Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression.
(41/908)
Alterations in normal protein biogenesis and the resulting accumulation of improperly folded proteins in the endoplasmic reticulum (ER) trigger a stress response that up-regulates the expression of ER chaperones, while coordinately repressing overall protein synthesis and causing cell-cycle arrest. Activation of this unfolded protein response (UPR) in mouse NIH 3T3 fibroblasts with the glycosylation inhibitor tunicamycin led to a decline in cyclin D- and E-dependent kinase activities and to G(1) phase arrest. Cyclin D1 protein synthesis was rapidly inhibited by tunicamycin treatment. However, the drug did not significantly affect the mitogen-dependent activities of the extracellular signal-activated protein kinases ERK1 and ERK2 or the level of cyclin D1 mRNA until much later in the response. Therefore, the UPR triggers a signaling pathway that blocks cyclin D1 translation despite continuous mitogenic stimulation. Enforced overexpression of cyclin D1 in tunicamycin-treated cells maintained cyclin D- and E-dependent kinase activities and kept cells in cycle in the face of a fully activated UPR. Translational regulation of cyclin D1 in response to ER stress is a mechanism for checkpoint control that prevents cell-cycle progression until homeostasis is restored. (+info)
Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex.
(42/908)
Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor alpha (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function. (+info)
A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum.
(43/908)
A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies. (+info)
Retinoic acid prevents phosphorylation of pRB in normal human B lymphocytes: regulation of cyclin E, cyclin A, and p21(Cip1).
(44/908)
The mechanisms underlying the growth-inhibitory effect of retinoids on normal human B lymphocytes are not well understood. We addressed this issue by examining the effect of retinoic acid on the cell cycle machinery involved in G1/S transition. When retinoic acid was administered to B cells stimulated into mid to late G1 by anti-IgM antibodies (anti-mu) and Staphylococcus aureus crude cell suspension (SAC), the phosphorylation of pRB required for S-phase entry was prevented in a time- and dose-dependent manner. Thus, 2-hour treatment with retinoic acid at the optimal concentration of 1 micromol/L prevented phosphorylation of pRB, and effects were noted at concentrations as low as 10 nmol/L. Based on our results, we suggest that the rapid effect of retinoic acid on pRB phosphorylation is due primarily to the reduced expression of cyclin E and cyclin A in late G1. This could lead to the diminished cyclin E- and cyclin A-associated kinase activities noted as early as 2 hours after addition of retinoic acid. Furthermore, our results imply that the transient induction of p21(Cip1) could also be involved. Thus, retinoic acid induced a rapid, but transient increased binding of p21(Cip1) to CDK2. The retinoic acid receptor (RAR) agonist TTNPB mimicked the key events affected by retinoic acid, such as pRB phosphorylation, cyclin E expression, and expression of p21(Cip1), whereas the RAR-selective antagonist Ro 41-5253 counteracted the effects of retinoic acid. This implies that retinoic acid mediates its growth-inhibitory effect on B lymphocytes via the nuclear receptors. (+info)
Adenovirus-mediated decorin gene transfer prevents TGF-beta-induced inhibition of lung morphogenesis.
(45/908)
Excessive transforming growth factor (TGF)-beta signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-beta could be useful therapeutic agents. Because exogenous TGF-beta ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-beta activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-beta. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-beta efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-beta-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-beta-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-beta could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-beta-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-beta during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-beta signaling in the developing lung. (+info)
Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases.
(46/908)
Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G(2)/M transition. In the present study, we have investigated the function of human Cdc25A phosphatase. Cell lines that express human Cdc25A in an inducible manner have been generated. Ectopic expression of Cdc25A accelerates the G(1)/S-phase transition, indicating that Cdc25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human Cdc25A. Activation of endogenous Cdc25A occurs during late G(1) phase and increases in S and G(2) phases. We further demonstrate that Cdc25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, Cdc25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G(1). Overexpression of Cdc25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, Cdc25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that Cdc25A is an important regulator of the G(1)/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets. (+info)
A dual specificity promoter system combining cell cycle-regulated and tissue-specific transcriptional control.
(47/908)
The expression of both proliferation-associated and cell type-specific genes is a hallmark of both cancer cells and tumor endothelial cells. The possibility to combine both features in a single transcriptional control unit would greatly increase the selectivity of vectors used for cancer gene therapy. Previous studies by our laboratory have shown that the transcription of several cell cycle genes is regulated by a novel cell cycle-regulated repressor, termed CDF-1. This repressor functions by blocking in resting cells the transcriptional activation by specific factors binding to the upstream activating sequence (UAS), most notably the CCAAT-box binding factor NF-Y/CBF. Based on this work we have developed a dual specificity promoter system that combines cell type specificity with cell cycle regulation. A chimeric transcription factor (Gal4/NF-Y) consisting of the transactivation domain of NF-Y and the DNA-binding domain of Gal4 is expressed from a tissue-specific promoter. Gal4/NF-Y can bind to a second promoter consisting of a minimal cyclin A promoter with multiple Gal4 binding sites replacing the normal UAS. This leads to the tissue-specific expression of Gal4/NF-Y whose stimulatory activity on the promoter is restrained in resting cells by the recruitment of the CDF-1 repressor to the promoter. The functionality of this system is demonstrated for the specific transcriptional targeting of proliferating melanoma cells, where cell cycle regulation was >20-fold and cell type specificity was >50-fold. (+info)
Differential expression of the cyclin-dependent kinase inhibitor P27 in primary hepatocytes in early-mid G1 and G1/S transitions.
(48/908)
P27, an inhibitor of cyclin-dependent kinases, plays an important role in the control of cell adhesion and contact inhibition-dependent cell cycle regulation. Hepatocytes, maintained in primary culture, offer a model of synchronized primary epithelial cells which retain a differentiated profile while stimulated to proliferate. We therefore investigated the pattern of endogenous p27 expression in cyclin rat hepatocytes isolated by collagenase perfusion followed by mitogenic stimulation. P27 was expressed in whole normal liver and freshly isolated hepatocytes. We then observed a sharp decrease in p27 levels, concomitant with the progression in early-mid G1, followed by reaccumulation in late G1 and the G1/S transition. Immunochemistry and BrdU labelling demonstrated nuclear localization of p27 and its expression in cells engaged in both G1 and S phase. P27 was detected in late G1 in complexes containing cyclins D1, E and A. Cyclin E- and A-associated kinase activities, however, were detected at the G1/S transition and depletion experiments confirmed that most active complexes were free of p27. Phosphorylated forms of p27 were detected in unstimulated and stimulated hepatocytes in both early-mid G1 and G1/S. Finally, two-dimensional gel electrophoresis showed evidence for several forms of p27 with a distinct profile of distribution in quiescent and stimulated hepatocytes. Collectively, our data offer a model in which p27 shows a biphasic profile of accumulation, with the early decrease possibly involved in the progression through early and mid G1. In contrast with most cell types tested so far, the late G1 accumulation did not impair formation of active cyclin E- and A associated kinases, and thus G1/S transition. (+info)