Calmodulin-dependent Protein Kinase Inhibitor Research Articles

Loss of Flot2 expression in deep cerebellar nuclei neurons of mice with Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is caused by a deficiency of the NPC1 or NPC2 gene, leading to storages of unesterified cholesterol and sphingolipids. Cerebellar ataxia is a main symptom of NPC and the deep cerebellar nuclei (DCN) is the sole signal output of the cerebellum. In this study, we explored the pathological changes in DCN neurons of Npc1 knockout mice (Npc1-). We first demonstrated that DCN neurons of Npc1- mice had prominent ganglioside GM2 accumulation in the late endosomes but not in the lysosomes. More importantly, Flot2 expression, a marker for the lipid rafts, was lost. Single-nucleus RNA sequencing analysis revealed a generalized reduction in gene expression in DCN neurons, though Camk1d, encoding one of the Ca2+/calmodulin-dependent protein kinases (CaMKs), increased in expression. We treated Npc1- mice with CaMK inhibitor KN-93, but CaMK1D expression increased further. We also fed Npc1- mice with two medications for NPC. We found that miglustat, a sphingolipid synthesis inhibitor, increased the expression of Flot2. Moreover, N-acetyl l-leucine (NALL), an experimental medicine for NPC, recovered Flot2 expression. Therefore, our data suggest that in Npc1- mice, GM2 sequestration and the loss of lipid rafts lead to cell dysfunction and symptoms of NPC.

Ca2+/CaM/CaMK signaling is involved in cadmium-induced osteoclast differentiation

Environmental cadmium (Cd) pollution can ultimately lead to chronic toxicity via food consumption. Previous studies have demonstrated that long-term low-dose Cd exposure decreases bone mineral density and bone mineralization. Cd may increase receptor activator of nuclear factor-κ B ligand (RANKL) expression by osteoclasts, and inhibit the expression of osteoprotegerin. However, the molecular mechanism underlying Cd toxicity toward osteoclasts is unclear. In this study, bone marrow monocytes were isolated from C57BL/6 mice and treated with macrophage colony-stimulating factor and RANKL to induce the formation of osteoclasts. The results show that low-dose Cd exposure induced osteoclast differentiation. Cd also increased the intracellular calcium concentration of osteoclasts by triggering release of calcium ions from the endoplasmic reticulum into the cytoplasm. Furthermore, the elevation of intracellular calcium levels was shown to activate the calmodulin (CaM)/calmodulin-dependent protein kinase (CaMK) pathway. NFATc1 is a downstream protein of CaM/CaMK signaling, as well as a key player in osteoclast differentiation. Overall, we conclude that Cd activates the CaM/CaMK/NFATc1 pathway and regulates osteoclast differentiation by increasing intracellular calcium concentration. Our data provide new insights into the mechanisms underlying osteoclast differentiation following Cd exposure. This study provides a theoretical basis for future investigations into the therapeutic application of CaMK inhibitors in osteoporosis induced by Cd exposure.

Role of AMP-activated protein kinase during postovulatory aging of mouse oocytes†

Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cyclic adenosine monophosphate (cAMP) decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

Glycated albumin stimulates expression of inflammatory cytokines in muscle cells

The effects of glycated albumin on the expression of inflammatory cytokines in differentiated myotubes were investigated. Glycated albumin stimulates the expression of TNF α, IL-1β, IL-6 and CCL-2 both at the mRNA and protein levels via the receptor of AGEs. Various cytokines demonstrated different kinetics of stimulation by glycated albumin. At a high glucose concentration, the stimulation effect was more pronounced than at a low one. At physiological concentrations of albumin and fructosamine, the stimulation effect of glycated albumin on inflammatory cytokine expression in myotubes was also observed. The induction of expression of all studied cytokines was sensitive to the inhibitors of JNK, p38 MAPK, MEK1/2, Src family protein kinases and NF-κB. At the same time, the induction of TNFα and IL-1β was diminished by the Ca2+/calmodulin-dependent protein kinase inhibitor, whereas the induction of IL-6 and CCL-2 was reduced by the inhibitor of phosphoinositide 3-kinase. Possible implications of observed stimulation of cytokine expression by glycated albumin in the development of diabetes mellitus symptoms are discussed.

Polycomb Group Proteins Recruit TET1 to Remove Aberrant DNA Methylation in Murine Embryonic Stem Cells

At the top of the hematopoietic hierarchy are hematopoietic stem cells (HSCs), which reside in the bone marrow and are characterized by the ability to self-renew or differentiate into various types of mature blood cells. The self-renewal capacity of HSCs relies on the accurate transmission of epigenetic marks to their progeny. Our lab has shown previously that, despite global hypomethylation, DNA hypermethylation frequently occurs on Polycomb group protein (PcG) target genes and many tumor suppressor genes in aged HSCs (Sun et al.Cell Stem Cell. 2014). At the same time, such epigenetic marks are correctly maintained in young HSCs. These observations indicate the presence of epigenetic maintenance systems that deteriorate with age. Currently, the molecular mechanisms through which aberrant DNA hypermethylation accumulates only with age are unclear.To address this gap in knowledge, we examined the maintenance and clearance of forced DNA methylation in murine embryonic stem cells (ESCs) using a CRISPR/dCas9-based targeted DNA methylation tool, dCas9-MQ1147 (Lei et al, Nature Communication. 2017). We forcibly methylated genes that are bound in ESCs by PcG, including Runx1, Gata2, and Hoxa loci. Surprisingly, we found that the exogenous methylation induced by dCas9-MQ1147 was efficiently removed from the target sites during cell proliferation, indicating that methylation status is predetermined and maintained by local genetic and epigenetic marks.To understand which demethylation mechanism is responsible for the removal of the exogenous aberrant DNA methylation in our model, we treated both human somatic cells and murine ESCs with cell cycle arrest drugs, including selective ATP-competitive inhibitor of CDK1 (Ro-3306), selective Ca2+/calmodulin-dependent protein kinase inhibitor (KN93), and microtubule formation inhibitor (Paclitaxel) after inducing targeted methylation. Following treatment, we observed that cell cycle arrest cannot delay passive demethylation at the hypermethylated sites, indicating the removel of aberrant methylation is DNA duplication independent. To further investigate the demethylation mechanism herein, we next validated the contribution of DNA hydroxymethylation enzyme Ten-eleven Translocation (TET) activity in the Tet1/2/3 triple knockout (TKO) mESCs. Time-dependent experiments showed that TKO mESCs had a significant exogenous methylation retention compared to their wild-type counterparts. These data indicate that TET family proteins are recruited to remove aberrant methylation from the unmethylated PcG binding region via TDG or base excision repair, but not inaccurate maintenance by DNA methyltransferase 1 (DNMT1).To determine which TET protein or proteins contribute to the maintenance of predetermined unmethylation status, we used prokaryote DNA methyltransferase MQ1 wild-type protein to generate genome-wide hypermethylation in wild-type mESCs. We detected that TET1 was the most highly upregulated TET protein, with over 5-fold upregulation, following induced hypermethylation. Furthermore, by applying the novel Degron targeted degradation technique, we specific remove the expressed MQ1 protein and found that the degradation of MQ1 led to the reduction of Tet1 overexpression. These data indicate that TET1 participates in the removal of aberrant DNA methylation in mESCs.Overall, this study suggests that a proofreading mechanism at the PcG-targeted region recognizes aberrant DNA methylation and recruits TET1 to restore its original unmethylated status. The dysregulation of this mechanism in aging HSCs may lead to the accumulation of methylation abnormalities during proliferation.This study sheds light on an important molecular mechanisms responsible for maintaining the epigenetic status in ESCs and provides insight into how aberrant DNA methylation accumulates in these cells over time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Silibinin sensitizes TRAIL-mediated apoptosis by upregulating DR5 through ROS-induced endoplasmic reticulum stress-Ca2+-CaMKII-Sp1 pathway.

In this study, we addressed how silibinin enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in various cancer cells. Combined treatment with silibinin and TRAIL (silibinin/TRAIL) induced apoptosis accompanied by the activation of caspase-3, caspase-8, caspase-9, and Bax, and cytosolic accumulation of cytochrome c. Anti-apoptotic proteins such as Bcl-2, IAP-1, and IAP-2 were inhibited as well. Silibinin also triggered TRAIL-induced apoptosis in A549 cells through upregulation of death receptor 5 (DR5). Pretreatment with DR5/Fc chimeric protein and DR5-targeted small interfering RNA (siRNA) significantly blocked silibinin/TRAIL-mediated apoptosis in A549 cells. Furthermore, silibinin increased the production of reactive oxygen species (ROS), which led to the induction of TRAIL-mediated apoptosis through DR5 upregulation. Antioxidants such as N-acetyl-L-cysteine and glutathione reversed the apoptosis-inducing effects of TRAIL. Silibinin further induced endoplasmic reticulum (ER) stress as was indicated by the increase in ER marker proteins such as PERK, eIF2α, and ATF-4, which stimulate the expression of CCAAT/enhancer binding protein homologous protein (CHOP). CHOP-targeted siRNA eliminated the induction of DR5 and resulted in a significant decrease in silibinin/TRAIL-mediated apoptosis. We also found that silibinin/TRAIL-induced apoptosis was accompanied with intracellular influx of Ca2+, which was stimulated by ER stress and the Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA). Ca2+/calmodulin-dependent protein kinase (CaMKII) inhibitor, K252a, blocked silibinin/TRAIL-induced DR5 expression along with TRAIL-mediated apoptosis. Accordingly, we showed that ROS/ER stress-induced CaMKII activated Sp1, which is an important transcription factor for DR5 expression. Our results showed that silibinin enhanced TRAIL-induced apoptosis by upregulating DR5 expression through the ROS-ER stress-CaMKII-Sp1 axis.

Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro

Considering the importance of calcium (Ca2+) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3′-5′-cyclic adenosine monophosphate (cAMP) and adenosine 5′-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca2+. Results of the present study indicated that, under greater extracellular Ca2+concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca2+ on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca2+, however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca2+ had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca2+ signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca2+ on boar sperm as well as the involvement of energy metabolism in this mechanism.

Paeoniflorin protects diabetic mice against myocardial ischemic injury via the transient receptor potential vanilloid 1/calcitonin gene-related peptide pathway.

BackgroundDiabetes mellitus has multiple complications including neuropathy and increases cardiovascular events. Paeoniflorin (PF), a monoterpene glycoside, plays an essential role in neuroprotection and ischemic heart disease. In this study, we aimed to investigate the hypothesis that PF protects mice with diabetes mellitus against myocardial ischemic injury, and determine its associated mechanisms.ResultsMyocardial infarction (MI) was generated in the streptozotocin-mediated diabetic mice, which were pretreated with either vehicle or PF, respectively. Myocardial infarct size, myocardial enzyme, cardiac function, circulating calcitonin gene-related peptide (CGRP) concentration, histological analysis and the expression of associated molecules were determined and compared among different experimental groups. Compared to diabetic hearts pretreated with vehicle, hearts pretreated with PF exhibited less tissue damage and better CGRP concentration in serum when subjected to myocardial ischemia. Transient receptor potential vanilloid 1(TRPV1) gene knockout attenuated PF-mediated cardioprotection. Moreover, a specific Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitor, KN-93, increased tissue damage and decreased CGRP activity in serum. Meanwhile, pretreated with PF increased the phosphorylation of cAMP response element binding protein (CREB).ConclusionsTaken together, these findings demonstrate that PF protects diabetic mice against MI at least partially via the TRPV1/CaMK/CREB/CGRP signaling pathway.

The Expression of Can and Camk is Associated with Lipogenesis in the Muscle of Chicken

Intramuscular fat (IMF) content in chickens significantly contributes to meat quality. The main objective of this study was to assess the expression of calcineurin (CaN) and Ca2+/calmodulin-dependent protein kinase (CaMK) in lipogenesis in chicken muscle. Chickens were slaughtered and sampled at 4, 8, and 16 weeks of age. IMF content and the expression of CaN subunits and CaMK isoforms were measured in the thigh muscle tissue. The results showed that the IMF contents were greater at 16 weeks compared with those at 4 and 8 weeks (p<0.05). Transcription of fatty acid synthase (FAS) and fatty acid translocase CD36 (FAT/CD36) mRNA significantly increased with age, from four to 16 weeks (p<0.05). The mRNA levels of CaN B and CaMK IV were significantly lower at 16 weeks than at four weeks (p<0.05), but CaMK II mRNA levels were significantly higher than at four weeks (p<0.05). In order to evaluate the role of CaMK and CaN in adipogenesis, SV cells were incubated in standard adipogenic medium for 24 h and treated with specific inhibitor of CaMK and CaN. The expressions of CCAAT/enhancer binding protein b (C/EBPb, sterol regulatory element-binding protein 1 (SREBP1), and peroxisome proliferation-activated receptor g (PPARγ) were dramatically enhanced by the CsA, CaN inhibitor (p<0.05). KN93, CaMK II inhibitor, dramatically repressed the expression of those lipogenic gene (p<0.05). These results indicated that CaN and CaMK had different effects on adipogenesis in the muscle of chickens.

Palpitations, potassium and the pump.

Palpitations, potassium and the pump.

Serine hydrolase inhibitors block necrotic cell death by preventing calcium overload of the mitochondria and permeability transition pore formation (756.2)

Perturbation of calcium signaling occurs during cell injury and disease and promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, necrotic cell death and LDH release that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2 (cPLA2) or calcium independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2‐deficient fibroblasts was inhibited by the cPLA2 inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium loading. Inhibitors of calmodulin‐dependent protein kinase II, a mitochondrial Ca2+ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and by H2O2. Pyrrophenone blocked MCU‐mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium loading but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and OAG but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not fatty acid release that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell permeable serine hydrolase inhibitors that block MCU‐mediated mitochondrial calcium overload, MPTP formation and necrotic cell death.Grant Funding Source: NIH HL34303, HL61378, HL50040, DA009789, DA033760, MH084512

Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons

Phosphorylation is considered a main mechanism modulating nNOS (neuronal nitric oxide synthase) function to reduce NO production. In the present study, the effects of nNOS phosphorylation on redox signalling, including that of NO, ROS (reactive oxygen species), and 8-nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate), a downstream messenger of redox signalling, were investigated. Invitro experiments revealed that a phosphorylation-mimic mutant of nNOS (Ser847 replaced with aspartic acid, 847D) increased uncoupling to produce a superoxide. In addition, nicotine, which triggers an influx of Ca2+, induced more ROS and 8-nitro-cGMP production in 847D-expressing PC12 cells than WT (wild-type)-expressing cells. Additionally, nicotine-induced phosphorylation of nNOS at Ser847 and increased ROS and 8-nitro-cGMP production in rat CGNs (cerebellar granule neurons). In CGNs, the NOS (nitric oxide synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and superoxide dismutase completely inhibited ROS and 8-nitro-cGMP production, whereas the CaMK (Ca2+/calmodulin-dependent protein kinase) inhibitor KN93 mildly reduced this effect. Nicotine induced HO-1 (haem oxygenase 1) expression in CGNs and showed cytoprotective effects against apoptosis. Moreover, 8-nitro-cGMP treatment showed identical effects that were attenuated by KN93 pre-treatment. The present paper provides the first substantial corroboration for the biological effects of nNOS phosphorylation at Ser847 on redox signalling, including ROS and intracellular 8-nitro-cGMP generation in neurons, which possibly play roles in neuroprotection.

Regulation of type 1 IP3 receptor expression by dopamine D2-like receptors via AP-1 and NFATc4 activation

Type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca2+ concentration. Although our recent report demonstrates the essential involvement of IP3R-1 up-regulation induced by dopamine D1-like and D2-like receptor (D1 and D2R) stimulation in psychological dependence, exact regulatory mechanisms of IP3R-1 expression by D2Rs have not yet been clarified. Mouse cerebral cortical neurons were treated with inhibitor of Ca2+-related signal transduction pathways coupling to D2Rs and used to analyze the mechanisms of IP3R-1 expression regulated by transcriptional factor. A selective D2R agonist, quinpirole, up-regulated IP3R-1 protein following its mRNA increase, which was significantly inhibited by gallein (a Gβγ modulator), U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor). Immunocytochemical assessment showed that quinpirole increased expression of both cFos and phosphorylated-cJun in nucleus and enhanced translocation of NFATc4 complex to nucleus from cytoplasm. In addition, quinpirole directly recruited bindings between AP-1 and IP3R-1 promoter region and between NFATc4 and IP3R-1 promoter region. These results indicate that D2Rs enhance IP3R-1 gene transcription via increased bindings of AP-1 and NFATc4 to IP3R-1 promoter region after Gβγ activation.

Dopamine D1 receptors regulate type 1 inositol 1,4,5‐trisphosphate receptor expression via both AP‐1‐ and NFATc4‐mediated transcriptional processes

Although our recent report demonstrates the essential involvement of up-regulation of a regulator of intracellular Ca(2+) concentration, type 1 inositol 1,4,5-trisphosphate receptors (IP(3) Rs-1), mediated via dopamine D1-like receptor (D1DR) stimulation in the cocaine-induced psychological dependence, the exact mechanisms of regulation of IP(3) R-1 expression by D1DRs have not yet been clarified. This study attempted to clarify these mechanisms using mouse cerebral cortical neurons. An agonist for phosphatidylinositide-linked D1DRs, SKF83959, induced dose- and time-dependently IP(3) R-1 protein up-regulation following its mRNA increase without cAMP production. U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor), significantly inhibited the SKF83959-induced IP(3) R-1 up-regulation. Furthermore, immunohistochemical examinations showed that SKF83959 increased expression of both cFos and cJun in nucleus as well as enhanced translocation of both calcineurin and NFATc4 complex to nucleus from cytoplasm. In addition, SKF83959 directly recruited binding of both AP-1 and NFATc4 to IP(3) R-1 promoter region. These results indicate that D1DR activation induces IP(3) R-1 up-regulation via increased translocation of AP-1 as well as NFATc4 in Gαq protein-coupled calcium signaling transduction pathway.

Effects of increased systolic Ca2+ and phospholamban phosphorylation during β-adrenergic stimulation on Ca2+ transient kinetics in cardiac myocytes

Previous studies demonstrated higher systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitudes result in faster [Ca(2+)](i) decline rates, as does β-adrenergic (β-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca(2+)](i) decline rate with β-AR stimulation, the increased systolic Ca(2+) concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca(2+) concentration ([Ca(2+)](o))], followed by high extracellular Ca(2+) (3 mM [Ca(2+)](o)), washout with 1 mM [Ca(2+)](o), followed by 1 μM isoproterenol (ISO) with 1 mM [Ca(2+)](o). ISO increased Ser(16) phosphorylation compared with 3 mM [Ca(2+)](o), whereas Thr(17) phosphorylation was similar. Ca(2+) transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM [Ca(2+)](o) and ISO. [Ca(2+)](i) decline was significantly faster with ISO compared with 3 mM [Ca(2+)](o). Interestingly, the faster decline with ISO was only seen during the first 50% of the decline. CaT time to peak was significantly faster with ISO compared with 3 mM [Ca(2+)](o). A Ca(2+)/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM [Ca(2+)](o) or ISO but normalized ISO's time to peak with 3 mM [Ca(2+)](o). Thus, during β-AR stimulation, the major factor for the faster CaT decline is due to Ser(16) phosphorylation, and faster time to peak is due to CAMKII activation.

Acid Activation of Trpv1 Leads to an Up-Regulation of Calcitonin Gene-related Peptide Expression in Dorsal Root Ganglion Neurons via the CaMK-CREB Cascade: A Potential Mechanism of Inflammatory Pain

Increased production of calcitonin gene-related peptide (CGRP) in sensory neurons is implicated in inflammatory pain. The inflammatory site is acidic due to proton release from infiltrating inflammatory cells. Acid activation of peripheral nociceptors relays pain signals to the CNS. Here, we examined whether acid activated the transient receptor potential vanilloid subtype 1 (Trpv1), a widely recognized acid-sensing nociceptor and subsequently increased CGRP expression. Chemically induced inflammation was associated with thermal hyperalgesia and increased CGRP expression in dorsal root ganglion (DRG) in rats. In organ cultures of DRG, acid (pH 5.5) elevated CGRP expression and the selective Trpv1 antagonist 5'-Iodoresiniferatoxin decreased it. Trpv1-deficient DRG showed reduced CGRP increase by acid. Of note, many of CGRP/Trpv1-positive DRG neurons exhibited the phosphorylation of cAMP response element-binding protein (CREB), a nociceptive transcription factor. Knockdown of CREB by small interfering RNA or a dominant-negative form of CREB diminished acid-elevated CGRP expression. Acid elevated the transcriptional activity of CREB, which in turn stimulated CGRP gene promoter activity. These effects were inhibited by a Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. In conclusion, our results suggest that inflammatory acidic environments activate Trpv1, leading to an up-regulation of CGRP expression via CaMK-CREB cascade, a series of events that may be associated with inflammatory pain.

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

α7 nAChR-mediated activation of MAP kinase pathways in PC12 cells

The α7 nicotinic acetylcholine receptor (α7 nAChR) plays a fundamental role in Ca2+-dependent activation of signaling pathways that can modulate intracellular events involved in learning and memory. Activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) are well documented Ca2+ signaling events, but these have not been well characterized in response to α7 nAChR-selective ligands. The present study examined activation of ERK1/2 and explored pathways leading to CREB phosphorylation utilizing α7 nAChR-selective ligands in PC12 cells endogenously expressing α7 nAChRs. Robust concentration-dependent increase in ERK1/2 phosphorylation was triggered by structurally diverse α7 nAChR agonists such as nicotine, choline, GTS-21, SSR-180711A and PNU-282987 in the presence of the positive allosteric modulator (PAM) PNU-120596. This effect was attenuated by selective α7 nAChR antagonists or by chelation of extracellular Ca2+. ERK1/2 phosphorylation was also attenuated by inhibitors of calmodulin-dependent protein kinase II (CaMKII), p38 MAP kinase and mitogen-activated protein kinase kinase1/2 (MEK1/2), indicating the involvement of these kinases upstream of ERK1/2. This was confirmed by direct measurement of p38 MAPK and MEK1/2 phosphorylation. These data suggest that α7 nAChR agonist-triggered Ca2+ transient in PC12 cells induces activation of CaMKII, leading to sequential phosphorylation of p38 MAPK, MEK1/2, ERK1/2 and CREB. Such mechanisms may endow the α7 nAChRs with roles in modulating Ca2+-dependent intracellular second messenger events implicated in diverse aspects of cognition.

Menthol Response and Adaptation in Nociceptive-Like and Nonnociceptive-Like Neurons: Role of Protein Kinases

Menthol-sensitive/capsaicin-insensitive neurons (MS/CI) and menthol-sensitive/capsaicin-sensitive neurons (MS/CS) are thought to represent two functionally distinct populations of cold-sensing neurons that use TRPM8 receptors to convey innocuous and noxious cold information respectively. However, TRPM8-mediated responses have not been well characterized in these two neuron populations. Using rat dorsal root ganglion neurons, here we show that MS/CI neurons had larger menthol responses with greater adaptation. In contrast, MS/CS neurons had smaller menthol responses with less adaptation. All menthol-sensitive neurons showed significant reduction of menthol responses following the treatment of cells with the protein kinase C (PKC) activator PDBu (Phorbol 12,13-dibutyrate). PDBu-induced reduction of menthol responses was completely abolished in the presence of PKC inhibitors BIM (bisindolylmaleimide) or staurosporine. When menthol responses were examined in the presence of protein kinase inhibitors, it was found that the adaptation was significantly attenuated by either BIM or staurosporine and also by the Ca2+/calmodulin-dependent protein kinase (CamKII) inhibitor KN62 (N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) in MS/CI neurons. In contrast, in MS/CS neurons menthol response was not affected significantly by BIM, staurosporine or KN62. In both MS/CI and MS/CS neurons, the menthol responses were not affected by PKA activators forskolin and 8-Br-cAMP (8-Bromoadenosine-3', 5'-cyclic monophosphate) or by protein kinase A (PKA) inhibitor Rp-cAMPs (Rp-Adenosine-3',5'-cyclic monophosphorothioate). Taken together, these results suggest that TRPM8-mediated responses are significantly different between non-nociceptive-like and nociceptive-like neurons.

Intracellular Metabotropic Glutamate Receptor 5 (mGluR5) Activates Signaling Cascades Distinct from Cell Surface Counterparts

G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca(2+) signatures leading to unique cellular responses. Specifically, activation of either cell surface or intracellular mGluR5 leads to JNK, Ca(2+)/calmodulin-dependent protein kinase (CaMK), and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas activation of only intracellular mGluR5 leads to ERK1/2 and Elk-1 phosphorylation. Using pharmacological and genetic approaches, the present findings support a role for CaMK kinase in mediating mGluR5-dependent cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas CaMKII is upstream of intracellular mGluR5-mediated Elk-1 phosphorylation. Consistent with models showing Elk-1 regulating cascades of gene expression, the known Elk-1 targets c-fos and egr1 were up-regulated following intracellular mGluR5 activation, whereas a representative non-Elk-1 target, c-jun, was not. These findings emphasize that glutamate not only serves as a neurotransmitter for cell surface receptors but, when transported into the cell, can also activate intracellular receptors such as mGluR5. Glutamate activation of intracellular mGluR5 serves an important role in the regulation of nuclear Ca(2+), transcriptional activation, and gene expression necessary for physiological processes such as synaptic plasticity.