Masa-Aki Ikeda

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.

Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27

Many lines of evidence indicate that connexin genes expressing gap junction (GJ) proteins inhibit tumor cell proliferation. However, the precise molecular mechanisms remain unclear. In this study, we show that overexpression of connexin43 (Cx43) suppressed proliferation of human osteosarcoma U2OS cells through inhibition of the cell cycle transition from G1 to S phase. This inhibition was attributed to a significant accumulation of the hypophosphorylated retinoblastoma (Rb) protein, which was causally related to decreases in the kinase activities of cyclin-dependent kinases (CDKs) 2 and 4. Enforced Cx43 expression markedly increased the level of the CDK inhibitor p27. This increase resulted from an increased synthesis and a reduced degradation of the p27 proteins, but not influence of the p27 mRNA. Moreover, we show that the Cx43-modulated GJ function was the main contributor to the elevation in p27 levels, in which cAMP was involved. These data suggest that Cx43 appears to inhibit proliferation of U2OS cells by increasing the levels of p27 proteins via post-transcriptional regulatory mechanisms.

Growth suppression of human carcinoma cells by reintroduction of the p300 coactivator

The p300 and closely related cAMP response element binding protein (CREB)-binding protein (CBP) acetyltransferases function as global transcriptional coactivators and play important roles in a broad spectrum of biological processes, including cell proliferation and differentiation. A role of p300/CBP in tumor suppression has been proposed from the fact that these coactivators are targeted by viral oncoproteins and that biallelic mutations of p300 have been identified in carcinomas. Here, we show that transcriptional response to the transforming growth factor β (TGF-β), an inhibitor of epithelial cell growth, was severely impaired in human carcinoma cell lines carrying p300 mutations accompanied by inactivation of the second allele, and that wild-type expression restored TGF-β-dependent transcriptional activity. Furthermore, reintroduction of wild-type p300 suppressed the growth of p300-deficient carcinoma cells, whereas p300 did not inhibit the growth of carcinoma cells examined, which have no detectable alterations in p300 protein and retain the TGF-β-dependent transcriptional response. In addition, tumor-derived mutants missing the bromodomain or glutamine-rich region, which are respectively important for chromatin interaction and coactivator activities, lost the suppressive activity. In contrast, CBP exhibited no or reduced ability to suppress the growth of p300-deficient carcinoma cells. These results provide experimental evidence to show that p300 acts as a suppressor of tumor cell growth and suggest a distinct role of p300 in suppression of epithelial tumors.

Essential roles for G1cyclin-dependent kinase activity in development of cardiomyocyte hypertrophy

Although cardiomyocytes undergo terminal differentiation soon after birth, irreversibly withdrawing from the cell cycle, growth stimulation induces cell hypertrophy. Such growth stimulation is also responsible for the upregulation of G1 cyclins and cyclin-dependent kinase (CDK) activity in proliferating cells. We sought to determine whether G1 CDK activity is involved in the hypertrophy of rat neonatal cardiomyocytes in culture. We show that serum stimulation promoted the G1 CDK activity without induction of DNA synthesis in cardiomyocytes. Furthermore, overexpression of CDK inhibitors p16(INK4a) and p21(CIP1/WAF1) by use of the adenovirus vector effectively prevented cell enlargement and depressed serum-induced protein synthesis and expression of skeletal alpha-actin and atrial natriuretic factor, genetic markers of cardiac hypertrophy. These results suggest that the G1 CDK activity promoted by serum stimulation is required for the induction of cardiomyocyte hypertrophy and provide novel evidence for understanding the regulation of cardiac hypertrophy by cell cycle regulators.Although cardiomyocytes undergo terminal differentiation soon after birth, irreversibly withdrawing from the cell cycle, growth stimulation induces cell hypertrophy. Such growth stimulation is also responsible for the upregulation of G1 cyclins and cyclin-dependent kinase (CDK) activity in proliferating cells. We sought to determine whether G1 CDK activity is involved in the hypertrophy of rat neonatal cardiomyocytes in culture. We show that serum stimulation promoted the G1 CDK activity without induction of DNA synthesis in cardiomyocytes. Furthermore, overexpression of CDK inhibitors p16 INK4a and p21 CIP1/WAF1 by use of the adenovirus vector effectively prevented cell enlargement and depressed serum-induced protein synthesis and expression of skeletal α-actin and atrial natriuretic factor, genetic markers of cardiac hypertrophy. These results suggest that the G1CDK activity promoted by serum stimulation is required for the induction of cardiomyocyte hypertrophy and provide novel evidence for understanding the regulation of cardiac hypertrophy by cell cycle regulators.

Distinct recruitment of E2F family members to specific E2F-binding sites mediates activation and repression of the E2F1 promoter

The activity of E2F transcription factors plays a crucial role in mammalian cell-cycle progression and is controlled by physical association with the pocket proteins (pRb and its related p107 and p130). The E2F1 promoter, which contains two overlapping E2F-binding sites, is activated at the G1/S transition in an E2F-dependent manner. Mutational experiments have shown that the distal E2F-binding site on the E2F1 promoter is required for transcriptional repression in the G0 phase, whereas the proximal E2F-binding site contributes to transcriptional activation at the G1/S boundary. Consistent with these results, chromatin immunoprecipitation assays have revealed that the E2F4/p130 repressor complex specifically binds to the distal E2F-binding site, whereas E2F1 and E2F3 activators preferentially bind to the proximal E2F-binding site. The assays also showed that the specific binding of E2F4/p130 complex to the distal site was dramatically impaired by a mutation introduced into the contiguous repression site (cell Cycle gene Homology Region; CHR). Taken together, these findings indicate that the two E2F-binding sites play distinct roles in the regulation of E2F1 transcription by interacting with different sets of E2F members and cooperating with the contiguous repressor element.

Neonatal estrogen exposure inhibits steroidogenesis in the developing rat ovary

Treatment of newborn female rats with estrogens significantly inhibits the growth and differentiation of the ovary. To understand the molecular mechanism of estrogen action in the induction of abnormal ovary, we examined the expression profiles of steroidogenic factor 1 (SF‐1) and several of its target genes in the developing ovaries after neonatal exposure to synthetic estrogen, estradiol benzoate (EB) by using reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. Morphologic examination indicated inhibitory effects of estrogen on the stratification of follicles and development of theca and interstitial gland during postnatal ovarian differentiation. The expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side‐chain cleavage cytochrome P450 (P450SCC), which are both essential for steroid biosynthesis, markedly decreased in theca and interstitial cells throughout the postnatal development of the EB‐treated ovary. However, expression of the transcriptional activator of the two genes, SF‐1 was unaffected in theca and interstitial cells, although the number of these cells was lower in the EB‐treated ovary than in the control ovary. The expression of the estrogen mediator, estrogen receptor‐α (ER‐α), diminished specifically in theca cells at P6 and recovered by P14 in the EB‐treated ovary. These results indicate that the effect of estrogens is mediated by means of ER‐α resulting in the down‐regulation of StAR and P450SCC genes during early postnatal development of the ovary. These results suggest that the abnormal ovarian development by neonatal estrogen treatment is closely correlated with the reduced steroidogenic activity, and the data obtained by using this animal model may account in part the mechanism for aberrant development and function of the ovary in prenatally estrogen‐exposed humans.

Increased Expression of Müllerian-Inhibiting Substance Correlates with Inhibition of Follicular Growth in the Developing Ovary of Rats Treated with E2 Benzoate

Mullerian-inhibiting substance (MIS) is an essential factor for male sexual differentiation. In the present study, we exam- ined whether the expression of MIS and several of its related transcription factors is altered in the ovaries of rats treated with the synthetic estrogen, E2 benzoate (EB; 10 g/0.02 ml), from postnatal day 1 (P1) to P5. The EB-treated rats had a significantly reduced number of layered follicles at P6 in com- parison with the control rats that were treated with vehicle alone. The expression levels of both MIS mRNA and protein in the granulosa cells of small growing follicles in the ovary at P6 were higher in the EB-treated rats than in the controls. These results indicate that the inhibitory effect of EB on the follic- ular stratification may correlate with the inappropriately in- creased expression level of MIS. Furthermore, the expression levels of one of its transcriptional activators, steroidogenic factor 1, and ER- in granulosa cells of small growing follicles were higher in EB-treated ovaries than in the control ovaries. These results suggest the role of MIS in the regulation of follicular growth and the possible involvement of steroido- genic factor 1and/or ER- in this molecular cascade may con- tribute to postnatal ovarian development. (Endocrinology 143: 304 -312, 2002)

Expression of cyclin D1 and CDK4 causes hypertrophic growth of cardiomyocytes in culture: A possible implication for cardiac hypertrophy

Differentiated cardiomyocytes have little capacity to proliferate and show the hypertrophic growth in response to alpha1-adrenergic stimuli via the Ras/MEK pathway. In this study, we investigated a role of cyclin D1 and CDK4, a positive regulator of cell cycle, in cultured neonatal rat cardiomyocyte hypertrophy. D-type cyclins including cyclin D1 were induced in cells stimulated by phenylephrine. This induction was inhibited by MEK inhibitor PD98059 and the dominant negative RasN17, but mimicked by expression of the constitutive active Ras61L. Over-expression of cyclin D1 and CDK4 using adenovirus gene transfer caused the hypertrophic growth of cardiomyocytes, as evidenced by an increase of the cell size as well as the amount of cellular protein and its rate of synthesis. However, the cyclin D1/CDK4 kinase activity was not up-regulated in cells treated by hypertrophic stimuli or in cells over-expressing the cyclin D1 and CDK4. Furthermore, a CDK inhibitor, p16, did not inhibit the hypertrophic growth of cardiomyocytes. These results clearly indicated that cyclin D1 and CDK4 have a role in hypertrophic growth of cardiomyocytes through a novel mechanism(s) which appears not to be related to its activity required for cell cycle progression.