Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.
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.
Tamamori-Adachi
Mimi
M
Department of Biochemical Genetics, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Ito
Hiroshi
H
Sumrejkanchanakij
Piyamas
P
Adachi
Susumu
S
Hiroe
Michiaki
M
Shimizu
Masato
M
Kawauchi
Junya
J
Sunamori
Makoto
M
Marumo
Fumiaki
F
Kitajima
Shigetaka
S
Ikeda
Masa-Aki
MA
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000256:Adenoviridae / Q000235:genetics; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001973:Bromodeoxyuridine / Q000493:pharmacokinetics; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019394:Ki-67 Antigen / Q000096:biosynthesis; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000502:physiology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D016160:Retinoblastoma Protein / Q000378:metabolism; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D019394:Ki-67 Antigen; D019913:Nuclear Localization Signals; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; C495903:Cdk4 protein, rat; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D001973:Bromodeoxyuridine