WNT unrelated activities in commercially available preparations of recombinant WNT3a

WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/β‐catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of β‐catenin, the central effector of the canonical WNT/β‐catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/β‐catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT‐independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand. J. Cell. Biochem. 111: 1077–1079, 2010. © 2010 Wiley‐Liss, Inc.     

The Mood‐Stabilizing Agent Valproate Inhibits the Activity of Glycogen Synthase Kinase‐3

Abstract : Valproic acid (VPA) is a potent broad‐spectrum anti‐epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein‐1 (AP‐1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c‐jun by glycogen synthase kinase (GSK)‐3β inhibits AP‐1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK‐3β. These results suggest that lithium may increase AP‐1 DNA binding activity by inhibiting GSK‐3β. In the present study, we sought to determine if VPA, like lithium, regulates GSK‐3. We have found that VPA concentration‐dependently inhibits both GSK‐3α and ‐3β, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH‐SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK‐3β‐mediated ³²P incorporation into two putative GSK‐3 substrates (~85 and 200 kDa), compatible with inhibition of endogenous GSK‐3β by VPA. Consistent with GSK‐3β inhibition, incubation of SH‐SY5Y cells with VPA results in a significant time‐dependent increase in both cytosolic and nuclear β‐catenin levels. GSK‐3β plays a critical role in the CNS by regulating various cytoskeletal processes as well as long‐term nuclear events and is a common target for both lithium and VPA ; inhibition of GSK‐3β in the CNS may thus underlie some of the long‐term therapeutic effects of mood‐stabilizing agents.     

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.     

GSK‐3β inhibition protects the rat heart from the lipopolysaccharide‐induced inflammation injury via suppressing FOXO3A activity

Sepsis‐induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK‐3β is involved in the modulation of sepsis. However, the signalling details of GSK‐3β regulation in endotoxin lipopolysaccharide (LPS)‐induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK‐3β phosphorylation at its active site (Y216) and up‐regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK‐3β inhibitors and further reversed through β‐catenin knock‐down. This pharmacological inhibition of GSK‐3β attenuated the LPS‐induced cell injury via mediating β‐catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK‐3β suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS‐induced model was also blocked by inhibition of GSK‐3β, which curbed both ERK and NF‐κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP‐activated protein kinase (AMPK). Our results demonstrate that GSK‐3β inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

In AβPP‐overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AβPP751 and GSK3β activation: effects mitigated by lithium and apparently relevant to sporadic inclusion‐body myositis

Muscle fiber degeneration in sporadic inclusion‐body myositis (s‐IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid‐β (Aβ)‐precursor protein 751 (AβPP751), Aβ, phosphorylated tau, and other ‘Alzheimer‐characteristic’ proteins. Proteasome inhibition is an important component of the s‐IBM pathogenesis. In brains of Alzheimer’s disease (AD) patients and AD transgenic‐mouse models, phosphorylation of neuronal AβPP695 (p‐AβPP) on Thr668 (equivalent to T724 of AβPP751) is considered detrimental because it increases generation of cytotoxic Aβ and induces tau phosphorylation. Activated glycogen synthase kinase3β (GSK3β) is involved in phosphorylation of both AβPP and tau. Lithium, an inhibitor of GSK3β, was reported to reduce levels of both the total AβPP and p‐AβPP in AD animal models. In relation to s‐IBM, we now show for the first time that (1) In AβPP‐overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3β activity and AβPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated‐AβPP, (ii) total amount of AβPP, (iii) Aβ oligomers, and (iv) GSK3β activity; and (c) lithium improves proteasome function. (2) In biopsied s‐IBM muscle fibers, GSK3β is significantly activated and AβPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3β inhibitors, might benefit s‐IBM patients.     

The natural agent rhein induces β‐catenin degradation and tumour growth arrest

The natural agent rhein is an ananthraquinone derivative of rhubarb, which has anticancer effects. To determine the mechanisms underlying the anticancer effects of rhein, we detected the effect of rhein on several oncoproteins. Here, we show that rhein induces β‐catenin degradation in both hepatoma cell HepG2 and cervical cancer cell Hela. Treatment of HepG2 and Hela cells with rhein shortens the half‐life of β‐catenin. The proteasome inhibitor MG132 blunts the downregulation of β‐catenin by rhein. The induction of β‐catenin degradation by rhein is dependent on GSK3 but independent of Akt. Treatment of HepG2 and Hela cells with GSK3 inhibitor or GSK3β knockdown abrogates the effect of rhein on β‐catenin. GSK3β knockdown compromises the inhibition of HepG2 and Hela cell growth by rhein. Furthermore, rhein dose not downregulate β‐catenin mutant that is deficient of phosphorylation at multiple residues including Ser33, Ser37, Thr41 and Ser45. Moreover, rhein induces cell cycle arrest at S phase in both HepG2 and Hela cells. Intraperitoneal administration of rhein suppresses tumour cells proliferation and tumour growth in HepG2 xenografts model. Finally, the levels of β‐catenin are reduced in rhein‐treated tumours. These data demonstrate that rhein can induce β‐catenin degradation and inhibit tumour growth.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Lithium chloride promotes proliferation of neural stem cells in vitro,possibly by triggering the Wnt signaling pathway

The objective of this study was to clarify the relationship between the effect and associated mechanisms of lithium chloride on neural stem cells (NSCs) and the Wnt signaling pathway. The expression of key molecules proteins related to the Wnt signaling pathway in the proliferation and differentiation of control NSCs and lithium chloride-treated NSCs was detected by Western blot analysis. Flow cytometry analysis was applied to study the cell cycle dynamics of control NSCs and NSCs treated with lithium chloride. The therapeutic concentrations of lithium chloride stimulated NSC proliferation. β-catenin expression gradually decreased, while Gsk-3β expression gradually increased (P?P?in vitro and preventing the cells from differentiating, which is potentially mediated by activation of the Wnt signaling pathway.