Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase-->AKT-->GSK-3beta-->cyclin D1-->pRB pathway to block uterine epithelial cell proliferation

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.     

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.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.     

Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin

Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.     

beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase

Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21(ras) activation and beta-catenin/cyclin D1 up-regulation. In the present paper, we examined beta-catenin intracellular distribution and its association with E-cadherin. We observed an increased association between E-cadherin and beta-catenin in the lysyl-oxidase down-regulated cells during serum starvation. Moreover, we found that beta-catenin cytoplasmic and nuclear levels were increased, suggesting a failure of its down-regulation by the APC-GSK-3beta system, in particular the GSK-3beta phosphorylation of ser-33/37 and thr-41 of beta-catenin. Finally, we investigated the mechanisms leading to the observed cyclin D1 up-regulation. We showed that in the antisense lysyl oxidase cells the cyclin D1 promoter was activated through the LEF and the ATF/CRE sites in the proximal promoter. While the promoter activation through LEF is compatible with beta-catenin signaling, we investigated the possibility that the CRE-dependent activation might be linked to the down-regulation of lysyl oxidase. In fact, up-regulation of lysyl oxidase in a COS-7 cell model showed a significant diminution of the CREB protein binding to the cyclin D1 promoter, leading to a dramatic inhibition of its activity and a significant down-regulation of cyclin D1 protein level in vivo. Finally, our study describes some major anomalies occurring in lysyl oxidase down-regulated fibroblasts, related to beta-catenin signaling and cyclin D1 expression.     

pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.     

Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca2+/calmodulin in PTEN-negative U-87MG human glioblastoma cells

Clozapine (CZP), a dibenzodiazepine derivative with a piperazinyl side chain, is in clinical use as an antipsychotic drug. This study investigated the effect of CZP on the modulation of the PI3K/Akt/GSK-3beta pathway in PTEN-negative U-87MG glioblastoma cells. Treatment with CZP rapidly inhibited the basal and EGF-induced phosphorylation of Akt. The inhibition of Akt resulted in the dephosphorylation of GSK-3beta and increased GSK-3beta kinase activity. A voltage-sensitive Ca(2+) channel blocker and calmodulin (CaM) antagonists inhibited Akt phosphorylation, whereas elevation of the intracellular Ca(2+) concentration prevented CZP-induced dephosphorylation of Akt and GSK-3beta, suggesting that Ca(2+)/CaM participates in the inhibition of Akt by CZP in U-87MG cells. In addition, similar to LY294002, CZP arrested cell cycle progression at G0/G1 phase, which was accompanied by decreased expression of cyclin D1. The reduction in the cyclin D1 level induced by CZP was abrogated by the inhibition of GSK-3beta, the inhibition of proteasome-dependent proteolysis, or an increase in the intracellular Ca(2+) concentration. These results suggest that the antipsychotic drug CZP modulates the PI3K/Akt/GSK-3beta pathway by counteracting Ca(2+)/CaM in PTEN-negative U-87MG glioblastoma cells.     

Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C₂-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.     

Involvement of GSK-3beta and DYRK1B in differentiation-inducing factor-3-induced phosphorylation of cyclin D1 in HeLa cells

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.     

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.     

The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.     

GSK-3b inhibition: At the crossroad between Akt and mTOR constitutive activation to enhance cyclin D1 protein stability in mantle cell lymphoma

Strategies able to down-regulate the aberrant expression of cyclin D1 may prove of therapeutic relevance in cancer patients. This is particularly true for mantle cell lymphoma (MCL) in which cyclin D1 is overexpressed as a consequence of the t(11;14)(q13;q32) translocation. We have recently demonstrated that an increased cyclin D1 stability also contributes to the high levels of this protein observed in MCL cells. This effect is mediated by a constitutive activation of PI3-K/Akt, which keeps GSK-3b inhibited. Here we show that inhibition of PI3-K/Akt induces a 40% decrease of cyclin D1 half-life as a result of accumulation of the dephosphorylated/active form of GSK-3b within the nucleus, where this kinase can phosphorylate cyclin D1 on Thr286 thereby promoting its nuclear export. Translocation of cyclin D1 into the cytoplasm is mediated by the nuclear exportin CRM1, whose association with cyclin D1 increases following PI3-K/Akt inhibition. Notably, rapamycin down-regulated GSK-3b Ser9 phosphorylation with concurrent nuclear export of cyclin D1 only in MCL cells in which GSK-3b is under the control of mTOR. These findings suggest that the ability to down-regulate cyclin D1 through GSK-3b may identify subsets of MCL patients who may benefit from the treatment with mTOR inhibitors and stimulate further studies to assess whether the inability to affect GSK-3b activity may constitute a clinically relevant resistance factor to mTOR inhibitors.