Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

Protein Inhibitor of Activated STAT 1 (PIAS1) Is Identified as the SUMO E3 Ligase of CCAAT/Enhancer-Binding Protein β (C/EBPβ) during Adipogenesis

It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

Hypoxia-inducible Factor-1α (HIF-1α) Protein Diminishes Sodium Glucose Transport 1 (SGLT1) and SGLT2 Protein Expression in Renal Epithelial Tubular Cells (LLC-PK1) under Hypoxia

In this work, we demonstrated the regulation of glucose transporters by hypoxia inducible factor-1α (HIF-1α) activation in renal epithelial cells. LLC-PK₁ monolayers were incubated for 1, 3, 6, or 12 h with 0% or 5% O₂ or 300 μm cobalt (CoCl₂). We evaluated the effects of hypoxia on the mRNA and protein expression of HIF-1α and of the glucose transporters SGLT1, SGLT2, and GLUT1. The data showed an increase in HIF-1α mRNA and protein expression under the three evaluated conditions (p < 0.05 versus t = 0). An increase in GLUT1 mRNA (12 h) and protein expression (at 3, 6, and 12 h) was observed (p < 0.05 versus t = 0). SGLT1 and SGLT2 mRNA and protein expression decreased under the three evaluated conditions (p < 0.05 versus t = 0). In conclusion, our results suggest a clear decrease in the expression of the glucose transporters SGLT1 and SGLT2 under hypoxic conditions which implies a possible correlation with increased expression of HIF-1α.     

Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile α Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors

Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.     

Cellular FLICE-like Inhibitory Protein (c-FLIP) and PS1-associated Protein (PSAP) Mediate Presenilin 1-induced γ-Secretase-dependent and -independent Apoptosis,Respectively

Presenilin 1 (PS1) has been implicated in apoptosis; however, its mechanism remains elusive. We report that PS1-induced apoptosis was associated with cellular FLICE-like inhibitory protein (c-FLIP) turnover and that γ-secretase inhibitor blocked c-FLIP turnover and also partially blocked PS1-induced apoptosis. A complete inhibition of PS1-induced apoptosis was achieved by knockdown of PS1-associated protein (PSAP), a mitochondrial proapoptotic protein that forms a complex with Bax upon induction of apoptosis, in the presence of γ-secretase inhibitor. PS1-induced apoptosis was partially inhibited by knockdown of caspase-8, Fas-associated protein with death domain (FADD), or Bid. However, knockdown of Bax or overexpression of Bcl-2 resulted in complete inhibition of PS1-induced apoptosis. These data suggest that PS1 induces apoptosis through two pathways: the γ-secretase-dependent pathway mediated by turnover of c-FLIP and the γ-secretase-independent pathway mediated by PSAP-Bax complex formation. These two pathways converge on Bax to activate mitochondria-dependent apoptosis. These findings provide new insight into the mechanisms by which PS1 is involved in apoptosis and the mechanism by which PS1 exerts its pathogenic effects. In addition, our results suggest that PS2 induces apoptosis through a pathway that is different from that of PS1.     

α/β-Hydrolase Domain Containing Protein 15 (ABHD15) – an Adipogenic Protein Protecting from Apoptosis

Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G₁, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent K_m for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower K_m constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.     

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.     

Heat Shock Protein 90-α Mediates Aldo-Keto Reductase 1B10 (AKR1B10) Protein Secretion through Secretory Lysosomes

Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Our previous studies have shown that AKR1B10 is secreted through a lysosome-mediated nonclassical pathway, leading to an increase in the serum of breast cancer patients. This study illuminates the regulatory mechanism of AKR1B10 secretion. The cytosolic AKR1B10 associates with and is translocated to lysosomes by heat shock protein 90α (HSP90α), a chaperone molecule. Ectopic expression of HSP90α significantly increased the secretion of endogenous AKR1B10 and exogenous GFP-AKR1B10 fusion protein when cotransfected. Geldanamycin, a HSP90α inhibitor, dissociated AKR1B10-HSP90α complexes and significantly reduced AKR1B10 secretion in a dose-dependent manner. We characterized the functional domain in AKR1B10 and found that helix 10 (amino acids 233–240), located at the C terminus, regulates AKR1B10 secretion. Targeted point mutations recognized that amino acids Lys-233, Glu-236, and Lys-240 in helix 10 mediate the interaction of AKR1B10 with HSP90α. Together, our data suggest that HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. This finding is significant in exploiting the use of AKR1B10 in cancer clinics.     

Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform α1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside or constitutively active α1AMPK (CA-α1) significantly inhibits; although inhibiting α1AMPK by short hairpin RNA knock-down or dominant-negative α1AMPK (DN-α1) increases LPS- and FFA-induced tumor necrosis factor α expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-α1 in macrophages significantly inhibits LPS- or FFA-induced NF-κB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-α1 increases the SIRT1 activator NAD⁺ content and SIRT1 expression in macrophages. Furthermore, α1AMPK activation mimics the effect of SIRT1 on deacetylating NF-κB, and the full capacity of AMPK to deacetylate NF-κB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.     

Multidrug Resistance Protein 1 (MRP1, ABCC1), a “Multitasking” ATP-binding Cassette (ABC) Transporter

The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C₄ have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases.     

Paraspeckle Protein 1 (PSPC1) Is Involved in the Cisplatin Induced DNA Damage Response—Role in G1/S Checkpoint

Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.     

High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation

Obesity and type 2 diabetes are risk factors of Alzheimer’s disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage.     

Modulation of Dynamin-related Protein 1 (DRP1) Function by Increased O-linked-β-N-acetylglucosamine Modification (O-GlcNAc) in Cardiac Myocytes

O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.     

COPS5 (Jab1) Protein Increases β Site Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation by Stabilizing RanBP9 Protein Levels

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer''s disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.     

Protein kinase C δ (PKCδ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition-mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.