Differential activation of ERK 1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions

The mechanisms by which mitogen-activated protein kinases (MAPK) respond to the input of UV-induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC-irradiated cells. The intracellular oxidative level of normal human fibroblast-like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N-acetyl-L-cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV-irradiation was significantly reduced. Among MAPK, extracellular-signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c-Jun N-terminal kinase (JNK) activation was inhibited in NAC-treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV-induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.     

PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.     

Role of Ca(2+) in diallyl disulfide-induced apoptotic cell death of HCT-15 cells

Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.     

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (β-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.     

Salidroside attenuates hydrogen peroxide-induced cell damage through a cAMP-dependent pathway

Salidroside, a major component of Rhodiola rosea L., has shown various pharmacological functions, including antioxidant effects, but the signal transduction pathway of its antioxidant effects is not very clear. In this study, we found that salidroside could attenuate hydrogen peroxide (H(2)O(2))-induced HL-7702 cell damage, inhibit H(2)O(2)-induced cytosolic free Ca2+ ([Ca2+]i) elevation, scavenge reactive oxygen species (ROS) and increase 3'-5'-cyclic adenosine monophosphate (cAMP) level in a dose-dependent manner, but it couldn't influence 3'-5'-cyclic guanosine monophosphate (cGMP) levels. Therefore, these results indicated that the antioxidant effects of salidroside were associated with down-regulation of [Ca2+]i, ROS occur via a cAMP-dependent pathway.     

A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca~(2+) indicator STDIn-AM

A new fluorescent Ca2+ indicator STDIn-AM for detecting [Ca2+]i transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDIn and fluo-3 is discussed in detail. Using the new Ca2+ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDIn is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca2+ indicator and can reflect changes of cytosol [Ca2+] more accurately than that of fluo-3. In addition, STDIn responds to the [Ca2+]i transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca2+channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca2+]i elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of     

Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 murine macrophage by 7-O-methyl-naringenin

7-O-Methylnaringenin, extracted from Rhododendron speciferum, belongs to the flavanone class of polyphenols. In the present study, we investigated the anti-inflammatory effects of 7-O-methylnaringenin on cytokine production by lipopoly-saccharide (LPS)-stimulated RAW 264.7 macrophages in vitro. The results showed that pretreatment with 10, 20 or 40 μg/mL of 7-O-methylnaringenin could downregulate tumour necrosis factor (TNF-α), interleukin (IL-6) and interleukin (IL-1β) in a dose-dependent manner. Furthermore, we investigated the signal transduction mechanisms to determine how 7-O-methylnaringenin affects RAW 264.7 macrophages. The activation of mitogen-activated protein kinases (MAPK) and IκBα were measured by Western blotting. The data showed that 7-O-methylnaringenin could downregulate LPS-induced levels of phosphorylation of ERK1/2, JNK and IκBα. These observations indicated that 7-O-methylnaringenin modulated inflammatory cytokine responses by blocking NF-?B, ERK1/2 and JNK/MAPKs activation.     

Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.     

Mechanistic studies of a calcium-dependent MRI contrast agent

Intracellular Ca(2+) plays an important role in signal transduction, and we are developing new MRI techniques to study its regulation in living animals. We have reported on an MRI contrast agent (DOPTA-Gd) where the relaxivity of the complex is controlled by the presence or absence of the divalent ion Ca(2+). By structurally modulating inner-sphere access of water to a chelated Gd(3+) ion, we observe a substantial and reversible change in T(1) upon the addition of Ca(2+) and not other divalent ions. Luminescence lifetime and NMRD measurements of the complex have been acquired, and several parameters contribute to the Ca(2+) dependent relaxivity change of DOPTA-Gd. The number of inner-sphere water molecules is more than doubled after the Ca(2+) concentration is increased. This finding strongly supports the proposed conformational change of DOPTA-Gd when Ca(2+) is bound. Relaxometric measurements confirm these results and provide an indication that second-sphere water molecules are probably responsible for paramagnetic relaxation enhancement in the absence of Ca(2+). After Ca(2+) is bound to DOPTA-Gd, the molecule undergoes a substantial conformational change that opens up the hydrophilic face of the tetraazacyclododecane macrocycle. This change dramatically increases the accessibility of chelated Gd(3+) ion to bulk solvent. The design of this class of calcium-activated MR contrast agent was based primarily on the assumption that the number of coordinated inner-sphere water molecules would be the dominating factor in observed relaxivity measurements. This result has been confirmed; however, careful mechanistic studies reveal that additional factors are involved in this process.     

RNA aptamers as pathway-specific MAP kinase inhibitors

BACKGROUND: In eukaryotic cells, many intracellular signaling pathways have closely related mitogen activated protein kinase (MAPK) paralogs as central components. Although MAPKs are therefore obvious targets to control the cellular responses resulting from the activation of these signaling pathways, the development of inhibitors which target specific cell signaling pathways involving MAPKs has proven difficult. RESULTS: We used an RNA combinatorial approach to isolate RNAs that inhibit the in vitro phosphorylation activity of extracellular regulated kinase 2 (ERK2). These inhibitors block phosphorylation by ERK1 and ERK2, but do not inhibit Jun N-terminal kinase or p38 MAPKs. Kinetic analysis indicates these inhibitors function at high picomolar concentrations through the steric exclusion of substrate and ATP binding. In one case, we identified a compact RNA structural domain responsible for inhibition. CONCLUSIONS: RNA reagents can selectively recognize and inhibit MAPKs involved in a single signal transduction pathway. The methodology described here is readily generalizable, and can be used to develop inhibitors of MAPKs involved in other signal transduction pathways. Such reagents may be valuable tools to analyze and distinguish homologous effectors which regulate distinct signaling responses.     

The influence of different InsP(3) receptor isoforms on Ca(2+) signaling in tracheal smooth muscle cells

In airway myocytes, like in many cells, Ca(2+) signaling is controlled by inositol 1,4,5-trisphosphate (InsP(3)) via InsP(3) receptors (InsP(3)R) located in the sarco-endoplasmic reticulum. Three types of InsP(3)R exist, labeled Types 1, 2, and 3, which differ in their gating kinetics. We analyze a possible impact of the different gating kinetics of Type 1 and Type 3 InsP(3)R on the time course of cytosolic Ca(2+) concentration in tracheal smooth muscle cells upon agonist stimulation. Previous experimental data in rat tracheal myocytes showed that upon gradually increased stimulation with acetylcholine (ACh), a contractile agonist that acts via InsP(3) production, signal spikes, several spikes with declining maxima, and sustained oscillations appear. Our model reproduces the time courses of cytosolic Ca(2+) measured in tracheal myocytes. Moreover, by postulating slight variations in the model parameters which determine the total number of receptors expressed and the ratio between Type 1 and Type 3 InsP(3)R, it offers an explanation to the experimental observation of qualitatively different responses of cells within a presumably homogeneous tissue.     

Intermolecular communication on a liposomal membrane: enzymatic amplification of a photonic signal with a gemini peptide lipid as a membrane-bound artificial receptor

A supramolecular system that can activate an enzyme through photo-isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self-assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH-dependent L-lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane-bound receptor induced photo-isomerization, which significantly altered the receptor's metal-binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu(2+) ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

The Newly Synthetized Chalcone L1 Is Involved in the Cell Growth Inhibition,Induction of Apoptosis and Suppression of Epithelial-to-Mesenchymal Transition of HeLa Cells

Over the past decades, natural products have emerged as promising agents with multiple biological activities. Many studies suggest the antioxidant, antiangiogenic, antiproliferative and anticancer effects of chalcones and their derivatives. Based on these findings, we decided to evaluate the effects of the newly synthetized chalcone L1 in a human cervical carcinoma cell (HeLa) model. Presented results were obtained by western blot and flow cytometric analyses, live cell imaging and antimigratory potential of L1 in HeLa cells was demonstrated by scratch assay. In the present study, we proved the role of L1 as an effective agent with antiproliferative activity supported by G2/M cell cycle arrest and apoptosis. Moreover, we proved that L1 is involved in modulating Transforming Growth Factor-β1 (TGF-β) signal transduction through Smad proteins and it also modulates other signalling pathways including Akt, JNK, p38 MAPK, and Erk1/2. The involvement of L1 in epithelial-to-mesenchymal transition was demonstrated by the regulation of N-cadherin, E-cadherin, and MMP-9 levels. Here, we also evaluated the effect of conditioned medium from BJ-5ta human foreskin fibroblasts in HeLa cell cultures with subsequent L1 treatment. Taken together, these data suggest the potential role of newly synthesized chalcone L1 as an anticancer-tumour microenvironment modulating agent.     

p38 mitogen-activated protein kinase activation by ultraviolet A radiation in human dermal fibroblasts

UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen-activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH2-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.     

A new recognition concept using dye sensitized solar cell configuration

A bifunctional compound 1 was designed and synthesized, in which the donor-π-conjugated-acceptor-cyanoacrylic acid configuration is suited for DSSC, while the tetraoxa-azacrown (donor) part also acts as the recognition unit for e.g. Ca(2+) and the output of DSSC is utilized as signal transduction, demonstrating a new recognition concept using DSSC.