GSK Inhibitors Research Articles

Glycogen synthase kinase 3 (GSK3) inhibition: a potential therapeutic strategy for Alzheimer's disease.

Alzheimer's disease (AD), the most common type of dementia among older adults, is a chronic neurodegenerative pathology that causes a progressive loss of cognitive functioning with a decline of rational skills. It is well known that AD is multifactorial, so there are many different pharmacological targets that can be pursued. According to estimates from the World Health Organization (WHO), 18 million individuals worldwide suffer from AD. Major initiatives to identify risk factors, enhance care giving, and conduct basic research to delay the beginning of AD were started by the USA, France, Germany, France, and various other nations. Widely recognized as a key player in the development and subsequent progression of AD pathogenesis, glycogen synthase kinase-3 (GSK-3) controls a number of crucial targets associated with neuronal degeneration. GSK-3 inhibition has been linked to reduced tau hyperphosphorylation, β-amyloid formation, and neuroprotective benefits in Alzheimer's disease. Lithium, the very first inhibitor ofGSK-3βthat was used therapeutically,has been successfully used for many years with remarkable results. A great variety of structurally varied strong GSK-3β blockers have been identified in recent years. The purpose of this thorough review is to cover the biological and structural elements of glycogen synthase kinase, as well as the medicinal chemistry aspects of GSK inhibitors that have been produced in recent years.

High-Throughput Chemical Screen on Acute Myeloid Leukemia Stem Cells Identifies Novel Anti-LSC Compounds

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence.High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells.From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and <30% inhibition on CD34+ CB cells. We also identified glucocorticoids, which were also identified in our prior small-scale anti-LSC screen where they were found to specifically drive human LSCs to terminally differentiate (Laverdière & Boileau, et al., 2018).We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo.We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. DisclosuresNo relevant conflicts of interest to declare.

Copper nanoparticles induce the formation of fatty liver in Takifugu fasciatus triggered by the PERK-EIF2α- SREBP-1c pathway

Copper nanoparticles (CuNPs), a new pollutant in water environments, were widely used in various industrial and commercial applications. This study indicated that the presence of CuNPs exposure under environmental related concentration is an inducing factor that contributes to the fatty liver formation in Takifugu fasciatus. Furthermore, we explored the fatty liver formation mechanism. The results shown, (1) the cloned genes related to endoplasmic reticulum stress (ERS) (GRP78, IRE-1α, PERK, and ATF-6α) were highly expressed in the liver of T. fasciatus. (2) after 30-days exposure, CuNPs accumulated in the endoplasmic reticulum of liver and induced the appearance of ERS, then activated unfolded protein response (UPR) signaling pathway. Furthermore, the SREBP-1c pathway that plays a key role in lipid synthesis was activated. (3) by using 4-PBA and GSK inhibitors to respectively stimulate ERS and PKR-like ER kinase (PERK) through in vitro experiments, we confirmed that CuNPs induced the fatty liver formation in T. fasciatus triggered by the PERK-EIF2α pathway by activating the SREBP-1c pathway to promote fatty liver formation. This study provides a new perspective for identifying the pathogens of fatty liver formation, and adds to the knowledge of the ecological safety data service of CuNPs in water

Mir-125b Regulates the Self-Renewal of Acute Myeloid Leukemia Stem Cells through PTPN18 and GSK3

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents &amp; Royalties.

Nono, a Bivalent Domain Factor, Regulates Erk Signaling and Mouse Embryonic Stem Cell Pluripotency

Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency.

Collaborative rewiring of the pluripotency network by chromatin and signalling modulating pathways

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) represents a profound change in cell fate. Here, we show that combining ascorbic acid (AA) and 2i (MAP kinase and GSK inhibitors) increases the efficiency of reprogramming from fibroblasts and synergistically enhances conversion of partially reprogrammed intermediates to the iPSC state. AA and 2i induce differential transcriptional responses, each leading to the activation of specific pluripotency loci. A unique cohort of pluripotency genes including Esrrb require both stimuli for activation. Temporally, AA-dependent histone demethylase effects are important early, whereas Tet enzyme effects are required throughout the conversion. 2i function could partially be replaced by depletion of components of the epidermal growth factor (EGF) and insulin growth factor pathways, indicating that they act as barriers to reprogramming. Accordingly, reduction in the levels of the EGF receptor gene contributes to the activation of Esrrb. These results provide insight into the rewiring of the pluripotency network at the late stage of reprogramming.

Targeting Y397 FAK scaffolding compared with FAK kinase inhibition: Efficacy and specificity for tumor cells.

e13547 Background: Focal Adhesion Kinase is a non-receptor kinase which is highly overexpressed and activated or autophosphorylated in many types of tumors and plays a major role in survival signaling and metastasis. Recently, our group developed a highly specific allosteric scaffolding FAK autophosphorylation inhibitor, Y15, that blocked FAK Y397 autophosphorylation, decreased cancer cell viability, and significantly decreased breast, neuroblastoma, pancreatic and glioblastoma tumor growth. In addition, Y15 was not toxic in mice. In this study, we compared side by side efficacy of Y15 with two other FAK inhibitors in clinical trials, Pfizer PF-04554878 and Glaxo GSK-2256098. Methods: MTT, clonogenicity and Western blotting assays were used to detect the effect of FAK inhibitors on different cancer and normal cells. Student’s t-test was used for statistical analysis and p&lt;0.05 was considered significant. Results: We tested different cancer cell lines: kidney 293T, colon SW620 and LoVo; lung A549; glioblastoma U251 and U87; breast: BT474, MDA-231, MDA-361, MDA-453, MDA-468 and T47D and pancreatic PANC-1 cancer cells with different doses of Y15, PF and GSK inhibitors by MTT assay and found that Y15 was the most effective at decreasing cancer cell viability in most cancer cell lines, with higher efficacy than PF and much higher efficacy than GSK inhibitor. The same was observed for clonogenicity assay. In addition, we found that Y15 did not significantly affect viability of normal colon (CCD-112) cells, while viability of these cells with PF and GSK decreased to 54% and 57% at a 2 microM dose, respectively, from 100% in untreated cells. In addition, Y15 significantly decreased Y397-FAK, Y418-Src, phospho- Ser 473-AKT and phospho-ERK1/2, while PF had less effect and GSK only a minimal effect in four different cancer cell lines: U251, A549, 293T and Lovo at 10 microM and 100 microM doses, showing high potency of the FAK Y397 scaffolding inhibitor. Conclusions: These data show that Y15, which inhibits the Y397 site of FAK, is more potent against cancer cells and less toxic to normal cells than FAK inhibitors that target the conserved ATP-binding site in the kinase domain.

Novel Tetrahydropyrido[1,2-a]isoindolone Derivatives (Valmerins): Potent Cyclin-Dependent Kinase/Glycogen Synthase Kinase 3 Inhibitors with Antiproliferative Activities and Antitumor Effects in Human Tumor Xenografts

The development of CDK and GSK3 inhibitors has been regarded as a potential therapeutic approach, and a substantial number of diverse structures have been reported to inhibit CDKs and GSK-3β in recent years. Only a few molecules have gone through or are currently undergoing clinical trials as CDK and GSK inhibitors. In this paper, we prepared valmerins, a new family containing the tetrahydropyrido[1,2-a]isoindone core. The fused heterocycle was prepared with a straightforward synthesis that was functionalized by a (het)arylurea. Twelve valmerins inhibited the CDK5 and GSK3 with an IC(50) < 100 nM. A semiquantitative kinase scoring was realized, and a cellular screening was done. At the end of our study, we investigated the in vivo potency of one valmerin. Mice exhibited good tolerance to our lead, which proved its efficacy and clearly blocked tumor growth. Valmerins appear also as good candidates for further development as anticancer agents.

Colchicine-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells.

The purpose of this study was to examine whether glycogen synthase kinase-3 (GSK-3) is involved in colchicine-induced cell death in PC12 cells by using GSK inhibitors. Colchicine increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. GSK-3 inhibitors such as alsterpaullone, SB216763, and AR-A014418 prevented colchicine-induced cell death and caspase-3 activation. These results suggest that colchicine induces caspase-dependent apoptotic cell death and that GSK-3 activation is involved in cell death in PC12 cells.

Glycogen synthase kinase 3 regulates PAX3–FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3–FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3–FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3–FKHR. Also, GSK3 phosphorylated PAX3–FKHR in vitro, suggesting that GSK3 might regulate PAX3–FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3–FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

Lithium reduces Gsk3b mRNA levels: implications for Alzheimer Disease

There is evidence of increased systemic expression of active GSK3B in Alzheimer's disease patients, which apparently is associated with the formation of senile plaques and neurofibrillary tangles. Due to its central role in the pathogenesis of AD, GSK3B is currently a promising target of the pharmaceutical industry. Whilst trials with specific GSK inhibitors in AD are under way, major attention has been focused on the neuroprotective effects of lithium. Whereas the direct and indirect inhibitory effects of lithium over GSK3 activity have been documented by several groups, its effects over Gsk3 transcription have not yet been addressed. We used quantitative PCR to evaluate the transcriptional regulation of Gsk3a and Gsk3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. We found a significant and dose-dependent reduction in the expression of Gsk3b, which was specific to hippocampal cells. This same effect was further confirmed in vivo by measuring Gsk3 expression in different brain regions and in peripheral leukocytes of adult rats treated with lithium. Our studies show that LiCl can modulate Gsk3b transcription in vitro and in vivo. This observation suggest new regulatory effects of lithium over Gsk3b, contributing to the better understanding of its mechanisms of action, offering a new and complementary explanation for Gsk3b modulation and reinforcing its potential for the inhibition of key pathological pathways in Alzheimer's disease.

Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful?

The glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase widely expressed in mammalian tissues. Ini-tially identified by its ability to modulate glycogen synthesis, GSK-3 turned out to be a multifunctional enzyme, able to phosphorylate many proteins, including members of the steroid receptor superfamily. Although GSK-3 was shown to phosphorylate the androgen receptor (AR), its effects on AR transcriptional activity remain controversial. Analysis of short hairpin RNA (shRNA)–mediated downmodulation of GSK-3 proteins in prostate cancer cells showed a reduction in AR transcriptional activity and AR protein levels. Pharmacological GSK-3 inhibitors such as the maleimide SB216763 or the aminopyrazole GSK inhibitor XIII inhibited AR-dependent reporter gene activity and AR expression in vitro. Analysis of androgen-induced nuclear translocation of the AR was performed in PC3 cells transfected with pAR-t1EosFP coding for EosAR, a green fluorescent AR fusion protein. When grown in pres-ence of androgens, EosAR was predominantly nuclear. Incubation with SB216763 before and after androgen treat-ment almost completely reduced nuclear EosAR. In contrast, the thiazole-containing urea compound AR-A014418 increased rather than decreased AR–expression/function. Although not all GSK inhibitors affected AR–stability/ function, our observations suggest a potential new therapeutic application for some of these compounds in pros-tate cancer.

Inhibitors of GSK-3 Prevent Corticosterone from Inducing COX-1 Expression in Cardiomyocytes

Our recent study has demonstrated that glucocorticoids (GCs) induce cyclooxygenase-1 (COX-1) gene expression in rat cardiomyocytes. While investigating the mechanism underlying corticosterone (CT) induced COX-1, we found that three structurally and mechanistically distinct GSK-3 inhibitors, LiCl, SB216763, and (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO), inhibited COX-1 transcription and protein induction. A genetic approach of expressing wild type GSK-3beta increased COX-1 promoter activity, which was abolished by LiCl. LiCl increased inhibitory GSK-3alpha/beta phosphorylation at Ser21/Ser9, while BIO or SB216763 prevented stimulatory phosphorylation at Tyr279/Tyr216 of GSK-3alpha/beta. GSK inhibitors failed to block nuclear translocation of glucocorticoid receptor (GR) or activation of glucocorticoid response element (GRE) by CT treatment. While Sp3 transcription factor mediates CT induced COX-1 expression, GSK inhibitors did not change the level of Sp3 protein or binding of Sp3 transcription factor to COX-1 promoter. The observed effect of GSK-3 inhibitors appears to be unique to COX-1 since LiCl or BIO does not prevent CT from inducing COX-2 gene. We conclude that GSK-3 inhibitors block CT from inducing COX-1 gene expression via a mechanism beyond GR and Sp3 transcription factor.

25-Hydroxycholesterol induces mitochondria-dependent apoptosis via activation of glycogen synthase kinase-3β in PC12 cells

25-Hydroxycholesterol (25-OH-chol) induces apoptosis in many cell types. The present study investigated the possible involvement of mitochondria-dependent apoptotic signalling molecules in the death of PC12 cells treated with 25-OH-chol. 25-OH-chol increased the production of reactive oxygen species and opened mitochondrial permeability transition pore, resulting in release of cytochrome c and subsequent activation of caspase-9 and -3. 25-OH-chol induced the activation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-3β (GSK-3β). The JNK inhibitor SP600125 attenuated the activation of caspase-9 and -3 and reduced 25-OH-chol-induced cell death. GSK inhibitors SB415286 and SB216763 significantly down-regulated JNK activity and attenuated the cytotoxicity of 25-hydroxycholesterol. However, SP600125 did not alter the activity of GSK-3β. The results indicate that 25-OH-chol induces cell death via activation of GSK-3β and subsequent up-regulation of JNK. Pharmacological intervention of GSK-3β-JNK-caspase signalling pathway may be useful for the reduction of cytotoxicity of oxysterols.

Efficacy of small‐molecule glycogen synthase kinase‐3 inhibitors in the postnatal rat model of tau hyperphosphorylation

Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low microM inhibitor concentrations. Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis.

Hypertonic Stress Activates Glycogen Synthase Kinase 3β-mediated Apoptosis of Renal Medullary Interstitial Cells, Suppressing an NFκB-driven Cyclooxygenase-2-dependent Survival Pathway

The survival of renal medullary interstitial cells (RMICs) requires their adaptation to rapid shifts in ambient tonicity normally occurring in the renal medulla. Previous studies determined that cyclooxygenase-2 (COX 2) activation is critical for this adaptation. The present studies find that these adaptive mechanisms are dampened by the simultaneous activation of an apoptotic pathway linked to a glycogen synthase kinase 3β (GSK 3β). Inhibition of GSK 3 by LiCl or specific small molecule GSK inhibitors increased RMIC survival following hypertonic stress, and transduction of RMICs with a constitutively active GSK 3β (AdGSK 3βA9) significantly increased apoptosis, consistent with a proapoptotic role of GSK 3β. Following GSK 3β inhibition, increased survival was accompanied by increased COX 2 expression and COX 2 reporter activity. In contrast, GSK 3β overexpression reduced COX 2 reporter activity. Importantly, enhanced RMIC survival produced by GSK 3β inhibition was completely dependent on COX 2 because it was abolished by a COX 2-specific inhibitor, SC58236. The signaling pathway by which GSK 3β suppresses COX 2 expression was then explored. GSK 3β inhibition increased both NFκB and β-catenin activity associated with decreased IκB and increased β-catenin levels. The increase in COX 2 following GSK 3β inhibition was entirely blocked by NFκB inhibition using mutant IκB adenovirus. However, adenoviral overexpression of β-catenin did not increase COX 2 levels. These findings suggest that GSK 3β negatively regulates COX 2 expression and that GSK 3β inhibitors protect RMICs from hypertonic stress via induction of NFκB-COX 2-dependent pathway.