AbstractMice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo. However, Cdk6−/− erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6−/− erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia – and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.
Cyclins and cyclin-dependent kinases (Cdk) are critical regulators of the cell cycle. Cyclin/Cdk complexes trigger cell-cycle progression with D-type cyclins and Cdk responsible for controlling the early G1 phase of the cell cycle. In the early G1 phase, cyclin-D-Cdk4/6 complexes phosphorylate and inactivate the retinoblastoma protein to activate E2F-dependent transcription.1 Cdk4 and Cdk6 are 71% identical at the amino acid level, are ubiquitously expressed and bind all three D-type cyclins.2 Only the combined deletion of Cdk4 and Cdk6 induces late embryonic lethality due to defects in hematopoiesis,43 while deletion of either gene alone is not fatal. Accordingly, Cdk4 and Cdk6 are considered to have redundant functions in regulating cell-cycle progression.
Nevertheless, the lack of either of the kinases leads to specific defects in particular types of cells. Cdk4 mice have defective postnatal pancreatic beta cells and pituitary cells,65 while the lack of Cdk6 leads to disorders in the hematopoietic compartment such as altered thymocyte development and lower numbers of red blood cells and cells of other hematopoietic lineages.874 Recent evidence indicates that Cdk6 is a key player in the differentiation of a variety of cell types, a function not shared by Cdk4. A decline in the level of Cdk6 is required for terminal differentiation of murine erythroid leukemia cells9 and Cdk6 expression in astrocytes has been associated with the expression of progenitor cell markers.10 Osteoblast differentiation has been described to be inhibited by overexpression of Cdk6 but not of Cdk4.11 A related study showed that Cdk6 protein levels are downregulated by Rankl-induced osteoclast differentiation.12 Moreover, Cdk6 inhibits the transcriptional activation of Runx1 and thus blocks myeloid differentiation.13 Cdk6 kinase activity is also required for thymocyte development, in which it functions by modulating the expression of Notch target genes.8 Cdk6 but not Cdk4 regulates transcription of key players in lymphoma formation and myeloid leukemia in a kinase-dependent and/or kinase-independent manner.1614 Cdk6 has also been ascribed a unique role in actin dynamics in astrocytes:1710 overexpression of Cdk6 in primary mouse astrocytes results in altered gene expression and modified cytoskeletal architecture, as well as in loss of stress fibers and enhanced motility.
Erythrocytes transport oxygen through the body. They have a unique biconcave shape that allows a certain flexibility when travelling through narrow capillary vessels.18 Changes of form are made possible by the delicate interplay between the fluid cell membrane and the structure of the membrane cytoskeleton.18 Mutations in genes encoding actin-binding proteins such as adducin, dematin, tropomyosin, tropomodulin and protein 4.1 are known to cause altered actin stability and increased erythrocyte fragility leading to anemia. The correct polymerization and organization of actin is vital for the strength and flexibility of erythrocytes.18 Besides, actin-remodeling proteins are required for regulating actin filament length. Among those, gelsolin, a Ca-dependent protein, severs pre-assembled actin filaments and is hence crucial in actin remodeling.19
We show that the anemic phenotype of mice lacking Cdk6 kinase activity is not attributable to a reduced production of erythrocytes but is the consequence of a reduced erythroid life span. Our findings reveal a novel role for Cdk6 as a stabilizer of the erythrocyte cytoskeleton via the deregulation of genes involved in cytoskeleton stability.2220
Mice were maintained under pathogen-free conditions at the Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna (Austria). C57BL/6 mice are referred to as Cdk6. Cdk64 and Cdk68 mice were generated on the C57BL/6 background. Mice aged 6–8 weeks were used unless otherwise indicated. Blood parameters were analyzed with a VetABC blood counter.
Bone marrow, spleen and fetal liver cells were stained for erythroid subsets using the antibodies listed below. Samples were analyzed by a FACSCantoII flow cytometer using FACSDiva software (Becton-Dickinson). Erythrocyte development in the bone marrow and spleen was analyzed as follows: Ter119CD71 (proerythroblasts; R1), Ter119CD71 (basophilic erythroblasts; R2), Ter119CD71 (late basophilic and polychromatophilic erythroblasts; R3) and Ter119CD71 (orthochromatophilic erythroblasts; R4). Cells were stained with the fluorescently labeled antibodies: Ter119-PE (eBioscience), CD71 Biotin (eBioscience), Streptavidin eflour 780 (eBioscience), CD49e PE (integrin α5), and CD49d FITC (integrin α4).
In vivo biotin labeling
The life span of red blood cells in vivo was measured by injecting 3mg EZ-Link-sulfo-NHS-biotin (Pierce, Rockford, IL, USA) into mice. Decay of the labeled red blood cells was measured by FACS analysis.23
Fresh blood from age-matched mice was washed twice in phosphate-buffered saline. Osmotic fragility was measured by mixing 25 μL blood with 2.5 mL saline solutions (with salt concentrations between 0 and 0.9% NaCl). After gentle mixing the suspension was incubated for 15 min at room temperature and centrifuged at 500 × g for 10 min. The osmotic fragility curve was obtained by plotting the measured absorbance of hemoglobin released into the supernatants at 540 nm for each solution against NaCl concentrations. Duplicates for each NaCl concentration point were read.
Measurement of membrane mechanical stability
Fresh murine blood was taken, diluted in 10 volumes of phosphate-buffered saline and subjected to a constant shear stress of 29G, 0.33 × 12 mm. The suspension was centrifuged at 500 × g for 10 min. The absorbance of hemoglobin supernatant was measured at 540 nm. Fresh human erythrocytes were diluted in 10 volumes of phosphate-buffered saline and incubated with palbociclib (manufactured by Pfizer) at indicated concentrations overnight at 37°C, in 5% CO2 and 95% humidity. Dimethyl sulfoxide (DMSO) was used as the vehicle control. The degree of resistance of red blood cells to mechanical stress was measured as above.
Venous blood was drawn from healthy volunteers after informed consent. Animal experiments were performed in accordance with protocols approved by Austrian law and the Animal Welfare Committee at the University of Veterinary Medicine, Vienna.
Statistical analysis was carried out using a two-tailed unpaired Student t test and one-way or two-way analysis of variance (ANOVA) as appropriate. Data are presented as mean values ± standard error of the mean (SEM) and were processed using GraphPad software.
Cdk6−/− mice have anemia accompanied by an increased number of Ter119+ cells in the bone marrow
Cdk6−/− mice are viable and fertile and display minor defects in hematopoiesis with anemia and reduced cellularity in thymus and spleen.4 The reduction in erythrocyte numbers is paralleled by an increase in mean cell volume and mean corpuscular hemoglobin.4 We confirmed these observations in C57BL/6 Cdk6 mice under our housing conditions (Figure 1A,B and Online Supplementary Figure S1A).
Anemia may be caused by enhanced hemolysis or altered iron metabolism. We analyzed levels of plasma iron, plasma hepcidin (a key iron regulator) and erythropoietin (a major mitogen for erythroid progenitors). No significant changes were detected although there was a tendency towards higher plasma iron and erythropoietin levels in Cdk6 mice (Online Supplementary Figure S1B–D). Likewise, the ability to form myeloid colonies was not altered among genotypes (Online Supplementary Figure S1E). Analysis of the maturation of erythroid progenitors in the bone marrow and spleen by FACS revealed minor differences: a profiling developed by Socolovsky et al.,24 showed slightly enhanced numbers of cells in the R2 (basophilic erythroblasts) and R4 (orthochromatophilic erythroblasts) fractions in the bone marrow but not in the spleen (Figure 1C,D). The increased amount of erythrocyte precursors became most evident from the highly significant increase in total Ter119 erythroid cells in the bone marrow of Cdk6 animals (Figure 1E). No differences were detected in the spleen (Online Supplementary Figure S2A) or during erythrocyte maturation in fetal liver erythropoiesis (at day E13) between Cdk6 and wildtype controls (Online Supplementary Figure S2B).
One explanation for the increased proportion of Ter119 cells in the bone marrow accompanying anemia in the peripheral blood is a reduced ability of mature red blood cells to exit the bone marrow. To enter the periphery, red cells downregulate surface integrins during erythropoiesis. FACS staining revealed no differences in integrin patterns: levels of downregulation of integrin α4 (Online Supplementary Figure S2C) and integrin α5 (Online Supplementary Figure S2D) were comparable irrespective of the genotype. The unaltered ability of Cdk6 erythrocytes to migrate efficiently from the bone marrow to the periphery was also visible from the elevated numbers of reticulocytes (which contain residual amounts of RNA that can be stained with thiazole orange) in the blood. Cdk6 mice had significantly more reticulocytes in the peripheral blood than had Cdk6 controls (Figure 1F) reflecting the enhanced production of red blood cells. In summary, a reduced erythroid development in the bone marrow does not account for anemia in Cdk6 mice.
Cdk6−/− mice respond normally to erythropoietic stress
The bone marrow reacts immediately to conditions of high need by increasing the production of erythrocytes through a process referred to as stress erythropoiesis. We reasoned that subtle changes in erythropoiesis may become more pronounced under conditions of stress. Two independent experimental settings were used to investigate whether Cdk6−/− mice respond to hypoxia by increasing erythroid production. First, we mimicked stress erythropoiesis in vitro: fetal liver erythroblasts (day E13) can be cultured and stimulated to proliferate and differentiate by use of distinct cytokines.25 Cdk6+/+ and Cdk6−/− erythroblasts displayed superimposable growth curves (Figure 2A) and similar morphology (Figure 2B). A proliferation assay using CellTrace Violet showed no differences among genotypes (Online Supplementary Figure S3A). Differentiating erythrocytes divide approximately four times before they mature to fully differentiated enucleated red cells.25 The kinetics of differentiation of Cdk6 and Cdk6+/+ erythroblasts were comparable, as shown by growth curves (Figure 2C), the Violet Cell proliferation assay (Online Supplementary Figure S3B) and benzidine staining (Online Supplementary Figure S3C).
The in vitro findings were echoed by in vivo studies: mice were exposed to phenylhydrazine which induces hemolytic anemia and forces the subsequent rapid expansion of the erythroid lineage in the bone marrow and spleen.2726 Cdk6 and Cdk6 mice showed a comparable recovery response when analyzed 10 days after receipt of a single dose of phenylhydrazine. Red blood cell count (Figure 2D) and hematocrit (Online Supplementary Figure S3D) were restored to normal levels. Massive increases in numbers of all premature stages (R1–R2) of the erythroid lineage were detectable in the bone marrow (Online Supplementary Figure S3E) and the spleen (Online Supplementary Figure S3F) 3 days after phenylhydrazine induction irrespective of the genotype. We only observed one subtle difference: whereas the proportion of Ter119 erythroid cells in the wildtype bone marrow increased from ~50% to ~60%, it remained consistently high at 60% in Cdk6 bone marrow (Figure 2E). This difference was not seen in the spleen: numbers of Ter119 cells increased to the same extent in Cdk6−/− and Cdk6 mice upon phenylhydrazine treatment (Figure 2F).
Cdk6−/− erythrocytes have a decreased life span in vivo
We next analyzed the possibility that the anemia in Cdk6 mice is linked to a shortened lifespan of the cells. Peripheral erythrocytes were labeled in vivo with EZ-Link sulfo-NHS-biotin and blood samples were taken every 10 days. In line with published data,2928 we found that under these conditions wildtype erythrocytes had an average half-life of 19 days, while that of erythrocytes from Cdk6 mice was significantly shorter (14 days; Figure 3A). The shortened half-life may be indicative of a lower mechanical stability of the cells as the long-term survival of mature erythrocytes in the periphery demands mechanical strength and flexibility. Freeze-thaw cycles also pose a challenge to the mechanical stability of cells. Notably, we found that erythroblasts from Cdk6 fetal livers did not survive a freeze-thaw cycle (Figure 3B). Changes in membrane stability may also be associated with reduced tolerance to osmotic stress which was tested using various concentrations of NaCl. However, osmotic sensitivity was not altered between Cdk6 and Cdk6 erythrocytes (Figure 3C).
Cdk6 is tethered to the cell membrane in erythrocytes
We next tested whether the absence of Cdk6 is associated with changes of the cytoskeleton. The major cytoskeletal components (spectrin, ankyrin, band 3 and protein 4.1/4.2) were present at comparable levels irrespective of the genotype (Online Supplementary Figure S4). However, phalloidin staining of fixed erythroblasts revealed a pronounced reduction in filamentous-actin (F-actin) polymerization upon loss of Cdk6 (Figure 4A and Online Supplementary Figure S5A,B) although the level of total beta-actin (B-actin, non-polymerized actin) in erythroblasts was unaffected (Figure 4B). In mature erythrocytes differences in phalloidin staining were still observed, albeit to a lesser extent: in wildtype erythrocyte cytoskeleton F-actin structures were symmetrical and had a pronounced ring shape whereas the actin oligomers in Cdk6−/− erythrocytes were distributed diffusely (Online Supplementary Figure S5C). In line, Cdk6-deficient lymphoid cells had a decreased filamentous to globular actin ratio suggesting the interplay between Cdk6 and actin structures is not restricted to erythroid cells (Online Supplementary Figure S5D).
Proliferating erythroblasts express both cell-cycle kinases Cdk4 and Cdk6 (Figure 4B). Mature erythrocytes still contain Cdk6 protein (Figure 4C) whereas Cdk4 is degraded during cell maturation. Cdk4 was only present at low levels in Cdk6 erythrocytes (Figure 4C). Erythrocyte membranes (ghosts) contain mainly cytoskeletal and membrane proteins but also some peripheral and membrane-associated proteins. It is therefore of particular interest that we detected significant amounts of Cdk6 but not Cdk4 in ghosts (Figure 4D and Online Supplementary Figure S5E). These data indicate that Cdk6 is maintained in mature erythrocytes; a proportion is associated with the membrane and may play an active role in regulating cytoskeletal proteins. These findings are in line with a model that Cdk6 is tethered to the cytoskeleton where it may be protected from degradation and have a membrane stabilizing role.17
Cdk6 exerts kinase-dependent effects on erythrocyte stability
Cdk6 may affect cytoskeleton and membrane stability in a kinase-dependent and/or kinase-independent manner. Mice harboring a kinase dead mutant of Cdk6 (Cdk6K43M/K43M)8 recapitulate the phenotype of Cdk6 animals: we found decreased red blood cell counts accompanied by an increase in mean cell volume (Figure 5A and Online Supplementary Figure S6A). No gross morphological alterations were detected (Figure 5B). Reminiscent of Cdk6−/− mice we found alterations in the stages of erythroid development in the bone marrow and an increased total number of Ter119 erythroid cells (Figure 5C,D). Analysis of erythroid progenitors in the spleen and numbers of reticulocytes in the peripheral blood were not significantly altered (Online Supplementary Figure S6B,C). However, as seen in Cdk6-deficiency, Cdk6K43M/K43M erythroid cells showed a decreased filamentous to globular actin ratio (Figure 5E). These data suggest a kinase-dependent function of Cdk6 in cytoskeletal integrity.
Cdk6 transcriptionally regulates genes involved in cytoskeleton organization in a kinase-dependent manner
To investigate which cytoskeleton components are altered upon loss of Cdk6 kinase activity, we performed quantitative polymerase chain reaction studies of a set of genes that had been implicated in cytoskeleton remodeling in the literature using erythroid progenitors with intact nuclei [Ter119CD71 (orthochromatophilic erythroblasts; R4)] (Figure 6A and Online Supplementary Figure S7A). In the absence of Cdk6 kinase activity we found significant changes in the mRNA levels of four genes: Gelsolin, Tubulin alpha-8, Baiap2 and Pip5k1b were reduced to ~50% (Figure 6A). Cdk6 directly regulates the transcription of Gelsolin and Baiap2 as we were able to further show enhanced binding of Cdk6 to the respective promoters by chromatin immunoprecipitation assays in the non-transformed mouse progenitor HPC7 cell line and in Bcr/Abl-transformed lymphoid cells (Figure 6B). Lymphoid cell expression of gelsolin, a known regulator of actin filament (de-)polymerization,19 was also decreased in Cdk6K43M/K43M mice (Online Supplementary Figure S7B).
Intrigued by the observation that Cdk6 regulates the expression of genes involved in cytoskeleton organization in all our model systems we investigated which components of the cytoskeleton or cell membrane are bound to Cdk6. We immunoprecipitated Cdk6 from wildtype peripheral blood followed by mass spectrometry and complemented the analysis by including Cdk6 immunoprecipitation from wildtype lymphoid cells. The detection of Cdk6 protein served as a positive control. Mass spectrometry verified a set of potential Cdk6 interactors (Online Supplementary Figure S8). In mature erythrocytes we confirmed the interaction with gelsolin and also detected a set of proteins that have been associated with anemia and/or erythroid cytoskeletal organization/dynamics (Figure 6C and Online Supplementary Figure S8). Tubulin was an attractive candidate in lymphoid cells (Online Supplementary Figure S8).
Cdk6−/− cells display an increased sensitivity to shear stress and drugs interfering with cytoskeleton organization
On this basis, we also reasoned that Cdk6 cells should be more susceptible to any pharmacological disturbance of the cytoskeleton. We perturbed either actin metabolism by the addition of jasplakinolide,19 which stabilizes actin filaments, or microtubule metabolism by the addition of two benzimidazoles (albendazole and thiabendazole),30 which block tubulin polymerization. The treatments showed that our prediction was respected: Cdk6 cells were significantly more sensitive (Figure 6D). Combined treatment with CDK6 kinase inhibitor and microtubule poison mimicked the absence of Cdk6 in wildtype cells: the Bliss additivity model revealed an in vitro synergistic cytotoxicity between palbociclib and albendazole (Online Supplementary Figure S9A).
Fragility of erythrocytes can be tested by exposing cells to shear stress: Cdk6 and Cdk6K43M/K43M erythrocytes reacted with an enhanced lysis (Figure 6E). Similar observations although not reaching the level of statistical significance were made upon CDK4/6 kinase inhibitor treatment in human erythroid cells (Online Supplementary Figure S9B). Collectively, these data indicate a global role for Cdk6 as a regulator of membrane and cytoskeleton stability which manifests itself as anemia in the red cell compartment.
Anemia is a frequent disorder with a variety of possible causes. It most frequently stems from reduced erythrocyte formation due to a lack of iron, vitamin B12 or folic acid3331 but may also be associated with increased or premature destruction of erythrocytes in the periphery.34 Anemia of this latter type is generally caused by alterations in the cytoskeleton and the membrane and a large number of erythrocyte disorders are associated with mutations in genes required for membrane and cytoskeleton stability.18 Such mutations render erythrocytes susceptible to mechanical stress and lead to a premature degradation of red cells. We now define a deficiency in Cdk6 kinase activity as the cause of a novel form of anemia characterized by enhanced mechanical instability of erythrocytes.
Mice lacking Cdk6 or its kinase activity (Cdk6K43M/K43M) suffer from a mild form of anemia with enhanced numbers of Ter119 cells in the bone marrow and increased numbers of reticulocytes in the peripheral blood. This observation is in line with enhanced erythropoiesis, which may be viewed as the organism’s attempt to compensate for the enhanced loss of erythrocytes. We did not detect any reduction of erythroblast proliferation or differentiation in in vitro systems that mimic stress erythropoiesis or when mice were treated with phenylhydrazine.
Cdk6 does not have a critical role in cell proliferation, since its function can be performed in its absence by Cdk4. Differentiation along the erythroid lineage is largely unaltered by the elimination of Cdk6. This finding is in contrast to a report that Cdk6 is involved in the differentiation of the murine erythroleukemia cell line, MEL. The apparent discrepancy probably relates to the different experimental systems used. Transformed cell lines harbor multiple mutations that may interfere with normal or regular differentiation and so may not mimic the situation in untransformed cells. For example, loss of CDK6 in MLL-transformed myeloid acute myelogenous leukemia cells induces differentiation35 while the myeloid compartment in Cdk6-deficient mice is normal and contains cells at all stages of maturation.4 Signaling and transcriptional control are rewired in transformed cells so that molecules that are not required in non-transformed cells may become important for differentiation or proliferation.
Erythroblast protein expression changes during differentiation. Certain proteins are degraded or removed with the nucleus at enucleation or by vesiculation during reticulocyte maturation.3836 Most proteins are degraded during development. Levels of Cdk4 decrease dramatically and the protein is not detectable in mature erythrocytes, although Cdk6 is still present. Together with the observation that Cdk6- but not Cdk4-deficient mice65 suffer from anemia, this provides strong evidence that Cdk6 has a function in murine erythrocytes that cannot be performed by Cdk4. Interestingly, in the absence of Cdk6, mature erythrocytes do contain detectable levels of Cdk4. This paradoxical finding can be explained by postulating that in the absence of Cdk6, Cdk4 can interact with cytoskeletal structures usually occupied by Cdk6, thereby being at least partially protected from degradation, although the interaction may not have any functional consequence.
Previous studies indicated that expression of cell-cycle components correlate with variations in size and cell divisions during erythropoiesis.39 A direct role has been assigned to cyclin D3: its levels orchestrate the number of cell divisions during terminal erythropoiesis, thereby controlling the number and size of erythrocyte progeny.40 In contrast, proliferation and differentiation of erythrocytes are not impaired in mice lacking Cdk6 and mature erythrocytes contain Cdk6. We thus speculated that the anemic phenotype might be linked to alterations in the cytoskeleton. Cdk6 has been reported to associate with the cytoskeleton in astrocytes, where forced overexpression of Cdk6 induces changes to the cytoskeletal organization.1710 Staining of erythroid cells for F-actin revealed a dependence on the presence of Cdk6, with Cdk6-deficiency associated with markedly lower levels of F-actin. A number of clinical conditions have been linked to impaired F-actin formation424118 and these may be associated with a shortened half-life of erythrocytes in the peripheral blood. We found an altered F:G-actin composition in transformed lymphoid cells, indicating that the role of Cdk6 in actin remodeling is not restricted to erythroid cells. A possible mechanism is suggested by the observation that Cdk6 interacts in mature erythrocytes with gelsolin, a known regulator of actin remodeling.19
The cytoskeleton of erythrocytes faces particular challenges. Erythrocytes must pass through narrow capillaries, placing special requirements on the stability and flexibility of the cytoskeleton.18 Our results point to a further unique feature of the erythrocyte cytoskeleton, namely its dependence on Cdk6 for structural integrity and flexibility. Cdk6 is tethered to the erythrocyte cytoskeleton and this finding is consistent with the localization of the protein when it is overexpressed in astrocytes.17
We and others have shown that Cdk6 directly regulates transcription in both kinase-dependent and kinase-independent manners, interacting with a variety of transcription factors including Stat and AP-1 as well as with nuclear factor-κB.431614 We now report that Cdk6 promotes the transcription of genes involved in cytoskeletal organization. The function depends on Cdk6 kinase activity: upon loss of Cdk6 kinase activity in erythroid progenitors (R4) there is a significant decrease in mRNA levels of Tubulin alpha-8 implicated in microtubule assembly and Baiap2, Gelsolin and Pip5k1b involved in actin dynamics. BAIAP2 participates in F-actin rearrangements when activated by small GTPases.21 Loss of gelsolin in mice has been shown to change the balance between polymerized and depolymerized actin in red blood cells.19 PIP5K1B has a function in the dynamics of the actin cytoskeleton44 and PIP5 kinases synthesize phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which binds to gelsolin and thereby promote actin polymerization.4745 Loss of Cdk6 kinase activity thus causes impaired actin remodeling, which is likely to result in increased fragility of mature erythrocytes.48 Interestingly, mass spectrometry analysis of both erythroid and lymphoid cells confirmed that Cdk6 interacts with a number of proteins involved in cytoskeletal organization, suggesting a global role of Cdk6 in cytoskeletal integrity.
Two sets of experiments show that our results have functional significance. First, loss of Cdk6 causes mechanical instability of red blood cells in the shear test, which mimics the entry of erythrocytes into the narrow capillaries of the body. Secondly, Cdk6-deficient cells are more sensitive to actin and microtubule inhibitors.
Our work defines Cdk6 as a unique member of the Cdk family with a potential dual function for the cytoskeleton. In erythroid cells, Cdk6 affects cytoskeletal stability by transcriptionally regulating a set of genes that control cytoskeletal organization. This function depends on Cdk6 kinase activity. Furthermore, Cdk6 has a direct structural role in erythrocytes. It is tethered to the cytoskeleton, where it may phosphorylate unknown proteins, be a stabilizing anchoring factor or simply be protected from degradation. Further research is needed to unravel the mechanisms behind this function and its consequences.
The phenotype of Cdk6−/− mice is recapitulated in Cdk6K43M/K43M animals. This finding may have consequences for therapies that target CDK6 kinase activity over a longer time. Inhibitors of the CDK4/6 kinases are being tested for use in the treatment of many forms of cancer.49 Our findings provide a possible explanation for the observation that patients receiving inhibitors of CDK4/6 kinases are prone to reduced erythroid stability which contributes to anemia.50
We thank Philipp Jodl and Sabine Fajmann for their excellent technical help. We are deeply indebted to Graham Tebb for numerous scientific discussions and editing the manuscript. We thank Anja Redlinghofer for technical assistance, Botond Cseh for support and Michael Freissmuth, Richard Moriggl and Helmut Dolznig for critical scientific input.
This work was made possible by the Austrian Science Fund (FWF) Grant SFB F47 (to SK and VS) and FWF Grant P 24297-B23 (to VS), by an ERC-advanced grant (to VS) and by EMBO ASTF 103.00-2011a (to RMS).
- ↵* These authors contributed equally to this work
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/102/6/995
- Received November 11, 2016.
- Accepted February 22, 2017.
- Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes Dev. 2004; 18:2699-2711. PubMedhttps://doi.org/10.1101/gad.1256504Google Scholar
- Meyerson M, Enders GH, Wu CL. A family of human cdc2-related protein kinases. EMBO J. 1992; 11(8):2909-2917. PubMedGoogle Scholar
- Kozar K, Sicinski P. Cell cycle progression without cyclin D-CDK4 and cyclin D-CDK6 complexes. Cell Cycle. 2005; 4(3):388-391. PubMedhttps://doi.org/10.4161/cc.4.3.1551Google Scholar
- Malumbres M, Sotillo R, Santamaría D. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell. 2004; 118(4):493-504. PubMedhttps://doi.org/10.1016/j.cell.2004.08.002Google Scholar
- Rane SG, Dubus P, Mettus RV. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat Genet. 1999; 22(1):44-52. PubMedhttps://doi.org/10.1038/8751Google Scholar
- Tsutsui T, Hesabi B, Moons DS. Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity. Mol Cell Biol. 1999; 19(10):7011-7019. PubMedhttps://doi.org/10.1128/MCB.19.10.7011Google Scholar
- Hu MG, Deshpande A, Enos M. A requirement for cyclin-dependent kinase 6 in thymocyte development and tumorigenesis. Cancer Res. 2009; 69(3):810-818. PubMedhttps://doi.org/10.1158/0008-5472.CAN-08-2473Google Scholar
- Hu MG, Deshpande A, Schlichting N. CDK6 kinase activity is required for thymocyte development. Blood. 2011; 117(23):6120-6131. PubMedhttps://doi.org/10.1182/blood-2010-08-300517Google Scholar
- Matushansky I, Radparvar F, Skoultchi AI. Manipulating the onset of cell cycle withdrawal in differentiated erythroid cells with cyclin-dependent kinases and inhibitors. Blood. 2000; 96(8):2755-2764. PubMedGoogle Scholar
- Ericson KK, Krull D, Slomiany P, Grossel MJ. Expression of cyclin-dependent kinase 6, but not cyclin-dependent kinase 4, alters morphology of cultured mouse astrocytes. Mol. Cancer Res. 2003; 1(9):654-664. PubMedGoogle Scholar
- Ogasawara T, Kawaguchi H, Jinno S. Bone morphogenetic protein 2-induced osteoblast differentiation requires Smad-mediated down-regulation of Cdk6. Mol Cell Biol. 2004; 24(15):6560-6568. PubMedhttps://doi.org/10.1128/MCB.24.15.6560-6568.2004Google Scholar
- Ogasawara T, Katagiri M, Yamamoto A. Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6). J Bone Miner Res. 2004; 19(7):1128-1136. PubMedhttps://doi.org/10.1359/jbmr.2004.19.7.1128Google Scholar
- Fujimoto T, Anderson K, Jacobsen SEW, Nishikawa S-I, Nerlov C. Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. EMBO J. 2007; 26(9):2361-2370. PubMedhttps://doi.org/10.1038/sj.emboj.7601675Google Scholar
- Kollmann K, Heller G, Schneckenleithner C. A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis. Cancer Cell. 2013; 24(2):167-181. PubMedhttps://doi.org/10.1016/j.ccr.2013.07.012Google Scholar
- Scheicher R, Hoelbl-Kovacic A, Bellutti F. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation. Blood. 2015; 125(1):90-102. PubMedhttps://doi.org/10.1182/blood-2014-06-584417Google Scholar
- Uras IZ, Walter GJ, Scheicher R. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6. Blood. 2016; 127(23):2890-2902. PubMedhttps://doi.org/10.1182/blood-2015-11-683581Google Scholar
- Slomiany P, Baker T, Elliott ER, Grossel MJ. Changes in motility, gene expression and actin dynamics: Cdk6-induced cytoskeletal changes associated with differentiation in mouse astrocytes. J Cell Biochem. 2006; 99(2):635-646. PubMedhttps://doi.org/10.1002/jcb.20966Google Scholar
- Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008; 112(10):3939-3948. PubMedhttps://doi.org/10.1182/blood-2008-07-161166Google Scholar
- Cantù C, Bosè F, Bianchi P. Defective erythroid maturation in gelsolin mutant mice. Haematologica. 2012; 97(7):980-988. PubMedhttps://doi.org/10.3324/haematol.2011.052522Google Scholar
- Tolias KF, Hartwig JH, Ishihara H. Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Curr Biol. 2000; 10(3):153-156. PubMedhttps://doi.org/10.1016/S0960-9822(00)00315-8Google Scholar
- Yamagishi A, Masuda M, Ohki T, Onishi H, Mochizuki N. A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein. J Biol Chem. 2004; 279(15):14929-14936. PubMedhttps://doi.org/10.1074/jbc.M309408200Google Scholar
- Govind S, Kozma R, Monfries C, Lim L, Ahmed S. Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J Cell Biol. 2001; 152(3):579-594. PubMedhttps://doi.org/10.1083/jcb.152.3.579Google Scholar
- de Jong K. Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia. Blood. 2001; 98(5):1577-1584. PubMedhttps://doi.org/10.1182/blood.V98.5.1577Google Scholar
- Socolovsky M, Nam H, Fleming MD. Ineffective erythropoiesis in Stat5a(−/−)5b(−/−) mice due to decreased survival of early erythroblasts. Blood. 2001; 98(12):3261-3273. PubMedhttps://doi.org/10.1182/blood.V98.12.3261Google Scholar
- von Lindern M, Deiner EM, Dolznig H. Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis. Oncogene. 2001; 20(28):3651-3664. PubMedhttps://doi.org/10.1038/sj.onc.1204494Google Scholar
- Dornfest BS, Naughton BA, Johnson R, Gordon AS. Hepatic production of erythropoietin in a phenylhydrazine-induced compensated hemolytic state in the rat. J Lab Clin Med. 1983; 102(2):274-285. PubMedGoogle Scholar
- Dornfest BS, Lapin DM, Adu S, Naughton BA. Dexamethasone suppresses the generation of phenylhydrazine-induced anemia in the rat. Proc Soc Exp Biol Med. 1992; 199(4):491-500. PubMedhttps://doi.org/10.3181/00379727-199-43385Google Scholar
- Neumann CA, Krause DS, Carman CV. Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression. Nature. 2003; 424(6948):561-565. PubMedhttps://doi.org/10.1038/nature01819Google Scholar
- Hoffmann-Fezer G, Mysliwietz J, Mörtlbauer W. Biotin labeling as an alternative nonradioactive approach to determination of red cell survival. Ann Hematol. 1993; 67(2):81-87. PubMedhttps://doi.org/10.1007/BF01788131Google Scholar
- Lacey E. Mode of action of benzimidazoles. Parasitol Today. 1990; 6(4):112-115. PubMedhttps://doi.org/10.1016/0169-4758(90)90227-UGoogle Scholar
- Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013; 368(2):149-160. PubMedhttps://doi.org/10.1056/NEJMcp1113996Google Scholar
- Abbaspour N, Hurrell R, Kelishadi R. Review on iron and its importance for human health. J Res Med Sci. 2014; 19(2):164-174. PubMedGoogle Scholar
- Brabin BJ, Premji Z, Verhoeff F. An analysis of anemia and child mortality. J Nutr. 2001; 131(2S–2):636S-645S. PubMedGoogle Scholar
- Alaarg A, Schiffelers RM, van Solinge WW, van Wijk R. Red blood cell vesiculation in hereditary hemolytic anemia. Front Physiol. 2013; 4:365. PubMedhttps://doi.org/10.3389/fphys.2013.00365Google Scholar
- Placke T, Faber K, Nonami A. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia. Blood. 2014; 124(1):13-23. PubMedhttps://doi.org/10.1182/blood-2014-02-558114Google Scholar
- Pasini EM, Kirkegaard M, Mortensen P. In-depth analysis of the membrane and cytosolic proteome of red blood cells. Blood. 2006; 108(3):791-801. PubMedhttps://doi.org/10.1182/blood-2005-11-007799Google Scholar
- Goodman SR, Daescu O, Kakhniashvili DG, Zivanic M. The proteomics and interactomics of human erythrocytes. Exp Biol Med (Maywood). 2013; 238(5):509-518. PubMedhttps://doi.org/10.1177/1535370213488474Google Scholar
- Goodman SR, Kurdia A, Ammann L, Kakhniashvili D, Daescu O. The human red blood cell proteome and interactome. Exp Biol Med (Maywood). 2007; 232(11):1391-1408. PubMedhttps://doi.org/10.3181/0706-MR-156Google Scholar
- Dolznig H, Bartunek P, Nasmyth K, Müllner EW, Beug H. Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control. Cell Growth Differ. 1995; 6(11):1341-1352. PubMedGoogle Scholar
- Sankaran VG, Ludwig LS, Sicinska E. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes Dev. 2012; 26(18):2075-2087. PubMedhttps://doi.org/10.1101/gad.197020.112Google Scholar
- Fowler VM. Regulation of actin filament length in erythrocytes and striated muscle. Curr Opin Cell Biol. 1996; 8(1):86-96. PubMedhttps://doi.org/10.1016/S0955-0674(96)80052-4Google Scholar
- Iolascon A, Perrotta S, Stewart GW. Red blood cell membrane defects. Rev Clin Exp Hematol. 2003; 7(1):22-56. PubMedGoogle Scholar
- Handschick K, Beuerlein K, Jurida L. Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-κB-dependent gene expression. Mol Cell. 2014; 53(2):193-208. PubMedhttps://doi.org/10.1016/j.molcel.2013.12.002Google Scholar
- van den Bout I, Divecha N. PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions. J Cell Sci. 2009; 122(Pt 21):3837-3850. PubMedhttps://doi.org/10.1242/jcs.056127Google Scholar
- Tolias KF, Hartwig JH, Ishihara H. Type Iα phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Curr. Biol.. 2000; 10(3):153-156. PubMedhttps://doi.org/10.1016/S0960-9822(00)00315-8Google Scholar
- Janmey PA, Stossel TP. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 325(6102):362-364. Google Scholar
- Janmey PA, Iida K, Yin HL, Stossel TP. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987; 262(25):12228-12236. PubMedGoogle Scholar
- Kalfa TA, Pushkaran S, Mohandas N. Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton. Blood. 2006; 108(12):3637-3645. PubMedhttps://doi.org/10.1182/blood-2006-03-005942Google Scholar
- Johnson N, Shapiro GI. Cyclin-dependent kinase 4/6 inhibition in cancer therapy. Cell Cycle. 2012; 11(21):3913. PubMedhttps://doi.org/10.4161/cc.22390Google Scholar
- Walker AJ, Wedam S, Amiri-Kordestani L. FDA approval of palbociclib in combination with fulvestrant for the treatment of hormone receptor-positive, HER2-negative metastatic breast cancer. Clin Cancer Res. 2016; 22(20):4968-4972. PubMedhttps://doi.org/10.1158/1078-0432.CCR-16-0493Google Scholar