Evaluation of zoledronate, cytochalasin-D, and desferrioxamine on osseointegration in an intra-medullary femoral implant model.

OBJECTIVE: The rise in primary and revision surgeries utilizing joint replacement implants suggest the need for more reliable means of promoting implant fixation. Zoledronate-(Zol), cytochalasin-D-(cytoD), and desferrioxamine-(DFO) have been shown to enhance mesenchymal stem cell (MSC) differentiation into osteoblasts promoting bone formation. The objective was to determine whether Zol, cytoD, and DFO can improve fixation strength and enhance peri-implant bone volume about intra-medullary femoral implants. METHODS: 48 Sprague-Dawley female rats were randomized into four treatments, saline-control or experimental: Zol-(0.8 µg/µL), cytoD-(0.05 µg/µL), DFO-(0.4 µg/µL). Implants were placed bilaterally in the femoral canals following injection of treatment solution and followed for 28 days. Mechanical push-out testing and micro-CT were our primary evaluations, measuring load to failure and bone volume. Qualitative evaluation included histological assessment. Data was analyzed with a one-way ANOVA with Holm-Sidak mean comparison testing. RESULTS: Significant results included pushout tests showing an increase in maximum energy for Zol (124%) and cytoD (82%); Zol showed an increase in maximum load by 48%; Zol micro-CT showed increase in BV/TV by 35%. CONCLUSIONS: Our findings suggest that locally applied Zol and cytoD enhance implant mechanical stability. Bisphosphonates and actin regulators, like cytoD, might be further investigated as a new strategy for improving osseointegration.

Cytochalasin D Promotes Osteogenic Differentiation of MC3T3-E1 Cells via p38-MAPK Signaling Pathway.

BACKGROUND: Bone defect caused by trauma, tumor resection, infection or congenital malformation is a common clinical disease. Bone tissue engineering is regarded as a promising way of bone defect reconstruction. Thus, agents that can promote osteogenesis have received great attention. Cytochalasin D (Cyto D), a metabolite derived from molds, proves to be able to modify actin, reorganize cytoskeleton, and then promote the osteogenic differentiation. OBJECTIVE: The purpose of this study was to explore the effect and mechanism of Cyto D on osteogenic differentiation of mouse pre-osteoblast MC3T3-E1 cells. METHODS: The optimum concentration of Cyto D was explored. The osteogenic differentiation of MC3T3-E1 cells induced by Cyto D was assessed by alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, western blotting and quantitative real-time polymerase chain reaction (RT-qPCR). In addition, a specific pathway inhibitor was utilized to explore whether MAPK pathways were involved in this process. RESULTS: The results showed that the optimized concentration of action was 10-2µg/ml. The expression of Runx2, OCN and OSX was up-regulated by the supplement of Cyto D. ALP activity, calcium deposition, and phosphorylation level of p38 protein were also improved. Inhibition of the pathway significantly reduced the activation of p38, and the expression of osteogenic-related genes. CONCLUSION: Cyto D can promote the osteogenic differentiation of MC3T3 cells via the p38-MAPK signaling pathway, but not the ERK1/2 or JNK, and it is a potential agent to improve the osteogenesis of MC3T3 cells.

Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund's adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund's adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites.

The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement.

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.

Interfacial behavior of immortalized hypothalamic mouse neurons detected by acoustic wave propagation.

The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 µM), colchicine (1 µM), cytochalasin B (10 µM) and cytochalasin D (2 µM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.

Microscopy-based HTS examines the mechanism of stress F-actin fiber disruption by cytochalasin D: orientation texture data collated with quantitative kinetic modeling.

We report a drug dose-response, end-point study of intracellular filamentous actin (F-actin) by automated fluorescence microscopy, complemented with theoretical kinetic simulation of drug action. We highlight the use of an advanced orientation-sensitive image processing procedure ( transform), specially tailored for the detection of ordered filamentous "patches" in cell images. To examine the extent of stress F-actin disruption caused by the drug, we compare the measured response based on the above transformation with the theoretical data obtained from a quantitative model. We show that the assay data are consistent with the first-order mass action kinetics predicted by a basic reaction model. As a concluding remark, we briefly discuss advantages, perspectives, and challenges of conventional fluorescent microscopy within the context of the quantitative high-throughput screening paradigm.

Fine particles that adsorb lipopolysaccharide via bridging calcium cations may mimic bacterial pathogenicity towards cells.

Fine particles (10(2)- to 10(3)-nm diameter) are potentially potent adjuvants in acquired immune responses but little is known about their interaction with pathogen-associated molecular patterns (PAMPs) and impact upon innate immunity. Here we show that 200-nm-sized, food-grade titanium dioxide avidly binds lipopolysaccharide (LPS) with bridging calcium cations, and the complex induces marked proinflammatory signalling in primary human mononuclear phagocytes. In particular, caspase 1-dependent interleukin-1beta (IL-1beta) secretion was induced at levels far greater than for the sum of the individual components, and without concomitant secretion of modulatory cytokines such as interleukin-1 receptor antagonist or transforming growth factor-beta1 (TGF-beta1). Secondly, the conjugate induced apoptotic-like cell death. These responses were inhibited by blockade of both phagocytosis and scavenger receptor uptake. Specific caspase 1-facilitated IL-1beta secretion and apoptosis following phagocytosis are features of cellular responses to certain invasive, enteric pathogens, and hence induction of these events may be mimicked by fine particle-LPS conjugates. The inadvertent adsorption of PAMPs to ingested, inhaled, or "wear" fine particulate matter provides a further potential mechanism for the proinflammatory nature of fine particles.

Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells.

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.

Hypoosmotic cell swelling as a novel mechanism for modulation of cloned HCN2 channels.

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.

Actin polymerization promotes the reversal of streaming in the apex of pollen tubes.

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.

Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation.

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

Protein kinase C activation downregulates human organic anion transporter 1-mediated transport through carrier internalization.

Organic anion transport in intact renal proximal tubule cells in animal model systems is downregulated by treatments that activate protein kinase C (PKC). How this downregulation is achieved is not yet known. Stimulation of PKC with sn-1,2-dioctanoylglycerol resulted in strong inhibition of p-aminohippurate transport mediated by the cloned human organic anion transporter 1 (hOAT1) expressed in Xenopus oocytes and HEK293 cells, as well as hOAT1 internalization in both expression systems. The sn-1,2-dioctanoylglycerol-induced transport inhibition was partially prevented by staurosporine. It was independent of the conserved canonical PKC consensus sites in hOAT1, however, and was unaffected by agents that destabilize actin filaments or microtubules, which altered baseline hOAT1-mediated p-aminohippurate uptake activity in oocytes. It is concluded that PKC-induced hOAT1 downregulation is achieved through carrier retrieval from the cell membrane and does not involve phosphorylation of the predicted classic hOAT1 PKC consensus sites.

High glucose-altered gene expression in mesangial cells. Actin-regulatory protein gene expression is triggered by oxidative stress and cytoskeletal disassembly.

High extracellular glucose plays a pivotal role in the pathophysiology of diabetic nephropathy. Here we report 200 genes, identified using suppression-subtractive hybridization, that are differentially expressed when human mesangial cells are propagated in high ambient glucose in vitro. The major functional classes of genes identified included modulators and products of extracellular matrix protein metabolism, regulators of cell growth and turnover, and a cohort of actin cytoskeleton regulatory proteins. Actin cytoskeletal disassembly is a prominent feature of diabetic nephropathy. The induction of actin cytoskeleton regulatory gene expression by high glucose was attenuated by the inhibitor of reactive oxygen species generation, carbonyl cyanide m-chlorophenylhydrazone but not by the protein kinase C inhibitor GF 109203X and was not mimicked by the addition of transforming growth factor beta. Enhanced expression of actin cytoskeleton regulatory genes was also observed following disruption of the mesangial cell actin cytoskeleton by cytochalasin D. In aggregate, these results suggest that the induction of genes encoding actin cytoskeleton regulatory proteins (a) is a prominent component of the mesangial cell transcriptomic response in diabetic nephropathy and (b) is dependent on oxidative stress, is independent of protein kinase C and transforming growth factor-beta, and represents an adaptive response to actin cytoskeleton disassembly.

The Arabidopsis gene tardy asynchronous meiosis is required for the normal pace and synchrony of cell division during male meiosis.

Male meiosis in higher organisms features synchronous cell divisions in a large number of cells. It is not clear how this synchrony is achieved, nor is it known whether the synchrony is linked to the regulation of cell cycle progression. Here, we describe an Arabidopsis mutant, named tardy asynchronous meiosis (tam), that exhibits a phenotype of delayed and asynchronous cell divisions during male meiosis. In Arabidopsis, two nuclear divisions occur before simultaneous cytokinesis yields a tetrad of haploid cells. In tam, cell divisions are delayed, resulting in the formation of abnormal intermediates, most frequently dyad meiotic products, or in rare cases, dyad pollen (two gametophytes within one exine wall). Temperature-shift experiments showed that the percentage of the abnormal intermediates increased at 27 degrees C. Analysis of tam and the tam/quartet1 double mutant showed that most of these abnormal intermediates could continue through the normal rounds of cell divisions and form functional pollen, though at a slower than normal pace. The asynchrony of cell division started at the G2/M transition, with cells entering metaphase at different time points, during both meiosis I and II. In addition, chromosome condensation defects and mis-segregation were sometimes observed in tam. These observations suggest that the TAM protein positively regulates cell cycle progression, perhaps by promoting the G2/M transition. We speculate that there is a signal, perhaps TAM, that couples the normal pace of cell cycle progression with the synchrony of cell division during male meiosis.

Potassium channel blocker activates extracellular signal-regulated kinases through Pyk2 and epidermal growth factor receptor in rat cardiomyocytes.

OBJECTIVES: We sought to determine whether potassium (K(+)) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes. BACKGROUND: Because KBs attenuate K(+) outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca(2+)) transient ([Ca(2+)](i)) in cardiomyocytes. Elevation of intracellular Ca(2+) levels can trigger various signaling events. Influx of Ca(2+) through L-type Ca(2+) channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown. METHODS: Primary cultured rat cardiomyocytes were stimulated with four different KBs-4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine-and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca(2+))(i) was monitored by the fluorescent calcium indicator Fluo-4. RESULTS: E-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca(2+))(i) by 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not. CONCLUSIONS: These findings indicate that KBs induce ERK activation, which starts with Ca(2+) entry through the L-type Ca(2+) channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

The aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression.