Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

The retinoids are reported to reduce 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 declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted 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 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended 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.

Induction of cyclin D1 by simian virus 40 small tumor antigen

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at −954 with additional contributions from a CRE site at −57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97–103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stress-activated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

α5β1 Integrin Controls Cyclin D1 Expression by Sustaining Mitogen-activated Protein Kinase Activity in Growth Factor-treated Cells

Cyclin D1 expression is jointly regulated by growth factors and cell adhesion to the extracellular matrix in many cell types. Growth factors are thought to regulate cyclin D1 expression because they stimulate sustained extracellular signal-regulated kinase (ERK) activity. However, we show here that growth factors induce transient ERK activity when added to suspended fibroblasts and sustained ERK activity only when added to adherent fibroblasts. Cell attachment to fibronectin or anti-α5β1 integrin is sufficient to sustain the ERK signal and to induce cyclin D1 in growth factor-treated cells. Moreover, when we force the sustained activation of ERK, by conditional expression of a constitutively active MAP kinase/ERK kinase, we overcome the adhesion requirement for expression of cyclin D1. Thus, at least in part, fibroblasts are mitogen and anchorage dependent, because integrin action allows for a sustained ERK signal and the expression of cyclin D1 in growth factor-treated cells.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor

Stimulation of dopamine D1 receptors has profound effects on addictive behavior, movement control, and working memory. Many of these functions depend on dopaminergic systems in the striatum and D1–D2 dopamine receptor synergies have been implicated as well. We show here that deletion of the D1 dopamine receptor produces a neural phenotype in which amphetamine and cocaine, two addictive psychomotor stimulants, can no longer stimulate neurons in the striatum to express cFos or JunB or to regulate dynorphin. By contrast, haloperidol, a typical neuroleptic that acts preferentially at D2-class receptors, remains effective in inducing catalepsy and striatal Fos/Jun expression in the D1 mutants, and these behavioral and neural effects can be blocked by D2 dopamine receptor agonists. These findings demonstrate that D2 dopamine receptors can function without the enabling role of D1 receptors but that D1 dopamine receptors are essential for the control of gene expression and motor behavior by psychomotor stimulants.

Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors

Dopamine D1, dopamine D2, and adenosine A2A receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D2 receptor antagonist eticlopride (0.1–2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A2A receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A2A receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D1 receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D1, D2, and A2A receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D2 antagonists in vivo.

Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes

The possible molecular basis for the previously described antagonistic interactions between adenosine A1 receptors (A1R) and dopamine D1 receptors (D1R) in the brain have been studied in mouse fibroblast Ltk− cells cotransfected with human A1R and D1R cDNAs or with human A1R and dopamine D2 receptor (long-form) (D2R) cDNAs and in cortical neurons in culture. A1R and D1R, but not A1R and D2R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A1R/D1R heteromerization disappeared after pretreatment with the D1R agonist, but not after combined pretreatment with D1R and A1R agonists. A high degree of A1R and D1R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A1R and D2R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A1R agonist caused coclustering (coaggregation) of A1R and D1R, which was blocked by combined pretreatment with the D1R and A1R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D1R and A1R agonists, but not with either one alone, substantially reduced the D1R agonist-induced accumulation of cAMP. The A1R/D1R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A1R of D1R receptor signaling in the brain. The persistence of A1R/D1R heteromerization seems to be essential for the blockade of A1R agonist-induced A1R/D1R coclustering and for the desensitization of the D1R agonist-induced cAMP accumulation seen on combined pretreatment with D1R and A1R agonists, which indicates a potential role of A1R/D1R heteromers also in desensitization mechanisms and receptor trafficking.

Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: Reversal by D1 antagonists

The conditioning of cocaine's subjective actions with environmental stimuli may be a critical factor in long-lasting relapse risk associated with cocaine addiction. To study the significance of learning factors in persistent addictive behavior as well as the neurobiological basis of this phenomenon, rats were trained to associate discriminative stimuli (SD) with the availability of i.v. cocaine vs. nonrewarding saline solution, and then placed on extinction conditions during which the i.v. solutions and SDs were withheld. The effects of reexposure to the SD on the recovery of responding at the previously cocaine-paired lever and on Fos protein expression then were determined in two groups. One group was tested immediately after extinction, whereas rats in the second group were confined to their home cages for an additional 4 months before testing. In both groups, the cocaine SD, but not the non-reward SD, elicited strong recovery of responding and increased Fos immunoreactivity in the basolateral amygdala and medial prefrontal cortex (areas Cg1/Cg3). The response reinstatement and Fos expression induced by the cocaine SD were both reversed by selective dopamine D1 receptor antagonists. The undiminished efficacy of the cocaine SD to elicit drug-seeking behavior after 4 months of abstinence parallels the long-lasting nature of conditioned cue reactivity and cue-induced cocaine craving in humans, and confirms a significant role of learning factors in the long-lasting addictive potential of cocaine. Moreover, the results implicate D1-dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala as substrates for cocaine-seeking behavior elicited by cocaine-predictive environmental stimuli.

Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at –52 and a novel site at –30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at –30 (–30–21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the –30–21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the –30–21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle.

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia.

To study the involvement of cyclin D1 in epithelial growth and differentiation and its putative role as an oncogene in skin, transgenic mice were developed carrying the human cyclin D1 gene driven by a bovine keratin 5 promoter. As expected, all squamous epithelia including skin, oral mucosa, trachea, vaginal epithelium, and the epithelial compartment of the thymus expressed aberrant levels of cyclin D1. The rate of epidermal proliferation increased dramatically in transgenic mice, which also showed basal cell hyperplasia. However, epidermal differentiation was unaffected, as shown by normal growth arrest of newborn primary keratinocytes in response to high extracellular calcium. Moreover, an unexpected phenotype was observed in the thymus. Transgenic mice developed a severe thymic hyperplasia that caused premature death due to cardio-respiratory failure within 4 months of age. By 14 weeks, the thymi of transgenic mice increased in weight up to 40-fold, representing 10% of total body weight. The hyperplastic thymi had normal histology revealing a well-differentiated cortex and medulla, which supported an apparently normal T-cell developmental program based on the distribution of thymocyte subsets. These results suggest that proliferation and differentiation of epithelial cells are under independent genetic controls in these organs and that cyclin D1 can modulate epithelial proliferation without altering the initiation of differentiation programs. No spontaneous development of epithelial tumors or thymic lymphomas was perceived in transgenic mice during their first 8 months of life, although they continue under observation. This model provides in vivo evidence of the action of cyclin D1 as a pure mediator of proliferation in epithelial cells.