Calcium-calmodulin Dependent Kinase Research Articles

Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Rad GTPase Deficiency Leads to Cardiac Hypertrophy

Rad (Ras associated with diabetes) GTPase is the prototypic member of a subfamily of Ras-related small G proteins. The aim of the present study was to define whether Rad plays an important role in mediating cardiac hypertrophy. We document for the first time that levels of Rad mRNA and protein were decreased significantly in human failing hearts (n=10) compared with normal hearts (n=3; P<0.01). Similarly, Rad expression was decreased significantly in cardiac hypertrophy induced by pressure overload and in cultured cardiomyocytes with hypertrophy induced by 10 micromol/L phenylephrine. Gain and loss of Rad function in cardiomyocytes significantly inhibited and increased phenylephrine-induced hypertrophy, respectively. In addition, activation of calcium-calmodulin-dependent kinase II (CaMKII), a strong inducer of cardiac hypertrophy, was significantly inhibited by Rad overexpression. Conversely, downregulation of CaMKIIdelta by RNA interference technology attenuated the phenylephrine-induced hypertrophic response in cardiomyocytes in which Rad was also knocked down. To further elucidate the potential role of Rad in vivo, we generated Rad-deficient mice and demonstrated that they were more susceptible to cardiac hypertrophy associated with increased CaMKII phosphorylation than wild-type littermate controls. The present data document for the first time that Rad is a novel mediator that inhibits cardiac hypertrophy through the CaMKII pathway. The present study will have significant implications for understanding the mechanisms of cardiac hypertrophy and setting the basis for the development of new strategies for treatment of cardiac hypertrophy.

HMGB1 release induced by liver ischemia involves Toll-like receptor 4–dependent reactive oxygen species production and calcium-mediated signaling

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.

SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process

In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na(+),K(+)-ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions. Here we report the existence of a salt-inducible kinase-1 (SIK1) that associates constitutively with the NK regulatory complex and is responsible for increases in its catalytic activity following small elevations in intracellular sodium concentrations. Increases in intracellular sodium are paralleled by elevations in intracellular calcium through the reversible Na(+)/Ca(2+) exchanger, leading to the activation of SIK1 (Thr-322 phosphorylation) by a calcium calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK alpha-subunit and an increase in its catalytic activity. A protein phosphatase 2A/phosphatase methylesterase-1 (PME-1) complex, which constitutively associates with the NK alpha-subunit, is activated by SIK1 through phosphorylation of PME-1 and its dissociation from the complex. These observations illustrate the existence of a distinct intracellular signaling network, with SIK1 at its core, which is triggered by a monovalent cation (Na(+)) and links sodium permeability to its active transport.

Abstract 615: LKB1 is Required for Statin-induced Activation of the AMP-activated Protein Kinase

Statins, lipid-lowering drugs, exert many beneficial effects on the cardiovascular system. Recent studies have demonstrated that clinically relevant concentrations of statins might exert their therapeutic effects by activating the AMP-activated kinase (AMPK) in cultured endothelial cells, but the mechanism remains elusive. The aim of the study was to determine the molecular mechanism by which statins activate AMPK. Confluent bovine aortic endothelial cells (BAEC) were exposed to simvastatins for 1 to 24 h. Exposure of BAEC to simvastatins caused a time-dependent and dose-dependent increase of AMPK-Thr172 phosphorylation. In parallel, simvastatins significantly increased the phosphorylation of both LKB1 at serine 428 and protein kinase C (PKC)-ζ at Thr410/403. Further, adenoviral overexpression of LKB1 kinase-dead (LKB1 D194A) mutants, but not adenoviruses encoding green fluorescence proteins (GFP), ablated simvastatin-induced AMPK-Thr172 phosphorylation, suggesting LKB1 is required for statin-enhanced AMPK activation. Conversely, neither inhibition of calcium calmodulin-dependent kinase kinase (CaMKK) with STO609 (1 μM), a CaMKK-specific inhibitor, nor transfection of CaMKK-specific siRNA altered simvastatin-enhanced AMPK Thr172 phosphorylation, suggesting that CaMKK is not required for statin-enhanced AMPK activation. Moreover, overexpression of PKC-ζ dominant negative mutants or PKC-ζ-specific siRNA abolished simvastatin-enhanced phosphorylation of both LKB1-Ser428 and AMPK-Thr172, implying that PKC-ζ is the upstream enzyme of LKB1. Finally, compared to lean mice, the phosphorylation of both AMPK-Thr172 and LKB1-Ser428 was significantly suppressed in the hearts and livers of diabetic DB/DB mice (n=4 or 6, p&lt;0.05). Treatment of diabetic DB/DB mice with fluvastatin (22.5 mg/kg/day in drinking water) for 4 months significantly increased the levels of total AMPK as well as AMPK-Thr172 phosphorylation in the livers and hearts of DB/DB mice. In addition, fluvastatin significantly increased detection of LKB1-Ser428 phosphorylation. We conclude that statins increase AMPK activation by increasing PKC-ζ-dependent LKB1 phosphorylation at Ser428 in vivo .

The expression of L‐type voltage‐gated calcium channels in retinal photoreceptors is under circadian control

Photoreceptors are non-spiking neurons, and their synapses mediate the continuous release of neurotransmitters under the control of L-type voltage-gated calcium channels (VGCCs). Photoreceptors express endogenous circadian oscillators that play important roles in regulating photoreceptor physiology and function. Here, we report that the L-type VGCCs in chick cone photoreceptors are under circadian control. The L-type VGCC currents are greater when measured during the subjective night than during the subjective day. Using antibodies against the VGCCalpha1C and VGCCalpha1D subunits, we found that the immunofluorescence intensities of both VGCCalpha1C and VGCCalpha1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCalpha1D, but not VGCCalpha1C, are rhythmic. Nocturnal increases in L-type VGCCs are blocked by manumycin A, PD98059, and KN93, which suggest that the circadian output pathway includes Ras, Erk, and calcium-calmodulin dependent kinase II. In summary, four independent lines of evidence show that the L-VGCCs in cone photoreceptors are under circadian control.

The influence of Pavlovian cues on instrumental performance is mediated by CaMKII activity in the striatum

Pavlovian cues associated with reward exert a powerful motivational influence on the performance of goal-directed actions. This motivational process depends critically on the ventral striatum, although little is known about the cellular and molecular mechanisms that mediate it. In the current experiments we examined the role of calcium calmodulin-dependent kinase II (CaMKII) by using transgenic mice that express a constitutively active form of this kinase. We found that controlled expression of active CaMKII in the striatum did not affect learning but did impair the motivation of goal-directed actions by Pavlovian cues associated with reward. Mutant mice learned to lever press for reward, remained sensitive to outcome devaluation and contingency degradation manipulations, and were able to acquire Pavlovian responses to cues paired with reward. However, Pavlovian cues were completely unable to motivate lever pressing in mutant mice. This was true even in mice trained with the CaMKII transgene turned off and then tested with it turned on. We were also able to suppress transgene expression in impaired mutants and fully restore the motivational effects of reward cues in these animals. Therefore, the current experiments demonstrate that normal CaMKII activity in the striatum is essential for the motivational effects of reward cues on goal-directed actions.

Males Afraid, Females Brave

There are different kinds of memories. In mice, differences have been noted in contextual fear memory formation and spatial memory formation, although both forms of memory appear to involve a calcium signal and the activation of calcium/calmodulin-dependent kinases (CaMKs). In mice, there are two isoforms of CaMK that have been implicated in memory formation (CaMKI and CaMKIV) and two upstream kinases that phosphorylate CaMK (CaM kinase kinases: CaMKKα and CaMKKβ). CaMKKβ has been implicated in spatial memory formation, and CaMKIV has been implicated in long-term potentiation and fear conditioning. Two groups, Blaeser et al . and Mizuno et al ., created mice deficient in CaMKKα function and found that the mice showed normal spatial memory, normal motor activity, and normal sensory perception; however, male mice were impaired in the formation of contextual fear memories. Synaptic transmission was not impaired in the CamKKα-deficient mice, nor was formation of hippocampal long-term potentiation. Blaeser et al . showed that, compared with wild-type mice, the CamKKα-deficient male mice exhibited decreased phosphorylation of CaMKIV and the transcriptional regulator CREB (a substrate of CaMKIV) in response to fear conditioning. Phosphorylation of CREB under basal conditions was increased in the mutant mice in certain brain regions; however, the fear-induced increases in phosphorylation were absent. Mizuno et al . investigated memory formation in both male and female mice deficient for CaMKKα and noted that females did not show impairment of contextual fear conditioning. (It was known that female mice tend to exhibit contextual fear conditioning to a lesser extent than do male mice.) In wild-type mice, contextual fear conditioning promoted an increase in brain-derived neurotrophic factor (BDNF) in males only and a decrease in BDNF in females (transcript abundance was measured by real-time polymerase chain reaction). In the CaMKKα-deficient mice, the increase in BDNF in males was compromised, but the decrease in females was unaffected. The gene encoding BDNF is a CREB target; however, expression of another gene regulated by CREB, the gene encoding nerve growth factor-inducible gene B (NGF-1B), was increased in response to fear conditioning in both males and females and was not affected by lack of CaMKKα. Thus, in addition to specific forms of memory requiring specific signaling cascades, there is an additional gender-specific layer of regulation. F. Blaeser, M. J. Sanders, N. Truong, S. Ko, L. J. Wu, D. F. Wozniak. M. S. Fanselow, M. Zhuo, T. A. Chatila, Long-term memory deficits in Pavlovian fear conditioning in Ca 2+ /calmodulin kinase kinase α-deficient mice. Mol. Cell. Biol. 26 , 9105-9115 (2006). [Abstract] [Full Text] K. Mizuno, L. Ris, A. Sánchez-Capelo, E. Godaux, K. P. Giese, Ca 2+ /calmodulin kinase kinase α is dispensable for brain development but is required for distinct memories in male, though not in female, mice. Mol. Cell. Biol. 26 , 9094-9104 (2006). [Abstract] [Full Text]

Phosphorylation of Gα11Protein Contributes to Agonist-Induced Desensitization of 5-HT2AReceptor Signaling

Agonist treatment causes desensitization of many G protein-coupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of Galpha q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT2A) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT(2A/2C) receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of Galpha q/11 proteins, as demonstrated by immunoprecipitation of Galpha q/11 and both incorporation of 32P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of Galpha q/11 proteins, suggesting that different mechanisms are involved in desensitization after short- and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in Galpha11 significantly reduced DOI-stimulated phosphorylation of Galpha11 and DOI-induced desensitization of 5-HT2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of Galpha q/11 proteins and DOI-induced desensitization of 5-HT2A receptors. Expression of Galpha11 S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of Galpha11 protein by PKC and CaMK contributes to agonist-induced homologous desensitization of 5-HT2A receptor signaling as well as heterologous desensitization. The phosphorylation of Galpha protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.

Threonine phosphorylation of integrin β 3 in calyculin A-treated platelets is selectively sensitive to 5′-iodotubercidin

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin β 3. Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44 mapk, p38 mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β 3. Inhibitors of p38 mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β 3 on thr 753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β 3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β 3 molecules and inhibition of platelet responses by protein phosphatase inhibitors.

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.

Inhibition of the CaM-Kinases augments cell death in response to oxygen radicals and oxygen radical inducing cancer therapies in MCF-7 human breast cancer cells

Many cancer treatments induce cell death through lethal oxidative stress. Oxidative stress also induces the activation of the calcium/calmodulin-dependent kinases (CaM-Ks), CaM-KII and CaM-KIV. In turn, the CaM-Ks are known to induce the activation of antiapoptotic signaling pathways, such as Akt, ERK, and NF-κB in many different cell types. The aim of this study was to determine the role of CaM-Kinases in resistance to hydrogen peroxide and three oxidative stress-inducing cancer therapies in MCF-7 breast cancer cells. We found that oxidative stress induced CaM-Kinase activity in MCF-7 breast cancer cells and that CaM-K inhibition increased hydrogen peroxide-induced cell death in MCF-7 human breast cancer cells. When MCF-7 cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy in the presence of a CaM-K inhibitor a greater level of cell killing was observed than when cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy alone. In support of this finding, CaM-K inhibition increased hydrogen peroxide-induced apoptosis in MCF-7 cells, as determined by increased number of apoptotic cells, DNA fragmentation, and PARP cleavage. Pharmacological and molecular inhibition indicated that CaM-KII was participating in hydrogen peroxide-induced ERK phosphorylation in breast cancer cells indicating a potential mechanism by which this sensitization occurs. This is the first time that CaM-K inhibition is reported to sensitize cancer cells to reactive oxygen intermediate inducing cancer treatments.

(n-3) Long-Chain Polyunsaturated Fatty Acids Prolong Survival following Myocardial Infarction in Rats

Many clinical studies report that (n-3) PUFAs decrease the incidence of sudden death in patients with coronary artery disease after myocardial infarction (MI). However, the mechanisms for the beneficial effects of (n-3) PUFAs are unknown. The objectives of the present study were to confirm the findings from clinical trials using an animal model of MI in which dietary intake could be closely controlled and to utilize the model to investigate molecular mechanisms for the beneficial effects of (n-3) PUFAs. Male rats were subjected to coronary ligation to induce MI and were randomly assigned to diets high in (n-6) (58% of lipid) or (n-3) (28% of lipid) PUFAs for 6 mo. A diet high in (n-3) PUFAs was associated with an improvement in 6-mo survival (89.2% vs. 64.9%, P = 0.013) compared with rats consuming a diet high in (n-6) PUFAs (n = 37/group). In a separate study (n = 5 rats/diet group), the (n-3) PUFA diet decreased the (n-6):(n-3) PUFA ratio in plasma (0.6 +/- 0.1 vs. 7.9 +/- 1.8, P < 0.05) and cardiac tissue (0.9 +/- 0.1 vs. 11.8 +/- 1.6, P < 0.05) of rats fed for 4 wk. The increased survival in the (n-3) diet group was associated with decreased cardiac activities of protein kinase A and calcium calmodulin-dependent kinase II by 33-38% (P < 0.05) and a 28% decrease (P < 0.05) in phosphorylation (activation) of the ryanodine receptor calcium release channel. Based upon our results, we speculate that decreased activities of protein kinases induced by diets high in (n-3) PUFAs are associated with a decrease in sudden death after MI in rats.

Second messenger and Ras/MAPK signalling pathways regulate CLOCK/CYCLE‐dependent transcription

The heterodimeric complex of the transcription factors CLOCK (CLK) and CYCLE (CYC) constitutes the positive element of the circadian clock in Drosophila and mammals. Phosphorylation of clock proteins represents an essential mechanism for promotion and control of the molecular oscillator. However, the kinases and signalling pathways that regulate CLK/CYC function remain largely elusive. In the present study we performed a chemical screen of kinase inhibitors in a cell culture reporter assay to identify functional regulators of CLK/CYC-dependent gene expression. These studies and analysis of constitutively active forms of kinases revealed that cyclic nucleotide/protein kinase A (PKA), calcium/calmodulin-dependent kinase (CaMK) II and Ras/mitogen-activated protein kinase (MAPK) regulate CLK/CYC activity. In vitro phosphorylation analysis showed a direct phosphorylation of CLK by CaMK II and p42 MAPK [extracellular signal-regulated kinase (ERK) 2], suggesting that these kinases regulate CLK/CYC-dependent transcription by direct phosphorylation of CLK.

Histopathological and molecular changes produced by hippocampal microinjection of domoic acid

The phytoplankton-derived neurotoxin, domoic acid (DOM), frequently causes poisoning of marine animals and poses an increasing threat to public health through contamination of seafood. In this study, we used stereotactic microinjection technique to administer varying amounts of DOM into the hippocampal CA1 region in order to examine potential histopathological changes after injection of sub-lethal concentrations to CA1 pyramidal neurons. Gross anatomical abnormalities in CA1 were observed at above 10 μM DOM (3 pmol in 0.3 μl saline). At 1 mM concentration, DOM produces both ipsilateral and contralateral neuronal cell death in CA1, CA3 as well as dentate gyrus subfields. Animal behavioral changes after microinjection were similar to those observed by previous studies through systemic DOM injection. Neuronal degeneration was paralleled by reduced glutamate receptor (NR1, GluR1 and GluR6/7) immunolabeling throughout the whole hippocampal formation. Pre-injection of the AMPA/KA receptor antagonist NBQX (10 μM, 0.3 μl) blocked 1 mM DOM-induced neuronal degeneration as well as behavioral symptoms. At concentrations lower than 10 μM, no histopathological changes were observed microscopically, nor were the levels of immunostaining of NR1, GluR1, GluR6/7 different. However, increased immunolabeling of autophosphorylated calcium–calmodulin-dependent kinase II (CaMKII, p-Thr286) and phosphorylated cAMP response element binding protein (CREB, p-Ser133) were observed at 24 h post-injection, suggesting that altered intracellular signal transduction mediated by GluRs might be an adaptive cellular protective mechanism against DOM-induced neurotoxicity.

Activation of kinase pathways in MCF-7 cells by 17β-estradiol and structurally diverse estrogenic compounds

17β-Estradiol (E2) activates non-genomic pathways in MCF-7 cells, and this study investigates the effects of structurally-diverse estrogenic compounds on activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC), PKA, and calcium calmodulin-dependent kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens, bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis( p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2′,3′,4′,5′-tetrachloro-4-biphenylol (HO-PCB-Cl 4). With the exception of resveratrol, all the compounds activated PI3-K and MAPK. Activation of PKC by the xenoestrogens was structure-dependent since resveratrol, kepone and HO-PCB-Cl 4 were inactive and only minimal activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl 4 was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of estrogen receptor-α-mediated non-genomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent and can result in activation or inhibition of kinase activities.

Intracellular Trafficking of Histone Deacetylase 4 Regulates Neuronal Cell Death

Histone deacetylase 4 (HDAC4) undergoes signal-dependent shuttling between the cytoplasm and nucleus, which is regulated in part by calcium/calmodulin-dependent kinase (CaMK)-mediated phosphorylation. Here, we report that HDAC4 intracellular trafficking is important in regulating neuronal cell death. HDAC4 is normally localized to the cytoplasm in brain tissue and cultured cerebellar granule neurons (CGNs). However, in response to low-potassium or excitotoxic glutamate conditions that induce neuronal cell death, HDAC4 rapidly translocates into the nucleus of cultured CGNs. Treatment with the neuronal survival factor BDNF suppresses HDAC4 nuclear translocation, whereas a proapoptotic CaMK inhibitor stimulates HDAC4 nuclear accumulation. Moreover, ectopic expression of nuclear-localized HDAC4 promotes neuronal apoptosis and represses the transcriptional activities of myocyte enhancer factor 2 and cAMP response element-binding protein, survival factors in neurons. In contrast, inactivation of HDAC4 by small interfering RNA or HDAC inhibitors suppresses neuronal cell death. Finally, an increase of nuclear HDAC4 in granule neurons is also observed in weaver mice, which harbor a mutation that promotes CGN apoptosis. Our data identify HDAC4 and its intracellular trafficking as key effectors of multiple pathways that regulate neuronal cell death.

Strain-dependent regulation of plasticity-related proteins in the mouse hippocampus

Inbred mouse strains have different genetic backgrounds that can result in impairment of synaptic plasticity and memory. Strain-dependent performance in behavioral and cognitive tasks is well-documented. Hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that may underlie some forms of learning and memory has been shown to differ significantly between inbred mouse strains. However, an effect of strain on the expression of proteins, critically involved in synaptic plasticity, learning and memory has not been described yet. We have been addressing this question by determining expressional levels of a panel of proteins involved in neuronal information processing in hippocampus of five mouse strains by immunoblotting. Four inbred strains (FVB/N, C57Bl/6J, 129S2/Sv and Balb/c), commonly used for generating genetically modified mice and for conventional experiments in pharmacology and toxicology and one outbred strain (OF1) have been selected. A significant effect of strain was detected for total and phosphorylated calcium–calmodulin dependent kinase IIα (CaMKII, pCaMKII), phosphorylated mitogen-activated protein kinase (pMAPK), total and phosphorylated calcium-responsive element binding 1 (creb, pcreb), early-growth response protein 1 (egr 1), brain derived neurotrophic factor (BDNF), drebrin and postsynaptic density-95 (PSD-95). These results may indicate genetic determination of synaptic plasticity-related mechanisms relevant for the molecular events mediating hippocampal information processing and storage. Data presented herein highlight the importance of careful selection of the mouse strain for studies of synaptic plasticity.

Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men

Serine/threonine kinase 33 (STK33/Stk33) is a recently discovered gene whose inferred amino acid sequence translation displays characters typical for a calcium/calmodulin dependent kinase (CAMK). In this study we analysed the STK33/Stk33 RNA and protein distribution and the localization of the protein. The STK33/Stk33 expression pattern resembles those of some related members of the CAMK group. STK33/Stk33 displays a nonubiquitous and, in most tissues, low level of expression. It is highly expressed in testis, particularly in cells from the spermatogenic epithelia. Moreover, significant expression is detected in lung epithelia, alveolar macrophages, horizontal cells in the retina and in embryonic organs such as heart, brain and spinal cord. A possible role of STK33/Stk33 in spermatogenesis and organ ontogenesis is discussed.

Calcium/Calmodulin-Dependent Kinase I and Calcium/Calmodulin-Dependent Kinase Kinase Participate in the Control of Cell Cycle Progression in MCF-7 Human Breast Cancer Cells

Calcium is universally required for cell growth and proliferation. Calmodulin is the main intracellular receptor for calcium. Although calcium and calmodulin are well known to be required for cell cycle regulation, the target pathways for their action remain poorly defined. Potential targets include the calcium/calmodulin-dependent kinases (CaM-K). The aim of this study was to determine the role of the CaM-Ks on cell proliferation and progress through the cell cycle in breast cancer cells. CaM-KI inhibition with either KN-93 or specific interfering RNA (siRNA) caused an arrest in the cell cycle in the human breast cancer cell line, MCF-7. This arrest occurred in the G(1) phase of the cell cycle. Supporting this finding, CaM-K inhibition using KN-93 also resulted in a reduction of cyclin D1 protein and pRb phosphorylation when cells were compared with control cultures. Furthermore, inhibition of the upstream activator of CaM-KI, CaM-KK, using siRNA also resulted in cell cycle arrest. In summary, CaM-KK and CaM-KI participate in the control of the G(0)-G(1) restriction check point of the cell cycle in human breast cancer cells. This arrest seems due to an inhibition in cyclin D1 synthesis and a reduction in pRb phosphorylation. To the best of our knowledge, this is the first time that CaM-KK has been reported to be involved in mammalian cell cycle regulation and that CaM-Ks are regulating breast cancer cell cycle.