SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G(1)-->S progression by controlling the expression and cellular compartmentalization of cyclin D

SSeCKS, first isolated as a G(1)-->S inhibitor that is downregulated in src- and ras-transformed cells, is a major cytoskeleton-associated PKC substrate with tumor suppressor and kinase-scaffolding activities. Previous attempts at constitutive expression resulted in cell variants with truncated ectopic SSeCKS products. Here, we show that tetracycline-regulated SSeCKS expression in NIH 3T3 cells induces G(1) arrest marked by extracellular signal-regulated kinase 2-dependent decreases in cyclin D1 expression and pRb phosphorylation. Unexpectedly, the forced reexpression of cyclin D1 failed to rescue SSeCKS-induced G(1) arrest. Confocal microscopy analysis revealed cytoplasmic colocalization of cyclin D1 with SSeCKS. Because the SSeCKS gene encodes two potential cyclin-binding motifs (CY) flanking major in vivo protein kinase C (PKC) phosphorylation sites (Ser(507/515)), we addressed whether SSeCKS encodes a phosphorylation-dependent cyclin scaffolding function. Bacterially expressed SSeCKS-CY bound cyclins D1 and E, whereas K-->S mutations within either CY motif ablated binding. Activation of PKC in vivo caused a rapid translocation of cyclin D1 to the nucleus. Cell permeable, penetratin-linked peptides encoding wild-type SSeCKS-CY, but not K-->S or phospho-Ser(507/515) variants, released cyclin D1 from its cytoplasmic sequestration and induced higher saturation density in cyclin D1-overexpressor cells or rat embryo fibroblasts. Our data suggest that SSeCKS controls G(1)-->S progression by regulating the expression and localization of cyclin D1. These data suggest that downregulation of SSeCKS in tumor cells removes gating checkpoints for saturation density, an effect that may promote contact independence.     

SSeCKS Promotes Tumor Necrosis Factor-α Autocrine via Activating p38 and JNK Pathways in Schwann Cells

Tumor necrosis factor-alpha (TNF-α) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-α could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-α production induced by TNF-α, overexpression of SSeCKS could promoted TNF-α autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-α. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.     

SSeCKS promote beta-amyloid-induced PC12 cells neurotoxicity by up-regulating tau phosphorylation in Alzheimer’s disease

In Alzheimer’s disease, Beta-amyloid peptide (Aβ) could induce tau hyperphosphorylation which is the major cause of neuron apoptosis. However, the underlying mechanisms in the process remain unclear. In this study, Aβ-induced apoptosis and tau phosphorylation were investigated in differentiated PC12 cells. This Aβ-induced tau phosphorylation paralleled with the increase of expression and phosphorylation of Src-suppressed protein kinase C substrate (SSeCKS). By knocking down the expression of SSeCKS, Aβ-induced apoptosis and tau hyperphosphorylation in PC12 cells were partially rescued, and were increased further due to the overexpression of SSeCKS in PC12 cells. Also, the cell apoptosis and tau hyperphosphorylation were strongly decreased when the cells were pretreated with the protein kinase C inhibitor, Gö6983. In addition, Aβ-induced tau phosphorylation was also partially decreased due to the overexpression of SSeCKS in PC12cells. In summary, our data indicate that SSeCKS may play a critical role in Aβ-induced PC12 cells apoptosis through its phosphorylation.     

PTEN induces cell cycle arrest by decreasing the level and nuclear localization of cyclin D1

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3'-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G(1)-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G(1)- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.     

Dictyostelium differentiation-inducing factor-3 activates glycogen synthase kinase-3beta 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.     

Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Calmodulin and cyclin D anchoring sites on the Src-suppressed C kinase substrate, SSeCKS

SSeCKS and its human orthologue, Gravin, are large scaffolding proteins that are thought to facilitate mitogenic control by anchoring key signal mediators such as protein kinase (PK) C, PKA, the plasma membrane associated isoform of alpha-1,4-galactosyltransferase (GalTase), beta2-adrenergic receptor, and cyclins. SSeCKS is also a major PKC substrate and phosphatidylserine-dependent PKC binding protein whose phosphorylation sites shares homology with a site in the MARCKS protein that encodes phosphorylation-sensitive calmodulin (CaM) binding activity. In the present study, we mapped the in vitro binding sites for CaM and cyclins on SSeCKS. Four CaM binding sites were identified by binding assays that conform to the so-called 1-5-10 motif. Notably, CaM binding was antagonized by prephosphorylation of SSeCKS by PKC. We also identified two major cyclin binding (CY) sites that overlap a major PKC phosphorylation site in SSeCKS (Ser(507/515)), and showed that cyclin D binding is attenuated if SSeCKS is prephosphorylated by PKC. These data suggest that the scaffolding activities of SSeCKS are modulated by mitogenically stimulated kinases such as PKC.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human astrocytoma cells by the pyrrolo-1,5-benzoxazepine,PBOX-21

The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D(3) protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D(3)-CDK4 leading to a decrease in the levels of active cyclin D(3)-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D(3). However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.     

ERK/MAPK pathway is required for changes of cyclin D1 and B1 during phorbol 12-myristate 13-acetate-induced differentiation of K562 cells

Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of human erythroleukemic K562 cells is characterized by growth arrest, morphological change, and expression of megakaryocyte-specific proteins. We examined the possible involvement of cell cycle regulators with PMA-induced growth arrest and megakaryocytic differentiation of K562 cells. The concentrations of cyclin D1 and p21Waf1/Cip1 were dramatically increased, whereas those of cyclin B1 and cdc2 were decreased, by PMA treatment. The concentrations of most cyclin-dependent kinases (Cdk2, Cdk4, and Cdk6), however, were unchanged by PMA treatment. PD98059, a specific inhibitor of MEK1, partially prevented the increase in cyclin D1 caused by PMA and fully reversed the down-regulation of cyclin B1 protein seen in response to PMA treatment. Thus, it is demonstrated here that the PMA-mediated changes of cyclin D1 and B1 are the result of a persistent increase in extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity.     

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1

We have previously shown that the mitogenic effect of endothelin-1 (ET-1) in primary astrocytes is dependent on activation of both extracellular signal-regulated kinase (ERK)- and cytoskeleton (CSK)-dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET-1-induced expression of cyclins D1 and D3 is dependent on the ERK- and CSK-dependent pathways, respectively; moreover, a decrease in the levels of the cyclin-dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell-cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D-type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D-type cyclin-associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET-1, are regulated by distinct signaling pathways.     

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.     

Permanent growth arrest in irradiated human fibroblasts

Exposure of human fibroblasts to doses of ionizing radiation sufficient to cause a permanent growth arrest repressed the expression of genes induced late during G(0)/G(1)-phase traverse, including both cyclin A and cyclin E. In addition, radiation prevented the cell cycle-dependent activation of cyclin D1-associated kinase activity and the subsequent phosphorylation of the RB tumor suppressor protein. Exposure to radiation did not alter the cellular levels of cyclin D1 protein, nor did it alter the formation of cyclin D1-CDK4 complexes. Surprisingly, the repression of cyclin D1-associated kinase activity in damaged mitogen-stimulated quiescent cells could not be accounted for by a relative increase in the association of CDKN1A (also known as p21(Cip1)) with cyclin D1 complexes, nor was cyclin D1 activity targeted by increased levels of CDKN1A in irradiated, logarithmically growing cultures under conditions where cyclin A activity was acutely repressed. Therefore, a radiation-induced permanent growth arrest is mediated by pathways that are distinct from those that cause cell cycle delay in damaged cells involving repression of cyclin-dependent kinase activity by CDKN1A.     

IGF-1 induces Pin1 expression in promoting cell cycle S-phase entry.

Insulin-like growth factor I (IGF-1) is a well-established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr-Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1(-/-)mouse embryo fibroblast (MEF) cells are defective in re-entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1(-/-) MEF cells display a delayed cell cycle S-phase entry in response to IGF stimulation and that IGF-1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF-1 is mediated via the phosphatidylinositol 3-kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF-1-induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1(-/-) MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1(-/-) MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF-1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1-S transition.