Beta-catenin is not activated by downregulation of PTEN in osteoblasts

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell’s attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in β-catenin expression in cells with PTEN knockdown. The distribution of β-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in β-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, β-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.     

The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway

Phosphoinositide-3 kinase (PI-3 kinase) and its downstream signaling molecules PDK-1 and Akt were analyzed in SK-N-SH and SK-N-BE(2) human neuroblastoma cell lines. When cells were stimulated with insulin, PI-3 kinase was activated in both cell lines, whereas the translocation of PDK-1 to the membrane fraction and phosphorylated Akt were observed only in SK-N-SH cells. Analyses of the insulin-mediated reactive oxygen species (ROS) generation and Phosphatase and Tensin homolog (PTEN) oxidation indicate that PTEN oxidation occurred in SK-N-SH cells, which can produce ROS, but not in SK-N-BE(2) cells, which cannot increase ROS in response to insulin stimulation. When SK-N-SH cells were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride before insulin stimulation, insulin-mediated translocation of PDK-1 to the membrane fraction and phosphorylation of Akt were remarkably reduced, whereas PI-3 kinase activity was not changed significantly. These results indicate that not only PI-3 kinase activation but also inhibition of PTEN by ROS is needed to increase cellular level of phosphatidylinositol 3,4,5-trisphosphate for recruiting downstream signaling molecules such as PDK-1 and Akt in insulin-mediated signaling. Moreover, the ROS generated by insulin stimulation mainly contributes to the inactivation of PTEN and not to the activation of PI-3 kinase in the PI-3 kinase/Akt pathway.     

Phosphorylation of PTEN and Akt in astrocytes of the rat hippocampus following transient forebrain ischemia

To ascertain whether the PTEN (phosphatase and tensin homolog deleted on chromosome 10)/Akt signaling pathway is activated during ischemic brain injury, we investigated the expression and phosphorylation of PTEN and Akt by immunohistochemistry in the rat hippocampus after transient forebrain ischemia. Weak immunoreactivity for PTEN and its phosphorylated form (p-PTEN) was constitutively expressed in hippocampal neurons and astrocytes of the control rats, but their upregulation was detected mainly in reactive astrocytes in the ischemic hippocampus. Increased immunoreactivity for PTEN and p-PTEN occurred specifically in astrocytes by day 1 and was sustained for more than 2 weeks. The spatiotemporal activation of Akt in the ischemic hippocampus mirrored that of p-PTEN expression. Post-ischemic activation of Akt, revealed by phosphorylated Akt (p-Akt) immunoreactivity, was first detected at day 1 and was maintained for at least 2 weeks. Double-labeling experiments revealed that the cells expressing PTEN, p-PTEN, or p-Akt were reactive astrocytes expressing glial fibrillary acidic protein. These results demonstrate the increased phosphorylation of PTEN and Akt in reactive astrocytes of the post-ischemic hippocampus, suggesting that the PTEN/Akt pathway is involved in the astroglial reaction in the rat hippocampus after transient forebrain ischemia.This research was supported by Korea Science and Engineering Foundation (R01-2002-000-00334-0(2002)).     

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.     

Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis. Components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt). Also, mutations occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. These pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of elevated activated Akt levels to phosphorylate and inactivate Raf-1. We have investigated the genetic structures and functional roles of these two signaling pathways in the malignant transformation and drug resistance of hematopoietic, breast and prostate cancer cells. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell-lineage-specific effects. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines (FDC-P1 and TF-1), a trait associated with tumorigenesis. In contrast, expression of activated PI3K or Akt does not abrogate the cytokine dependence of these hematopoietic cell lines, but does have positive effects on cell survival. However, activated PI3K and Akt can synergize with activated Raf to abrogate the cytokine dependence of another hematopoietic cell line (FL5.12) which is not transformed by activated Raf expression by itself. Activated Raf and Akt also confer a drug-resistant phenotype to these cells. Raf is more associated with proliferation and the prevention of apoptosis while Akt is more associated with the long-term clonogenicity. In breast cancer cells, activated Raf conferred resistance to the chemotherapeutic drugs doxorubicin and paclitaxel. Raf induced the expression of the drug pump Mdr-1 (a.k.a., Pgp) and the Bcl-2 anti-apoptotic protein. Raf did not appear to induce drug resistance by altering p53/p21^Cip−1 expression, whose expression is often linked to regulation of cell cycle progression and drug resistance. Deregulation of the PI3K/PTEN/Akt pathway was associated with resistance to doxorubicin and 4-hydroxyl tamoxifen, a chemotherapeutic drug and estrogen receptor antagonist used in breast cancer therapy. In contrast to the drug-resistant breast cancer cells obtained after overexpression of activated Raf, cells expressing activated Akt displayed altered (decreased) levels of p53/p21^Cip−1. Deregulated expression of the central phosphatase in the PI3K/PTEN/Akt pathway led to breast cancer drug resistance. Introduction of mutated forms of PTEN, which lacked lipid phosphatase activity, increased the resistance of the MCF-7 cells to doxorubicin, suggesting that these lipid phosphatase deficient PTEN mutants acted as dominant negative mutants to suppress wild-type PTEN activity. Finally, the PI3K/PTEN/Akt pathway appears to be more prominently involved in prostate cancer drug resistance than the Raf/MEK/ERK pathway. Some advanced prostate cancer cells express elevated levels of activated Akt which may suppress Raf activation. Introduction of activated forms of Akt increased the drug resistance of advanced prostate cancer cells. In contrast, introduction of activated forms of Raf did not increase the drug resistance of the prostate cancer cells. In contrast to the results observed in hematopoietic cells, Raf may normally promote differentiation in prostate cells which is suppressed in advanced prostate cancer due to increased expression of activated Akt arising from PTEN mutation. Thus in advanced prostate cancer it may be advantageous to induce Raf expression to promote differentiation, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK-induced proliferation. These signaling and anti-apoptotic pathways can have different effects on growth, prevention of apoptosis and induction of drug resistance in cells of various lineages which may be due to the expression of lineage-specific factors.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.     

Relevance and Therapeutic Possibility of PTEN-Long in Renal Cell Carcinoma

PTEN-Long is a translational variant of PTEN (Phosphatase and Tensin Homolog). Like PTEN, PTEN-Long is able to antagonize the PI3K-Akt pathway and inhibits tumor growth. In this study, we investigated the role PTEN-Long plays in the development and progression of clear cell renal cell carcinoma (ccRCC) and explored the therapeutic possibility using proteinaceous PTEN-Long to treat ccRCC. We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt). Gain of function experiments showed overexpression of PTEN-Long in the ccRCC cell line 786-0 suppressed PI3K-Akt signaling, inhibited cell proliferation, migration and invasion, and eventually induced cell death. When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage. Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model. Our results implicated that understanding the roles of PTEN-Long in renal cell carcinogenesis has therapeutic significance.     

Akt participation in the Wnt signaling pathway through Dishevelled

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.     

Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH''s anabolic actions on bone.     

Modeling of Tumor Progression in NSCLC and Intrinsic Resistance to TKI in Loss of PTEN Expression

EGFR signaling plays a very important role in NSCLC. It activates Ras/ERK, PI3K/Akt and STAT activation pathways. These are the main pathways for cell proliferation and survival. We have developed two mathematical models to relate to the different EGFR signaling in NSCLC and normal cells in the presence or absence of EGFR and PTEN mutations. The dynamics of downstream signaling pathways vary in the disease state and activation of some factors can be indicative of drug resistance. Our simulation denotes the effect of EGFR mutations and increased expression of certain factors in NSCLC EGFR signaling on each of the three pathways where levels of pERK, pSTAT and pAkt are increased. Over activation of ERK, Akt and STAT3 which are the main cell proliferation and survival factors act as promoting factors for tumor progression in NSCLC. In case of loss of PTEN, Akt activity level is considerably increased. Our simulation results show that in the presence of erlotinib, downstream factors i.e. pAkt, pSTAT3 and pERK are inhibited. However, in case of loss of PTEN expression in the presence of erlotinib, pAkt level would not decrease which demonstrates that these cells are resistant to erlotinib.     

Binding and phosphorylation of par-4 by akt is essential for cancer cell survival

Activation of the PI3K-Akt pathway by loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) function, increased growth factor signaling, or oncogene expression renders cancer cells resistant to apoptotic signals and promotes tumor growth. Although Akt acts as a global survival signal, the molecular circuits of this pathway have not been completely established. We report that Akt physically binds to the pro-apoptotic protein Par-4 via the Par-4 leucine zipper domain and phosphorylates Par-4 to inhibit apoptosis. Suppression of Akt activation by the PI3K-inhibitor PTEN or LY294002, Akt expression by RNA-interference, or Akt function by dominant-negative Akt caused apoptosis in cancer cells. Apoptosis induced by inhibiting Akt was blocked by inhibition of Par-4 expression, but not by inhibition of other apoptosis agonists that are Akt substrates, suggesting that inhibition of the PI3K-Akt pathway leads to Par-4-dependent apoptosis. Thus, Par-4 is essential for PTEN-inducible apoptosis, and inactivation of Par-4 by Akt promotes cancer cell survival.     

Over-expression of PTEN on proliferation and apoptosis in canine mammary tumors cells

Phosphatase and tensin homolog (PTEN) is an important tumor-suppressor gene which constitutes an important PI3K/Akt pathway by regulating the signaling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth has been gaining increasing attention. However, the role of PTEN in regulating apoptosis of canine mammary tumors cells still needs further investigation. In this experiment, the effect of PTEN on proliferation and apoptosis in canine mammary tumors (CMT) cells was analyzed. As a result, gene and protein expression levels of apoptosis-related genes were detected. Eukaryotic expression vector pcDNA3.1+-PTEN were successfully constructed and stably transferred into canine CMT cells after geneticin (G418) selection. After pcDNA3.1+-PTEN transfection, compared with control group, the cells proliferation was inhibited and the cell apoptosis was increased in CMT cells. The expression of p-Akt was decreased and the apoptosis-related genes, such as caspase-3, caspase-9, and Bax, were increased. These data serve to demonstrate the function of PTEN on apoptosis and gene regulatory in PI3K/Akt pathway in CMT cells. Collectively, our data link the tumor-suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signaling molecules whose signal target is the functional inactivation of the apoptosis gene product.     

Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.     

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.