-Red-O staining and expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) and aP2 indicated the generation of adipocytes in cultures submitted to the adipogenic protocol. Contracting cells were also generated. Cells positive for sarcomeric actinin and negative for cardiac troponin inhibitor (cTpnI) indicated generation of skeletomyocytes, and cTpnI positive cells revealed generation of cardiomyocytes. Levels of cTpnI and of the skeletal marker MyoD were almost similar in both protocols, whereas no Oil-Red-O staining was associated with the cardiomyogenic protocol. Addition of 10(-7) M OT to the adipogenic protocol did not affect Oil-Red-O staining and PPARgamma expression. Interestingly, Oct3/4 pluripotency marker disappeared in the adipogenic protocol but remained expressed in the cardiomyogenic one. P19 cells thus have an adipogenic potential non affected by 10(-7) M OT. RA/T3+insulin combination generates a larger spectrum of mesodermal cell derivatives and is a more potent morphogenic treatment than OT. P19 cells could help investigating mechanisms of cell fate decision during development.

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide.

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CPs putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.

THE BLUE COPPER CERULOPLASMIN INDUCES AGGREGATION OF NEWLY DIFFERENTIATED NEURONS: A POTENTIAL MODULATOR OF NERVOUS SYSTEM ORGANIZATION

Ceruloplasmin (CP) is a copper-dependent ferroxidase. It regulates iron metabolism and is involved in inflammation, angiogenesis, and protection against oxidative stress. CP also modulates K(+) channel activity in neuroblastoma cells and affects cardiodynamics of isolated hearts. Considering the presence of CP in the nervous system and the importance of iron ions and K(+) channels in neuronal activity, we postulated a role of CP in neuronal development. This hypothesis was tested using the P19 mouse embryonal carcinoma cell line, a model of neuronal differentiation. Addition of CP to the culture medium of newly differentiated P19 neurons induced cell aggregation within 24 h. This effect was concentration-dependent half-maximal at 50 nM, and not associated with necrosis, apoptosis or changes in secretory function. Deglycosylated CP was aggregative but not denatured CP, copper salts, His(2)Cu complex, or other copper enzymes or serum proteins. CP-induced aggregation was less pronounced with aging neurons and seemed not to involve K(+) channels. Immunocytofluorescence analysis demonstrated that digoxigenin-labeled CP bound to P19 neurons and the proportion of responding neurons decreased with aging. The interaction of digoxigenin-labeled CP with neurons was half-maximal at 120 nM by enzyme-linked immunosorbent assay and displaced by unlabeled CP. Our data indicate a specific aggregative action of CP on young neurons in vitro, possibly involving CP receptors. A potential developmental role of CP in nervous system organization is thus demonstrated.

RhoB expression is induced after the transient upregulation of RhoA and Cdc42 during neuronal differentiation and influenced by culture substratum and microtubule integrity.

RhoGTPases are important intracellular signalling switches in the regulation of cytoskeleton organization. They likely have an important role in ontogenesis because cytoskeletal rearrangements accompany cell differentiation and specialization. Western blotting showed that protein expression of RhoA, RhoB and Cdc42 RhoGTPases dramatically increased, in a programmed manner, during neuronal differentiation of P19 mouse embryonal carcinoma cells with retinoic acid. RhoA and Cdc42 expression were sequentially upregulated and peaked during the commitment period while that of RhoB was induced in post-mitotic neurons. Although RhoB had a higher expression on matrices allowing cell spreading and neurite elongation, it was distributed throughout cell volume by immunocytofluorescence and associated with various cell compartments by centrifugal subfractionation, suggesting a role not restricted at neurites at this stage of differentiation. RhoA and Cdc42 were mainly cytosolic and RhoB particulate in the P19 cell model. Treatment of cells with cytoskeleton disruptors showed that poisons of microtubules but not of actin filaments or neurofilaments increased the cytosolic level of RhoB. The results indicate that RhoA, Cdc42 and RhoB must intervene at specific stages of neuronal development and there exists a relationship between RhoB expression/distribution, the microtubule network and the extracellular matrix during this process.