Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells

We have recently reported the chondrogenic effect of bone morphogenetic protein-2 (BMP-2) in high density cultures of the mouse multipotent mesenchymal C3H10T1/2 cell line and have shown the functional requirement of the cell-cell adhesion molecule N-cadherin in BMP-2-induced chondrogenesis in vitro (Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Differentiation 59, 25-34; Haas, A. R., and Tuan, R. S. (1999) Differentiation 64, 77-89). Furthermore, BMP-2 treatment also results in an increased protein level of beta-catenin, a known N-cadherin-associated Wnt signal transducer (Fischer, L., Haas, A., and Tuan, R. S. (2001) Signal Transduction 2, 66-78), suggesting functional cross-talk between the BMP-2 and Wnt signaling pathways. We have observed previously that BMP-2 treatment up-regulates expression of Wnt-3A in high density cultures of C3H10T1/2 cells. To assess the contribution of Wnt-3A to BMP-2-mediated chondrogenesis, we have generated C3H10T1/2 cell lines overexpressing Wnt-3A and various forms of glycogen synthase kinase-3beta (GSK-3beta), an immediate cytosolic component of the Wnt signaling pathway, and examined their response to BMP-2. We show that overexpression of either Wnt-3A or kinase-dead GSK-3beta enhances BMP-2-mediated chondrogenesis. Furthermore, Wnt-3A overexpression results in decreases in both N-cadherin and GSK-3beta protein levels, whereas Wnt-3A as well as kinase-dead GSK-3beta overexpression increase total and nuclear levels of both beta-catenin and LEF-1. Direct cross-talk between Wnts and BMP-2 was also indicated by the up-regulated interaction between beta-catenin and SMAD-4 in response to BMP-2. These results suggest that Wnt-3A acts in a manner opposite to that of other Wnts, such as Wnt-7A, which were previously identified as inhibitory to chondrogenesis, and is the first BMP-2-regulated, chondrogenesis-enhancing member of the Wnt family.     

Unrestrained glycogen synthase kinase-3 beta activity leads to activated T cell death and can be inhibited by natural adjuvant

Activated T cell death (ATCD) after peak clonal expansion is required for effective homeostasis of the immune system. Using a mouse model of T cell clonal expansion and contraction, we found that regulation of the proapoptotic kinase glycogen synthase kinase (GSK)-3beta plays a decisive role in determining the extent to which T cells are eliminated after activation. Involvement of GSK-3beta in ATCD was tested by measuring T cell survival after GSK-3beta inhibition, either ex vivo with chemical and pharmacological inhibitors or in vivo by retroviral expression of a dominant-negative form of GSK-3. We also measured amounts of inactivating phosphorylation of GSK-3beta (Ser9) in T cells primed in the presence or absence of LPS. Our results show that GSK-3beta activity is required for ATCD and that its inhibition promoted T cell survival. Adjuvant treatment in vivo maintained GSK-3beta (Ser9) phosphorylation in activated T cells, whereas with adjuvant-free stimulation it peaked and then decayed as the cells became susceptible to ATCD. We conclude that the duration of GSK-3beta inactivation determines activated T cell survival and that natural adjuvant stimulation decreases the severity of clonal contraction in part by keeping a critical proapoptotic regulatory factor, GSK-3beta, inactivated.     

Glycogen synthase kinase 3beta is a negative regulator of growth factor-induced activation of the c-Jun N-terminal kinase

The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3beta (GSK-3beta) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3beta also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3beta decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3beta null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3beta-/- MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3beta in GSK-3beta-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3beta through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3beta kinase activity was required for GSK-3beta to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3beta serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3beta in signal transduction and cellular responses to growth factors.     

Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3 beta phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3 beta phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3beta antibody. Endogenous IGF-I stimulated GSK-3 beta phosphorylation and inhibited GSK-3 beta activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3 beta phosphorylation and the resulting GSK-3 beta inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced beta-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3 beta inhibitor. Endogenous IGF-I inhibited beta-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3 beta activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.     

Identification of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) as a novel downstream target of phosphatidylinositol 3-kinase/AKT/GSK-3beta pathway

The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/AKT, which is involved in cell survival, proliferation, and growth, has become a major focus in targeting cancer therapeutics. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was previously identified as a gene induced by several anti-tumorigenic compounds including nonsteroidal anti-inflammatory drugs, peroxisome proliferator-activated receptor gamma ligands, and dietary compounds. NAG-1 has been shown to exhibit anti-tumorigenic and/or pro-apoptotic activities in vivo and in vitro. In this report, we showed a PI3K/AKT/glycogen synthase kinase-3beta (GSK-3beta) pathway regulates NAG-1 expression in human colorectal cancer cells as assessed by the inhibition of PI3K, AKT, and GSK-3beta. PI3K inhibition by LY294002 showed an increase in NAG-1 protein and mRNA expression, and 1l-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor) also induced NAG-1 expression. LY294002 caused increased apoptosis, cell cycle, and cell growth arrest in HCT-116 cells. Inhibition of GSK-3beta, which is negatively regulated by AKT, using AR-A014418 and lithium chloride completely abolished LY294002-induced NAG-1 expression as well as the NAG-1 promoter activity. Furthermore, the down-regulation of GSK-3 gene using small interference RNA resulted in a decline of the NAG-1 expression in the presence of LY294002. These data suggest that expression of NAG-1 is regulated by PI3K/AKT/GSK-3beta pathway in HCT-116 cells and may provide a further understanding of the important role of PI3K/AKT/GSK-3beta pathway in tumorigenesis.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Inhibition of glycogen synthase kinase-3beta is sufficient for airway smooth muscle hypertrophy

We examined the role of glycogen synthase kinase-3beta (GSK-3beta) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3beta, each of which inhibit GSK-3beta activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-beta, a proasthmatic cytokine. GSK-3beta inhibition increased mRNA expression of alpha-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon) attenuated LiCl- and SB216763-induced protein synthesis and expression of alpha-actin and SM22, indicating that eIF2B is required for GSK-3beta-mediated airway smooth muscle hypertrophy. eIF2Bepsilon siRNA also blocked CT-1- but not TGF-beta-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3beta-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3beta, blocked protein synthesis and alpha-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-beta. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased alpha-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3beta phosphorylation. These data suggest that inhibition of the GSK-3beta/eIF2Bepsilon translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-beta-induced hypertrophy does not depend on GSK-3beta/eIF2B signaling.     

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.     

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.     

Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of alpha v beta 3 and alpha 5 beta 1 integrins

Protein kinase B (PKB)/Akt is known to promote cell migration, and this may contribute to the enhanced invasiveness of malignant cells. To elucidate potential mechanisms by which PKB/Akt promotes the migration phenotype, we have investigated its role in the endosomal transport and recycling of integrins. Whereas the internalization of alpha v beta 3 and alpha 5 beta 1 integrins and their transport to the recycling compartment were independent of PKB/Akt, the return of these integrins (but not internalized transferrin) to the plasma membrane was regulated by phosphatidylinositol 3-kinases and PKB/Akt. The blockade of integrin recycling and cell spreading on integrin ligands effected by inhibition of PKB/Akt was reversed by inhibition of glycogen synthase kinase 3 (GSK-3). Moreover, expression of nonphosphorylatable active GSK-3 beta mutant GSK-3 beta-A9 suppressed recycling of alpha 5 beta 1 and alpha v beta 3 and reduced cell spreading on ligands for these integrins, indicating that PKB/Akt promotes integrin recycling by phosphorylating and inactivating GSK-3. We propose that the ability of PKB/Akt to act via GSK-3 to promote the recycling of matrix receptors represents a key mechanism whereby integrin function and cell migration can be regulated by growth factors.     

Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3 beta (GSK-3 beta), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3 beta is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3 beta and inhibition of its protein kinase activity, indicating that GSK-3 beta is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3 beta inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3 beta, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3 beta reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3 beta caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3 beta, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3 beta function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3 beta-independent pathways.     

Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability

Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta, inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of Dvl-1(DeltaPDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.     

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.     

Phospholipase C-independent activation of glycogen synthase kinase-3beta and C-terminal Src kinase by Galphaq

It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.     

GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.