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Lineberger Comprehensive Cancer Center,1 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,3 Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 606072
Received 3 March 2004/ Returned for modification 9 April 2004/ Accepted 10 May 2004
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ABSTRACT |
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INTRODUCTION |
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The physiologic significance of evolving a separate family of CDK inhibitors and multiple members within each family in mammalian cells is presumed to meet increasing needs for integrating more intricate and multifaceted cell growth signals, both intracellular and extracellular, into a single cell cycle control machinery. Supporting this notion are the observations that the expression of individual CDK inhibitor genes is activated selectively by different checkpoint pathways, displays distinct temporary and spatial patterns during both in vitro cell differentiation and in vivo embryonic development, and is maintained differentially in different adult and senescent tissues. Further support for different physiologic functions of individual CDK inhibitor genes in vivo comes from the genetic analyses of mutant strains of mice with targeted mutation in each of seven individual CDK inhibitor genes. Various different phenotypes were observed in these mice, ranging from lack of major defects after the loss of the p15 or the p19 gene (27, 55), compromised DNA damage response after inactivation of the p21 gene (4, 8), increase of tumor development resulting from loss of function of p16 (25, 45), and severe developmental defects after p57 mutation (54) to widespread hyperplastic cell proliferation and organomegaly in p18 and p27 null mice (14, 15, 23, 27, 37). Elucidation of the molecular pathways linking various cell growth signals to the expression of individual CDK inhibitor genes, however, remains a major challenge and has only been validated in a few exceptional cases. Two examples are the p53-mediated activation of p21 gene expression following DNA damage (11) and transforming growth factor ß-mediated induction of p15Ink4b (17).
Biochemically, CDK inhibitors within each family act almost indistinguishably in binding to and regulating CDK enzymes but differ among the families. The CIP/KIP proteins share a unique N-terminal sequence motif comprising two subdomains for binding cooperatively to and forming ternary complexes with CDK and cyclin subunits. The CIP/KIP proteins diverge in their C-terminal sequences. The INK4 proteins, on the other hand, consist essentially of four or five tandem copies of ankyrin repeats and few additional sequences. Unlike CIP/KIP inhibitors, which are capable of interacting with multiple CDK-cyclin complexes, the only binding partners and functional targets identified thus far for INK4 proteins are two very closely related catalytic CDK subunits, CDK4 and CDK6. Such functional dependency of INK4 on CDK4 or CDK6, however, has not yet been tested genetically.
Ectopic overexpression of individual INK4 genes causes a G1 cell cycle arrest with a correlative dependency on the intact Rb pathway (16), and loss of either Rb function or a combination of p107 and p130 functions effectively canceled the G1 arrest due to INK4 overexpression (3, 24, 35). These findings provide evidence that, at least in cultured cells, the function of INK4, and CDK4 and CDK6 by extension, in controlling the G1-to-S transition is dependent on the presence of both an intact Rb and p107-p130 functions. Hence, there may exist in vivo a linear INK4-CDK4/CDK6 (CDK4/6)-Rb G1 control pathway in mammalian cells. Delineating the pathway(s) for p21 family inhibitors in vivo is more complicated and remains somewhat perplexing; this pathway is attributed largely to their interaction with both the CDK and cyclin subunits and with multiple CDK-cyclin complexes. It was initially observed that p21 levels undergo an increase immediately following mitogenic stimulation of serum-starved human fibroblasts, before declining at the G1-S boundary (29), and that CDK and cyclin can be found in active CDK-cyclin complexes at a one-to-one ratio when expressed at a low concentration (18, 53). Later studies found that the assembly and kinase activity of CDK4-cyclin D correlate concomitantly with the binding of CIP/KIP proteins (26) and were reduced in mouse embryonic fibroblasts (MEFs) lacking p21 and p27 (6). A titration model—cyclin Ds-CDK4/6 complexes act as activators of cyclin Es-CDK2 complexes by titrating CIP/KIP proteins away from, and thus releasing the inhibition of, cyclin Es-CDK2 complexes—was proposed to accommodate these observations, which seemingly contradict the classification of CIP/KIP as a CDK inhibitor. According to this model, CIP/KIP genes can be considered to act genetically downstream of cyclin Ds-CDK4 complexes in an INK4-cyclin Ds/CDK4-CIP/KIP-cyclin Es/CDK2-Rb pathway. Results challenging both notions—that p21 only stoichiometrically inhibits cyclin A-CDK2 and that p21-p27 deficiency reduced cyclin D-CDK4 assembly and activity—were reported (1, 19), leaving the mechanistic role of CIP/KIP proteins in regulating cyclin Ds-CDK4/6 and cyclin Es-CDK2 complexes at an incompletely understood and somewhat confusing state at present.
The p18Ink4c and p27Kip1 genes represent two of the most extensively studied CDK inhibitor genes in mice. Loss of function of either gene resulted in similar and widespread defects in cell proliferation and organ development, providing suitable models for investigating the functional and mechanistic differences between the two families of CDK inhibitors in living animals. In this study, we set forth to test genetically in double-mutant mice the following questions: is the function of INK4 to inhibit CDK4, and how does the loss of Cdk4 and a gene from either CDK inhibitor family affect each other phenotypically in mice?
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MATERIALS AND METHODS |
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Analysis of fertility and diabetes. Mating ability and fertility of mice were determined by keeping each mutant and a wild-type fertile partner together in a cage and checking daily for the presence of a vaginal plug. Females with plugs were separated and monitored throughout pregnancy, delivery, and nursing. Blood glucose level was monitored in the morning (8 to 10 a.m.) by using an automatic glucose monitor (Glucometer Elite; Bayer). Each mouse was analyzed twice for blood glucose on two consecutive days for every time point.
Histopathology. Tissues of most organs were removed, fixed in 10% neutral buffered formalin, and examined histologically by two pathologists after hematoxylin-eosin staining. Lesions were photographed, and additional sections were taken for immunohistochemical analyses.
Pancreatic islet size determination. Pancreatic islet size (cells per islet section) was measured as previously reported with minor modifications (33). Briefly, islet size was determined in at least three cut sections from matched pancreatic regions of three animals per genotype at each stage of development. Sections were more than 500 µm from each other to avoid overestimating larger islets in this analysis.
Immunohistochemistry. To measure proliferating and mitotic cells, sections were blocked with normal goat serum in phosphate-buffered saline (PBS) and incubated with either a polyclonal antibody against mitosis-specific phosphorylated histone H3 (5 µg ml–1; Upstate Biotechnology) or a polyclonal antibody against Ki67 (1:1,000; NCL-Ki67; Novocastra Laboratories) for 1 h and with a biotin-conjugated secondary antibody (Vector Laboratories) for 30 min. Immunocomplexes were detected with the Vectastain ABC alkaline phosphatase kit according to the manufacturer's instructions (Vector Laboratories). For apoptosis assays, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were carried out using the in situ ApopTag kit (Intergen) according to the manufacturer's protocol.
MEFs and flow cytometry procedures. Primary MEFs were isolated from embryonic day 13.5 embryos. Early-passage MEFs (younger than passage 4) from individual embryos were plated in 100-mm plates and incubated in Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). For serum starvation, asynchronous cultures at approximately 50% confluence were washed with PBS and serum starved in DMEM containing 0.1% FBS for 72 h. Cells were restimulated by addition of DMEM with 10% FBS. For bromodeoxyuridine (BrdU) labeling, MEFs were grown in media with or without serum containing 10 µM BrdU for 1 h. Nuclei were isolated following trypsinization and fixation with 95% ethanol by incubation in 0.08% pepsin (Sigma)-0.1 N HCl for 20 min at 37°C. Nuclear DNA was denatured by incubation in 2 N HCl for 20 min at 37°C, followed by neutralization with Na2B4O7, pH 8.5. Incorporated BrdU was detected with anti-BrdU-fluorescein isothiocyanate (Becton Dickinson; 1:10 dilution) in 10 mM HEPES (pH 7.3)-150 mM NaCl-4% FBS-0.5% Tween 20. MEFs were then harvested and resuspended in PBS containing 1% FBS and incubated with propidium iodide (5 µg ml–1) and RNase A (0.1 mg ml–1) for 30 min at 37°C. The samples were analyzed with a B-D FACScan (Becton Dickinson), and the data were processed with Summit software (version 3.0; BD Biosciences).
Northern blot analysis. MEFs at early passage were serum deprived (0.1% FBS) for 3 days and released from quiescence by serum stimulation (10% FBS). Total RNA was prepared from cells at different time points after serum stimulation as well as from an asynchronized cell population. RNA samples were resolved on a 1% agarose gel, transferred to a nitrocellulose filter, and hybridized with a probe derived from full-length mouse p18Ink4c cDNA. The blot was then stripped and rehybridized with a mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe.
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RESULTS |
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Growth retardation of p18–/–; Cdk4–/– mice became obvious during prepuberty, was persistent through the entire experimental duration of 9 weeks, and was observed in both sexes. The body weights of both p18–/–; Cdk4–/– and p18+/–; Cdk4–/– mice were lower than those of either p18–/– or wild-type mice and were statistically indistinguishable from those of Cdk4–/– single null mice (Fig. 1A and C). Thus, loss of Cdk4 led to a body weight phenotype that was completely dominant over loss of p18.
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Both male and female p18–/–; Cdk4–/– mice are infertile. All p18–/–; Cdk4–/– mice, both males (n = 8) and females (n = 10), that we examined are infertile, and most (more than 90%) of Cdk4–/– males and all female mutants are sterile, as reported previously. In contrast to testes enlargement caused by p18 loss, the testes of 5-month-old p18–/–; Cdk4–/– mice were small and had sizes comparable to those of testes of Cdk4–/– mice (Fig. 2A). Histological analysis revealed that, while p18–/– male mice developed Leydig cell hyperplasia, the p18–/–; Cdk4–/– male mouse, like the Cdk4–/– mouse, contains abnormal seminiferous tubules with degeneration of a significant fraction of primary spermatocytes and reduced Leydig cells. Very few normal spermatozoa were found in the lumen of seminiferous tubules of both Cdk4–/– and p18–/–; Cdk4–/– mice at an age of 5 months (Fig. 2A). Hence, the seminiferous tubules in mice lacking both Cdk4 and p18 genes exhibited defects similar to those found in Cdk4–/– mice. Leydig cell proliferation associated with p18 loss was inhibited by Cdk4 loss.
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Loss of p27 also resulted in the development of ovarian enlargement but impaired the function of ovulation and caused female sterility (14, 23, 37, 42, 47), indicating that p18 and p27 play different roles in ovulation and granulosa cell proliferation. p27–/–; Cdk4–/– female (as well as male) mice are infertile.
Development of pituitary hyperplasia in p18–/– mice requires CDK4 function. A hallmark defect in mice with reduced or complete loss of Rb function is the hyperplastic phenotype of the pituitary (20, 21, 28, 31, 51). Loss of p18 results in the development of the same hyperplastic phenotype in the intermediate lobe of the pituitary (15, 27) (Fig. 3A) and, conversely, loss of Cdk4 causes a pituitary hypoplasia and lactotroph dysfunction (36), offering an excellent in vivo setting to determine the genetic interaction between these two Rb regulators. We confirmed that pituitaries of Cdk4 null adult mice (4 months old) were significantly smaller and extremely hypoplastic relative to wild-type pituitaries in all three lobes: the intermediate lobe, the anterior lobe, and the neurohypophysis (Fig. 3A). The pituitaries of p18–/–; Cdk4–/– mice were as small as Cdk4 null pituitaries and also showed significant hypoplasia, which might be responsible for the infertility and defect in formation of CL in p18–/–; Cdk4–/– mice. Like those from pituitaries of Cdk4 single-knockout mice, the intermediate lobes from p18–/–; Cdk4–/– pituitaries were hypoplastic, indicating that the development of pituitary hyperplasia in p18–/– mice requires a functional CDK4. Late in life (>9 months), p18–/– mice developed pituitary adenomas in the intermediate lobe. Because of early death of p18–/–; Cdk4–/– mice, we were not able to demonstrate that loss of Cdk4 would prevent the development of pituitary adenomas.
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Both Ki-67 and phosphor-H3 staining revealed an increase of cell proliferation in the p18–/– pituitary and a decrease of cell proliferation to almost the basal level in the Cdk4–/– pituitary (Fig. 3B). Increase of both Ki-67- and phosphor-H3 positive cells in the p18–/– pituitary is not restricted to the intermediate lobe; it is also evidently in both the anterior lobe and the neurohypophysis (Fig. 3B), revealing a previously unrecognized function of p18 in these parts of the pituitary. Consistent with these results, we also found that p18 is expressed in all three lobes of the pituitary (data not shown). As apoptosis in both cell types was not significantly increased, an increase of cell proliferation, but not the size of both anterior lobe and neurohypophysis, suggests that a separate compensating mechanism may maintain an overall normal tissue size in these parts of pituitary. The numbers of both Ki-67- and phosphor-H3-positive cells in the p18–/–; Cdk4–/– pituitary were similar to those seen in the wild type, indicating that loss of the Cdk4 gene inhibited hyperproliferation caused by p18 loss.
Loss of p18 does not rescue diabetes of Cdk4-deficient mice. Cdk4-deficient mice displayed normoglycemia at 3 weeks of age and developed diabetes around 7 weeks of age (40, 49) (Fig. 4A). Histological analysis revealed that by the age of 3 weeks the number and size of pancreatic islets were not affected by the loss of CDK4. However, by the ages of 7 and 16 weeks the number of pancreatic islets was significantly decreased. Pancreases of wild-type mice showed 82.6, 16.6, and 0.8% small, medium, and large islets, respectively (Fig. 4B and C). The number of islets from Cdk4–/– mice was 50% less than the number from wild-type littermate mice (Fig. 4B and data not shown). Analysis of the sizes of the islets found in Cdk4–/– pancreases of mice at 16 weeks of age revealed that 96.8% were small (1 to 40 cells/islet section), that only 3.2% were medium sized (41 to 400 cells/islet section), and that there were no large-sized islets (>400 cells/islet section). These results suggest that not only islet number but also the size of the islets were significantly decreased in Cdk4-deficient mice. In contrast, the number of islets from p18–/– mice (16 weeks of age) was approximately 40% higher than the number from wild-type littermate mice. Although most of the islets of p18–/– mice (72.1%) were small, there were significantly more medium-sized (23.1%) and especially large-sized islets (4.6%) than were found in wild-type mice (Fig. 4B). Pancreases of p18–/–; Cdk4–/– mice are very similar to those of Cdk4–/– mice; islets were sparsely located in the p18–/–; Cdk4–/– pancreas, and the number of islets was comparable to the number in Cdk4–/– mice. For the p18–/–; Cdk4–/– islets, 95.5% were small and only 4.5% could grow to medium size. No large-sized islets were found in p18–/–; Cdk4–/– pancreases (Fig. 4C). Consistent with the defects in pancreatic development and function, p18–/–; Cdk4–/– mice also developed diabetes from about 7 weeks of age (Fig. 4A). Loss of p18 did not rescue defective pancreatic islet (ß-cell) development. Instead, loss of Cdk4 abolished all hyperplastic growth of pancreatic islet ß cells caused by p18 loss.
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To determine the functional interaction between CDK4 and its two regulators, p18 and p27, we derived p18–/–; Cdk4–/– and p27–/–; Cdk4–/– double-mutant MEFs and determined their cell cycle kinetics for serum deprivation and stimulation. Consistent with a previous report (49), the p27–/–; Cdk4–/– MEFs displayed cell cycle kinetics intermediate between those of Cdk4–/– and p27–/– MEFs, indicating that deletion of p27 partially restored the delayed S-phase entry caused by Cdk4 loss (Fig. 7A and B). Surprisingly, p18–/–; Cdk4–/– MEFs exhibited kinetics more similar to that of p18–/– MEFs: 20.1 and 25.4% of cells were stained positive for BrdU at 18 and 21 h post-serum stimulation. These results are consistent with the idea that p18 plays a critical role in controlling the serum response in MEFs by regulating not only CDK4 but also another target, likely CDK6, and that delay in S phase entry due to loss of Cdk4 could be compensated by an increase of CDK6 activity.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (52), were reported to bind with p19Ink4d and p16Ink4a, respectively, although the functional consequence and physiologic significance of both interactions remain unclear. Generation and characterization of p18- and Cdk4-deficient mutant mouse strains and development of a series of consistent phenotypes in these two strains of mice provide an opportunity to determine the functional dependency of INK4 on CDK4 in vivo. In this paper, we provide the first genetic evidence that the developmental function of an INK4 protein is dependent on CDK4. We have examined in p18–/–, Cdk4–/–, and p18–/–; Cdk4–/– mice body weight and six cell types, tissues, or organs which developed highly penetrant phenotypes because of the loss of p18 alone: testis, ovary, pituitary, endocrine pancreas, kidney, and adrenal gland. In all six cases, simultaneous loss of Cdk4 virtually canceled all p18 loss-induced defects. These results provide direct evidence that the function of p18Ink4c is dependent on CDK4 in vivo. Many of these hyperproliferative defects associated with p18 loss were also developed in p27-deficient mice to a similar extent with a nearly complete penetrance, including body weight increase; pituitary hyperplasia and tumor development; and enlargement of thymus, spleen, testis, and ovary (14, 23, 37). Notably, the body weight increase, or the gigantism phenotype, caused by p27 loss was only partially canceled in p27–/–; Cdk4–/– double-mutant mice (Fig. 1). This result is consistent with and provides genetic support for the model that, while INK4 regulates only CDK4 and CDK6, CIP/KIP proteins have an additional target, CDK2. The role of p27 (and p21) in the regulation of Cdk4 is currently controversial (38), and it has been suggested that p27 has opposing roles as an activator (6) and as an inhibitor (1). Our results that the body weights of p27–/–; Cdk4–/– double-mutant mice and the cell cycle kinetics of p27–/–; Cdk4–/– MEFs exhibited a phenotype intermediate between that of either single mutant are in agreement with an oppose function of these two genes and support the notion that p27 is an inhibitor of Cdk4. In addition to tumor suppression, the Rb pathway controls cell differentiation, tissue development, and organ size. Of various p18 loss-caused defects that were canceled by the simultaneous loss of Cdk4, the body weight increase, pancreatic islet number increase and size enlargement, and ovary enlargement are defects in animal development and organ size regulation, not tumor growth, indicating that the functional dependency of p18 on Cdk4 is not restricted to tumor suppression and may extend into other cellular processes.
The only defect caused by p18 loss that was not significantly alleviated by the loss of Cdk4 is the accelerated G1-to-S progression of MEFs following serum deprivation and stimulation: p18–/–; Cdk4–/– MEFs displayed cell cycle kinetics more similar to that of p18–/– MEFs (Fig. 7). In the same assay, the p27–/–; Cdk4–/– MEFs exhibited intermediate cell cycle kinetics, indicating, again, that deletion of Cdk4 partially reduced accelerated G1-to-S progression caused by p27 loss. The most likely explanation for the lack of significant rescue of p18 loss-caused defects by Cdk4 loss is that in MEFs, unlike other adult tissues and differentiated cells, Cdk6 may play a significant role in regulating G1 progression following serum starvation-stimulation, either in its own right or when induced in a compensatory manner after Cdk4 loss, therefore sensitizing the MEFs to p18 loss. Consistent with this idea, CDK6 is readily detectable in MEFs and CDK6-p27 complexes were noticeably increased in Cdk4 null MEFs (49), suggesting a compensatory regulation of p27, and thus Cdk2, by CDK6 in the absence of CDK4. The in vivo function of CDK6 in the whole animal and how widely CDK6 compensates for CDK4 loss in other cell types and tissues remain unclear. Our results that Cdk4 loss canceled all six defects caused by p18 loss that we have examined suggest that Cdk6 plays a relatively minor role in vivo in mediating the function of p18.
Wild-type MEFs accumulate in either G1 phase of the cell cycle or a quiescence state in response to serum deprivation and then reenter the cell cycle and travel through the G1 phase with consistent kinetics: DNA replication starts approximately 14 to 16 h after serum stimulation. Loss of p18 accelerated G1 progression and initiation of DNA replication (Fig. 7), revealing a previously unrecognized function of p18 in regulation of the serum response in MEFs. Supporting this function of p18 is the observation that p18 mRNA is induced during reentry of serum-deprived MEFs into the cell cycle (Fig. 7). The mechanism responsible for p18 induction during G1 progression remains to be determined but does not appear to be dependent on the function of E2F1. Notably, similar percentages of p18–/– and wild-type MEFs (7 to 8%) were stained positive for BrdU after 72 h of serum starvation (Fig. 7), indicating that p18–/– MEFs were arrested in the G0 quiescent state as efficiently as wild-type MEFs. Likewise, loss of p27, which also accelerated G1 progression following serum stimulation, did not impair the ability of MEFs to enter and maintain a stable quiescence upon serum deprivation. Differently, in response to serum deprivation, the Rb–/–; p107–/–; p130–/– triple-deficient MEFs continue to incorporate BrdU and then undergo apoptotic cell death (7, 43), indicating an essential function of pocket proteins, and by extension implying a function of a CDK inhibitor(s), in causing and/or maintaining G1 arrest under growth-inhibiting conditions. Lack of a significant defect in entering into and maintaining G1 arrest upon serum starvation in p18-deficient MEFs is consistent with its decreased low-level expression in quiescent cells (Fig. 7). It remains to be determined whether, during the cellular response to growth factor deprivation-stimulation, p18 and p27 alone are sufficient to cause and maintain a G1 arrest, but both are required for a proper reentry of quiescent cells into the proliferation cycle. Alternatively, our results suggest that another CDK inhibitor protein separately controls the entry into and maintenance of G1 arrest while p18 and p27 regulate the reentry into the cell cycle.
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ACKNOWLEDGMENTS |
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Y.X. is supported in part by a U.S. Department of Defense Career Development Award (DAMD17-99-1-9574). This study was supported by NIH grants CA100204 to H.K. and CA65572 and CA68377 to Y.X.
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FOOTNOTES |
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