In this study, we demonstrated that CDK4−/− mice survive embryogenesis, suggesting that CDK4 is not essential for mouse development. Furthermore, examination of CDK4−/− fibroblasts suggested that CDK4 is dispensable during continuous proliferation. However, our analysis with quiescent CDK4−/− fibroblasts indicated that CDK4 plays a rate-limiting role when quiescent cells enter the cell cycle. Growth retardation, reproductive dysfunction, and degeneration of pancreatic islets in CDK4−/− mice further suggest that CDK4 plays a critical role in in vivo regulation of cell cycle progression, the lack of which other CDKs cannot completely compensate for.
The dwarfism-like phenotype of CDK4
-null mice is reminiscent of those in cyclin D1-deficient mice (13
) and transgenic mice with high copy numbers of the Rb gene (4
). It also presents an image opposite to that of the gigantism-like phenotypes of mice deficient in the CDK inhibitor p27 (25
) and mice lacking p18INK4c
, an inhibitor of CDK4 and CDK6 (14
). These observations suggest that the genetic pathway involving p27, p18, cyclin D1, CDK4, and Rb may be important for regulation of animal growth (46
). Perturbed G1
progression in serum-stimulated CDK4−/−
embryonic fibroblasts further implies that growth retardation of CDK4−/−
mice is associated with defective cell cycle regulation during development. However, it is also possible that the dwarfism in CDK4−/−
mice is due to another problem, e.g., a neurological or an endocrine disorder.
The gonads are highly proliferative organs. In the adult testis, germ cells continuously proliferate and differentiate into spermatids in response to hormonal signals. Disturbed control of proliferation in the gonads could lead to reduced or abrogated fertility. We have shown that CDK4 deficiency leads to reproductive dysfunction in both males and females. The testicular atrophy in CDK4−/−
males showed some similarity to that in mice lacking E2F-1
). Phosphorylation of Rb by cyclin D-CDK4 activates the transcriptional activity of E2F-1 by releasing E2F-1 from sequestration (37
). Thus, this genetic pathway seems to be critical for proper regulation of spermatogenesis.
In contrast with proliferating spermatogonia in the testis, oocytes in the adult ovary are arrested in meiosis, while somatic granulosa cells in the follicle proliferate and differentiate in response to gonadotropins (50
). Maintenance of the oocyte function depends on interactions between oocytes and granulosa cells via cell-cell contact and paracrine factors. Furthermore, upon ovulation, granulosa cells differentiate into luteal cells, which secrete steroid and peptide hormones required for the maintenance of pregnancy. CDK4−/−
females were infertile, and their ovaries exhibited disturbed cellularity in corpora luteum and apparently abnormal luteinization. These morphological changes in CDK4−/−
ovaries looked different from those in infertile cyclin D2−/−
). cyclin D2−/−
ovaries had only small follicles and are defective in FSH-dependent proliferation of granulosa cells. In contrast, the absence of CDK4 seems to allow granulosa cells to proliferate to form large follicles, although the kinetics of differentiation may be disturbed. This difference between cyclin D2−/−
ovaries implies that in the absence of CDK4, cyclin D2 may activate other CDKs to somehow promote granulosa cell proliferation. Female mice lacking p27 are also infertile in association with hyperproliferation of granulosa cells during luteal differentiation (25
). These studies recapitulate the importance of the pathway involving cyclin D2, CDK4, and p27 in the homeostasis of the proliferation and differentiation of granulosa cells.
We have found that a majority of CDK4−/−
mice develop diabetes mellitus, associated with degenerative changes in pancreatic islets. This spontaneous development of nonobese diabetes resembles that in NOD mice or BB rats, which are animal models of autoimmune type I diabetes (8
). However, CDK4−/−
pancreas displayed no sign of insulitis, the characteristic inflammation of islets due to autoimmunity. The marked decrease in cell numbers of CDK4−/−
pancreatic islets and the presence of apoptotic cells suggest that CDK4 plays a critical role for the postnatal proliferation (and possibly for the maintenance) of these endocrine islet cells. The loss of islets, and especially of insulin-secreting B cells, leads to the development of diabetes (56
). It is presently unclear why approximately 30% of CDK4-null mice do not develop diabetes. Although diabetes could affect postnatal growth, all CDK4−/−
mice display similar extents of growth retardation whether or not they develop symptomatic diabetes. In addition, postnatal death often observed by 4 weeks of age does not seem to be related to diabetes, since no CDK4−/−
mice developed glucosuria until 5 to 6 weeks. We are currently investigating the mechanism of the incomplete penetrance of diabetes and its relationship to other phenotypes of these mutant animals.
The delayed cell cycle entry of CDK4−/−
fibroblasts with impaired Rb phosphorylation indicates that CDK4 participates in the rate-limiting mechanism for the G0
-S transition. Diminished phosphorylation of Ser-780 of Rb, a specific target of cyclin D-dependent kinases (24
), further implies that in fibroblasts, CDK4 plays a central role in the initiation of Rb phosphorylation. Although CDK6 is bound to D-type cyclins, Rb kinase activity detected in CDK6 immunoprecipitates is very weak in both wild-type and CDK4−/−
). The question of whether CDK6 is essential for proliferation without CDK4 awaits future experiments to inactivate CDK6 in CDK4−/−
cells by gene targeting or other methods.
Our data also recapitulate the significance of CDK4-p27 interaction for the control of cyclin E-CDK2 activation. A larger amount of p27 that is free from sequestration could set a higher inhibitory threshold for the cyclin E-CDK2 activity. Therefore, p27 may act as an intermediate coordinator of CDK4 and CDK2 activation. The partial restoration of the kinetics of the G0-S transition in CDK4−/− p27−/− fibroblasts supports the hypothesis that CDK4 functions as an upstream regulator of p27, although the enhanced proliferation of CDK4−/− p27−/− cells may involve other regulatory mechanism(s) independent of the CDK4-p27 interaction, such as redistribution of other CDK inhibitors and modifications of CDK proteins. The mechanism of this enhanced S-phase entry is complex, since elevated CDK2 activity by p27 elimination might also compensate for the absence of CDK4, especially in Rb phosphorylation. We have observed that in serum-stimulated CDK4−/− p27−/− cells, the amounts of hyperphosphorylated Rb are slightly increased compared with those in CDK4−/− p27+/+ cells (data not shown).
In proliferating cells, a majority of p27 molecules are in complex with cyclin D-dependent kinases (44
), which does not necessarily inhibit the kinase activity (5
). In contrast, cyclin E-CDK2 and cyclin A-CDK2 appear to be inhibited whenever they bind to p27 (5
). Moreover, studies with inducible expression of p27 or in vitro-purified proteins have demonstrated that the amount of p27 required for inhibition of cyclin D-CDK4 is much larger than that required for the inhibition of cyclin A-CDK2 (5
). Our study together with these other studies suggests the significance of the p27-regulatory action of CDK4 in the G0
-S transition. The p27 reservoir function of CDK4 may allow multiple modes of control of G1
-CDK activity in response to various extracellular signals. For instance, transforming growth factor β decreases the expression of CDK4 (12
) and induces the CDK4-specific inhibitor p15INK4b
). These changes will disrupt the cyclin D-CDK4-p27 complex, leading to the inhibition of cyclin E-CDK2 and cyclin A-CDK2. Growth factor deprivation, differentiation, contact inhibition, or loss of anchorage will also decrease the expression of cyclin D and/or CDK4 (23
), resulting in the mobilization of p27 to the CDK2 complexes. In contrast, when cells enter the cell cycle from quiescence, mitogenic signals facilitate complex formation of cyclin D-CDK4 (7
), which will sequester p27 and allow the timely activation of cyclin E-CDK2.
Growth retardation and reproductive dysfunction in CDK4-null mice present an opposite image to the phenotype of mice deficient in p27, such as gigantism and gonadal hyperplasia (14
). These observations imply that p27 and CDK4 genetically counteract to regulate animal growth and reproductive function, which may depend on the stoichiometric interaction between these two molecules. Interestingly, 7-week-old mice deficient in both p27 and CDK4 (n
= 3) are 18 to 33% smaller than wild-type litter mice, whereas CDK4-null mice with intact p27 are 35 to 57% smaller than wild-type controls (24a
). CDK4- and p27-null mice provide unique models in which we can further investigate interactions of cell cycle-regulatory proteins and their roles in development and oncogenesis.