Notch induces quiescent cardiomyocytes to reenter the cell cycle
Neonatal rat ventricular cardiomyocytes (NRVCs) normally exit the cell cycle and enter G0
(Burton et al., 1999
), but infection with a recombinant adenovirus encoding the N2ICD
) dramatically changed the cell cycle profile of cardiomyocytes, increasing the percentage of NRVCs in S/G2
/M phase 2.4-fold 48 h after infection (which corresponds to postnatal day 5 [P5]; ). In contrast, control NRVCs infected with an adenovirus encoding β-galactosidase (Ad-βGal) had no effect over background (). Ad-N2ICD
also increased the incidence of Ki67+
NRVCs nearly 10-fold (), whereas Ad-βGal had no significant effect (). Notch signaling was confirmed by the induction of hairy and enhancer of split 1 (Hes1) promoter–dependent luciferase activity in Ad-N2ICD
–infected cells () and the expression of high levels of recombinant N2ICD
(V5-tagged) protein in the nuclei of Ad-N2ICD
–infected cardiomyocytes (, inset; and ). Endogenous Notch2 in cardiomyocytes declined postnatally (not depicted) such that it was no longer detectable by P5 (), which indicates that exogenous reactivation of Notch2 can direct cell cycle entry.
Figure 1. Activated Notch induces cell cycle reentry in quiescent cardiomyocytes. (A–D) NRVCs were either uninfected (A and E) or infected with Ad-βGal (B and F) or Ad-N2ICD (C and G). 48 h after infection, cells were stained with mouse anti-MF20 (more ...)
Similarly, mESC lines were created with a dual-cassette vector that produced N2ICD under control of the ventricular cardiomyocyte-specific myosin light chain 2V (MLC2V) promoter and the blastocidinr gene for drug selection from the stem cell promoter Rex. N2ICD showed extended replicative capacity of cardiomyocytes to at least day 21 after initiation of differentiation, yielding overgrowths in the cultures (), whereas control mESCs harboring only Rex-Blar showed the normal cessation of cell division by day 12–16.
Notch2ICD induces expression and nuclear translocation of cyclin D1
Neonatal mouse ventricular cardiomyocytes (NMVCs) were prepared from RBP-JκFlox/Flox
mice to probe whether cell cycle reentry required RBP-Jκ. Excision of exons 6 and 7 of RBP-Jκ by Cre recombinase eliminates function (Tanigaki et al., 2002
), and introduction of a Cre-EGFP fusion protein blocked the ability of N2ICD
to stimulate cell cycle reentry, demonstrating the dependence on RBP-Jκ ().
Figure 2. Cell cycle reentry requires RBP-Jκ. (A) Examples of RBP-JκFlox/Flox NMVCs infected with Ad-Cre-EGFP to remove RBP-Jκ, then reinfected 12 h later with Ad-N2ICD, as indicated, cultured 48 h, and stained with MF20 (red, Alexa 594), (more ...)
A potential transcriptional target of RBP-Jκ is cyclin D1. Cyclin D1 and other D-type cyclins (D2 and D3) translocate to the nucleus, where they function with Cdk4 and 6 to phosphorylate Rb and cause progression through the cell division cycle (DeGregori, 2004
). A time-dependant up-regulation of cyclin D1 was apparent beginning ~6–9 h after Ad-N2ICD
infection (). Cyclin D1 induction precedes cell cycle entry () by ~24 h, which correlates well with the duration of G1
phase observed for rat cells (i.e., 24 h in primary cultures of endothelial cells; Solodushko and Fouty, 2007
). Also, the level of the G1
/S-phase cyclin, cyclin E, increased at 36 h (), which correlates with the observed cell cycle profile () and confirms the S-phase entry of NRVCs.
Figure 3. Notch induces expression and nuclear localization of cyclin D1 and phosphorylation of Rb before cell cycle reentry. (A) The DNA content of the cardiac cells analyzed by flow cytometry (as in ) at the indicated times after infection. One of two experiments (more ...)
Although low serum was included in the preceding experiments to enhance cell survival, its absence did not prevent cell cycle reentry by N2ICD (18.3% S+G2+M phase cells in uninfected cells vs. 44.4% in infected cells), which indicates that Notch-induced cell cycle reentry is independent of serum factors, consistent with induction of cyclin D1 by RBP-Jκ. Moreover, even high (10%) serum was insufficient to stimulate cell cycle entry, see the following paragraph.
Cyclin D1 levels were elevated within the nuclei of Ad-N2ICD
–infected NRVCs, visualized by coimmunofluorescent staining with the V5 epitope on N2ICD
, but were absent in nuclei of control Ad-βGal–infected NRVCs (arrows indicate examples in ). Nuclear localization of cyclin D1 was accompanied by phosphorylation of Rb at Ser807/811, Ser795, and Ser 780 in extracts prepared with 10 mM EDTA to inhibit kinase activity after lysis (, right). Although phenylephrine (PHE) and 10% FCS induced cyclin D1 (), both failed to stimulate its characteristic nuclear localization (, inset, arrowhead in the N2ICD
panel indicates a characteristic nuclear pattern), Rb phosphorylation () or DNA synthesis (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200806104/DC1
). Lysates prepared in the absence of EDTA to preserve kinase activity after dissolution of the cell membranes showed that PHE or FCS can stimulate Cdk activity (, left). Because Rb phosphorylation occurred under these conditions but not in the presence of EDTA, we concluded that PHE and FCS alone cannot stimulate Rb phosphorylation because the components are not colocalized in the nucleus. This raises the possibility that the N2ICD
might have a unique role in localizing the cyclin D1–Cdk complex to the nucleus.
Cyclin D1 nuclear localization requires notch ICD
To distinguish induction of cyclin D1 gene expression from nuclear localization, we transfected NRVCs to express a constitutively active form of RBP-Jκ made by fusion to the VP16 transactivation domain and a MYC epitope tag (pRBP-Jκ-VP16). RBP-Jκ-VP16 activates transcription of Notch-dependent genes, including pHes1-luciferase in cardiomyocytes (). RBP-Jκ-VP16 induced the accumulation of cyclin D1 in the cytosol but not in the nucleus, as had been seen with N2ICD (), and did not stimulate cell cycle entry (), which confirms that elevated cyclin D1 expression is insufficient to promote cell cycle entry. As a control, a DNA-binding mutant (DBM) control of RBP-Jκ (pRBP-Jκ-DBM) that does not activate pHes1-luciferase () did not induce cyclin D1 (). PHE or 10% FCS also induced only cytosolic accumulation of cyclin D1 (). The failure of activated RBP-Jκ, PHE, or 10% FCS to localize cyclin D1 to the nucleus indicated a specific requirement for N2ICD.
Figure 4. RBP-Jκ is insufficient to promote nuclear localization of cyclin D1. (A and B) Examples of NRVCs were transfected with 0.8 μg of plasmids encoding a myc epitope-tagged, constitutively active version of RBP-Jκ in pCDNA3 (pRBP-Jκ-VP16; (more ...)
N2ICD also stimulated nuclear localization of cyclin D1 in RBP-JκFlox/Flox NMVCs after Cre excision (). Ad-Cre-EGFP on P1 effectively excised RBP-Jκ 1 d later (). As shown in , N2ICD cannot induce cyclin D1 in the absence of RBP-Jκ, therefore 10% FCS was added 1 d before harvesting the cells to induce cyclin D1 (). Ad-βgal–treated cells showed the characteristic cytoplasmic and perinuclear localization of cyclin D1 (), whereas Ad-N2ICD () and the sequential treatment with Ad-Cre-EGFP and Ad-N2ICD () both induced nuclear localization. Closed arrows () indicate examples of Cre-EGFP+, N2ICD+ cells with nuclear localization of cyclin D1. Prominent nuclear expression of cyclin D1 was seen in the rare cells that expressed N2ICD but had not received Cre-EGFP (indicated by open arrows). In contrast, cyclin D1 was cytoplasmic and perinuclear in cells that had Cre-EGFP but did not express N2ICD (, indicated by asterisks). Collectively, RBP-Jκ is required for Notch to induce cyclin D1 transcription but not for nuclear localization.
Figure 5. Nuclear localization of cyclin D1 is RBP-Jκ independent. (A) NMVCs were isolated from RBP-JκFlox/Flox pups on the day of birth (P1) and treated as indicated until processing for immunostaining (the equivalent of P6). 10% FCS was added (more ...)
Notch-regulated nuclear export versus import of cyclin D1
Nuclear localization of cyclin D1 is controlled by a fine balance between import, through an unknown mechanism, and export through the chromosome region maintenance 1 (CRM1)/exportin-1 transporter. Cyclin D1 phosphorylation by glycogen synthase kinase-3β (GSK3β) at Thr286
is thought to trigger export to the cytosol and degradation by the proteasome (Diehl et al., 1998
To test if N2ICD
regulates export, we first compared its action to 6-bromoindirubin-3′-oxime (BIO), a small molecule inhibitor of GSK3β. BIO induced cell cycle reentry in a dose-dependent manner, albeit less dramatically than what is seen with N2ICD
(), as previously described (Tseng et al., 2006
). The inactive control compound MetBIO failed to promote cell cycle reentry of NRVCs. BIO, but not MetBIO, also caused nuclear accumulation of cyclin D1 () and phosphorylation of nuclear Rb (), which is suggestive that retention of cyclin D1 in the nucleus contributes to cell cycle entry by BIO.
Figure 6. Notch-induced nuclear accumulation of cyclin D1 in NRVCs is not caused by inhibition of export. (A) NRVCs were treated for 36 h with the GSK3β inhibitor BIO or control MetBIO before staining with MF20 and analysis of DNA content of cardiomyocytes (more ...)
Unlike BIO, N2ICD did not inhibit GSK3β, which was determined by monitoring luciferase activity from the T cell factor–dependent luciferase activity from the pTOPflash reporter plasmid that senses stabilized β-catenin, a direct target of GSK3β (). Moreover, the levels of phospho–Thr286–cyclin D1 levels were not inhibited in Ad-N2ICD–infected NRVCs but were in fact up-regulated compared with controls and, more importantly, with PHE- or 10% FCS-treated NRVCs, which also expressed high levels of Cyclin D1 (). The increase in phospho–Thr286–cyclin D1 correlated with increased levels of nuclear cyclin D1 available for phosphorylation by GSK3β.
As an independent test for regulated export, cells were treated with leptomycin B (LMB) to specifically inhibit CRM1/exportin-1. LMB enhanced nuclear localization of cyclin D1 beyond that achieved by N2ICD
alone (). Furthermore, LMB stimulated nuclear cyclin D1 only in cells that expressed N2ICD
and not in control, uninfected cells (compare N2ICD+
cells [, indicated by arrows] to uninfected cells [, indicated by circles]). These data are consistent with the idea that N2ICD
acts upstream of CRM1/exportin-1. LMB did not increase the number of S/G2
/M cells (unpublished data) because inhibition of CRM1/exportin-1 inhibition prevents cell cycle progression (Yoshida et al., 1990
). The results with LMB, together with the lack of an inhibitory effect of N2ICD
on GSK3β or the phosphorylation of cyclin D1, suggest that N2ICD
does not control export or degradation but might stimulate nuclear import.
Activation of the DNA damage checkpoint in older NRVCs
Activated Notch effectively stimulated P2 NRVCs to enter mitosis, as revealed by phosphorylation of histone 3 (His3) on Ser10
-infected P2 NRVCs vs. 44.5% for uninfected; P < 0.001, two-tailed Student's t
test). The presence of phospho-Ser10
is a hallmark for entry into mitosis and is required for chromatin condensation (e.g., Adams et al., 2001
; Crosio et al., 2002
). In striking contrast, older P5 NRVCs completely failed to phosphorylate His3 (0.57% phospho-Ser10
; ), which revealed a postnatal decline in the ability to traverse the G2
/M interphase. A similar postnatal decline in cell division was noted by Collesi et al. (see p. 117
of this issue) in response to stimulation by exogenous Jagged.
Figure 7. Ad-N2ICD–infected NRVCs arrest at the G2/M interphase. (A and B) 3 d after birth, NRVCs were infected with Ad-N2ICD or Ad-β-gal, or not infected, and, 12 h later, treated with aphidicolin or nocodazole for an additional 36, 48, or 60 h. (more ...)
To understand the nature of the block, we probed the cell cycle status of treated cells using the DNA polymerase inhibitor aphidicolin or the mitotic spindle assembly inhibitor nocodazole for various times to block the cell cycle (see , inset, for experimental design). As expected, aphidicolin, which blocks cells at the G1/S interphase, reduced the percentage of Ad-N2ICD–expressing NRVCs that had progressed into S, G2, or M phases of the cell cycle (), confirming entry into S phase. Nocodazole interferes with microtubules and causes cells to arrest at the G2/M interphase. Importantly, nocodazole did not alter the percentage of cardiomyocytes in S/G2/M phase (), which is suggestive of the fact that cells were already arrested at this point. To test this idea, the nocodazole was washed out during the last 12 h of culture. Normally, cycling cells resume progression after removal of nocodazole; however, this had no effect on the P5 NRVCs, which indicates that they were likely arrested at or before G2/M. In contrast, the residual noncardiomyocytes (MF20− cells) in the preparations arrested at G2/M upon nocodazole treatment and were released by its removal (), which demonstrates that the Notch-induced block was specific to the cardiomyocytes in the cell preparation.
AuroraB directly phosphorylates Ser10 of His3. As shown in , Ad-N2ICD dramatically increased AuroraB expression in the nuclei of P5 NRVCs but completely failed to induce His3 Ser10 phosphorylation in control uninfected or Ad-βGal–infected NRVCs. A blockade at the level of AuroraB confirmed the G2/M arrest and suggested activation of the DNA damage checkpoint.
Caffeine effectively overrides DNA damage G2
/M checkpoint activation and can permit entry of arrested cells into M phase by inhibiting the ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinases, and most likely other DNA checkpoint components (e.g., Kastan et al., 1991
; Sarkaria et al., 1999
). Entry into M phase in the presence of caffeine is therefore diagnostic of checkpoint activation. Caffeine treatment alone did not increase the level of phospho-Ser10
-His3 in uninfected P5 NRVCs and in fact caused a minor reduction (). In contrast, N2ICD
increased the number of phospho-His3+ NRVCs by 3.5-fold. Overriding checkpoint activation pushed cells into mitosis and resulted in cell death, visualized by an 87-fold increase in the number of TUNEL-positive cardiomyocytes (), which is consistent with induction of programmed cell death when the checkpoint is overridden in cells with DNA replication defects (Belka, 2006
). The mitosis-inducing kinase Cdc2 is a key component of the DNA damage checkpoint pathway downstream of ATM/ATR and Chk1, and it shows inhibitory Tyr15
phosphorylation with checkpoint activation. shows an increase in phospho-Tyr15
-Cdc2 in N2ICD
-stimulated NRVCs, as well as a possible increase in the total Cdc2 protein levels, providing further evidence of DNA damage checkpoint activation after the N2ICD
-induced cell cycle. As expected, phospho-Tyr15
-Cdc2 returned to basal levels after caffeine treatment of N2ICD
-stimulated P5 cardiomyocytes. We conclude that activation of DNA damage checkpoint causes the G2
/M arrest and is triggered as a consequence of improper or incomplete DNA synthesis during S phase.
Figure 8. DNA damage checkpoint activation implicated in G2/M arrest. (A–E) NRVCs were either infected with Ad-N2ICD or left uninfected. 24 h later, 10 mM caffeine (C, D, H, and I) or control (A, B, F, and G) media were added. Cells were then cultured for (more ...)