Cyclin D


Table of contents

Cyclin D and apoptosis

Cyclin D1 is involved in the regulation of neuronal cell death. During neuronal apoptosis, Cyclin D1-dependent kinase activity is stimulated, due to an increase in Cyclin D1 levels. Artificial elevation of Cyclin D1 levels is sufficient to induce apoptosis, even in non-neural cell types. Cyclin D1-induced apoptosis, like neuronal apoptosis, can be inhibited by 21 kDa E1B, Bcl2 and pRb, but not by 55 kDa E1B. Overexpression of the Cyclin D-dependent kinase inhibitor p16INK4 protects neurons from apoptotic cell death, demonstrating that activation of endogenous Cyclin D1-dependent kinases is essential during neuronal apoptosis. These data support a model in which neuronal apoptosis results from an aborted attempt to activate the cell cycle in terminally differentiated neurons (Kranenburg, 1996).

Cyclin D1-deficient mice have small eyes with thin retinas. There is a lower level of retinal cell proliferation and a unique pattern of photoreceptor cell death. Death is first observed in scattered clusters of cells in the retina. It then appears to spread from these few cells to nearby photoreceptors, eventually producing extensive holes in the photoreceptor layer. These holes appear to be filled with interneurons from the inner nuclear layer. Deaths mainly occur during the second to fourth postnatal weeks. Other models of photoreceptor degeneration in rodents differ in that they occur more uniformly across the retina, with death proceeding over a longer period of time until all, or nearly all, of the photoreceptors degenerate. Expression of a bcl-2 transgene cannot prevent the death (Ma, 1998).

D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. This study reports that D-cyclins perform a rate-limiting antiapoptotic function in vivo. Acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. Adult hematopoietic stem cells are particularly dependent on D-cyclins for survival, and they are especially sensitive to cyclin D loss. Surprisingly, it was found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. This analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis (Choi, 2014).

Transcriptional regulation of Cyclin D

Integrin-mediated cell adhesion to the extracellular matrix is required for normal cell growth. Cyclin D1 is a key regulator of G1-to-S phase progression of the cell cycle. Integrin signaling through focal adhesion kinase (FAK) plays a role in the regulation of cell cycle progression, which correlates with changes in the expression of cyclin D1 and the cdk inhibitor, p21, induced by FAK. The roles of both cyclin D1 and p21 in the regulation of cell cycle progression by FAK have been investigated. Overexpression of a dominant-negative FAK mutant DeltaC14 suppresses cell cycle progression in p21(-/-) cells as effectively as in the control p21(+/+) cells. Furthermore, overexpression of ectopic cyclin D1 can rescue cell cycle inhibition by DeltaC14. These results suggested that cyclin D1, but not p21, is the primary functional target of FAK signaling pathways in cell cycle regulation. The mechanisms underlying the regulation of cyclin D1 expression by FAK signaling have been investigated. Using Northern blotting and cyclin D1 promoter/luciferase assays, FAK signaling is shown to regulate cyclin D1 expression at the transcriptional level. Using a series of cyclin D1 promoter mutants in luciferase assays as well as electrophoretic mobility shift assays (EMSA), it has been shown that the EtsB binding site mediates cyclin D1 promoter regulation by FAK. Finally, it has been shown that FAK regulation of cyclin D1 depends on integrin-mediated cell adhesion and is likely to occur through its activation of the Erk signaling pathway. Together, these studies demonstrate that transcriptional regulation of cyclin D1 by FAK signaling pathways contributes to the regulation of cell cycle progression in cell adhesion (Zhao, 2001).

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. The transcription factor KLF8 has been identified as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant DeltaC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated directly by KLF8 binding to the GT box A and by an indirect mechanism, through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, these data provide further insight into molecular mechanisms for FAK to regulate cell cycle progression (Zhao, 2003).

Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. DNA microarrays have been used to identify targets of Shh in these cells; Shh was found to activate a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis (Oliver, 2003).

The FoxO forkhead transcription factors FoxO4 (AFX), FoxO3a (FKHR.L1), and FoxO1a (FKHR) (homologs of Drosophila Foxo) represent important physiological targets of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) signaling. Overexpression or conditional activation of FoxO factors is able to antagonize many responses to constitutive PI3K/PKB activation including its effect on cellular proliferation. The FoxO-induced cell cycle arrest is partially mediated by enhanced transcription and protein expression of the cyclin-dependent kinase inhibitor p27(kip1). A p27(kip1)-independent mechanism has been identified that plays an important role in the antiproliferative effect of FoxO factors. Forced expression or conditional activation of FoxO factors leads to reduced protein expression of the D-type cyclins D1 and D2 and is associated with an impaired capacity of CDK4 to phosphorylate and inactivate the S-phase repressor pRb. Downregulation of D-type cyclins involves a transcriptional repression mechanism and does not require p27(kip1) function. Ectopic expression of cyclin D1 can partially overcome FoxO factor-induced cell cycle arrest, demonstrating that downregulation of D-type cyclins represents a physiologically relevant mechanism of FoxO-induced cell cycle inhibition (Schmidt, 2002).

Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing transcription of cell cycle regulators, including cyclin D1. Evidence is provided that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, protein-DNA interactions have been identified within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which has been termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. The finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter (Hilton, 2003).

A missense mutation within the histone acetyltransferase (HAT) domain of the TATA binding protein-associated factor TAF1 induces ts13 cells to undergo a late G(1) arrest and decreases cyclin D1 transcription. TAF1 mutants (Delta844-850 and Delta848-850, from which amino acids 844 through 850 and 848 through 850 have been deleted, respectively) deficient in HAT activity are unable to complement the ts13 defect in cell proliferation and cyclin D1 transcription. Chromatin immunoprecipitation assays revealed that histone H3 acetylation is reduced at the cyclin D1 promoter but not the c-fos promoter upon inactivation of TAF1 in ts13 cells. The hypoacetylation of H3 at the cyclin D1 promoter is reversed by treatment with trichostatin A (TSA), a histone deacetylase inhibitor, or by expression of TAF1 proteins that retain HAT activity. Transcription of a chimeric promoter containing the Sp1 sites of cyclin D1 and c-fos core remain TAF1 dependent in ts13 cells. Treatment with TSA restores full activity to the cyclin D1-c-fos chimera at 39.5 degrees C. In vivo genomic footprinting experiments indicate that protein-DNA interactions at the Sp1 sites of the cyclin D1 promoter are compromised at 39.5 degrees C in ts13 cells. These data have led to a hypothesis that TAF1-dependent histone acetylation facilitates transcription factor binding to the Sp1 sites, thereby activating cyclin D1 transcription and ultimately G(1)-to-S-phase progression (Hilton, 2005).

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the unique abilities of differentiation into any cell type of the organism (pluripotency) and indefinite self-renewal. H Rem2 GTPase, a suppressor of the p53 pathway, is up-regulated in hESCs and, by loss- and gain-of-function studies, that it is a major player in the maintenance of hESC self-renewal and pluripotency. Rem2 mediates the fibroblastic growth factor 2 (FGF2) signaling pathway to maintain proliferation of hESCs. Rem2 effects are mediated by suppressing the transcriptional activity of p53 and cyclin D(1) to maintain survival of hESCs. Importantly, Rem2 does this by preventing protein degradation during DNA damage. Given that Rem2 maintains hESCs, it was also shown to be as efficient as c-Myc by enhancing reprogramming of human somatic cells into iPSCs eightfold. Rem2 does this by accelerating the cell cycle and protecting from apoptosis via its effects on cyclin D(1) expression/localization and suppression of p53 transcription. The effects of Rem2 on cyclin D(1) are independent of p53 function. These results define the cell cycle and apoptosis as a rate-limiting step during the reprogramming phenomena. These studies highlight the possibility of reprogramming somatic cells by imposing hESC-specific cell cycle features for making safer iPSCs for cell therapy use (Edel, 2010).

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, a mouse embryonic stem (ES) cell line was engineered in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells (Das, 2010).

Post-transcriptional regulation of Cyclin D Levels and Degradation of Cyclin D

The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. Cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 is less than 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination, either in vitro or in vivo. It is markedly stabilized (t1/2 ~3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells. Although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated (Diehl, 1997a).

The retinoids are reported to reduce the incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein, but not mRNA expression. Exogenous cyclin D1 protein also declines after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms are active in this regulation of cyclin D1 expression. Treatment with ubiquitin-dependent proteasome inhibitors calpain inhibitor I and lactacystin each prevent this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, does not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appear after proteasome inhibitor treatments, suggesting that ubiquitinated species are present. To learn whether RA directly promotes degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degrades more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depends on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation (Langenfeld, 1997).

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells results in rapid loss of cyclin D1 protein that is completely reversible by calpain inhibitors. Actinomycin D and lovastatin induce rapid loss of cyclin D1 in prostate and breast cancer cells that is reversible by calpain inhibitors but not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increases the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates results in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increases cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation (Choi, 1997).

The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 reversibly arrests mouse and human cells in the G1 phase of the cell cycle. The stimulation of Ca(2+)-independent (autonomous) CaMK-II enzymatic activity, a barometer of in situ activated CaMK-II, is prevented by the same KN-93 concentrations that cause G1 phase arrest. KN-93 causes the retinoblastoma protein pRB to become dephosphorylated and the activity of both cdk2 and cdk4, two potential pRb kinases, to decrease. Neither the activity of p42MAP kinase, an early response G1 signaling molecule, nor the phosphorylation status or DNA-binding capability of the transcription factors serum response factor and cAMP responsive element-binding protein is altered during this G1 arrest. The protein levels of cyclin-dependent kinase 2 (cdk2) and cdk4 are unaffected during this G1 arrest and the total cellular levels of the cdk inhibitors p21cip1 and p27kip1 are not increased. Instead, the cdk4 activity decreases resulting from KN-93 are the result of a 75% decrease in cyclin D1 levels. In contrast, cyclin A and E levels are relatively constant. Cdk2 activity decreases are primarily the result of enhanced p27kip1 association with cdk2/cyclin E. All of these phenomena are unaffected by KN-93's inactive analog, KN-92, and are reversible upon KN-93 washout. The kinetics of recovery from cell cycle arrest are similar to those reported for other G1 phase blockers. These results suggest a mechanism by which G1 Ca2+ signals can be linked via calmodulin-dependent phosphorylations to the cell cycle-controlling machinery through cyclins and cdk inhibitors (Morris, 1998).

Cyclin D-Cdk targets

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF) has been analyzed. Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, Ser484 has been identified as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation (Voit, 1999).

Rb forms a repressor complex containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the cell cycle, which in turn regulates exit from G1 and from S phase, respectively (Zhang, 2000).

D-type cyclin-cdk4 complexes, which are only active in proliferating cells, can suppress the skeletal muscle differentiation program in proliferating myoblasts. Cyclin D-cdk activity can block the activity of the MEF2 family of transcriptional regulators, which are crucial regulators of skeletal muscle gene expression. Cyclin D-cdk activity blocks the association of MEF2C with the coactivator protein GRIP-1 and thereby inhibits the activity of MEF2. During skeletal muscle differentiation, GRIP-1 is localized to punctate nuclear structures and can apparently tether MEF2 to such structures. Cotransfection of GRIP-1 can both potentiate the transcriptional activity of a Gal4-MEF2C construct and induce MEF2C localization to punctate nuclear structures. Consistent with the absence of punctate nuclear GRIP-1 in proliferating myoblasts, it was found that ectopic cyclin D-cdk4 expression disrupts the localization of both GRIP-1 and MEF2C to these punctate subnuclear structures. These findings indicate that cyclin D-cdk4 activity represses skeletal muscle differentiation in proliferating cells by blocking the association of MEF2 with the coactivator GRIP-1 and concomitantly disrupts the association of these factors with punctate nuclear subdomains within the cell (Lazaro, 2002).

Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. This study shows that cyclin D1 deficiency increases mitochondrial size and activity that is rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1; (Drosophila homolog Erect wing), which induces nuclear-encoded mitochondrial genes, is repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function (Wang, 2006).

Cyclin D functions as a transcriptional regulator

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. This study developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, several proteins involved in transcription were uncovered. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, it was found that in developing mouse retinas - an organ that critically requires cyclin D1 function - cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. This approach, which termed 'genetic-proteomic', can be used to study the in vivo function of essentially any protein (Bienvenu, 2010).

Cyclin D and development

In many organisms, initiation and progression through the G1 phase of the cell cycle requires the activity of G1-specific cyclins (cyclin D and cyclin E) and their associated cyclin-dependent kinases (CDK2, CDK4, CDK6). The Caenorhabditis elegans genes cyd-1 and cdk-4, encoding proteins similar to cyclin D and its cognate cyclin-dependent kinases, respectively, are necessary for proper division of postembryonic blast cells. Animals deficient for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G1 cell cycle arrest. Moreover, ectopic expression of cyd-1 and cdk-4 in transgenic animals is sufficient to activate an S-phase reporter gene. No embryonic defects associated with depletion of either of these two gene products is observed, suggesting that their essential functions are restricted to postembryonic development. It is proposed that the cyd-1 and cdk-4 gene products are integral parts of the developmental control of larval cell proliferation through the regulation of G1 progression (Park, 1999).

Gastrulation in rodents is associated with an increase in the rate of growth and with the start of differentiation within the embryo proper. In an effort to understand the role played by the cell cycle control in these processes, expression of cyclin D1, D2, and D3 (three major positive regulators of the G1/S transition) has been investigated by in situ hybrization and RT-PCR. Cyclin D1 and D2 transcripts are first detected in the epiblast at gastrulation, when a proliferative burst occurs, and subsequently in its differentiated derivatives within the embryo proper, indicating that activation of their expression takes place prior to the differentiation of epiblast progenitors. In contrast, cyclin D3 transcript is undetectable in the epiblast itself and its expression is activated exclusively in extraembryonic tissues of both epiblast and trophoblast origin. During neurulation, expression of each cyclin D RNA is dynamically regulated along the anterior-posterior axis. In the hindbrain, cyclin D1 and D2 show distinct segment-specific restricted expression and this pattern is conserved between mouse and chick. These results strongly suggest that D-type cyclins act as developmental regulators (Wianny, 1998).

Formation of brain requires deftly balancing primary genesis of neurons and glia; accurate detection when sufficient cells of each type have been produced; timely shutdown of proliferation and complete removal of excess cells. The region and cell type-specific expression of cell cycle regulatory proteins, such as demonstrated for cyclin D2, may contribute to these processes. If so, regional brain development should be affected by alteration of cyclin expression. To test this hypothesis, the representation of specific cell types was examined in the cerebellum of animals lacking cyclin D2. The loss of this cyclin primarily affects two neuronal populations: granule cell number is reduced and stellate interneurons are nearly absent. Differences between null and wild-type siblings are obvious by the second postnatal week. Decreases in granule cell number arise from both reduction in primary neurogenesis and increase in apoptosis of cells that fail to differentiate. The dearth of stellate cells in the molecular layer indicates that emergence of this subpopulation requires cyclin D2 expression. Surprisingly, Golgi and basket interneurons, thought to originate from the same precursor pool as stellate cells, appear unaffected. These results suggest that cyclin D2 is required in the cerebellum not only for proliferation of the granule cell precursors but also for proper differentiation of granule and stellate interneurons (Huard, 1999).

Thyroid hormone is a major regulator of postnatal brain development, but the precise molecular mechanisms underlying its action in this organ remain poorly understood. Microarray analysis was used to identify new target genes in brain. Thyroid hormone treatment of hypothyroid Pax8-/- knockout mice, which lack thyroid follicular cells, elicited a very limited global effect on brain transcripts. This analysis mainly identified cyclin D2 as a new thyroid hormone target gene in the cerebellum of hypothyroid mice. Thyroid hormone receptor (TRalpha and/or TRß) knockout mice studies provide further genetic evidence that cyclin D2 is likely to mediate the antiapoptotic effect exerted by thyroid hormone on the cerebellum external granular layer neuroblasts but that this transcriptional activation is not directly exerted by the thyroid hormone receptors (Poquet, 2003).

Modulation of cell-cycle dynamics is required to regulate the number of cerebellar GABAergic interneurons and their rhythm of maturation

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, mice lacking the G1 phase-active cyclin D2 were examined. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. This study found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2-/- interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry (Leto, 2011).

Cyclin D regulates sexually dimorphic cell division in C. elegans

The C. elegans somatic gonadal precursor cell (SGP) divides asymmetrically to establish gonad-specific coordinates in both sexes. In addition, the SGP division is sexually dimorphic and initiates sex-specific programs of gonadogenesis. Wnt/MAPK signaling determines the gonadal axes, and the FKH-6 transcription factor specifies the male mode of SGP division. This paper demonstrated that C. elegans cyclin D controls POP-1/TCF asymmetry in the SGP daughters as well as fkh-6 and rnr expression in the SGPs. Although cyclin D mutants have delayed SGP divisions, the cyclin D defects are not mimicked by other methods of retarding the SGP division. EFL-1/E2F has an antagonistic effect on fkh-6 expression and gonadogenesis, which is relieved by cyclin D activity. It is proposed that cyclin D and other canonical regulators of the G1/S transition coordinate key regulators of axis formation and sex determination with cell cycle progression to achieve the sexually dimorphic SGP asymmetric division (Tilmann, 2005).

Asymmetric cell divisions are a widespread mechanism for generating diverse cell types during animal development. Model asymmetric divisions include those of the C. elegans zygote, the C. elegans EMS blastomere, and the Drosophila neuroblast and sensory organ precursor. This study has embarked on an in depth analysis of a different asymmetric division, that of the somatic gonadal precursor cell (SGP) in C. elegans. This division establishes the proximal-distal axis of the gonad of both sexes, and it is sexually dimorphic. By teasing apart the molecular regulators of the SGP division, it wll be learned how precursor cells establish an organ coordinate system and how asymmetric divisions can be modulated during development to generate distinct organs (Tilmann, 2005).

The C. elegans embryo generates a four-celled gonadal primordium that appears the same in XX hermaphrodites and XO males. Within the primordium, one SGP resides at each of the two opposite poles, and two germline precursors lie between. During the first larval stage (L1), the SGP divides asymmetrically in both sexes to generate proximal and distal daughters that establish gonadal axes. The SGP division is also sexually dimorphic with respect to both size and fate of its daughters. The hermaphrodite SGP makes distal and proximal daughters of roughly equal size, but the male SGP produces a smaller distal daughter and a larger proximal daughter. In addition, the SGP daughters exhibit sex-specific behaviors (e.g., migration) and generate sex-specific regulatory cells that control gonad elongation and germline proliferation. Therefore, the SGP asymmetric division initiates sex-specific programs of gonadogenesis that generate a double-armed ovotestis in hermaphrodites and a single-armed testis in males (Tilmann, 2005).

Two major pathways of regulation converge to regulate the SGP division. The first is gender neutral: the Wnt/MAPK pathway specifies the distal SGP daughter fate in both sexes. The terminal regulators of this pathway are POP-1/TCF and SYS-1/β-catenin, a DNA binding protein and its transcriptional coactivator, respectively. In the early embryo, activated Wnt/MAPK signaling promotes nuclear export of POP-1 so that the daughter cell receiving the Wnt signal has less nuclear POP-1 than its sister, a phenomenon called POP-1 asymmetry. A similar situation is observed after the SGP division: the distal daughter is specified by Wnt/MAPK activation and contains less nuclear POP-1 than its proximal sister. In mutants lacking POP-1, SYS-1, or upstream components of the Wnt/MAPK pathway, distal-specific cells are not made, and extra proximal-specific cells are sometimes seen; by contrast, gonads with excess SYS-1 produce extra distal cells and lack proximal cells. Therefore, the Wnt/MAPK pathway establishes the proximal-distal axes of both hermaphrodite and male gonads (Tilmann, 2005).

The second pathway controls the sexual dimorphism of the SGP division. Most important for this work are two transcription factors. FKH-6 is a forkhead transcription factor that specifies the male-specific SGP division during the first larval stage of development. FKH-6 controls sex determination, specifically in the SGPs, and does not affect sex determination in other tissues or at other times of gonadal development; however, it does have a second and more poorly defined late larval role in hermaphrodite gonadogenesis. TRA-1 is the C. elegans GLI transcription factor that acts in virtually all tissues to specify the female fat. The XO gonad is feminized and disorganized in fkh-6 null mutants, and the XX gonad is masculinized in tra-1 null mutants. The SGP division in fkh-6; tra-1 double mutants is hermaphrodite-like, indicating that TRA-1 acts upstream of FKH-6. Therefore, FKH-6 is the terminal regulator of the SGP male fate (Tilmann, 2005).

In this paper, the single C. elegans cyclin D gene, cyd-1, is identifed as a key regulator of axis formation and sex determination in the gonad. Cyclin D functions during L1, and it appears to be specific to the SGP and its immediate daughters. The cyd-1(q626) mutation is a missense allele, and its effects can be mimicked by cyd-1 RNAi. Cyclin D has its effect on gonadogenesis via the canonical cell cycle machinery, including the cyclin-dependent kinase 4 (cdk-4), Rb (lin-35), E2F (efl-1), DP (dpl-1), the CDK inhibitors (cki-1 and cki-2), and cyclin E (cye-1). Cyclin D affects axis determination in both sexes by promoting POP-1 asymmetry and controls the sex-specific cell size asymmetry of the male SGP division, apparently by relieving E2F repression of the fkh-6 gonad-specific sex determination gene. Cyclin D also affects rnr::GFP expression, specifically in SGPs, and slows the SGP cell cycle. However, other methods of slowing the SGP cell cycle do not mimic cyd-1 defects. It is proposed that cyclin D and regulators of the G1/S transition coordinate both specification of distal-proximal organ axes and specification of sexual fate with the cell cycle (Tilmann, 2005).

Cyclin-dependent kinases (CDKs) and their cyclin binding partners are best known as regulators of the cell cycle. Of particular importance to this work is cyclin D, which has been implicated in the control of the G1/S transition. In C. elegans, cyclin D and its CDK-4 partner promote the G1/S transition during most larval cell divisions Cyclin D and CDK-4 counteract LIN-35/Rb, EFL-1/E2F, DPL-1/DP, and CKI-1, which, in turn, inhibit cyclin E and CDK-2. Therefore, the cell cycle machinery governing the G1/S transition has been conserved in C. elegans (Tilmann, 2005).

cyd-1, the single C. elegans cyclin D gene, controls the asymmetric division of the SGP, a highly regulated division that establishes organ axes and embarks on sex-specific programs of gonadogenesis. Specifically, cyclin D affects both POP-1/TCF asymmetry and fkh-6 expression in the early gonad. POP-1/TCF asymmetry is controlled by Wnt/MAPK signaling, and, in the SGP daughters, it specifies gonadal axes. The FKH-6 transcription factor is a sex-determining gene that specifies the male gonadal fate. Therefore, cyd-1 affects two key regulators of early gonadogenesis (Tilmann, 2005).

The cyd-1 defects are specific for the SGP division. SGP divisions are delayed, but other larval blast cells divide normally; fkh-6 and rnr expression are affected in SGPs, but other cells express these genes normally; and POP-1 asymmetry is abolished in the SGPs, but it occurs normally in non-gonadal blast cells. Why are the SGPs affected so specifically in cyd-1(q626) mutants? One possibility might have been that cyd-1(q626) identifies a domain that mediates an SGP-specific control. However, gonad defects typical of cyd-1(q626) mutants were also observed after cyd-1 RNAi, which depletes cyclin D but does not eliminate it. It is suggested that cyd-1(q626) is a partial loss-of-function allele, and that the SGP division is particularly sensitive to the level of cyclin D activity. Interestingly, the cyd-1(cc600) mutation also acts in a cell type-specific manner, affecting only the division of embryonically derived coelomocytes, Therefore, individual cell divisions appear to have distinct requirements for cyd-1 activity (Tilmann, 2005).

The SGP cell division is delayed in cyd-1 mutants, raising the possibility that the cyclin D effects on POP-1 asymmetry and fkh-6 expression might be nonspecific. Cell cycle length has been implicated as a factor in both cell fate specification and proper asymmetric divisions both in C. elegans and Drosophila. However, delays of the SGP division by other methods did not mimic the cyd-1 effect. Thus, hydroxyurea treatment, which slows S phase and delays cell divisions, had no apparent effect on gonadal development. Furthermore, gon-4 RNAi, which delays the SGP division specifically, did not change the sex-specific asymmetry of the SGP division, POP-1 asymmetry, or fkh-6 expression. Therefore, a simple delay in cell division is not likely to explain the cyclin D effects on the SGP asymmetric division (Tilmann, 2005).

Cyclin D regulates three molecular markers in the SGPs or their daughters. In the SGP itself, cyclin D affects expression of both fkh-6::GFP and rnr::GFP, a marker of S phase. Since fkh-6 and rnr reporters are expressed with similar timing in the SGPs, an attractive idea is that cyd-1 coordinates expression of both fkh-6 and rnr as the cell progresses from G1 into S phase. In addition to its control of fkh-6 and rnr in the SGPs, cyclin D affects POP-1 asymmetry in the SGP daughters. The linkage between the cyclin D control of POP-1 asymmetry and the G1/S transition is less evident. POP-1 asymmetry is first observed in SGP daughter cells soon after the SGP division. One possibility is that CYD-1 controls events in the SGP mother cell to subsequently affect POP-1 asymmetry in her daughters. If this is the case, it is noted that fkh-6 is not required, because POP-1 asymmetry is not abolished in fkh-6 null mutants. Alternatively, CYD-1 might influence POP-1 asymmetry in the daughters themselves. Attempts were made to distinguish between these two possibilities with temperature shifts, but the experiment did not have sufficient resolution. Regardless, the CYD-1 regulation of POP-1 and fkh-6 appear to be distinct, suggesting that cyclin D is a common regulator of both gonadal regulators (Tilmann, 2005).

It was found that cyclin D works together with other major regulators of the G1/S transition to control the SGP asymmetric division. Most importantly, RNAi depletion of efl-1, which encodes an E2F-related transcription factor, suppressed the cyd-1 defects. This suppression was observed in both sexes, suggesting an effect of E2F on both POP-1 asymmetry and fkh-6 expression. Although the specific Wnt/MAPK signaling components regulated are not known, several lines of evidence suggest that fkh-6 is controlled antagonistically by cyclin D and E2F: an fkh-6 reporter was either not expressed at all in cyd-1 mutant SGPs or expressed late; by contrast, fkh-6 expression was restored in cyd-1; efl-1(RNAi) SGPs and was seen earlier than normal in efl-1(RNAi) SGPs. Therefore, cyclin D and E2F have opposite effects on fkh-6 expression. Given the gonadal feminization by depletion of cyclin D, one might think a priori that depletion of E2F might masculinize the hermaphrodite gonad. However, in wild-type L1s, fkh-6 is typically expressed in both hermaphrodite and male SGPs, and E2F depletion did not masculinize the hermaphrodite gonad. The simplest explanation is that E2F normally represses fkh-6 expression, and that cyclin D relieves that repression. Although a canonical E2F binding sequence was identified in the fkh-6 promoter, biochemical experiments will be required to learn whether fkh-6 is a direct target of E2F repression. It has been concluded that the G1/S regulatory machinery controls fkh-6 expression (Tilmann, 2005).

Cyclin D is not an essential regulator of all metazoan cell cycles, a conclusion based on RNAi, deletion, and nonsense mutants of the single cyclin D gene in C. elegans; on deletion mutants of the single cyclin D gene in Drosophila; and on a triple knockout of the three cyclin D genes in mice. Instead, cyclin D appears to be essential for a variety of developmental functions: regulation of the G1/S transition in most larval cell divisions in C. elegans, control of cell growth and cell number in Drosophila, and regulation of heart development and expansion of hematopoietic stem cells in mice. A caveat to interpreting these deletion studies is that other cyclins may fill in for cyclin D, an idea supported by genetic interactions with CDK-2 in Drosophila. Therefore, the scope of biological functions mediated by cyclin D and the G1/S cell cycle machinery more generally are poorly understood in metazoans (Tilmann, 2005).

Over the past ten years, it has become clear that G1/S cell cycle regulators are intimately linked with controls of cell fate. An early example was myoD, which drives muscle myoblasts out of the cell cycle by induction of p21(Waf1/Cip). In addition, myoD and cyclin D1 physically interact with each other and antagonize each other’s activities. Furthermore, Drosophila cyclin D/CDK-4 binds and stabilizes STAT92E, a control critical for embryonic segmentation, and mammalian cyclin E/CDK2 binds and phosphorylates cytosolic β-catenin, leading to its rapid degradation during G1. An effect of G1/S regulators on asymmetric divisions has also been seen in Drosophila. Most strikingly, cyclin E controls the asymmetric division of an NB6-4 neuronal precursor. However, other components of the cell cycle machinery were not involved in this asymmetric division, and, therefore, this Drosophila control may be distinct from that reported here. While these previous results have forged a link between the cell cycle machinery and controls of cell fate, the current findings extend this idea and suggest that cyclin D and other regulators of the G1/S transition coordinate the activity of multiple cell fate determinants during a single asymmetric cell division (Tilmann, 2005).

Cyclin D and coordination of cell cycle progression in spinal cord development

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. The integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation was investigated. In chicken embryo, it was found that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. Loss- and gain-of-function experiments demonstrate that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to the understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains (Labjois, 2004)

Cyclin D and germ cells

Protein levels of cyclin D3 are highly detectable only in thymus and testis in rats. Since testis offer unique opportunities to examine the cell cycle in vivo, the temporal and spatial expression of cyclin D3 and proliferating cell nuclear antigen (Drosophila homolog: PCNA, the DNA synthesis processivity factor) were studied in the rat testis during development. The protein levels of cyclin D3 protein in testis from 7 days to 3 months old are almost constant and then decrease gradually thereafter. The protein levels of cyclin D1 and PCNA are high in the testis of 7- and 14-day-old rats and decrease during testicular development. In the seminiferous tubules of 7-day-old newborns, cyclin D3 is surprisingly located in the cytoplasm of stem cells that have bigger nuclei than the nuclei of surrounding cells. Interestingly, cyclin D3 immunopositive cells do not immunostain with PCNA in nuclei. In the adult testis, anti-cyclin D3 antibody strongly stains the cytoplasm of early stage primary spermatocytes, lightly stains pachytene spermatocytes, but does not stain elongated spermatids. There is no detectable cyclin D3 in Sertoli cells, interstitial cells, or fibroblasts within seminiferous tubules, or in blood vessels within the interstitial matrix. The known cyclin D3 partner, cyclin dependent kinase 4, is located mainly in nuclei of spermatogonia and in early stage primary spermatocytes. Strong PCNA immunopositive staining is located in the nuclei of spermatogonia in adult testis. These results indicate that cyclin D3 is detectable in meiotically active male germ cells (PCNA-negative cells), but is conspicuously absent from mitotically active spermatogonia (PCNA-positive cells). Moreover, in contrast to in vitro reports, cyclin D3 is not located in the nucleus, but rather in the cytoplasm of male germ cells in vivo. Taken together, the presence of cyclin D3 in spermatocytes and its location in the cytoplasm leads to the speculation that cyclin D3 may have functions in male germ cells other than mitosis (Kang, 1997).

Table of contents

Cyclin D: Biological Overview | Regulation | Developmental Biology | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.