PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro‐inflammatory, suggesting that the establishment of endothelial polarity elicits an anti‐inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR‐3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow‐induced spatial distribution of PAR‐3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow‐dependent polarity to the flow axis, but is not necessary for flow‐induced anti‐inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.     

Phosphoinositide 3‐kinases in cell migration

Cell migration is essential for many biological processes in animals and is a complex highly co‐ordinated process that involves cell polarization, actin‐driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3‐kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro‐migratory stimuli, and the diverse ways in which PI3K‐mediated signal transduction contributes to different aspects of cell migration.     

GSK3β isoform‐selective regulation of depression,memory and hippocampal cell proliferation

Abnormally active glycogen synthase kinase‐3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood‐relevant behaviors, impairing performance in several cognitive tasks and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine‐to‐alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression‐like behavior. Three cognitive measures impaired in GSK3α/β knockin mice showed differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not in GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and co‐ordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of overexpression or disease pathology, is sufficient to impair mood regulation, novel object recognition and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results show that hyperactivity of the two GSK3 isoforms execute non‐redundant effects on these processes.     

Lectin BS‐I inhibits cell migration and invasion via AKT/GSK‐3β/β‐catenin pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is most common malignant cancer worldwide; however, the mortality rate of HCC remains high due to the invasion and metastasis of HCC. Thus, exploring novel treatments to prevent the invasion of HCC is needed for improving clinical outcome of this fatal disease. In this study, we identified lectin from Bandeiraea simplicifolia seeds (BS‐I) binds to metastasis‐associated HCC cell surface glycans by a lectin microarray and inhibits HCC cell migration and invasion through downregulating the matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and urokinase‐type plasminogen activator (uPA) production. These effects of BS‐I were mediated by inhibiting the activation of AKT/GSK‐3β/β‐catenin pathway and depended on specificity of lectin BS‐I binding to GalNAc. GSK3β inhibitors rescued BS‐I‐mediated inhibition of migration and invasion of HCC cell. Further, we identified that lectin BS‐I interacts with sGrp78, affects membrane localization of sGrp78 and attenuates the binding of sGrp78 and p85 to inhibit the activation of AKT/GSK‐3β/β‐catenin pathway. Overexpression of Grp78 or P85 rescues BS‐I‐mediated inhibition of migration and invasion of HCC cell. These findings demonstrated for the first time that BS‐I can act as a novel potential drug to prevent the invasion of HCC.     

Phosphorylation of LCRMP-1 by GSK3β promotes filopoda formation, migration and invasion abilities in lung cancer cells

LCRMP-1, a novel isoform of CRMP-1, can promote cancer cell migration, invasion and associate with poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). However, the underlying regulatory mechanisms of LCRMP-1 in cancer cell invasiveness still remain obscure. Here, we report that GSK3β can phosphorylate LCRMP-1 at Thr-628 in consensus sequences and this phosphorylation is crucial for function of LCRMP-1 to promote filopodia formation, migration and invasion in cancer cells. Impediment of Thr-628 phosphorylation attenuates the stimulatory effects of LCRMP-1 on filopodia forming, migration and invasion abilities in cancer cells; simultaneously, kinase-dead GSK3β diminishes regulation of LCRMP-1 on cancer cell invasion. Furthermore, we also found that patients with low-level Ser-9-phosphorylated GSK3β expression and high-level LCRMP-1 expression have worse overall survival than those with high-level inactive GSK3β expressions and low-level LCRMP-1 expressions (P<0.0001). Collectively, these results demonstrate that GSK3β-dependent phosphorylation of LCRMP-1 provides an important mechanism for regulation of LCRMP-1 on cancer cell invasiveness and clinical outcome.     

Alterations in the temporal expression and function of cadherin‐7 inhibit cell migration and condensation during chondrogenesis of chick limb mesenchymal cells in vitro

Endochondral bone formation requires a complex interplay among immature mesenchymal progenitor cells to form the cartilaginous anlagen, which involves migration, aggregation and condensation. Even though condensation of chondrogenic progenitors is an essential step in this process, its mechanism(s) has not been well studied. Here, we show that cadherin‐7 plays a central role in cellular condensation by modulating cell motility and migration. In this study, many mesenchymal cells failed to migrate, and precartilage condensation was inhibited, after knockdown of endogenous cadherin‐7 levels. Exposure of mesenchymal cells to SB203580 (a specific inhibitor of p38MAPK), LiCl (an inhibitor of GSK‐3β) or overexpression of β‐catenin resulted in inhibition of cadherin‐7 levels and, subsequently, suppression of cell migration. Collectively, our results suggest that cadherin‐7 controls cell migration in chick limb bud mesenchymal cells, and that p38MAPK and GSK signals are responsible for regulating cadherin‐7‐mediated cell migration. J. Cell. Physiol. 221: 161–170, 2009. © 2009 Wiley‐Liss, Inc     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

Combinatorial cell-specific regulation of GSK3 directs cell differentiation and polarity in Dictyostelium

In Dictyostelium, the interaction of secreted cAMP with specific cell surface receptors regulates the activation/de-activation of GSK3, which mediates developmental cell patterning. In addition, Dictyostelium cells polarize in response to extracellular cAMP, although a potential role for GSK3 in this pathway has not been investigated. Previously, we had shown that ZAK1 was an activating tyrosine kinase for GSK3 function in Dictyostelium and we now identify ZAK2 as the other tyrosine kinase in the cAMP-activation pathway for GSK3; no additional family members exist. We also now show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 separately regulate GSK3 in distinct differentiated cell populations, and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrate that efficient polarization of Dictyostelium towards cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration and cell differentiation, pathways that extend the complexity of GSK3 signaling throughout the development of Dictyostelium.     

Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

Deciphering what governs inflammation and its effects on tissues is vital for understanding many pathologies. The recent discovery that glycogen synthase kinase-3 (GSK3) promotes inflammation reveals a new component of its well-documented actions in several prevalent diseases which involve inflammation, including mood disorders, Alzheimer’s disease, diabetes, and cancer. Involvement in such disparate conditions stems from the widespread influences of GSK3 on many cellular functions, with this review focusing on its regulation of inflammatory processes. GSK3 promotes the production of inflammatory molecules and cell migration, which together make GSK3 a powerful regulator of inflammation, while GSK3 inhibition provides protection from inflammatory conditions in animal models. The involvement of GSK3 and inflammation in these diseases are highlighted. Thus, GSK3 may contribute not only to primary pathologies in these diseases, but also to the associated inflammation, suggesting that GSK3 inhibitors may have multiple effects influencing these conditions. Special issue dedicated to John P. Blass.     

糖原合酶激酶3β以细胞周期蛋白D1依赖性方式引发人肺腺癌细胞A549细胞周期阻滞

对糖原合酶激酶3β（glycogen synthase kinase 3β,6SK3β）在细胞增殖中的作用研究,在不同细胞系和不同刺激因素作用下得出了不同结论,本文旨在探讨GSK3β在人肺腺癌细胞系A549细胞生长中的直接作用。A549细胞瞬时转染持续激活型S9A-GSK3β以及显性负突变型KM-GSK3β两种GSK3β突变型质粒,改变GSK3β活性。24 h后,分别进行细胞计数,流式细胞术及Western blot检测。结果显示,增强GSK3β活性可导致细胞数量下降,G．期细胞百分比升高。细胞周期蛋白D1表达水平被GSK3β下调。结果提示,GSK3β可能以细胞周期蛋白D1依赖性方式引发A549细胞的G,期阻滞,从而发挥生长抑制效应。     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1''s central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

The influence of glycogen synthase kinase 3 in limiting cell addition in the mammalian ear

In the vestibular organs of the inner ear, an early postnatal decline in the capacity for cell proliferation appears to be responsible for limits to hair cell regeneration that are unique to mammals. We have investigated the time course of that decline in cell proliferation and its potential regulation by glycogen synthase kinase-3 (GSK3). Our immunoblots have revealed that inactive GSK3 beta decreases postnatally in the murine utricular epithelium, as E-cadherin and the active forms of GSK3 alpha and GSK3 beta each increase. In cultured utricular epithelia, pharmacological inhibition of GSK3 by LiCl and SB-216763 increased cell proliferation across a range of postnatal ages. LiCl treatments also led to increased levels of beta-catenin and Snail and decreased expression of E-cadherin. Transfection with a dominant-negative GSK3 beta enhanced proliferation in these epithelia in a cell-autonomous manner, while overexpression of wild-type GSK3 beta markedly reduced it. The evidence from these measurements and experimental manipulations indicates that the balance of active and inactive forms of GSK3 helps to determine whether mammalian vestibular supporting cells will proliferate; permitting proliferation during early development when inactive GSK3 predominates and progressively inhibiting proliferation, and thereby limiting the capacity for hair cell regeneration as more GSK3 becomes active during the first week of postnatal maturation.     

PI3K/Akt-dependent phosphorylation of GSK3β and activation of RhoA regulate Wnt5a-induced gastric cancer cell migration

Wnt5a, a non-transforming Wnt family member, plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in gastric cancer progression remains poorly defined. In this study, we found that Wnt5a dose-dependently stimulated the migration of human gastric cancer cells (SGC-7901), with the maximal effect at 100 ng/mL, via enhancing phosphorylation of PI3K/Akt and GSK3β and activating RhoA. Pharmaceutical inhibition of PI3K with LY294002 or Akt siRNA significantly decreased Wnt5a-induced GSK3β phosphorylation and consequently cell migration. Additionally, GSK3β siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Analogously, pre-treatment with LiCl, which induced phosphorylation of GSK3β at Ser9, increased Wnt5a-induced cell migration. Finally, ectopic expression of dominant negative RhoA (N19) suppressed Wnt5a-induced cell migration. Taken together, we demonstrated for the first time that Wnt5a promoted gastric cancer cell migration via the PI3K/Akt/GSK3β/RhoA signaling pathway. These findings could provide a rationale for designing new therapy targeting gastric cancer metastasis.     

Altering PI3K—Akt signalling in zebrafish embryos affects PTEN phosphorylation and gastrulation

Background information. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a negative regulator of the PI3K (phosphoinositide 3‐kinase)–Akt (also called protein kinase B) signalling pathway and is essential for embryogenesis, but its function in early vertebrate embryos is unclear. Results. To address how PTEN functions in early embryos, we overexpressed one of the four zebrafish PTEN isoforms at the 1–2‐cell stage. Overexpression of Ptena454 alters phospho‐Akt levels and impairs cell movements associated with gastrulation. Heat shocking embryos increases phospho‐Akt levels and lowers phospho‐Ptena454 levels. Inhibiting CK2 (protein kinase CK2) activity reduces phospho‐Pten levels and augments the effects due to Ptena454 overexpression. Low phospho‐Akt and corresponding low phospho‐GSK‐3 (glycogen synthase kinase‐3) and high phospho‐Pten levels accompany wortmannin or LY294002 treatment, which inhibit PI3K activity. Conclusions. These results suggest that Ptena454 regulation is correlated to changes in phospho‐Akt levels. We propose a model in which homoeostasis in rapidly dividing and migrating embryonic cells depends on a counterbalance between pro‐survival signalling employing CK2 and GSK‐3 and the pro‐apoptotic activity of Ptena454.     

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells

Lithium is a well‐established non‐competitive inhibitor of glycogen synthase kinase‐3β (GSK‐3β), a kinase that is involved in several cellular processes related to cancer progression. GSK‐3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK‐3β‐independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. J. Cell. Biochem. 117: 458–469, 2016. © 2015 Wiley Periodicals, Inc.