Given that deregulated expression of mammalian Plks is closely associated with cell proliferation and oncogenesis (3
), we generated Plk3 knockout mice and examined their phenotypic characteristics. We first designed a targeting construct in which a neomycin resistance cassette was inserted between mouse Plk3 exons 1 and 9. The neomycin resistance gene was flanked by Plk3 gene sequences at both the 5′ and 3′ ends (). The mutant allele was expected to be devoid of exons 2 to 8 after homologous recombination (). Mouse ES cells were transfected with the linearized targeting vector and stable, G418-resistant transfectants were selected. An analysis of DNA isolated from stable ES cell transfectants by PCR revealed that one Plk3
allele was mutated (). Mutation of the Plk3
allele was confirmed by Southern blot analysis (). Whereas a 6.9-kbp fragment was detected in wild-type ES cell DNA, an additional fragment of 3.4 kbp was detected in DNA isolated from mutant ES cells, consistent with the predicted sizes for these alleles (). Two independent 129/SvJ-derived ES cell clones were injected into C57BL/6 blastocysts, which were subsequently implanted into pseudopregnant female mice. The resulting chimeric mice were backcrossed to wild-type C57BL/6 animals. Disruption of the Plk3
gene was confirmed by a PCR strategy using mouse tail DNA (). Heart RNA was isolated from four wild-type and two Plk3-null mice and subjected to quantitative real-time PCR analysis. Plk3
mRNA expression was not detected in Plk3
-null mouse tissue (). Combined, these results indicate that our mice were Plk3
Figure 1 Disruption of mouse Plk3 locus. A, schematic presentation of mouse Plk3 gene structure showing the relative sizes and positions of 15 exons. Structures of the targeting vector and the targeted mutant allele are also shown. TK, thymidine kinase gene; (more ...)
During the first year of breeding, we obtained >300 live animals from crosses of Plk3+/−
mice. Genotype analysis of live births from Plk3+/−
intercrosses revealed that wild-type animals, heterozygotes, and homozygotes were born at a ratio of 1:1.8:0.85. The slightly lower numbers than expected for heterozygotes and homozygotes suggest that there were some disadvantages for embryonic development of Plk3-deficient
mice. Because Plk2−/−
newborn mice are smaller than wild-type or Plk2+/−
), we measured the weight of newborn mice of Plk3+/−
intercrosses and followed their postnatal weight up to 2 months. We did not observe a significant weight difference between Plk3+/+
, and Plk3−/−
mice (data not shown). However, Plk3−/−
mice at 20 months of age were somewhat larger than their age- and sex-matched littermates (Supplementary Fig. S1A
). Because a previous report indicated that Plk4
haploinsufficiency results in enhanced carcinogenesis (24
), we studied tumor development in Plk3
-deficient mice. No significantly elevated levels of tumor formation were observed in young (<12 months) Plk3+/−
mice. In contrast, ageing Plk3−/−
mice (>18 months) developed tumors in various organs at a much enhanced rate compared with that of wild-type littermates (). Whereas the incidence of lung cancer was the highest, other cancers including those of kidney, liver, and uterus were also observed (, arrowheads
; Supplementary Table S1
Figure 2 Plk3 deficiency results in enhanced tumorigenesis. A, tumor incidences of age-matched wild-type and Plk3−/− mice. B, representative liver, lung, uterus, and kidney tumors that developed in aged Plk3−/− mice. Arrowheads (more ...)
Histologic analysis revealed that kidney typically developed adenocarcinoma composed of tubuloglandular architecture with heavy inflammatory infiltration. Numerous eosinophilic crystalloids filled tumor cells near the luminal surface (, arrowheads). The adenocarcinoma cells exhibited large nuclei with prominent nucleoli and moderate to abundant eosinophilic cytoplasm (). Lung tumors were mostly adenocarcinomas and the tumor cells had crowded large hyperchromatic nuclei with prominent nucleoli and scant basophilic cytoplasm (, arrowheads). Uterine tumors were leiomyosarcomas, which were well circumscribed but not encapsulated; also, the tumors were highly cellular with cytologic atypia and active mitosis (, arrowheads). Liver cancer exhibited bridging necrosis with dilated sinus and congested vessels. These tumors had regenerative hepatocytes with prominent nucleoli, binuclei or multiple nuclei, and active bile duct hyperplasia with mild cholestasis ().
Many tumors in Plk3−/− mice were large in size and vascularized (, arrowheads), suggesting active tumor angiogenesis. To confirm this possibility, we stained paired lung tumor sections with an antibody to vWF, an endothelial cell marker. We observed that tumor blood vessel density, manifested as vWF-positive areas/foci, was much higher in Plk3−/− tumors than that of Plk3+/+tumors ().
Plk3 activation is highly responsive to oxidative stress (11
);we therefore examined HIF-1α expression in cultured MEFs because this transcription factor plays a critical role in supporting cancer angiogenesis. Paired MEFs were maintained under hypoxic conditions (1% O2
) for various times. Western blot analysis revealed low HIF-1α expression in untreated Plk3+/+
MEFs but HIF-1α expression was rapidly induced after exposure to hypoxia (). However, HIF-1α levels were induced to a greater extent in Plk3−/−
MEFs compared with Plk3+/+
MEFs. HIF-1 β expression, on the other hand, was not induced in wild-type nor Plk3−/−
MEFs (). The increase in HIF-1α levels was correlated with the appearance of slow-mobility HIF-1α forms in Plk3−/−
MEFs, which were especially prominent in cells exposed to hypoxia for 24 h (). Although HIF-1α was also induced in wild-type MEFs after an extended exposure to hypoxia, the magnitude of induction in these cells was much smaller than that observed in Plk3
-null MEFs ().
Figure 3 Superinduction of HIF-1 α in Plk3−/− MEFs under hypoxic conditions. A, paired wild-type (W) and Plk3−/− (H) MEFs were cultured under hypoxic conditions for various times as indicated. Equal amounts of cell lysates (more ...)
Nickel ions are known to deplete cellular iron and ascorbic acid, resulting in an oxidative stress condition similar to hypoxia (25
). Nickel also directly inhibits HIF prolyl hydroxylase (PHD; ref. 26
), which otherwise “tags” HIF proteins for recognition by von Hippel-Lindau (VHL) ubiquitin E3 ligase for polyubiquitination and subsequent degradation by the proteasome (27
). To determine whether Plk3
deficiency compromised the HIF-1α degradative pathway, paired Plk3−/−
and wild-type MEFs were treated with NiCl2 for various times. Nickel ion treatment significantly enhanced HIF-1α, but not HIF-1β, levels in both types of MEFs; however, HIF-1α was induced to a much greater extent in Plk3−/−
MEFs than that in wild-type MEFs (). Because VEGF-A is a direct transcriptional target of HIF-1α, we also measured the level of VEGF-A in conditioned medium of paired MEFs treated with nickel ion. ELISA analysis revealed that VEGF-A levels were significantly higher in Plk3−/−
MEF medium after 16 and 24 h of nickel treatment ().
In hypoxia, HIF-1α is stabilized due to the inhibition of PHD that requires O2
). To determine whether protein stabilization was responsible for the increased HIF-1α levels in Plk3−/−
MEFs, paired MEFs were treated with nickel ions and/or MG132, a specific proteasome inhibitor. MG132 alone stabilized HIF-1α to a much higher level in Plk3
-null MEFs than in wild-type MEFs; in combination with nickel, the proteasome inhibitor superinduced HIF-1α and the magnitude of induction was much greater in Plk3−/−
MEFs (). These combined studies therefore strongly suggest that Plk3
deficiency renders cells hypersensitive to the induction of HIF-1α by hypoxia.
To further confirm that Plk3 is directly involved in regulating HIF-1α production, we made use of a series of human Plk3 and Plk1 expression constructs that have been described (19
). Plk3 consists of a KD and a PBD. Plasmid constructs were made expressing KD, PBD, and FL Plk3 as GFP fusion proteins (). The various expression constructs were transfected into HeLa cells for 24 h and then treated with nickel for 4 h. Indirect fluorescence microscopy revealed that, whereas little HIF-1α was detected in untreated HeLa cells, nickel treatment greatly stimulated the accumulation of this protein in the nucleus (). Expression of Plk3-KD, but not Plk3-PBD, drastically suppressed the induction of nuclear HIF-1α by nickel; on the other hand, expression of Plk1-KD did not affect the induction of HIF-1α (). Interestingly, ectopically expressed Plk3-FL did not have a noticeable effect on suppression of nuclear accumulation of HIF-1α (). Expression of Plk3-KD, but not Plk3-PBD, also significantly suppressed the induction of HIF-1α by cobalt ions (Supplementary Fig. S2A and B
). These data suggest that Plk3 plays a specific role in regulating the hypoxic response signaling pathway and that active Plk3 may be necessary for the destabilization and nuclear exclusion of HIF-1α.
Figure 4 Ectopic expression of the Plk3-KD suppresses nuclear accumulation of HIF-1α in HeLa cells. A, schematic presentation of Plk3 domain structure and several Plk3 and Plk1 expression constructs. B, HeLa cells treated with NiCl2 for 4 h were stained (more ...)
Expression of Plk3-KD greatly suppressed NiCl2- or CoCl2-mediated induction of HIF-1α, suggesting that the kinase activity may be required for negative regulation of this transcription factor. To test this possibility, we made a kinase-defective Plk3-KD by mutating a residue (Thr219) that is important for kinase activation. As expected, expression of this mutant construct significantly suppressed the accumulation of nuclear HIF-1α (). On the other hand, we noticed that there existed a population of Plk3-KDT219D–expressing cells containing a significant amount of nuclear HIF-1α after nickel treatment (). This is likely due to a basal level of the kinase activity associated with this construct (data not shown).
Figure 5 HIF-1α expression is negatively correlated with Plk3 activity. A, HeLa cells transfected with GFP-Plk3-KD or GFP-Plk3-KDT219D expression constructs for 24 h and then treated with NiCl2 for 4 h were stained with antibodies to HIF-1α (red) (more ...)
To further confirm the effect of Plk3 kinase activity on suppressing HIF-1α induction, we transfected HEK293 cells with various GFP-tagged Plk3 expression constructs for 24 h followed by treatment with nickel for 4 h. Equal amounts of cell lysates were blotted for HIF-1α. Blotting with the anti-GFP antibody revealed that transfected constructs were expressed with anticipated sizes at comparable levels (). Transfection of Plk3-KD greatly reduced the level of HIF-1α compared with that of Plk3-PBD-transfected or GFP-transfected control; consistent with the notion that the kinase activity is required for the inhibition, transfection of Plk3-KD did not significantly suppress the level of HIF-1α induced by nickel (;Supplementary Table S2
). Interestingly, transfection of Plk3-FL construct also reduced the level of HIF-1α (; supplementary Table S2
), suggesting that FL Plk3 is capable of inhibiting the steady-state level of HIF-1α.
Because Plk3 is constitutively expressed, we asked whether hypoxic stress affected expression of endogenous Plk3. Immuno-blot analysis showed that Plk3, which was mostly cytosolic, was significantly down-regulated on treatment with NiCl2;on the other hand, expression of Plk1, which was mostly nuclear, was not affected by the nickel ion treatment (). As expected, HIF-1α expression was induced and translocated into the nucleus after NiCl2 treatment (). HIF-1β expression was not induced by nickel; however, it was translocated into the nucleus on hypoxic stress ().
Activated Plk3 seems to undergo degradation via the ubiquitin-proteosome pathway (20
). Through expressing various Plk3 mutants as well as wild-type Plk3, we found that mutations of a leucine-rich region in the Plk3 COOH-terminal tail greatly stabilized ectopically expressed Plk3 (). To determine whether ectopic expression of a stable Plk3 protein affected HIF-1α accumulation after hypoxic stress, we transfected HeLa cells with Plk3 wild-type (Plk3-WT) and Plk3-4LA (Plk3 with four leucine mutations) expression constructs for 24 h before treatment with NiCl2 for 8 h. HIF-1α expression was greatly induced by nickel ion in cells transfected with the Plk3-WT construct; however, the induction of HIF-1α was significantly suppressed in cells transfected with the Plk3-4LA expression construct ().
Figure 6 Ectopic expression of a Plk3 COOH-terminal region mutant suppresses NiCl2-mediated nuclear accumulation of HIF-1α in HeLa cells. A, amino acid sequence of the relevant mouse Plk3 region (numbering based on the 648-amino acid Plk3 isoform). WT (more ...)
Given the newly identified role of Plk3 in response to hypoxic stress as well as the known regulators in the HIF-1α signaling pathway, we propose the following model for Plk3 function (Supplementary Fig. S3
). Plk3 can potentially inhibit HIF-1α by physical interaction and direct phosphorylation. HIF-1 is a phosphoprotein (25
), although the exact role of phosphorylation remains to be elucidated. PHD, which is inhibited under hypoxia or by treatment with nickel or cobalt ions, catalyzes prolyl hydroxylation of HIF-1α, promoting its association with VHL and subsequent ubiquitination. In theory, Plk3 could up-regulate the function of PHD and/or VHL, resulting in enhanced degradation of HIF-1α by the proteasomal pathway. On the other hand, it remains a possibility that Plk3 has unknown targets, which in turn modulate the activities of components in the HIF-1α signaling pathway.