Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells

We have previously shown that sphingosine inhibits depolarisation-induced calcium influx through voltage-operated calcium channels (VOCCs) in GH(4)C(1) cells, whereas sphingosine-1-phosphate (S1P) does not. In the present study we investigated whether sphingosine kinase modulates VOCC activity in GH(4)C(1) cells by removing inhibitory sphingosine. Sphingosine and the structurally similar sphingosine kinase inhibitor dimethylsphingosine (DMS) both rapidly attenuated the calcium influx evoked by depolarisation. The inhibitory effect declined over time to a greater extent in cells treated with sphingosine than in cells treated with DMS, indicating that sphingosine is being metabolised more rapidly. When the specific sphingosine kinase inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKi) was added to the cells after depolarisation there was likewise a reduction of the calcium response. This inhibitory effect was slow and reached a plateau about 3 min after application. In contrast, the sphingosine-mediated inhibition was immediate, suggesting that the SKi-induced inhibition was due to build-up of cellular sphingosine. In experiments on cells overexpressing sphingosine kinase, the inhibitory effect of sphingosine was reversed faster than in control cells. The effect was not due to the produced S1P, since S1P did not have any effect on VOCCs even at concentrations as high as 50 microM. In patch-clamp experiments the calcium entry through VOCCs was attenuated in GH(4)C(1) cells overexpressing a kinase-dead sphingosine kinase, compared with cells overexpressing the wild type sphingosine kinase. In addition, in cells treated with SKi the calcium entry through VOCCs was attenuated compared with control cells. Our results provide compelling evidence that sphingosine kinase regulates the function of voltage-operated calcium channels in GH(4)C(1) cells, not through its catalytic product, but by removal of the substrate sphingosine.     

Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.     

Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr(286) results in calmodulin (CaM) trapping, a >10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca(2+)], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca(2+)], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe(293) in the kinase and Glu(120) and Met(124) in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe(293) and Asn(294) in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

Cannabinoid receptor interacting protein 1a interacts with myristoylated Gαi N terminus via a unique gapped β-barrel structure

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB₁ cannabinoid receptor G-protein coupling in part by altering the selectivity for Gα_i subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid–binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gα_i N terminus. However, CRIP1a could not bind the nonmyristolyated Gα_i peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB₁ receptor.     

BMAL1 regulates mitochondrial homeostasis in renal ischaemia‐reperfusion injury by mediating the SIRT1/PGC‐1α axis

The regulation of renal function by circadian gene BMAL1 has been recently recognized; however, the role and mechanism of BMAL1 in renal ischaemia‐reperfusion injury (IRI) are still unknown. The purpose of this study was to clarify the pathophysiological role of BMAL1 in renal IRI. We measured the levels of BMAL1 and mitochondrial biogenesis‐related proteins, including SIRT1, PGC‐1α, NRF1 and TFAM, in rats with renal IRI. In rats, the level of BMAL1 decreased significantly, resulting in inhibition of SIRT1 expression and mitochondrial biogenesis. In addition, under hypoxia and reoxygenation (H/R) stimulation, BMAL1 knockdown decreased the level of SIRT1 and exacerbated the degree of mitochondrial damage and apoptosis. Overexpression of BMAL1 alleviated H/R‐induced injury. Furthermore, application of the SIRT1 inhibitor EX527 not only reduced the activities of SIRT1 and PGC‐1α but also further aggravated mitochondrial dysfunction and partially reversed the protective effect of BMAL1 overexpression. Moreover, whether in vivo or in vitro, the application of SIRT1 agonist resveratrol rescued the mitochondrial dysfunction caused by H/R or IRI by activating mitochondrial biogenesis. These results indicate that BMAL1 is a key circadian gene that mediates mitochondrial homeostasis in renal IRI through the SIRT1/PGC‐1α axis, which provides a new direction for targeted therapy for renal IRI.     

Sphingosine kinase 1 regulates pro-inflammatory responses triggered by TNFalpha in primary human monocytes

Monocytes play an important role in inflammation, angiogenesis, and atherosclerosis. During these processes monocytes release pre-formed proinflammatory mediators from granules, and synthesize de novo cytokines and chemokines important in the amplification of the inflammatory response. One of the most prominent triggers of inflammatory responses is the cytokine TNFalpha. However, the intracellular signaling cascades triggered by TNFalpha are not fully understood. In this study we investigated the roles of SPHK on the TNFalpha-triggered responses on human primary monocytes. We show that TNFalpha rapidly triggers S1P generation and activation of SPHK. Moreover, our data shows that SPHK1 is the isoform activated by TNFalpha, and plays an essential role on the TNFalpha-triggered intracellular Ca2+ signals, degranulation, cytokine production, and activation of NFkappaB, thus suggesting a pivotal role for SPHK1 on the proinflammatory responses triggered by TNFalpha.     

Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.     

Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.     

Targeting SYK of monocyte-derived macrophages regulates liver fibrosis via crosstalking with Erk/Hif1α and remodeling liver inflammatory environment

Liver fibrosis is a danger signal indicating a huge risk of liver cancer occurrence, but there is still no effective clinical means to regulate the progress of liver fibrosis. Although a variety of drugs targeting SYK have been developed for tumors and autoimmune diseases, the mechanism and specific efficacy of SYK’s role in liver fibrosis are not yet clear. Our studies based on chronic CCL4, bile duct ligation, and subacute TAA mouse models show that SYK in monocyte-derived macrophages (MoMFs) is fully dependent on phosphorylation of Erk to up-regulate the expression of Hif1α, thereby forming the crosstalk with SYK to drive liver fibrosis progress. We have evaluated the ability of the small molecule SYK inhibitor GS9973 in a variety of models. Contrary to previous impressions, high-frequency administration of GS9973 will aggravate CCL4-induced liver fibrosis, which is especially unsuitable for patients with cholestasis whose clinical features are bile duct obstruction. In addition, we found that inhibition of MoMFs SYK impairs the expression of CXCL1, on one hand, it reduces the recruitment of CD11bhiLy6Chi inflammatory cells, and on the other hand, it promotes the phenotype cross-dress process of pro-resolution MoMFs, thereby remodeling the chronic inflammatory environment of the fibrotic liver. Our further findings indicate that on the basis of the administration of CCR2/CCR5 dual inhibitor Cenicriviroc, further inhibiting MoMFs SYK may give patients with fibrosis additional benefits.Subject terms: Mechanisms of disease, Monocytes and macrophages     

Glycyrrhizin regulates rat TMJOA progression by inhibiting the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway

To investigate the role of glycyrrhizin on the progression of temporomandibular joint osteoarthritis (TMJOA) and the underlying mechanism by regulation of the high‐mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE)/toll‐like receptor 4 (TLR4)‐nuclear factor kappa B (NF‐κB)/protein kinase B (AKT) pathway. After a rat model of TMJOA was built by intra‐articular injection of monosodium iodoacetate, glycyrrhizin was intragastrically administered at low concentration (20 mg/kg) or high concentration (50 mg/kg). Micro‐computed tomography, histological and immunohistochemical analysis were used to reveal the progression of TMJOA. Rat TMJ chondrocytes and disc cells were cultured in inflammatory condition with different doses of glycyrrhizin. Western blot was used to evaluate the effect of glycyrrhizin on the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway. Administration of glycyrrhizin alleviated cartilage degeneration, lowered the levels of inflammatory and catabolic mediators and reduced the production of HMGB1, RAGE and TLR4 in TMJOA animal model. Increased production of RAGE and TLR4, and activated intracellular NF‐κB and/or AKT signalling pathways in chondrocytes and disc cells were found in inflammatory condition. Upon activation, matrix metalloprotease‐3 and interleukin‐6 were upregulated. Glycyrrhizin inhibited not only HMGB1 release but also RAGE and TLR4 in inflammatory condition. Glycyrrhizin alleviated the pathological changes of TMJOA by regulating the HMGB1‐RAGE/TLR4‐NF‐kB/AKT signalling pathway. This study revealed the potential of glycyrrhizin as a novel therapeutic drug to suppress TMJ cartilage degradation.     

The serine/threonine/tyrosine kinase STY46 defends against hordeivirus infection by phosphorylating γb protein

Protein phosphorylation is a common post-translational modification that frequently occurs during plant–virus interaction. Host protein kinases often regulate virus infectivity and pathogenicity by phosphorylating viral proteins. The Barley stripe mosaic virus (BSMV) γb protein plays versatile roles in virus infection and the coevolutionary arms race between plant defense and viral counter-defense. Here, we identified that the autophosphorylated cytosolic serine/threonine/tyrosine (STY) protein kinase 46 of Nicotiana benthamiana (NbSTY46) phosphorylates and directly interacts with the basic motif domain (aa 19–47) of γb in vitro and in vivo. Overexpression of wild-type NbSTY46, either transiently or transgenically, suppresses BSMV replication and ameliorates viral symptoms, whereas silencing of NbSTY46 leads to increased viral replication and exacerbated symptom. Moreover, the antiviral role of NbSTY46 requires its kinase activity, as the NbSTY46_T436A mutant, lacking kinase activity, not only loses the ability to phosphorylate and interact with γb but also fails to impair BSMV infection when expressed in plants. NbSTY46 could also inhibit the replication of Lychnis ringspot virus, another chloroplast-replicating hordeivirus. In summary, we report a function of the cytosolic kinase STY46 in defending against plant viral infection by phosphorylating a viral protein in addition to its basal function in plant growth, development, and abiotic stress responses.

The cytosolic serine/threonine/tyrosine kinase STY46 negatively regulates Barley stripe mosaic virus replication by phosphorylating and interacting with cb protein.     

Remifentanil up‐regulates HIF1α expression to ameliorate hepatic ischaemia/reperfusion injury via the ZEB1/LIF axis

Ischaemia/reperfusion (I/R)‐induced hepatic injury is regarded as a main reason of hepatic failure after transplantation or lobectomy. The current study aimed to investigate how the opioid analgesic remifentanil treatment affects I/R‐induced hepatic injury and explore the possible mechanisms related to HIF1α. Initially, an I/R‐induced hepatic injury animal model was established in C57BL/6 mice, and an in vitro hypoxia‐reoxygenation model was constructed in NCTC‐1469 cells, followed by remifentanil treatment and HIF1α silencing treatment. The levels of blood glucose, lipids, alanine transaminase (ALT) and aspartate transaminase (AST) in mouse serum were measured using automatic chemistry analyser, while the viability and apoptosis of cells were detected using CCK8 assay and flow cytometry. Our results revealed that mice with I/R‐induced hepatic injury showed higher serum levels of blood glucose, lipids, ALT and AST and leukaemia inhibitory factor (LIF) expression, and lower HIF1α and ZEB1 expression (P < .05), which were reversed after remifentanil treatment (P < .05). Besides, HIF1α silencing increased the serum levels of blood glucose, lipids, ALT and AST (P < .05). Furthermore, hypoxia‐induced NCTC‐1469 cells exhibited decreased HIF1α and ZEB1 expression, reduced cell viability, as well as increased LIF expression and cell apoptosis (P < .05), which were reversed by remifentanil treatment (P < .05). Moreover, HIF1α silencing down‐regulated ZEB1 expression, decreased cell viability, and increased cell apoptosis (P < .05). ZEB1 was identified to bind to the promoter region of LIF and inhibit its expression. In summary, remifentanil protects against hepatic I/R injury through HIF1α and downstream effectors.     

Macrophage contributes to radiation-induced anti-tumor abscopal effect on transplanted breast cancer by HMGB1/TNF-α signaling factors

Objectives: The roles of innate immunity including macrophages in radiation-induced abscopal effect (RIAE) are ambiguous. In this study, we evaluated the role of macrophage in RIAE and the interaction of cytokines in tumor microenvironment after irradiation.Materials and Methods: Transplanted tumor of breast cancer cells in BalB/C mice, severe combined immunodeficiency (SCID) mice and non-obese diabetic (NOD)-SCID mice were irradiated with fractionation doses to observe anti-tumor abscopal effect. The underlying mechanism of RIAE was investigated by treating the mice with TNF-α inhibitor or macrophage depletion drug and analyzing the alteration of macrophage distribution in tumors. A co-culture system of breast cancer cells and macrophages was applied to disclose the signaling factors and related pathways involved in the RIAE.Results: The growth of nonirradiated tumor was effectively suppressed in mice with normal or infused macrophages but not in mice with insufficiency/depletion of macrophage or TNF-α inhibition, where M1-macrophage was mainly involved. Investigation of the bystander signaling factors in vitro demonstrated that HMGB1 released from irradiated breast cancer cells promoted bystander macrophages to secret TNF-α through TLR-4 pathway and further inhibited the proliferation and migration of non-irradiated cancer cells by PI3K-p110γ suppression.Conclusions: HMGB1 and TNF-α contributes to M1-macrophages facilitated systemic anti-tumor abscopal response triggered by radiotherapy in breast cancer, indicating that the combination of immunotherapy and radiotherapy may has important implication in enhancing the efficiency of tumor treatment.