Retinoid X receptor agonists inhibit phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 cells into macrophages

Although metformin has been used to treat type 2 diabetes for several decades, the mechanism of its action on glucose metabolism remains controversial. To further assess the effect of metformin on glucose metabolism this work was undertaken to investigate the acute actions of metformin on glycogenolysis, glycolysis, gluconeogenesis, and ureogenesis in perfused rat livers. Metformin (5 mM) inhibited oxygen consumption and increased glycolysis and glycogenolysis in livers from fed rats. In perfused livers of fasted rats, the drug (concentrations higher than 1.0 mM) inhibited oxygen consumption and glucose production from lactate and pyruvate. Gluconeogenesis and ureogenesis from alanine were also inhibited. The cellular levels of ATP were decreased by metformin whereas the AMP levels of livers from fasted rats were increased. Taken together our results indicate that the energy status of the cell is probably compromised by metformin. The antihyperglycemic effect of metformin seems to be the result of a reduced oxidative phosphorylation without direct inhibition of key enzymatic activities of the gluconeogenic pathway. The AMP-activated protein kinase cascade could also be a probable target for metformin, which switches on catabolic pathways such as glycogenolysis and glycolysis, while switches off ATP consuming processes.     

Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos mRNA in a human neuroblastoma cell line.

Regulation of cholecystokinin (CCK) and the proto-oncogene c-fos mRNA expression was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated either with the tumor promoting phorbol-ester phorbol-12-myristate-13-acetate (PMA), the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c-fos mRNA was determined by Northern-blot analysis with CCK and c-fos specific antisense RNA probes after 4-24 h of drug treatment. Treatment with PMA and IBMX for 4-24 hours transiently raised the CCK mRNA level approximately 1.5-3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK mRNA abundance 1.5-6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c-fos mRNA level approximately 3-5.5 times. The drug combination showed a pronounced synergistic effect and raised the c-fos mRNA level approximately 3-20 times as compared to controls. Apparently, CCK and c-fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP-dependent mechanisms in SK-N-MC cells.     

PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA)

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.     

Evidence for cyclooxygenase-1 association with caveolin-1 and -2 in cultured human embryonic kidney (HEK 293) cells

The purpose of this study was to confirm protein-protein interaction between cyclooxygenase-1 (COX-1) and caveolins. The interaction of cyclooxygenase-1 and caveolins in the cultured human embryonic kidney (HEK 293) cells was investigated using immuno-precipitation and Western blot analysis. In HEK 293 cells, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Caveolae rich membranous fractions from the HEK 293 cells contained both COX-1 and caveolin-1 or caveolin-2 in same fractions. The experiments of immuno-precipitation showed complex formation between the COX-1 and caveolin-1 or caveolin-2 in the HEK 293 cells. Confocal microscopic results also support co-localization of COX-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with cylooxygenase-1 in caveolae suggested that caveolin would play an important role in regulating the function of COX-1.     

c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.     

Effect of phorbol 12-myristate 13-acetate (PMA) on the phosphoinositol (PI) system in Tetrahymena. Study of the 32P incorporation and breakdown of phospholipids

Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased ³²P incorporation into phospholipids namely phosphatadyl-inositol (PI); phosphatidyl-inositol-4-phosphate (PIP); phosphatidyl-inositol-4,5-bis-phosphate (PIP₂); phosphatidyl-acid (PA); phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE) particularly at the 20–30th min after treatment. The ratio of members of the phosphoinositol system, especially PIP and PI, related to the total phospholipid content was increased. PMA (2 × 10^?7 M ) was the most effective of the three concentrations tested. The results call attention to the presence of a working phosphoinositol system in Protozoa.     

Role of phorbol ester receptors in the 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-induced down-regulation of colony-stimulating factor (CSF-1) binding to murine peritoneal exudate macrophages

Treatment of murine peritoneal exudate macrophages (PEM) by tumor-promoting phorbol esters (TPA) results in a rapid loss of binding activity to radioactive-labeled colony-stimulating factor ([125I]-CSF-1) on the cell surface. The inhibitory effect of TPA on PEM is transient; treated cells recover full [125I]-CSF-1 binding activity in less than 6 hr at 37 degrees C either in the presence or after the removal of added TPA. The role of phorbol ester receptors in the induction of [125I]-CSF-1 binding inhibition was studied. The biologically active ligand [3H]-phorbol 12,13-dibutyrate ([3H]-PDBu) bound specifically to cultured murine PEM. At 0 degree C, stable and equilibrium binding occurred after 2-3 hr. Scatchard analysis revealed linear plots with a dissociation constant and receptor number per cell of 20.9 nM and 3.9 X 10(5)/cell, respectively. Treatment of PEM with biologically active phorbol esters at 37 degrees C rapidly inhibited the binding activity of [3H]-PDBu on cell surface (down-regulation) and rendered these cells refractory to the TPA-induced [125I]-CSF-1 binding inhibition by the subsequent TPA treatment. The inhibition of phorbol ester binding activity on TPA-treated PEM is caused by a reduction in the total number of available phorbol ester receptors rather than by a decrease in receptor affinity as judged by Scatchard analysis. The disappearance of [3H]-PDBu binding activity is reversible and transient. However, unlike CSF-1 receptors the restoration of phorbol ester receptors on TPA-treated PEM is a very slow process; a prolonged incubation of up to 72 hr after the removal of TPA was required for PEM to regain fully its [3H]-PDBu binding activity. Furthermore, the degree of TPA-induced CSF-1-receptor down-regulation is closely associated with the number of available phorbol ester receptors present on PEM at the time of treatment. Thus, the refractoriness to TPA diminished as the phorbol ester receptors on PEM recovered. A 72-hr incubation time at 37 degrees C was needed for PEM to lose their refractoriness and again become fully sensitive to TPA-induced CSF-1-receptor down-regulation. This study provides evidence that the loss of CSF-1-receptors induced by TPA treatment requires the presence of phorbol ester receptors and proceeds presumably via a co-internalization of both CSF-1 and phorbol ester receptors; the refractoriness to TPA is thereby induced by a transient loss of available phorbol ester receptors.     

Transient expression of recombinant ACKR4 (CCRL1) gene,an atypical chemokine receptor in human embryonic kidney (HEK 293) cells

ACKR4 also called CCX-CKR, CCRL1 as a member of atypical chemokine receptors, regulates the biological responses by clearance or transporting homeostatic chemokines such as CCL19, CCL21, CCL25, and CXCL13. Since these chemokines are involved in cancer development and metastasis, ACKR4 could have inhibition roles in cancer cell proliferation and invasion. Forming complexes with chemokine receptors by ACKR4 as in the case of hCXCR3 which lead to chemotaxis prevention is the other function of this protein is. However, as an atypical chemokine receptor, ACKR4 is less well-characterized compared to other members. Here, as the first step in understanding the molecular mechanisms of ACKR4 action, transfectants in HEK293T cell, was generated. In this study, ACKR4 coding sequence was cloned and human embryonic kidney 293T cells were used for recombinant production of ACKR4 protein. The liposome-mediated transfection with ACKR4 CDs, were detected in ACKR4 positive cells as early as 48 h post-transfection. The production of ACKR4 protein was confirmed using RT-PCR, dot blot, western blot, and flow cytometry. ACKR4 may represent a novel molecular target in cancer therapy, which might provide a chance for new therapeutic strategy. Therefore, the first step in the understanding of the molecular mechanisms of ACKR4 action is generation ACKR4-HEK293T recombinant cells.     

Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA

We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca2+]i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B2 release induced by collagen as well as the 'weak agonists', ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10-30 s) with PMA (400 nM) before agonist addition caused an inhibition (60-100%) of 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation in response to maximally effective doses of ADP (10 microM), adrenaline (10 microM) and PAF (0.5 microM) but potentiated collagen-induced 5-hydroxy[14C]tryptamine secretion and [3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20-50%) in the extent of collagen-induced 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation as seen earlier with thrombin, although collagen-induced [3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclo-oxygenase inhibitor, indomethacin (10 microM), abolished 5-hydroxy[14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5-10 micrograms/ml) -induced secretion by 50-90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by 'thromboxane-dependent' agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.     

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.     

Activation of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12-myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via the glucose transporter-1 (GLUT-1)

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.     

Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation

Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.     

cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3 - cotransporter pNBC1 in human embryonic kidney (HEK293) cells

Background

The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fission yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity.

Results

In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins.

Conclusion

The estradiol-regulatable hormone binding domain provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.     

Chitinase 3-like protein 2 (CHI3L2, YKL-39) activates phosphorylation of extracellular signal-regulated kinases ERK1/ERK2 in human embryonic kidney (HEK293) and human glioblastoma (U87 MG) cells

Human cartilage chitinase 3-like protein 2 (CHI3L2, YKL-39) is secreted by articular chondrocytes, also synoviocytes, lung, and heart. Increased levels of YKL-39 have been demonstrated in synovial fluids of patients with rheumatoid or osteoarthritis as well as in some other pathologies and in malignant tumors, particularly in glioblastomas. It belongs to glycosyl hydrolase family 18 and the most closely related to human cartilage glycoprotein 39 (HC gp-39 or chitinase 3-like protein 1, CHI3L1 or YKL-40), which as it was shown previously, promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts. Dose-dependent growth stimulation was observed when the fibroblastic cell line was exposed to YKL-40 in a concentration range from 0.1 to 2 nM, which is similar to the effective dose of the well characterized mitogen, insulinlike growth factor I. The use of selective inhibitors of the mitogen-activated protein kinase (MAP kinase) signaling pathway indicates that both, YKL-40 and IGF-I are involved in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2). Thus YKL-40 initiates a signaling cascade which leads to increased cell proliferation, suggesting that this protein could play some role in the inhibition of apoptosis. We report here that YKL-39, which as YKL-40 has significantly increased expression in glioblastomas, also activates signal-regulated kinases ERK1/ERK2 in human embryonic kidney (HEK293) and human glioblastoma (U87 MG) cells.     

Cytosolic retention of phosphorylated extracellular signal-regulated kinase and a Rho-associated kinase-mediated signal impair expression of p21(Cip1/Waf1) in phorbol 12-myristate-13- acetate-induced apoptotic cells

In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21(Cip1/Waf1) was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21(Cip1/Waf1) induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21(Cip1/Waf1) requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21(Cip1/Waf1) promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21(Cip1/Waf1) expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21(Cip1/Waf1) induction in PMA-induced proapoptotic TF-1 and D2 cells.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.