CaM Antagonists Research Articles

Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

BackgroundCalmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR.ResultsWe provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified.ConclusionOur results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.Graphical abstractCalmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration.

The aim of this study was to investigate the effects of CaM antagonist, PTZ, and TFP on cell proliferation and migration of colon cancer cells and its impact on POPDC protein expression. The 50% inhibitory concentration (IC50) of PTZ and TFP in SW1116, SW480, HCT-15, and COLO205 colon cancer cell lines are measured using MTT. Western blot and immunocytochemistry were used to determine the expression of PCNA, cyclin D1 (CD1), and POPDC proteins. Cell migration was observed using a scratch wound-healing assay. Treatment with PTZ and TFP inhibited colon cancer cells growth in a dose-dependent manner. PTZ and TFP significantly inhibited the activation of proliferation markers, PCNA and CD1, and the migration of colon cancer cells. Furthermore, POPDC protein was significantly suppressed in all cell types of colon cancer, particularly in SW480. Finally, the CaM antagonist upregulates the POPDC1 expression in colon cancer cells. These findings suggest that CaM antagonists suppress colon cancer cells proliferation via downregulation of CD1 and PCNA. In addition, POPDC protein could be used as a biomarker in colon cancer, and CaM antagonist could be used to regulate POPDC1 expression. This study suggests that targeting POPDC1 with CaM inhibition could be a potential therapeutic strategy for colon cancer treatment.<br />.

Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²⁺, CaM and Depolarization of Plasma Membrane Potential.

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca2+ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca2+ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca2+ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca2+ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca2+ and H+ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca2+-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

The interaction of salicylic acid and Ca2+ alleviates aluminum toxicity in soybean (Glycine max L.)

Both calcium ion (Ca2+) and salicylic acid (SA) influence various stress responses in plants. In acidic soils, aluminum (Al) toxicity adversely affects crop yield. In this study, we determined the influences of Ca2+ and SA on root elongation, Al accumulation, and citrate secretion in soybean plant. We also investigated the activity of antioxidative enzymes in Al-exposed soybean roots. Root elongation was severally inhibited when the roots were exposed to 30 μM Al. The Al-induced inhibition of root elongation was ameliorated by Ca2+ and SA but aggravated by Ca2+ channel inhibitor (VP), CaM antagonists (TFP), Ca2+ chelator (EGTA), and SA biosynthesis inhibitor (PAC). Furthermore, 1.0 mM CaCl2 and 10 μM SA reduced the accumulation of Al in roots, but their inhibitors stimulated the accumulation of Al in roots. Citrate secretion from these roots increased with the addition of either 1.0 mM CaCl2 or 10 μM SA but did not increase significantly when treated with higher Ca2+ concentration. Enzymatic analysis showed that Ca2+ and SA stimulated the activities of superoxidase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) in Al-treated roots. In addition, SA restored the inhibition of Ca2+ inhibitors on root elongation and Al content. Thus, both Ca2+ and SA contribute to Al tolerance in soybean. Furthermore, Ca2+ supplements rapidly increased Al-induced accumulation of free-SA or conjugated SA (SAG), while Ca2+ inhibitors delayed the accumulation of SA for more than 8 h. Within 4 h of treatment, SA increased cytosolic Ca2+ concentration in Al-treated roots, and upregulated the expression of four genes that possibly encode calmodulin-like (CML) proteins. These findings indicate that SA is involved in Ca2+-mediated signal transduction pathways in Al tolerance.

Ca2+ and CaM are involved in Al3+ pretreatment-promoted fluoride accumulation in tea plants (Camellia sinesis L.)

Tea plant (Camellia sinensis (L.) O. kuntze) is known to be a fluoride (F) and aluminum (Al3+) hyper-accumulator. Previous study showed that pre-treatment of Al3+ caused a significant increase of F accumulation in tea plants. However, less is known about the intricate network of Al3+ promoted F accumulation in tea plants. In this study, the involvement of endogenous Ca2+ and CaM in Al3+ pretreatment-promoted F accumulation in tea plants was investigated. Our results showed that Al3+ induced the inverse change of intracellular Ca2+ fluorescence intensity and stimulated Ca2+ trans-membrane transport in the mature zone of tea root. Also, a link between internal Ca2+ and CaM was found in tea roots under the presence of Al3+. In order to investigate whether Ca2+ and CaM were related to F accumulation promoted by Al3+ pretreatment, Ca2+ chelator EGTA and CaM antagonists CPZ and TFP were used. EGTA, CPZ, and TFP pretreatment inhibited Al3+-induced increase of Ca2+ fluorescence intensity and CaM content in tea roots, and also significantly reduced Al3+-promoted F accumulation in tea plants. Taken together, our results suggested that the endogenous Ca2+ and CaM are involved in Al3+ pretreatment-promoted F accumulation in tea roots.

Dissecting blue light signal transduction pathway in leaf epidermis using a pharmacological approach.

Blue light signalling pathway in broad bean leaf epidermal cells includes key membrane transporters: plasma- and endomembrane channels and pumps of H (+) , Ca (2+) and K (+) ions, and plasma membrane redox system. Blue light signalling pathway in epidermal tissue isolated from the abaxial side of fully developed Vicia faba leaves was dissected by measuring the effect of inhibitors of second messengers on net K(+), Ca(2+) and H(+) fluxes using non-invasive ion-selective microelectrodes (the MIFE system). Switching the blue light on-off caused transient changes of the ion fluxes. The effects of seven groups of inhibitors were tested in this study: CaM antagonists, ATPase inhibitors, Ca(2+) anatagonists or chelators, agents affecting IP3 formation, redox system inhibitors, inhibitors of endomembrane Ca(2+) transport systems and an inhibitor of plasma membrane Ca(2+)-permeable channels. Most of the inhibitors had a significant effect on steady-state (basal) net fluxes, as well as on the magnitude of the transient ion flux responses to blue light fluctuations. The data presented in this study suggest that redox signalling and, specifically, plasma membrane NADPH oxidase and coupled Ca(2+) and K(+) fluxes play an essential role in blue light signal transduction.

Involvement of calcium and calmodulin signaling in adaptation to heat stress-induced oxidative stress in Solanum lycopersicum L. leaves

Heat stress is a common form of stress suffered by plants. Therefore, plants have evolved mechanisms to cope with the problems caused by high temperatures. In this study, the involvement of calcium ion and calmodulin (Ca 2+ -CaM) in the protection against heat stress-induced oxidative damage in tomato ( Solanum lycopersicum L.) seedlings was investigated. Treatment of tomato leaves with short-term heat stress led to significant increase in the level of malondialdehyde (MDA) and protein oxidation (in terms of carbonyl groups), two important parameters of oxidative stress. However, such oxidative processes were mitigated by pretreatment with Ca 2+ and aggravated by pretreatment with the Ca 2+ chelator ethylene glycol-bis(β-amino ethyl ether)-N,N,N’,N’-tetraacetid acid (EGTA), the Ca 2+ channel blockers lanthanum chloride (LaCl 3 ), or the CaM antagonists chlorpromazine (CPZ), trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesul-fonamide (W7). Besides, Ca 2+ pretreatment significantly reduced the accumulation of reactive oxygen species (ROS), O 2 .- and H 2 O 2 and remarkably induced the total activities of the antioxidant enzymes superoxide dismutase (SOD) and ascorbate peroxidase (APX) under heat stress, while EGTA, LaCl 3 , CPZ, TFP and W7 pretreatment reversed them. The results obtained here suggest that Ca 2+ -CaM signaling pathways are involved in resistance to heat stress-induced oxidative stress by regulation of antioxidant mechanisms in tomato leaves. Key words: Calcium, calmodulin, heat stress, oxidative stress, Solanum lycopersicum L.

Identification and Inhibitory Properties of a Novel Ca2+/Calmodulin Antagonist

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.

Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.

Abstract 3003Poster Board II-980Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures:No relevant conflicts of interest to declare.

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes

Cardiac hypertrophy impairs Ca 2+ handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca 2+ release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca 2+-dependent signaling and the phosphorylation of phospholamban, which regulates Ca 2+ uptake during myocardial relaxation and is in turn regulated by Ca 2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72 h and remained elevated for at least 96 h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca 2+ mobilization all peaked 48 h after these treatments. By 96 h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca 2+ mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca 2+ regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.

Development of a new Ca2+/calmodulin antagonist and its anti-proliferative activity against colorectal cancer cells

We previously identified a cellular target of a cell cycle inhibitor HBC as Ca2+/calmodulin (Ca2+/CaM) through chemical genetics approach. Using the mechanism-based drug design, we developed a new Ca2+/CaM antagonists based on the structure of HBC. The compound, (4-{3,5-bis-[2-(4-hydroxy-3-methoxy-phenyl)-vinyl]-4,5-dihydro-pyrazol-1-yl}-phenyl)-(4-methyl-piperazin-1-yl)-methanone (referred as HBCP), binds to Ca2+/CaM in vitro and inhibits the proliferation of HCT15 colon cancer cells. HBCP induced sustained phosphorylation of ERK1/2 and subsequently activated p21WAF1 expression in HCT15 cells. Moreover, HBCP reversibly induced the G0/G1 cell cycle arrest in the cells. These data demonstrate that HBCP is a new potent Ca2+/CaM antagonist and can be applied for CaM related therapeutic uses.

Effect of natural and synthetic benzyl benzoates on calmodulin

The present investigation describes the effect of the spasmolytic benzylbenzoates 1– 9 from Brickellia veronicifolia on CaM using a functional in vitro enzymatic assay. Bovine brain PDE1 was used as a monitoring enzyme. The most active natural inhibitors of the system CaM–PDE1 were benzyl benzoates 3– 5, which inhibited the activity of PDE1 in a concentration-dependent manner. In addition, three series of analogs of compound 4, compounds 10a– 32a, were prepared and assayed. The benzyl benzoates from the first series, namely 10a– 24a, possess no substituents on ring B but different number and position of hydroxyl or methoxy groups in ring A. The second group ( 25– 32a), on the other hand, possesses an A ring identical to that on compound 4, but different substituents in Ring B. The most active compounds were 14a, 15a and 30a. These compounds were two to six times more potent than chlorpromazine, a well known CaM inhibitor. Benzyl benzoates 14a and 15a have methoxyl groups at C-2/C-4 and C-3/C-4 in ring A, respectively; while 30a, in addition to the methoxyl groups at C-2/C-6 of ring A, hold a benzoyloxy moiety at C-3′ of ring B. Kinetic studies revealed that compounds 3, 4, 14a, 15a and 30a behave as competitive CaM antagonists.

Calmodulin is essential for angiogenesis in response to hypoxic stress in endothelial cells

Angiogenesis, the formation of new blood vessels that is regulated by hypoxia, is a critical process for the growth and spread of tumors. Multiple phases of this process, including migration, adhesion, and formation of new capillary tubes, are needed for optimal tumor growth. Here, a new regulatory function for Ca2+-CaM in the vascular endothelium is described. Ca2+-CaM activation induced by hypoxia in endothelial cells is essential for angiogenic cellular responses. Inhibition of Ca2+-CaM activity suppressed endothelial cell migration, adhesion on collagen I substrate, invasion and impaired in vitro endothelial cell differentiation into tube-like structures. We also reported that CaM is co-distributed with the actin structures in the lamellipodia in migrating cells, whereas the actin cytoskeleton rearrangement induced by hypoxia was disrupted and HIF-1 transcriptional activity was decreased when treated with CaM antagonists into cultures. These data indicate that Ca2+-CaM activation is more closely associated with the regulation of angiogenic key events, especially in response to hypoxic stress.

Heterologous expression and biochemical characterization of two calcium-dependent protein kinase isoforms CaCPK1 and CaCPK2 from chickpea

In plants, calcium-dependent protein kinases (CPKs) constitute a unique family of enzymes consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. We isolated two cDNAs encoding calcium-dependent protein kinase isoforms (CaCPK1 and CaCPK2) from chickpea. Both isoforms were expressed as fusion proteins in Escherichia coli. Biochemical analyses have identified CaCPK1 and CaCPK2 as Ca(2+)-dependent protein kinases since both enzymes phosphorylated themselves and histone III-S as substrate only in the presence of Ca(2+). The kinase activity of the recombinant enzymes was calmodulin independent and sensitive to CaM antagonists W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazoilum. Phosphoamino acid analysis revealed that the isoforms transferred the gamma-phosphate of ATP only to serine residues of histone III-S and their autophosphorylation occurred on serine and threonine residues. These two isoforms showed considerable variations with respect to their biochemical and kinetic properties including Ca(2+) sensitivities. The recombinant CaCPK1 has a pH and temperature optimum of pH 6.8-8.6 and 35-42 degrees C, respectively, whereas CaCPK2 has a pH and temperature optimum of pH 7.2-9 and 35-42 degrees C, respectively. Taken together, our results suggest that CaCPK1 and CaCPK2 are functional serine/threonine kinases and may play different roles in Ca(2+)-mediated signaling in chickpea plants.

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

ABSTRACTThe involvement of calcium and different calmodulin isoforms (Ca2+‐CaM) in heat shock (HS) signal transduction in Arabidopsis (Arabidopsis thaliana) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β‐glucuronidase (GUS) activity was up‐regulated by the addition of CaCl2 and down‐regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl2 not only increased the GUS activity after HS, but also up‐regulated the GUS activity under non‐HS conditions. These results provide additional support for the involvement of the Ca2+‐CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up‐regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2.

Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe ( 35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e−56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC 30) was identified and wild type DdTK1 plus two constructs (DdTKΔC36, DdTKΔC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTKΔN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca 2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113 ± 0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2 ± 0.8%) and W13 (96.6 ± 0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca 2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.

Calmodulin and Calmodulin-dependent Kinase IIα Regulate Osteoblast Differentiation by Controlling c-fos Expression

Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.

Calmodulin is involved in heat shock signal transduction in wheat.

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.

Calmodulin antagonists and cAMP inhibit ionizing-radiation-enhancement of double-strand-break repair in human cells

Ionizing radiation (IR) enhances double-strand-break (DSB)-repair fidelity in plasmids processed in normal lymphoblasts but not in lymphoblasts from ataxia telangiectasia (A–T) patients. Putatively, signal-transduction pathways mediate this DNA-repair induction. Because IR inhibition of DNA synthesis is defective in A–T cells and is mediated by a calmodulin (caM)-dependent pathway, we evaluated the involvement of caM-dependent pathways in DSB-repair induction. Human lymphoblasts were γ-irradiated with or without treatment with caM antagonists and the cells' abilities to repair shuttle pZ189 carrying a single DSB (linDNA) were assessed. In untreated controls, IR enhanced DSB-rejoining fidelity if transfection occurred promptly but diminished fidelity if transfection was delayed. Treatment with two caM antagonists, W-7 and W-13, prior to irradiation blocked this IR-enhancement of DSB-rejoining fidelity. Vinpocetine, a caM-dependent phosphodiesterase inhibitor, and 8-bromo-cAMP also inhibited IR enhancement of repair fidelity, but caM-dependent protein kinase II inhibitor KN62 had no effect. Other protein kinase inhibitors, staurosporine and genistein, also did not inhibit IR enhancement of DSB repair fidelity. However, staurosporine blocked the twofold reduction in DSB-repair fidelity seen if linDNA transfection was delayed 2 h after irradiation. These findings point to the involvement of caM/cAMP-dependent pathway(s) in mediating IR-enhancement of DSB-rejoining fidelity in mammalian cells.

The effect of external Ca2+ and Ca(2+)-channel modulators on red-light-induced swelling of protoplasts of Phaseolus radiatus L.

Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean (Phaseolus radiatus L.) was observed only when Ca2+ ions were present in the medium. The optimal CaCl2 concentration was 250 microM. Swelling response declined when Ca2+ was supplied into the medium after red light irradiation. The Ca(2+)-chelator EGTA eliminated the red-light-induced swelling and 45Ca2+ accumulation in the protoplasts. In contrast, A23187, a Ca(2+)-ionophore, could mimic the effect of red light in darkness. These results indicate that Ca2+ may play a role in light signal transduction. In addition, swelling response was prevented by TFP and CPZ (both are CaM antagonists), implying the involvement of CaM in red-light-induced and Ca(2+)-dependent protoplast swelling.