Cyclin D1 Degradation Research Articles

Mirk/dyrk1B Kinase Destabilizes Cyclin D1 by Phosphorylation at Threonine 288

The phosphorylation of cyclin D1 at threonine 286 by glycogen synthase kinase 3beta (GSK3beta) has been shown to be required for the ubiquitination and nuclear export of cyclin D1 and its subsequent degradation in the proteasome. The mutation of the nearby residue, threonine 288, to nonphosphorylatable alanine has also been shown to reduce the ubiquitination of cyclin D1, suggesting that phosphorylation at threonine 288 may also lead to degradation of cyclin D1. We now demonstrate that the G(0)/G(1)-active arginine-directed protein kinase Mirk/dyrk1B binds to cyclin D1 and phosphorylates cyclin D1 at threonine 288 in vivo and that the cyclin D1-T288A construct is more stable than wild-type cyclin D1. Transient overexpression of Mirk in nontransformed Mv1Lu lung epithelial cells blocked cells in G(0)/G(1). Depletion of endogenous Mirk by RNA interference increased cyclin D1 protein levels but not mRNA levels, indicating that Mirk destabilizes cyclin D1 protein. Destabilization was confirmed by induction of a stable Mirk transfectant of Mv1Lu cells, which blocked cell migration (Zou, Y., Lim, S., Lee, K., Deng, X., and Friedman, E. (2003) J. Biol. Chem. 278, 49573-49581), and caused a decrease in the half-life of endogenous cyclin D1, concomitant with an increase in Mirk expression. In vitro cyclin D1 was phosphorylated in an additive fashion by Mirk and GSK3beta. Mirk-phosphorylated cyclin D1 mutated at the GSK3beta phosphorylation site and was capable of phosphorylating cyclin D1 in the presence of the GSK3beta inhibitor LiCl. Mirk may function together with GSK3beta to assist cell arrest in G(0)/G(1) by destabilizing cyclin D1.

Cyclin D1 as a target for chemoprevention

Lung cancer is the leading cause of cancer mortality. Chemoprevention is an attractive strategy to combat this major public health problem. Pre-clinical and clinical studies have identified diverse candidate chemopreventive agents that affect cellular proliferation, differentiation, apoptosis and tumor angiogenesis, among other pathways. These pharmacological agents are undergoing testing through use of pre-clinical models and clinical trials. These studies have uncovered cyclin D1 as a chemoprevention target and a surrogate marker of chemopreventive response in the lung. Chemoprevention of tobacco-carcinogen transformed human bronchial epithelial (HBE) cells appears to be due at least partly to degradation of cyclin D1. These studies of cultured HBE cells were extended to the in vivo setting by examination of preneoplastic bronchial lesions that established the frequent aberrant expression of cyclin D1 in lung carcinogenesis. Certain retinoids, natural and synthetic derivatives of vitamin A, repress cyclin D1, but activation of the epidermal growth factor receptor (EGFR) induces cyclin D1. Retinoids and specific chemopreventive agents can activate the proteasome-dependent degradation of cyclin D1 and also repress EGFR expression, thereby reducing cyclin D1 levels. These actions oppose the mitogenic effects of cyclin D1. This is hypothesized to trigger G1 arrest and thereby permit repair of carcinogenic damage of genomic DNA. These and other pre-clinical and clinical studies that will be reviewed here indicate that cyclin D1 and perhaps other cyclins are attractive pharmacological targets for lung cancer chemoprevention.

Regulation of p27 in the process of neuroblastoma N2A differentiation.

Neuronal differentiation implies morphological and biochemical changes to generate a specialized neuron. N2A neuroblastoma cells can be promoted to undergo differentiation associated to neurites outgrowth, a process linked to the arrest of cell division. Using N2A cells as a model, we investigated the detailed molecular aspects on the involvement of p27 in dibutyryl cAMP-induced neuronal differentiation. In the undifferentiated N2A phenotype, an unusually high level of accumulated p27 protein mass was evidenced. Data suggest that in proliferating cells, p27 could be sequestered by direct interaction with cyclin D1, thus preventing its inhibitory action on cell cycle Cdks. Studies also indicate that p27 is functionally active and that its loss of action on Cdks in proliferating cells is due to its strong association with cyclin D1. Therefore, when cell differentiation is triggered, the action of p27 on Cdks seems to depend on both p27 and cyclin D1 degradation during the early steps of differentiation followed by late events of re-synthesis of active p27. In this context, an overexpression of p27 after N2A transfection with a mouse p27 clone induces the outgrowth of neurites associated with a decrease in cyclin D1 expression. On the other hand, treatment of N2A undifferentiated cells with c-myc antisense oligonucleotides led to a decrease in p27 and cyclin D1 levels, similar events as those in early stages of cell differentiation. Studies suggest that blockage in c-myc expression triggers early events in neuronal differentiation. These studies are of the utmost importance to elucidate regulatory mechanisms of molecules that play a critical role in the transition from a proliferating phenotype to differentiated cells.

Dictyostelium Differentiation-inducing Factor-3 Activates Glycogen Synthase Kinase-3β and Degrades Cyclin D1 in Mammalian Cells

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

Reduced cyclin D1 ubiquitination in UVB-induced murine squamous cell carcinomas

Ubiquitination of cyclin D1 signals for its proteosomal degradation. To assess the possibility that reduced cyclin D1 proteolysis is a putative mechanism for its accumulation during UVB-induced skin tumorigenesis, ubiquitination activity of cyclin D1 was assessed in UVB-induced murine SCCs. Cyclin D1 was rapidly ubiquitinated by control skin extract, whereas ubiquitination of cyclin D1 was significantly reduced in SCCs. Mutant cyclin D1, in which residues important for GSK3β-mediated degradation of cyclin D1 are altered to non-phosphorylatable alanine, was not ubiquitinated. We also observed phosphorylation-dependent inactivation of GSK3β in SCCs. Our results indicate reduced ubiquitination of cyclin D1 in UVB-induced murine SCCs and suggest that inactivation of GSK3β-dependent cyclin D1 degradation pathway contributes to the accumulation of cyclin D1 in UVB-induced murine SCCs.

Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells are known to express D-type cyclins at very low levels and these levels increase dramatically during in vitro and in vivo differentiation. Here, we investigate some of the signalling pathways regulating expression of cyclin D1 and progression to S phase, the Ras/Extracellular signal-regulated protein kinase (ERK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. We demonstrate that ERK phosphorylation is fully dispensable for the regulation of cyclin D1 level and for the progression from G1 to S phase in ES cells. By contrast, PI3-kinase activity is required for both. Differentiation induced by retinoic acid results in the gain of ERK-dependent control of cyclin D1 expression and of S phase progression. Differentiation is also paralleled by an increase in PI3-kinase activity. This leads (a) to an increase in the p70 S6 kinase-dependent regulation of the steady-state level of cyclin D1, and (b) to a concomitant decrease in the GSK3beta-dependent rate of cyclin D1 degradation. Altogether, these multiple pathways account for the dramatic increase in the level of cyclin D1 protein which parallels ES cell differentiation. Our studies suggest that PI3-kinase is an important regulator of the ES cell cycle and that its activity is not regulated by mitogen stimulation.

Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Activation and Cyclin/Cyclin-dependent Kinase Degradation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.

DNA Damage Invokes Mismatch Repair-dependent Cyclin D1 Attenuation and Retinoblastoma Signaling Pathways to Inhibit CDK2

DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G(2)/M progression. This damage led to RB dephosphorylation/activation and subsequent RB-dependent attenuation of cyclin A and CDK2 activity. In both Rb+/+ and Rb -/- cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.

Retinoic Acid-mediated Growth Arrest Requires Ubiquitylation and Degradation of the F-box Protein Skp2

The mechanism by which all-trans retinoic acid (ATRA) leads to a G(1) arrest of the cell cycle remains unclear. We show here that the decrease in D-type cyclin levels observed following ATRA treatment correlates with an increase in the rate of cyclin D1 ubiquitylation in both T-47D and MCF-7 breast cancer cell lines. However, MCF-7 cells are more resistant to ATRA than T-47D cells indicating that cyclin D1 degradation is not sufficient for ATRA-mediated arrest. We found a striking difference between these cells in that while ATRA induces an elevation in the cdk inhibitor p27 in T-47D cells, this is not observed in the ATRA-resistant MCF-7 cells. Furthermore, we demonstrate that ATRA promotes the ubiquitylation of Skp2, an F-box protein that targets p27 for degradation. Moreover, overexpression of Skp2 in T-47D cells prevents accumulation of p27 and promotes resistance to ATRA. In addition, overexpression of cyclin D1 in T-47D cells also promotes ATRA resistance. We found that the mechanism of ATRA-induced ubiquitylation of cyclin D1 and Skp2 is independent of CUL-1 expression and that ATRA can rescue cyclin D1 degradation in the uterine cell line SK-UT-1, where D-type cyclins are stabilized due to a specific defect in proteolysis. These data suggest that ATRA induces a novel pathway of ubiquitylation and that the degradation of the F-box protein Skp2 is the mechanism underlying p27 accumulation and cyclin E-cdk2 inactivation following ATRA treatment.

Cyclin proteolysis as a retinoid cancer prevention mechanism.

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT.

Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are deficient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF(Skp2) ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF(Skp2) ubiquitin ligase complex affects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF(Skp2) complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that differential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway.

Antitumor bicyclic hexapeptide RA-VII modulates cyclin D1 protein level.

A bicyclic hexapeptide, RA-VII or O-methyl deoxybouvardin, isolated from Rubia cordifolia, is known to inhibit protein biosynthesis in vitro and in vivo. We here demonstrate that the treatment of human colon cancer DLD-1 cells with RA-VII induces cell growth inhibition associated with a partial G1 arrest and a rapid decrease (below 2 h) in the level of cyclin D1 protein. Since cycloheximide, another protein synthesis inhibitor, neither decreased the amount of cyclin D1 in the cells nor arrested cells in G1 phase, it is unlikely that this RA-VII-induced reduction of cyclin D1 was fully dependent on its direct inhibitory effect of protein synthesis. Northern blot analysis revealed that RA-VII did not affect the level of cyclin D1 mRNA. Meanwhile, pre-treatment of cells with lactacystin, a proteasome inhibitor, abolished the RA-VII-induced decrease in cyclin D1. Moreover, RA-VII still decreased cyclin D1 protein in the presence of cycloheximide. These results indicate that the RA-VII-induced cyclin D1 decrease depends on cyclin D1 degradation via the ubiquitin-proteasome pathway and does not require additional protein synthesis. RA-VII might actively proceed the degradation process of cyclin D1 via the ubiquitin-proteasome pathway in DLD-1 cells.

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts.

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H2O2) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incubated with 3 mM or higher H2O2 concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expression of cyclin D1 and D2 upon H2O2 treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H2O2 was not due to induction of protein synthesis. These results indicate that H2O2 reversibly inhibits the ubiquitin-proteasome dependent degradation of cyclin D1 and D2, probably by transiently inhibiting ubiquitination and/or the proteasome.

A potential mechanism for fumonisin B1-mediated hepatocarcinogenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3β activity

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.

Oncogenic Ras-mediated Cell Growth Arrest and Apoptosis are Associated with Increased Ubiquitin-dependent Cyclin D1 Degradation

The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-Ras(Val-12) in Rat-1 fibroblasts resulted in G(1) growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-Ras(Val-12) results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-Ras(Val-12) expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G(1). Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-Ras(Val-12) resulted in activation of Akt kinase and inactivation of glycogen-synthase-3beta kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3beta kinase pathway.

Distinct Initiation and Maintenance Mechanisms Cooperate to Induce G1 Cell Cycle Arrest in Response to DNA Damage

DNA damage causes stabilization of p53, leading to G1 arrest through induction of p21 cip1. As this process requires transcription, several hours are needed to exert this response. We show that DNA damage causes an immediate and p53-independent G1 arrest, caused by rapid proteolysis of cyclin D1. Degradation is mediated through a previously unrecognized destruction box in cyclin D1 and leads to a release of p21 cip1 from CDK4 to inhibit CDK2. Interference with cyclin D1 degradation prevents initiation of G1 arrest and renders cells more susceptible to DNA damage, indicating that cyclin D1 degradation is an essential component of the cellular response to genotoxic stress. Thus, induction of G1 arrest in response to DNA damage is minimally a two step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability.

Ubiquitination of Free Cyclin D1 Is Independent of Phosphorylation on Threonine 286

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.

Beta2-adrenergic receptor agonists and cAMP arrest human cultured airway smooth muscle cells in the G(1) phase of the cell cycle: role of proteasome degradation of cyclin D1.

Hyperplasia of airway smooth muscle (ASM) contributes to the airway hyperresponsiveness that characterizes asthma. We have investigated the relationship between cAMP-induced growth arrest of ASM cells and thrombin-stimulated, extracellular-regulated protein kinase (ERK) activity, cyclin D1, and the restriction protein retinoblastoma. The beta(2)-adrenergic receptor agonist albuterol (100 nM) inhibited DNA synthesis after incubation with ASM for periods as brief as 1 h when these coincided with the timing of the restriction point. Inhibition of thrombin-stimulated DNA synthesis by albuterol (1-100 nM), 8-bromo-cAMP (300 microM), or prostaglandin E(2) (1 microM) was accompanied by a reduction in cyclin D1 protein levels. The ERK kinase inhibitor PD98059 (3-30 microM) attenuated thrombin-stimulated ERK phosphorylation and activity and the increase in cyclin D1 protein levels, as did albuterol (1-100 nM) or 8-bromo-cAMP (300 microM). In contrast, neither albuterol (100 nM) nor PD98059 (30 microM) reduced cyclin D1 mRNA levels between 4 and 20 h after thrombin addition, which suggests that elevation of cAMP regulates cyclin D1 by a post transcriptional mechanism. The proteasome inhibitor MG132 (30 and 100 nM) and the calpain I inhibitor N-acetyl-Leu-Leu-leucinal (10 microM) attenuated the reduction in thrombin-stimulated cyclin D1 levels in ASM exposed to albuterol (100 nM), 8-bromo-cAMP (300 microM), or the phosphodiesterase inhibitor isobutylmethylxanthine (100 microM). Thus, the cAMP-induced arrest of ASM in the G(1) phase of the cell cycle is associated with a proteasomal degradation of cyclin D1 protein and a reduced protein retinoblastoma phosphorylation that prevents passage through the restriction point.

Retinoic Acid Promotes Ubiquitination and Proteolysis of Cyclin D1 during Induced Tumor Cell Differentiation

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G(1) arrest. In contrast, retinoic acid receptor gamma-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G(1) and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor gamma restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Muscarinic Cholinergic Receptors Activate Both Inhibitory and Stimulatory Growth Mechanisms in NIH3T3 Cells

Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.