Protein Kinase Activation Research Articles

Probiotic Characteristics and Anti-Inflammatory Effects of Limosilactobacillus fermentum 664 Isolated from Chinese Fermented Pickles.

Limosilactobacillus fermentum (L. fermentum) is widely used in industrial food fermentations, and its probiotic and health-promoting roles attracted much attention in the past decades. In this work, the probiotic potential of L. fermentum 664 isolated from Chinese fermented pickles was assessed. In addition, the anti-inflammatory properties and mechanisms were investigated using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results indicated that L. fermentum 664 demonstrated excellent acid and bile salt tolerance, adhesion capability, antimicrobial activity, and safety profile. L. fermentum 664 downregulated the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) stimulated with LPS. Moreover, L fermentum 664 inhibited the nuclear translocation of the nuclear factor κB (NF-κB) and the activation of mitogen-activated protein kinases (MAPKs) induced by LPS. This action was associated with a reduction in reactive oxygen species (ROS) levels and an enhanced expression of heme oxygenase-1 (HO-1) protein. Additionally, whole genome sequencing indicated that L. fermentum 664 contained genes that encode proteins with antioxidant and anti-inflammatory functions, including Cytochrome bd ubiquinol oxidase subunit I (CydA), Cytochrome bd ubiquinol oxidase subunit II (CydB), and NAD(P)H dehydrogenase quinone 1 (NQO1). In conclusion, our study suggested that L. fermentum 664 has the potential to become a probiotic and might be a promising strategy for the prevention of inflammation.

The novel ROCK2 selective inhibitor NRL-1049 preserves the blood-brain barrier after acute injury

Endothelial blood-brain barrier (BBB) dysfunction is critical in the pathophysiology of brain injury. Rho-associated protein kinase (ROCK) activation disrupts BBB integrity in the injured brain. We aimed to test the efficacy of a novel ROCK2 inhibitor in preserving the BBB after acute brain injury. We characterized the molecular structure and pharmacodynamic and pharmacokinetic properties of a novel selective ROCK2 inhibitor, NRL-1049, and its first metabolite, 1-hydroxy-NRL-1049 (referred to as NRL-2017 hereon) and tested the efficacy of NRL-1049 on the BBB integrity in rodent models of acute brain injury. Our data show that NRL-1049 and NRL-2017 both inhibit ROCK activity and are 44-fold and 17-fold more selective towards ROCK2 than ROCK1, respectively. When tested in a mouse model of cortical cryoinjury, NRL-1049 significantly attenuated the increase in water content. Interestingly, 60% of the mice in the vehicle arm developed seizures within 2 hours after cryoinjury versus none in the NRL-1049 arm. In spontaneously hypertensive rats, NRL-1049 attenuated the dramatic surge in Evans Blue extravasation compared with the vehicle arm after transient middle cerebral artery occlusion. Hemorrhagic transformation was also reduced. We show that NRL-1049, a selective ROCK2 inhibitor, is a promising drug candidate to preserve the BBB after brain injury.

Cutin-derived oligomers induce hallmark plant immune responses.

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix-cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and mitogen-activated protein kinase activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of pattern-triggered immunity. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitor compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses. Further investigation is needed to understand how cutin breakdowns are perceived and to explore their potential use in agriculture.

Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

BackgroundAcupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture. MethodsAcupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed. ResultsAcupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration. ConclusionAcupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Akt and AMPK activators rescue hyperexcitability in neurons from patients with bipolar disorder

Bipolar disorder (BD) is a multifactorial psychiatric illness affecting ∼1% of the global adult population. Lithium (Li), is the most effective mood stabilizer for BD but works only for a subset of patients and its mechanism of action remains largely elusive. In the present study, we used iPSC-derived neurons from patients with BD who are responsive (LR) or not (LNR) to lithium. Combined electrophysiology, calcium imaging, biochemistry, transcriptomics, and phosphoproteomics were employed to provide mechanistic insights into neuronal hyperactivity in BD, investigate Li's mode of action, and identify alternative treatment strategies. We show a selective rescue of the neuronal hyperactivity phenotype by Li in LR neurons, correlated with changes to Na+ conductance. Whole transcriptome sequencing in BD neurons revealed altered gene expression pathways related to glutamate transmission, alterations in cell signalling and ion transport/channel activity. We found altered Akt signalling as a potential therapeutic effect of Li in LR neurons from patients with BD, and that Akt activation mimics Li effect in LR neurons. Furthermore, the increased neural network activity observed in both LR & LNR neurons from patients with BD were reversed by AMP-activated protein kinase (AMPK) activation. These results suggest potential for new treatment strategies in BD, such as Akt activators in LR cases, and the use of AMPK activators for LNR patients with BD. Supported by funding from ERA PerMed, Bell Brain Canada Mental Research Program and Brain & Behavior Research Foundation.

Excessive endometrial PlGF- Rac1 signalling underlies endometrial cell stiffness linked to pre-eclampsia

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).

Inhibition of p38α MAPK-MK2 axis alleviate inflammatory response in various inflammation-related disease models

Abstract Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated by p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. Preclinical studies indicate that targeting MK2 may be a much safer approach than blocking p38a MAPK activation because that dissect the pathway downstream of p38α MAPK without affecting additional cellular functions. 141-A was identified as a highly potent and selective p38α MAPK-MK2 allosteric inhibitor that exhibited nanomolar-level inhibition on p38α MAPK activation of MK2 while sparing the p38α MAPK activation of ATF2 and PRAK. 141-A potently reduced various cytokine production (TNFα, IL-1β, IL-6, IL-8 and IL-17) in human peripheral blood mononuclear cells (PBMC) and displayed excellent efficacy in adjuvant-induced arthritis (AIA), psoriasis and MSU-induced air pouch rat models with 1/6 of the systematic exposure of CDD450. The compound exhibits favorable physicochemical and pharmacokinetic properties, along with an excellent preclinical toxicity profile, which may enhance therapeutic efficacy without causing side effects. 141-A is currently under IND-enabling studies and P1 trial is planned in early 2025.

Oral Administration of Bifidobacterium lactis Ameliorates Cognitive Deficits in Mice Intracerebroventricularly Administered Amyloid Beta via Regulation the Activation of Mitogen-activated Protein Kinases.

Probiotics are functional microorganisms that exhibit various biological activities, such as allergic reactions, inflammation, and aging. The aim of this study is to evaluate the effects of Bifidobacterium lactis CBT BL3 (BL) on the amyloid beta (Aβ)-mediated cognitive impairments. Oral administration of live BL to intracerebroventricularly Aβ-injected mice significantly attenuated short- and long-term memory loss estimated using the Y-maze and Morris water maze tests. We found that expression of apoptosis-related proteins such as caspase-9, caspase-3, and cleaved poly (ADP-ribose) polymerase was significantly elevated in the brain tissues of Aβ-injected mouse brains when compared to that of the control mouse group. Interestingly, these expression levels were significantly decreased in the brain tissue of mice fed BL for 6 wk. In addition, the abnormal over-phosphorylation of mitogen-activated protein kinases (MAPKs) such as ERK1/2, p38 MAPK, and JNK in the brain tissue of intracerebroventricularly Aβ-injected mice was significantly attenuated by oral administration of BL. Taken together, the results indicate that Aβ-induced cognitive impairment may be ameliorated by the oral administration of BL by controlling the activation of MAPKs/apoptosis in the brain. This study strongly suggests that BL can be developed as a functional probiotic to attenuate Aβ-mediated cognitive deficits.

Oxya chinensis sinuosa (OC) Extracts Protects ARPE-19 Cells against Oxidative Stress via Activation of the Mitogen-Activated Protein Kinases (MAPKs)/ Nuclear Factor-κB (NF-κB) Pathway.

Oxya chinensis sinuosa (OC) is a well-known edible insect. Several researches on the health benefits of OC consumption have been performed to date; however, their effect on eye health remains largely unknown. This study aimed to assess the protective effects of OC extracts on the oxidative stress on the retinal pigment epithelium (RPE) cells. Oxidative damage has been identified as one of the key regulatory factors in age-related macular degeneration. H2O2-induced reactive oxygen species (ROS) production, a well-known oxidative stress factor, can cause cell death in retinal pigment epithelia cells. In this study, we found that three OC extracts effectively prevented H2O2-induced ROS production and subsequent death of ARPE-19 cells in a dose-dependent manner. In addition, the OC extracts inhibited the phosphorylation of mitogen-activated protein kinases including p38, JNK, and ERK. The OC extracts restored IκBα degradation induced by H2O2, indicating that OC extracts suppressed the activation of nuclear factor-κB. Furthermore, the three OC extracts were shown to have antioxidant effects by up-regulating the intracellular expression of key antioxidant proteins such as SOD, NQO, and HO-1. Here we demonstrated the antioxidant and anti-apoptotic effects of the OC extracts on ARPE-19, indicating their potential role in improving eye health. These results suggest that three OC extracts plays a critical role in oxidative stress-induced cell death protects in ARPE-19 cells.

IM156, a new AMPK activator, ameliorates polymicrobial sepsis by regulating inflammatory responses

Abstract IM156, a biguanide with a higher potency of AMP-activated protein kinase (AMPK) activation than metformin, exhibits inhibitory activity against angiogenesis and cancer. In this study, we explored the anti-inflammatory effects of IM156 against polymicrobial sepsis. Administration of IM156 significantly improved the survival rate and tissue damage in cecal ligation and puncture (CLP)-induced sepsis. Mechanistically, IM156 markedly reduced the number of bacterial cells in the peritoneal fluid and peripheral blood in a CLP-induced sepsis model. IM156 also inhibited splenocyte apoptosis and the production of inflammatory cytokines, including IL-1β, IL-6, and IL-10, in CLP mice. Furthermore, IM156 strongly inhibited the reactive oxygen species (ROS) generation by enhancing antioxidative effects, which can lead to reduced neutrophil extracellular trap (NET) formation in response to lipopolysaccharide. In conclusion, these findings collectively indicate that IM156 can suppress the inflammatory response and provide protection against polymicrobial sepsis. These results suggest the potential of IM156 as a novel therapeutic agent for sepsis control.

Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

CaMKIIα mediates spermidine-induced memory enhancement in rats: A potential involvement of PKA/CREB pathway

Memory consolidation is associated with the regulation of protein kinases, which impact synaptic functions and promote synaptogenesis. The administration of spermidine (SPD) has been shown to modulate major protein kinases associated with memory improvement, including the Ca2+-dependent protein kinase (PKC) and cAMP-dependent protein kinase (PKA), key players in the cAMP response element-binding protein (CREB) activation. Nevertheless, the initial mechanism underlying SPD-mediated memory consolidation remains unknown, as we hypothesize a potential involvement of the memory consolidation precursor, Ca2+/calmodulin-dependent protein kinase II-α (CaMKIIα), in this process. Based on this, our study aimed to investigate potential interactions among PKC, PKA, and CREB activation, mediated by CaMKIIα activation, in order to elucidate the SPD memory consolidation pathway. Our findings suggest that the post-training administration of the CaMKII inhibitor, KN-62 (0.25 nmol, intrahippocampal), prevented the memory enhancement induced by SPD (0.2 nmol, intrahippocampal) in the inhibitory avoidance task. Through western immunoblotting, we observed that phosphorylation of CaMKIIα in the hippocampus was facilitated 15 min after intrahippocampal SPD administration, resulting in the activation of PKA and CREB, 180 min after infusion, suggesting a possible sequential mechanism, since SPD with KN-62 infusion leads to a downregulation in CaMKIIα/PKA/CREB pathway. However, KN-62 does not alter the memory-facilitating effect of SPD on PKC, possibly demonstrating a parallel cascade in memory acquisition via PKA, without modulating CAMKIIα. These results suggest that memory enhancement induced by SPD administration involves crosstalk between CaMKIIα and PKA/CREB, with no PKC interaction.

Lactucopicrin promotes fatty acid β-oxidation and attenuates lipid accumulation through adenosine monophosphate-activated protein kinase activation in free fatty acid-induced human hepatoblastoma cancer cells.

With its annually increasing prevalence, non-alcoholic fatty liver disease (NAFLD) has become a serious threat to people's life and health. After a preliminary research, we found that Lactucopicrin has pharmacological effects, such as lowering blood lipids and protecting the liver. Further research showed its significant activation for fatty acid β-oxidase hydroxyacyl-coenzyme A (CoA) dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), so we hypothesized that Lactucopicrin could ameliorate lipid accumulation in hepatocytes by promoting fatty acid β-oxidation. In this study, free fatty acid (FFA)-induced human hepatoblastoma cancer cells (HepG2) were used to establish an invitro NAFLD model to investigate the molecular basis of Lactucopicrin in regulating lipid metabolism. Staining with Oil red O and measurements of triglyceride (TG) content, fatty acid β-oxidase (FaβO) activity, reactive oxygen species (ROS) content, mitochondrial membrane potential, and adenosine triphosphate (ATP) content were used to assess the extent to which Lactucopicrin ameliorates lipid accumulation and promotes fatty acid β-oxidation. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot methods were used to explore the regulatory effects of Lactucopicrin on factors related to fatty acid β-oxidation. Results showed that Lactucopicrin downregulated phosphorylated mammalian target of rapamycin (P-mTOR) by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway and upregulated the messenger RNA (mRNA) and protein expression levels of coactivators (peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)), transcription factors (peroxisome proliferator-activated receptor α (PPARα) and peroxisome proliferator-activated receptor γ (PPARγ)), and oxidative factors (carnitine palmitoyltransferase 1A (CPT1A) and HADHA). This phenomenon resulted in a significant increase in FaβO activity, ATP content, and JC-1 and a significant decrease in ROS level, TG content, and intracellular lipid droplets. With the addition of Dorsomorphin, all the effects of Lactucopicrin intervention were suppressed. In summary, Lactucopicrin promotes fatty acid β-oxidation by activating the AMPK pathway, thereby ameliorating FFA-induced intracellular lipid accumulation in HepG2 cells.

Fortify the defense frontline: MAPKs phosphorylate receptor-like cytoplasmic kinase to maintain plant resistance in soybean

Mitogen-activated protein kinase (MAPK) activation is one of the significant immune events that respond to pathogens in plants. A MAPK cascade often contains a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK/MKK), and a MAPK. The well-characterized MAPK cascade, to date, is the MAPKKK3/4/5-MKK4/5-MPK3/6 module. Soybean cyst nematodes (SCN) is one of the most devastating soybean pathogens. However, the early immune components contributing to soybean resistance to SCN and the role of the MAPK cascade in the soybean–SCN interaction remain unclear. A recent study published in Plant Cell discovered that GmMPK3/6 phosphorylates a receptor-like cytoplasmic kinase (RLCK), CDG1-LIKE1 (GmCDL1), and maintains the stability of GmCDL1 in soybean. Remarkably, GmCDL1 enhances GmMPK3/6 activation and resistance to SCN by phosphorylating GmMAPKKK5 and activating the GmMAPKKK5-GmMKK4-GmMPK3/6 cascade. In addition, two L-type lectin receptor kinases (LecRKs), GmLecRK02g and GmLecRK08g, are involved in the GmCDL1 function after the perception of SCN. taken together, this study not only discovers a complete early immune pathway that responds to SCN infection in soybean, but also reveals a molecular mechanism by which plants maintain the activation of the MAPK cascade and resistance.

Quinic Acid Alleviates Behavior Impairment by Reducing Neuroinflammation and MAPK Activation in LPS-Treated Mice.

Compared to other organs, the brain has limited antioxidant defenses. In particular, the hippocampus is the central region for learning and memory and is highly susceptible to oxidative stress. Glial cells are the most abundant cells in the brain, and sustained glial cell activation is critical to the neuroinflammation that aggravates neuropathology and neurotoxicity. Therefore, regulating glial cell activation is a promising neurotherapeutic treatment. Quinic acid and its derivatives possess anti-oxidant and anti-inflammatory properties. Although previous studies have evidenced quinic acid's benefit on the brain, in vivo and in vitro analyses of its anti-oxidant and anti-inflammatory properties in glial cells have yet to be established. This study investigated quinic acid's rescue effect in lipopolysaccharide (LPS)-induced behavior impairment. Orally administering quinic acid restored social impairment and LPS-induced spatial and fear memory. In addition, quinic acid inhibited proinflammatory mediator, oxidative stress marker, and mitogen-activated protein kinase (MAPK) activation in the LPS-injected hippocampus. Quinic acid inhibited nitrite release and extracellular signal-regulated kinase (ERK) phosphorylation in LPS-stimulated astrocytes. Collectively, quinic acid restored impaired neuroinflammation-induced behavior by regulating proinflammatory mediator and ERK activation in astrocytes, demonstrating its potential as a therapeutic agent for neuroinflammation-induced brain disease treatments.

TAK1-binding protein 2 (TAB2) and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to the lysine 63-linked polyubiquitin chain through TAK1-binding protein 2 (TAB2) and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and messenger RNA (mRNA) levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in the TLR signaling pathway.

Influence of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses

Background/purposeDual-cure resin-cements are used for various dental restorations. However, whether the curing modes of these resin-cements influence gingival inflammation remains unclear. Hence, herein, we evaluated the effects of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses. Materials and methodsSpecimens were prepared using two dual-cure resin-cements—RelyX Unicem 2 (RU) and G-CEM ONE (GO)—by light-cure or self-cure modes. Degree of conversion (DC) and monomer elution of the resin-cements were measured using Fourier-transform infrared spectroscopy and high-performance liquid chromatography, respectively. Human gingival fibroblasts (GFs) and macrophages were cultured on resin-cements, and inflammatory cytokine levels, intracellular reactive oxygen species (ROS) generation, and mitogen-activated protein (MAP) kinase activation were evaluated. ResultsLight-cured (LC) resin-cements exhibited significantly higher DC and lower monomer elution than did self-cured (SC) resin-cements. Triethyleneglycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) were substantially eluted from RU and GO, respectively. Neither LC resin-cement exhibited cytotoxicity and enhanced pro-inflammatory cytokine expression in GFs and macrophages. However, both SC resin-cements significantly decreased cell numbers and promoted cellular inflammatory responses. SC generated higher intracellular ROS levels compared to that seen with LC, and different patterns of MAP kinase activation were observed between SC–RU and SC–GO. ConclusionCompared with LC dual-cure resin-cements, SC dual-cure resin-cements show stronger cytotoxicity and elicit greater inflammatory responses in gingival cells owing to residual monomers (e.g., TEGDMA and UDMA) by activating MAP kinases in GFs and macrophages. Clinicians should ensure adequate light irradiation during prosthesis cementation and make efforts to remove the excess cement.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Abstract 3079: AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth

Abstract Cancer cells undergo cellular adaptation through metabolic reprogrammin to sustain survival and rapid growth under various stress conditions. However, it remains unknown how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment. We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify regulatory mechanisms of S/G deprivation induced metabolic flexibility, we employed high throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. The high throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and poor prognosis of GBM patients. Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells and highlight the potential to target SSP for treating human GBM. Citation Format: Hye Jin Yun, Jong-Ho Lee. AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3079.