Mimi Tamamori-Adachi

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.

Abstract— Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons

Cyclin D-dependent kinases phosphorylate the retinoblastoma (Rb) protein and play a critical role in neuronal cell cycle control and apoptosis. Here we show that cyclin D1 became predominantly cytoplasmic as primary cortical progenitor cells underwent cell cycle withdrawal and terminal differentiation. Furthermore, ectopically expressed cyclin D1 sequestered in the cytoplasm of postmitotic neurons, whereas it efficiently entered the nucleus of proliferating progenitor cells. Cytoplasmic cyclin D1 were complexed with cyclin-dependent kinase 4 (CDK4), and also with CDK inhibitors, p27KipI or p21CipI, which positively regulate assembly and nuclear accumulation of the cyclin D1-CDK4 complex. Although overexpression of p21CipI promoted cyclin D1 nuclear localization, inhibition of either glycogen synthase kinase 3β- or CRM1-mediated cyclin D1 nuclear export did not, suggesting that the inhibition of its nuclear import, rather than the acceleration of nuclear export, contributes to cytoplasmic sequestration of cyclin D1 in postmitotic neurons. In differentiated progenitor cells, nuclear localization of ectopic cyclin D1 induced apoptosis, and the DNA-damaging compound camptothecin caused nuclear accumulation of endogenous cyclin D1, accompanied by Rb phosphorylation. These results indicate that nuclear accumulation of cyclin D1 is inhibited in postmitotic neurons and suggest a role of its subcellular localization in neuronal death and survival.

Cyclin A/cdk2 Activation Is Involved in Hypoxia-Induced Apoptosis in Cardiomyocytes

Abstract— Cardiomyocytes are terminally differentiated cells characterized as withdrawal cell-cycle machinery, but nonetheless they are known to express cell-cycle regulators. Because many proteins related to the cell cycle induce apoptosis in proliferating cells, we examined the involvement of these proteins in the apoptosis pathway in cardiomyocytes. Primary rat cardiomyocytes were exposed to a severe hypoxic condition to induce apoptosis. The apoptosis rate of cardiomyocytes increased to ≈40% under 24 hours of hypoxia as evaluated by the TUNEL method. The cyclin A protein level assessed by immunoblot analysis accumulated in a time-dependent manner in cardiomyocytes, but there was no increase in nonmyocytes. Hypoxia increased the activity of cyclin A–associated kinase but not the activity of cyclin E–associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus, suggesting that cyclin A and its associated kinase play significant roles in the apoptosis of cardiomyocytes. To investigate the cyclin A–mediated apoptosis, we infected cultured cells with cyclin A adenovirus. Apoptosis was induced in 63±12% of the infected cardiomyocytes in contrast to only 12±3% of the LacZ-infected control cells. In addition, the cells in the hypoxic condition showed an increase in caspase-3 activity and a subsequent decrease in p21cip1/waf1 protein, which is partly cleaved by caspase-3. These findings confirm that cyclin A–associated kinase mediates hypoxia-induced apoptosis in cardiomyocytes, and they also suggest that additional elements of the cell-cycle–dependent machinery participate in this mechanism.

Age-associated increase in heterochromatic marks in murine and primate tissues.

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin‐associated proteins. We found increased levels of the heterochromatin‐associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age‐associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age‐associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene‐induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.

Distinct roles of GSK-3α and GSK-3β phosphorylation in the heart under pressure overload

Glycogen synthase kinase-3 (GSK-3) is a master regulator of growth and death in cardiac myocytes. GSK-3 is inactivated by hypertrophic stimuli through phosphorylation-dependent and -independent mechanisms. Inactivation of GSK-3 removes the negative constraint of GSK-3 on hypertrophy, thereby stimulating cardiac hypertrophy. N-terminal phosphorylation of the GSK-3 isoforms GSK-3α and GSK-3β by upstream kinases (e.g., Akt) is a major mechanism of GSK-3 inhibition. Nonetheless, its role in mediating cardiac hypertrophy and failure remains to be established. Here we evaluated the role of Serine(S)21 and S9 phosphorylation of GSK-3α and GSK-3β in the regulation of cardiac hypertrophy and function during pressure overload (PO), using GSK-3α S21A knock-in (αKI) and GSK-3β S9A knock-in (βKI) mice. Although inhibition of S9 phosphorylation during PO in the βKI mice attenuated hypertrophy and heart failure (HF), inhibition of S21 phosphorylation in the αKI mice unexpectedly promoted hypertrophy and HF. Inhibition of S21 phosphorylation in GSK-3α, but not of S9 phosphorylation in GSK-3β, caused phosphorylation and down-regulation of G1-cyclins, due to preferential localization of GSK-3α in the nucleus, and suppressed E2F and markers of cell proliferation, including phosphorylated histone H3, under PO, thereby contributing to decreases in the total number of myocytes in the heart. Restoration of the E2F activity by injection of adenovirus harboring cyclin D1 with a nuclear localization signal attenuated HF under PO in the αKI mice. Collectively, our results reveal that whereas S9 phosphorylation of GSK-3β mediates pathological hypertrophy, S21 phosphorylation of GSK-3α plays a compensatory role during PO, in part by alleviating the negative constraint on the cell cycle machinery in cardiac myocytes.

Stress response gene ATF3 is a target of c-myc in serum-induced cell proliferation.

The c‐myc proto‐oncogene encodes a transcription factor that promotes cell cycle progression and cell proliferation, and its deficiency results in severely retarded proliferation rates. The ATF3 stress response gene encodes a transcription factor that plays a role in determining cell fate under stress conditions. Its biological significance in the control of cell proliferation and its crosstalk regulation, however, are not well understood. Here, we report that the serum response of the ATF3 gene expression depends on c‐myc gene and that the c‐Myc complex at ATF/CREB site of the gene promoter plays a role in mediating the serum response. Intriguingly, ectopic expression of ATF3 promotes proliferation of c‐myc‐deficient cells, mostly by alleviating the impeded G1‐phase progression observed in these cells, whereas ATF3 knockdown significantly suppresses proliferation of wild‐type cells. Our study demonstrates that ATF3 is downstream of the c‐Myc signaling pathway and plays a role in mediating the cell proliferation function of c‐Myc. Our results provide a novel insight into the functional link of the stress response gene ATF3 and the proto‐oncogene c‐myc.

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity

OBJECTIVE Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators. METHODS AND RESULTS Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21(cip1/waf1) protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21(cip1/waf1) protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results. CONCLUSION Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21(cip1/waf1) accumulation.

A splice variant of stress response gene ATF3 counteracts NF-κB-dependent anti-apoptosis through inhibiting recruitment of CREB-binding protein/p300 coactivator

Activating transcription factor (ATF) 3 plays a role in determining cell fate and generates a variety of alternatively spliced isoforms in stress response. We have reported previously that splice variant ATF3ΔZip2, which lacks the leucine zipper region, is induced in response to various stress stimuli. However, its biological function has not been elucidated. By using cells treated with tumor necrosis factor-α and actinomycin D or cells overexpressing ATF3ΔZip2, we showed that ATF3ΔZip2 sensitizes cells to apoptotic cell death in response to tumor necrosis factor-α, at least in part through suppressing nuclear factor (NF)-κB-dependent transcription of anti-apoptotic genes such as cIAP2 and XIAP. ATF3ΔZip2 interacts with a p65 (RelA)-cofactor complex containing CBP/p300 and HDAC1 at NF-κB sites of the proximal promoter region of the cIAP2 gene in vivo and down-regulates the recruitment of CBP/p300. Our study revealed that ATF3ΔZip2 counteracts anti-apoptotic activity of NF-κB, at least in part, by displacing positive cofactor CBP/p300 and provides insight into the mechanism by which ATF3 regulates cell fate through alternative splicing in stress response.

Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4: Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation

Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.