Neuronal Differentiation of P19 Embryonal Carcinoma Cells Modulates Kinin B2 Receptor Gene Expression and Function

University of Leipzig, Leipzig, Saxony, Germany
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(20):19576-19586. DOI: 10.1074/jbc.M502513200


Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins.
These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests
that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of
nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo
neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression
was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and
peaked on days 8 and 9. Measurement of [Ca2+]i in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application,
but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture
medium, and respond to bradykinin application with a transient increase in [Ca2+]i. However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic
bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response
induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1–M3 muscarinic acetylcholine receptors,
indicating crucial functions of the B2BKR during neuronal differentiation.


Available from: Paromita Majumder
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    • "The effect of BK was thought to be mostly related to regulation of inflammation and blood pressure, but now it is recognized to involve regulation of synaptic functions and neuronal differentiation [20], [21], [22], [23]. B2BKR expression is not limited to endothelial cells in the brain, but is also present along differentiation of rat neural progenitor cells [22], [23]. "
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    ABSTRACT: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Bradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.
    PLoS ONE 02/2012; 7(2):e30755. DOI:10.1371/journal.pone.0030755 · 3.23 Impact Factor
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    • "In adulthood, it plays a role in neuronal differentiation within the dentate gyrus [10], in the maintenance of motor neurons [11] and in mammalian nerve regeneration [12]. Indeed, RA is the most used morphogen to produce neural progenitor cells (NPCs) and neurons from stem cells in vitro [13], [14], [15], [16]. "
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    ABSTRACT: The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.
    PLoS ONE 06/2011; 6(6):e20667. DOI:10.1371/journal.pone.0020667 · 3.23 Impact Factor
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    • "Bradykinin is a 9-amino acid peptide with a wide range of biological actions mediated through B1 and B2 subtypes of G-protein-coupled receptors (reviewed by (Calixto et al., 2004). Activation of these receptors induces a transient increase in the cytosolic concentration of calcium caused by both its mobilization from intracellular stores and influx from extracellular environment (Kristensson et al., 1993 ; Martins et al., 2005). This has been associated with neuroprotection after ischemia (Ping et al., 2005; Yan-Feng et al., 2008; Danielisova et al., 2008). "
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    ABSTRACT: PrP(C) is highly expressed in both the central and peripheral nervous systems from early stages of development and in adulthood. Its major conformational change and conversion into an abnormal form (PrP(Sc)) has been associated with the generation of prions, the infectious agent of transmissible spongiform encephalopathies (TSEs). The massive neurodegeneration presented by individuals suffering from these diseases has been associated with the gain of neurotoxic activity of PrP(Sc). On the other hand, major neurodegeneration is also observed in transgenic mice expressing PrP(C) molecules deleted of specific domains, which points to important functional domains within this molecule, and supports the hypothesis that loss-of PrP(C) function may contribute to the pathogenesis of TSEs. Furthermore, a large body of data demonstrates direct or indirect interaction of PrP(C) with extracellular matrix proteins, soluble factors, transmembrane proteins, G-protein coupled receptors and ions channels. The ability of PrP(C) to drive the assembly of multi-component complexes at the cell surface is likely the basis for its neurotrophic functions. These properties indicate that PrP(C) may be relevant for not only the spongiform encephalopathies, but also as an ancillary component of the pathogenesis of other neurodegenerative diseases, and therefore amenable to therapeutic targeting.
    Current issues in molecular biology 09/2009; 12(2):63-86. · 5.75 Impact Factor
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