Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity

Abstract

Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.

Figure 1.

Selective inhibition of CDK1 activity induced differentiation of TS cells to TG cells. TS cells and ES cells were cultured in the presence of the CDK1 inhibitor RO3306 for the indicated times (days) and photographed at 10× magnification.

Figure 2.

Selective inhibition of CDK1 activity mimicked FGF4 deprivation induced endoreduplication in TS cells, but not in ES cells. TS cells endoreduplicated their genome either upon FGF4 deprivation (A), or addition of RO3306 to the culture medium (B), as evidence by FACS analysis at the times (days) indicated. 2N DNA content indicates cells in G1 phase. 4N indicates cells in either G2 or M phase. Endocycles (>4N) were detected as peaks in the FACS profiles that represented cells with integral multiples of diploid (2N) DNA content. (C) Addition of RO3306 to ES cells at first induced endocycles (1 d), and then apoptosis (days 2 and 3), as evidenced by the appearance of cells with <2N DNA content in the FACS profile. Treatment of either TS or ES cells with DMSO (the solvent used to dissolve RO3306) had no effect (data not shown). The positions of diploid (2N) and tetraploid (4N) cells are indicated. (D) To quantify the data in A–C, the percentage of cells with >4N DNA content was plotted as a function of time either after FGF4 deprivation or treatment with RO3306. (E) Examples of TS cells and TG cells (6 d of FGF4 deprivation) stained with the TS-specific antibodies TROMA-1 (anti-cytokeratin EndoA: blue). Nuclei were stained with propidium iodide (red).

Figure 3.

Endoreduplication in TS cells was triggered by selective inhibition of CDK1, not by arresting cells in mitosis. (A) TS cell cultures were washed free of unattached cells and then cultured in the presence of 200 nM nocodazole (Sigma, M1404) for 8 h. Mitotic cells were released into the medium by sharp tapping of the dishes and recovered by centrifugation. One portion of the mitotic cells was again cultured in TS-cell medium supplemented with nocodazole while the other portion was cultured in the presence of both nocodazole and RO3306. Total cells were harvested on the indicated day, stained with propidium iodide, and analyzed by FACS to determine the ploidy levels. (B) TS cells were treated with 20 μM roscovitine (Sigma, R7772) for 3 d before FACS analysis.

Figure 4.

Endoreduplication required CDK2. (A) Wild-type and Cdk2^−/− TS cells were subjected to FGF4 deprivation for up to 6 d, during which time both cell lines differentiated into TG cells. The percentage of cells with >4N DNA content were determined by FACS analysis, and the percentage of cells with >4N DNA content was plotted as a function of time. (B) Cdk2^−/− and wild-type (wt) TS cells were cultured in TS medium containing FGF4 and RO3306 for 3 d before subjecting them to FACS analysis (shaded profiles). As a control, cells were cultured in TS medium containing FGF4 and DMSO for 3 d (solid line, open space).

Figure 5.

FGF4 deprivation rapidly suppressed CDK1 activity in TS cells with concomitant expression of CDK-specific inhibitors p21 and p57. (A) TS cells were differentiated into TG cells by FGF4 deprivation for the indicated times. Cell lysates were prepared, CDK1 protein was immunoprecipitated (IP), and the immunoprecipitate was assayed for its ability to phosphorylate histone H1. Coomassie-stained histone H1 protein used for the kinase assay is shown at the bottom. (B) TS cells were differentiated into TG cells by FGF4 deprivation (−FGF4), and total cell lysates were subjected to Western immunoblotting analysis for the indicated protein. “+FGF4, +RO” indicates TS cells that were cultured for 3 d in the presence of RO3306. (C) p21 p27 and p57 were independently immunoprecipitated from lysates of TS cells and of TG cells 24 h after removal of FGF4. CDK1 was detected by Western immunoblotting analysis. (D) Results in these and other experiments were quantified plotted as a function of time that TS cells were deprived of FGF4. (●) Relative change in CDK1 activity; (◽) relative change in p57 protein level; (◦) relative change in p21 protein level; (◆) fraction of cells with 4N DNA content or greater.

Figure 6.

p57 was required for endoreduplication during TS cell differentiation. (A) p21^−/− and p57^−/− TS cells were differentiated into TG cells by FGF4 deprivation for the indicated time (days), and subjected to FACS analysis. (B) The fraction of cells with >4N DNA content was determined from the data in A and Figure 2A (wt) as a function of time. (C) Cdk2^−/−, p21^−/−, and p57^−/− TS cells were differentiated into TG cells by FGF4 deprivation for the time indicated (days). Total cell lysates were prepared and analyzed for the proteins indicated by Western immunoblotting analysis. (D) p21^−/−, p57^−/−, and wild-type TS cells were cultured for 3 d in the presence of RO3306 and then analyzed by FACS (shaded profiles). The same cells were also cultured in parallel with DMSO instead of RO3306 (solid line, open profile).

Figure 7.

p57 blocked mitosis in TG cells. (A) Equal numbers of wild-type (wt) and p57^−/− TS cells were differentiated into TG cells by FGF4 deprivation for the times indicated. Cells were then harvested and counted in triplicate. (B) Fraction of wild-type, p21^−/−, and p57^−/− cells with two or more nuclei present at the times indicated. Several fields of 100 cells were scored for each time point. (C) FACS analyses of p57^−/− TS cells before (solid line, open space) and at 10 d after FGF4 deprivation (shaded profile). Phase contrast images of p57^−/− TS cells at 3 d (D) and 10 d (E,F) of FGF4 deprivation.

Figure 8.

p57 protein levels oscillated during endoreduplication in TG cells. (A) TG cells after 3 d of FGF4 deprivation were stained with DAPI to identify nuclei and with anti-p57 antibodies to identify p57 protein. (B) TG cells after 3 d of FGF4 deprivation were cultured for 1 h in the presence of BrdU and then stained with both anti-BrdU antibodies (in situ cell proliferation kit, Roche) and anti-p57 antibodies.

Figure 9.

Differentiation of TS cells into TG cells suppressed the DNA damage response. (A) Total cell extracts were prepared from wild-type TS cells before (0 h) and up to 48 h after FGF4 deprivation, and then subjected to Western immunoblotting analysis for the proteins indicated. TS cells also were cultured in the presence RO3306 for 48 h and analyzed for CDK1 protein (+FGF4 +RO). (B) ES cells were treated with RO3306 for the indicated times and then analyzed as in panel A. (C) TS cells were subjected to FGF4 deprivation for 3 d in order to produce TG cells. Then TS cells and TG cells were cultured for 3 d either in the presence (shaded bars) or absence (open bars) of 10 mM caffeine (Sigma) to inhibit ATR, or 200 nM UCN-01 (Sigma) to inhibit CHK1. Total cells were harvested and the fraction of cells with <2N DNA content was determined by FACS.

Figure 10.

Mammalian endocycles. In TS cells, the transition from mitotic cell cycles to endocycles occurs when FGF4 deprivation induces expression of p57, a protein that selectively inhibits CDK1, the enzyme responsible for initiating mitosis. This allows cells to transit from G2 phase to G1 phase without passing through mitosis (M phase). Thus, mammalian endocycles consist of a Gap phase that is defined by the presence of p57 and an S phase that is defined by its absence. FGF4 deprivation also induces expression of p21, but the role of p21 is to suppress CHK1 and thereby prevent apoptosis in response to incomplete DNA replication. In addition, p21 may also suppress CDK2–CCNE activity during the Gap phase. CDK2–CCNE is required to initiate DNA replication during endocycles. Inhibition of CDK1 activity has at least two additional consequences. Inhibition of CDK1 prevents assembly of APC–Cdc20, the enzyme that targets cyclins A and B for degradation (∅), and it prevents inactivation of the Orc1 subunit by phosphorylation when S phase is completed. Thus, p57 allows ORC assembly and binding to DNA replication origins under conditions that prevent these events during mitotic cell cycles. Furthermore, CDC6, which is phosphorylated by CDK2 during S phase, will become dephosphorylated in the presence of p57 and p21. These events allow initiation of pre-RC assembly. Geminin (Gem), a specific inhibitor of CDT1 during S phase, is targeted for degradation by the APC–Cdh1 ubiquitin ligase, an enzyme that is active during the late M and early G1 phases of mitotic cell cycles. CDT1 can then complete pre-RC assembly by loading MCM helicases. Since CDK1 activity is required to activate APC–Cdc20, this ubiquitin ligase will be inactive. CDK2–CCNE activity then concurrently promotes destruction of p57 and p21 by activating the SCF–Skp2 ubiquitin ligase, restores geminin expression by preventing assembly of APC–CDH1, and initiates DNA replication.