Misae Kubota

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.

jumonji Downregulates Cardiac Cell Proliferation by Repressing cyclin D1 Expression

Spatiotemporal regulation of cell proliferation is necessary for normal tissue development. The molecular mechanisms, especially the signaling pathways controlling the cell cycle machinery, remain largely unknown. Here, we demonstrate a negative relationship between the spatiotemporal patterns of jumonji (jmj) expression and cardiac myocyte proliferation. cyclin D1 expression and cell proliferation are enhanced in the cardiac myocytes of jmj-deficient mutant embryos. In contrast, jmj overexpression represses cyclin D1 expression in cardiac cells, and Jmj protein binds to cyclin D1 promoter in vivo and represses its transcriptional activity. cyclin D1 overexpression causes hyperproliferation in the cardiac myocytes, but the absence of cyclin D1 in jmj mutant embryos rescues the hyperproliferation. Therefore, Jmj might control cardiac myocyte proliferation and consequently cardiac morphogenesis by repressing cyclin D1 expression.

Activation of Protein-tyrosine Phosphatase SH-PTP2 by a Tyrosine-based Activation Motif of a Novel Brain Molecule

BIT (a rain mmunoglobulin-like molecule with yrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (yrosine-based ctivation otifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in λgt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.

BIT, an immune antigen receptor‐like molecule in the brain1

We previously found a brain‐specific glycoprotein in the rat brain. It postnatally increases and is rich in the mature brain. We cloned cDNA of this protein. It is composed of a signal peptide, a V‐type immunoglobulin domain, two C1‐type immunoglobulin domains, a transmembrane segment and a cytoplasmic region containing two tyrosine‐based activation motifs (TAM) that are variants of the antigen receptor signaling motifs. The overall structure is similar to those of immune antigen receptors. This molecule, BIT (brain immunoglobulin‐like molecule with TAMs), is a major endogenous substrates of brain tyrosine kinases in vitro. Cerebral cortical neurons could extend their neurites on BIT‐coated substrate and anti‐BIT monoclonal antibody specifically inhibited the effect. These findings and our recent study concerning BIT signal transduction mechanism suggest that BIT, an immune antigen receptor‐like molecule of the brain, functions as a membrane signaling molecule that may participate in cell–cell interaction.

Tyrosine Phosphorylation and Association of BIT with SHP‐2 Induced by Neurotrophins

Abstract: BIT (brain immunoglobulin‐like molecule with tyrosine‐based activation motifs) is a membrane glycoprotein that has two cytoplasmic TAMs (tyrosine‐based activation motifs). We previously reported that tyrosine‐phosphorylated TAMs of BIT interact with the Src homology 2 domain‐containing protein tyrosine phosphatase SHP‐2 both in vitro and in transfected cells, and this association results in a potent stimulation of the phosphatase activity of SHP‐2. Both BIT and SHP‐2 are highly expressed in the mammalian brain, and they may play important roles in the regulation of synaptic function. In this study, we found that nerve growth factor (NGF) treatment of PC12 cells leads to the tyrosine phosphorylation of BIT and a subsequent complex formation between BIT and SHP‐2. Furthermore, brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) also induced the tyrosine phosphorylation of BIT and the association with SHP‐2 in primary cultured rat neurons. Our results suggest that the BIT‐SHP‐2 signaling pathway is a novel signal transduction mechanism of neurons that acts in response to neurotrophic factors such as NGF, BDNF, and NT‐3.

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

Expression of CD47/integrin‐associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/SHP substrate 1 (BIT/SHPS‐1) is involved in brain‐derived neurotrophic factor (BDNF)‐promoted neuronal cell survival. Here, we report the apoptosis‐inducing effect of CD47/integrin‐associated protein (IAP), the heterophilic binding partner of BIT/SHPS‐1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL)‐positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad‐spectrum caspase inhibitor Z‐VAD‐fmk. Furthermore, we observed that co‐expression of CD47/IAP with BIT/SHPS‐1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase‐dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP‐induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS‐1 and by BDNF.

Increased level of neurokinin-1 tachykinin receptor gene expression during early postnatal development of rat brain

Substance P is known to elicit diverse actions via activating multiple subtypes of tachykinin receptors, and these actions appear to be involved not only in synaptic transmission but also in synaptic plasticity during development of the mammalian central nervous system. The availability of sensitive quantitation of individual tachykinin receptor subtypes is crucial for elucidating the physiological function specifically mediated by activation of a particular receptor subtype. We thus attempted to develop an assay to determine the level of messenger RNA molecule encoding the neurokinin-1-type tachykinin receptor and apply it for assessment of developmental changes in the neurokinin-1 receptor gene expression in the rat brain to explore the role of tachykinin receptors during ontogeny. The assay was designed to use a competitive reverse transcription-polymerase chain reaction co-amplifying endogenous neurokinin-1 receptor messenger RNA and internal standard, which enabled specific quantification of the number of neurokinin-1 receptor transcripts, ranging from 3.1 x 10(3) to 1.3 x 10(5) molecules/microgram total RNA. The levels of neurokinin-1 receptor gene expression were examined in three different brain regions of the rat aged 0-56 days after birth. The order of neurokinin-1 receptor messenger RNA expression was hippocampus > cerebral cortex > > cerebellum at all ages examined except postnatal day 0, where its expression was more abundant in the cerebral cortex than in the hippocampus. From postnatal day 3 onward, the hippocampus contained 140-160% of the cortical levels. Although the tachykinin receptor expression in the cerebellum was too low to be accurately assessed by conventional techniques, our assay enabled us to determine the amount of cerebellar neurokinin-1 receptor messenger RNA that changed in the range 7-23% of the cortical level during postnatal development. A prominent feature revealed by this assay is that the neurokinin-1 receptor gene expression in the rat brain is developmentally regulated. The hippocampus displayed a transient peak of neurokinin-1 receptor messenger RNA at postnatal day 3 and a subsequent gradual decrease. In the cerebral cortex, the amount of the message was highest at birth, and was followed by a moderate decrease during postnatal development. At 56 days after birth, the expression levels in both brain regions were down-regulated to approximately 50% of their maximal levels. The transitory pattern of gene expression was also observed in the cerebellum. The results of this study demonstrate that the reverse transcription-polymerase chain reaction-based assay is useful to quantitate precisely the neurokinin-1 tachykinin receptor message in limited tissue samples derived from discrete brain regions. Together with previous findings, the increased level of neurokinin-1 receptor messenger RNA expression in immature rat brain shown by the present analysis suggests that the neurokinin-1-type tachykinin receptor may play a role in the synaptic plasticity associated with morphological and functional development of the mammalian CNS.

Repression of cyclin D1 expression is necessary for the maintenance of cell cycle exit in adult mammalian cardiomyocytes.

Background: How cell cycle exit is maintained in adult mammalian cardiomyocytes is largely unknown. Results: Cyclin D1 expression causes cell cycle reentry in >40% of adult mouse cardiomyocytes. Conclusion: Silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit. Significance: One of the mechanisms regulating cell cycle exit in mammalian cardiomyocytes has been uncovered. The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

Strain diversity of mouse bit

The time course and spatial propagation of action potential (AP)-evoked increases in intracellular Ca2* concentration ([Ca”],) were studied in cultured bullfrog sympathetic neurones. The cells were loaded with Oregon Green 488 BAPTA-1 of relatively low concentration (10pM) through patch pipettes for [Ca”], measurement. The line-scan imaging with a confocal laser microscope revealed fast and slow phases of the inward spread of Ca2’ wave from the submembrane region elicited by a single AP or repetitive APs in both the cytosol and nucleus. Ryanodine (1 OpM) or thapsigargin (1 PM) effectively blocked the fast phase without affecting voltage-activated Ca2’ current during a short depolarizing pulse (5-l Oms) at plasma membrane. These results suggest that a single AP as well as repetitive APs can activate Ca2* -induced Ca2’ release (CICR) and then it facilitates the inward spread of Ca2’ wave from the submembrane region, indicating the physiological role of CICR in bullfrog sympathetic neurones.