B) receptors in middle cerebral arteries (MCA) after organ culture. This upregulation is dependent on mitogen-activated protein kinases and possibly protein kinase C (PKC). The aim of this study was to examine the effect of PKC inhibitors with different profiles on the upregulation of contractile ETB receptors in rat MCA. Artery segments were incubated for 24 hours at 37°C. To investigate involvement of PKC, inhibitors were added to the medium before incubation. The contractile endothelin-mediated responses were measured and real-time PCR was used to detect endothelin receptor mRNA levels. Furthermore, immunohistochemistry was used to demonstrate the ETB receptor protein distribution in the MCA and Western blot to measure which of the PKC subtypes that were affected by the inhibitors.Previous studies have shown that there is a time-dependent upregulation of contractile endothelin B .The PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and PKC inhibitor 20–28 attenuated the ETB receptor contraction and expression in MCA smooth muscle cells following organ culture. The PKC inhibitor 20–28 affects the protein levels, while Ro-32-0432 and bisindolylmaleimide I affect the mRNA levels, suggesting differences in activity profile. Since ETB receptor upregulation is seen in cerebral ischemia, the results of the present study provide a way to interfere with the vascular involvement in cerebral ischemia.The results show that PKC inhibitors are able to decrease the ET A) receptors are situated on the smooth muscle cells in arteries where they give rise to contractions, while the ETB receptors are primarily seen on endothelial cells. An activation of the endothelial ETB receptors will lead to dilatation [B receptors located on the smooth muscle cells of cerebral arteries. We have revealed that this upregulation, when occurring in organ culture, is dependent on mitogen-activated protein kinases (MAPKs) [The endothelins constitute a group of vasoactive peptides mainly produced by vascular endothelial cells . There alatation . Intereslatation , experimlatation  and expelatation  there is (MAPKs)  and poss (MAPKs) ,8.PKC was first discovered in 1977 -12. The B receptor upregulation [The PKC inhibitors Ro-31-8220 and Ro-31-7549, used in previous studies of ETgulation ,14. ConsB receptors in rat middle cerebral arteries (MCAs) during 24 hours of organ culture. This duration has proven to be an optimal time for obtaining an upregulation of contractile ETB receptors [B receptors on the arterial smooth muscle cells. Finally, Western blot was used to examine the levels of different PKC subtypes in the arteries after incubation with the PKC inhibitors.The aim of this study was therefore to establish the involvement of PKC in the upregulation of contractile ETeceptors . SubsequB receptor upregulation. Bisindolylmaleimide I (Bis I), Ro-32-0432 and PKC inhibitor 20–28 (PKCi 20–28) all significantly affected the sarafotoxin 6c  induced contractility. In addition, Ro-32-0432 decreased the ETB receptor mRNA levels, as did Bis I to some extent. PKCi 20–28 had no such effect. However, PKCi 20–28 did reduce the amount of ETB receptor protein in the arteries and also decreased the protein amount of the five PKC subtypes tested .The general PKC inhibitors tested, all of which are cell permeable, had different effects on the vascular ET+-induced contractions did not differ between the control group and the arteries incubated with each of the PKC inhibitors (data not shown). Previous studies have shown that S6c does not induce contraction of fresh MCA segments [B receptor. In the present study, the S6c induced contraction was significantly decreased in the arteries cultured with the PKC inhibitors Bis I, Ro-32-0432 and PKCi 20–28 as compared to control  only affects the remaining contractile ETA receptors [Ksegments . After ool Table , fig. 1.eceptors .50 value of the ET-1 concentration-response curve .A receptor and ETB receptor mRNA levels significantly as compared to control  and Bis I , however it was not significant.Immunohistochemistry showed a clear decrease of ETWestern blot experiments were carried out using antibodies directed specifically against phosphorylated, and thereby activated, PKC isoforms . These tests showed that PKCi 20–28 was able to decrease the protein amount of all five PKC subtypes tested, although the decrease was most prominent for PKCδ , which could explain why chelerythrine chloride fails to inhibit the ETB receptor upregulation in the present study.This study shows that each of the three general PKC inhibitors attenuates the organ culture induced upregulation of the ETcificity . Furthercificity . Our gro50 = 10 nM) [50 = 9 nM), but also βI, βII and γ [Bis I and Ro-32-0432 are both competitive inhibitors for the ATP-binding site of PKC ,19, but = 10 nM) , and the= 10 nM) . Ro-32-0B receptor mediated contraction.The third inhibitor tested, the PKC inhibitor 20–28, is a pseudosubstrate peptide, which mimics a particular domain of the PKC that keeps the enzyme in an inactive state . The pepB receptor mRNA relative to that seen in the control arteries. However, PKCi 20–28 failed to exhibit this effect on the mRNA level. Instead, PKCi 20–28 diminished ETB receptor protein expression. One reason for this discrepancy could be that Ro-32-0432 and Bis I exert their inhibitory effects earlier in the production of ETB receptors in the arteries, possibly upstream from gene level, causing a decrease of the ETB receptor mRNA. PKCi 20–28 on the other hand, affects the ETB receptor protein production and not the mRNA levels and hence at a translational level. In concert the PKCi 20–28 decreased the amount of ETB receptor protein as shown by immunohistochemistry, and marginally in the case of Bis I and Ro-32-0432. Nevertheless, a small decrease could be enough to diminish the functional response. Thus, the results could be interpreted to show a multiple role for PKC in the upregulation of ETB receptors. Another explanation for the difference between the mRNA levels and the protein levels in the case of PKCi 20–28 is that the inhibiting effect may not be as prolonged as for the two other inhibitors and subsequently, at the time point measured, the ETB receptor mRNA might be restored to its normal level.The real-time PCR showed that Ro-32-0432 and, to a certain extent, Bis I blocked the elevated levels of ETA receptor mRNA in rat cerebral arteries is not altered by 24 hours of organ culture [A receptor mRNA levels after incubation. This was not accompanied by a diminished ETA receptor mediated contraction. This discrepancy could be due to the fact that the mRNA levels do not necessarily reflect the presence of functional receptors, and at a later time point, the ETA receptor density will decrease.The ET culture . Still, B receptor alteration after organ culture [Previous studies with the PKC inhibitors Ro-31-8220 and Ro-31-7549 indicate that the conventional PKC subtypes  are involved in the ET culture . For exa culture , and PKC culture . FurtherB receptor upregulation in the MCA [B receptor upregulation in the cerebral arteries.We have previously shown that in both experimental focal ischemia and subarachnoid hemorrhage there is an ET the MCA ,5. This B receptor contraction and expression in smooth muscle cells of MCA following 24 hours of organ culture. A similar upregulation of contractile ETB receptors has been seen in an experimental model of focal ischemia. The results of the present study can therefore be of significance in finding new therapeutic targets in cerebral ischemia.This study demonstrates that PKC inhibitors are able to decrease the ETThe experimental procedures were approved by the Ethics Committee for Laboratory Animal Experiments at Lund University (Application number M 217-03).2 and decapitated. The right and left MCA were removed and dissected free from surrounding tissue. The arteries were cultured for 24 hours in Dulbeccos modified Eagle's medium (DMEM), supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (25 μg/ml), at 37°C in humidified 5% CO2 in air. This induces a maximal ETB receptor upregulation [i values and on previous studies using the inhibitors. Pilot experiments were done to determine the optimal doses in this setting.Male Wistar rats  were anesthetized with CO2 in O2, resulting in a pH of 7.4. The arteries were given an initial tension of 1.2 mN, and were allowed to adjust to this level of tension for 1 hour. The contractile capacity was determined by exposure to a potassium-rich (63.5 mM) buffer . Concentration-response curves for the agonists S6c (ETB receptor agonist) and ET-1 (ETA and ETB receptor agonist) were obtained by cumulative application (10-12-10-6.5 M). Following sarafotoxin 6c administration, the endothelin ETB receptors are desensitized, leaving only endothelin ETA receptors to interact with endothelin-1 [After incubation, the arteries were cut into cylindrical segments. The segments were mounted on two 40 μm diameter stainless steel wires in a Mulvany-Halpern myograph  ,28. One thelin-1 .max values represent the maximum vascular contraction as response to S6c or ET-1 and were calculated as percentage of the contractile response towards 63.5 mM K+. The pEC50 values represent the negative logarithm of the concentration which elicits half-maximum response. Data are expressed as mean values ± S.E.M. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn's post-hoc test. P < 0.05 is considered significant. There were 3–5 rats in each group, with 1–4 segments from each. n refers to the number of arterial segments.The ETotal cellular RNA was extracted from each MCA using the FastRNA Pro Green Kit following the suppliers' instructions. The resulting pellet was finally washed with 75% ethanol, air-dried and redissolved in diethylpyrocarbonate-treated water. Reverse transcription of total RNA to cDNA was carried out using the GeneAmp RNA PCR kit in a Perkin-Elmer DNA Thermal cycler, using random hexamers as primers. The reaction mixture was incubated at 25°C for 10 minutes, 42°C for 30 minutes, heated to 99°C for 5 minutes and chilled to 5°C for 5 minutes.® Green kit with the cDNA synthesized above as template in a 50 μl reaction volume. A no template control was included in all experiments. This technique consists of an optic imaging system that is able to monitor the amount of DNA in each PCR cycle via the detection of a fluorescent dye binding to double-stranded DNA. The DNA levels of the genes of interest are compared to an endogenous standard. In this study, specific primers for the rat ETA and ETB receptors were designed as follows: ETA receptor, forward: 5'-ATTGCCCTCAGCGAACAC-3'; reverse: 5'-CAACCAAGCAGAAGACGGTC-3', ETB receptor forward: 5'GATACGACAACTTCCGCTCCA-3'; reverse: 5'-GTCCACGATGAGGACAATGAG-3'. Elongation factor-1 (EF-1) mRNA was used as an endogenous standard. The EF-1 primers were designed as follows: forward; 5'-GCAAGCCCATGTGTGTTGAA-3'; reverse: 5'-TGATGACACCCACAGCAACTG-3'. The real-time PCR was carried out as follows: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample was examined in duplicates.Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System  using the GeneAmp SYBRA receptor and ETB receptor mRNA was calculated as relative to the amount of EF-1 mRNA in the same sample by the formula: X0/R0 = 2CtR-CtX, where X0 = original amount of endothelin receptor mRNA, R0 = original amount of elongation factor-1 mRNA, CtR = CT-value for elongation factor-1 and CtX = CT-value for the endothelin receptor. Each sample was examined in duplicates and the mean values were used.The amount of ETn = 6–7, where n is the number of MCAs used.Data are expressed as mean values ± S.E.M. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn's post-hoc test. P < 0.05 is considered significant. B receptor antibody, diluted 1:400, goat-antimouse endothelin ETA receptor antibody, diluted 1:100 and mouse-antirat CD31 antibody, diluted 1:200. The secondary antibodies used were donkey-antirabbit Cy™3 conjugated, diluted 1:100, donkey-antigoat Cy™2 conjugated, diluted 1:100, and donkey-antimouse Cy™5 conjugated, diluted 1:100. All dilutions were done in phosphate buffered saline with 10% fetal calf serum. The antibodies were then detected at the appropriate wavelength in a confocal microscope . As control, only addition of secondary antibodies was used.After incubation for 24 hours, the MCAs were placed onto Tissue TEK, frozen and subsequently sectioned into 10 μm slices. The primary antibodies used were rabbit-antihuman endothelin ETThe images were analysed using the ImageJ software . The fluAfter incubation, the MCAs were collected and placed on ice, homogenized in lysis-buffer with protease- and phosphatase inhibitors . After 20 minutes incubation in lysis buffer on ice, homogenates were centrifuged at 4500 g for 10 min at 4°C and supernatant collected. Total protein concentration was determined using a BioRad DC kit  and the absorbance was measured at 750 nm on a Genesys 10 spectrophotometer .Lysates were dissolved in Tris-glycine SDS sample buffer  and boiled for 5 minutes. Equal amounts of protein (10 μg/lane) were loaded on a 8% Tris-glycine gel  and separated by SDS-polyacrylamide gel electrophoresis. Molecular weight markers  were loaded on each gel for protein band identification. After separation, proteins were transferred to a nitrocellulose membrane . Subsequently the membrane was blocked with 6.5% non-fat milk in Tween-TBS overnight at 4°C. Membranes were then incubated with the following primary antibodies: rabbit polyclonal anti-PKCα phosphospecific (1:1000 dilution), rabbit polyclonal anti-PKCδ phosphospecific (1:1000 dilution), rabbit polyclonal anti-PKCε phosphospecific (1:1000 dilution), rabbit polyclonal anti-PKCβI phosphospecific (1:1000 dilution), rabbit polyclonal anti-PKCγ phosphospecific (1:1000 dilution) or mouse polyclonal β-actin overnight at 4°C, followed by 3 × 5 minutes wash with Tween-TBS. Thereafter, the membranes were incubated with the appropriate secondary antibody: goat anti-rabbit IgG-horseradish peroxidase or goat anti-mouse IgG-horseradish peroxidase (1:5000) for 1 hour at room temperature, followed by 5 × 5 minutes wash with Tween-TBS. Levels of β-actin were used to confirm equal loading of the lanes. The membranes were developed using the Supersignal Dura kit  and visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer .Protein lysates from the four different groups were compared. MCAs from three animals were pooled for each sample and each group consisted of three samples. Quantification of band density was performed with the electrophoresis computer analysis program Fujifilm Science Lab Image Gauge 4.0. The immunoblot optical density values were determined with repeated measurements and are presented as percentage activity in the groups treated with PKC inhibitors compared to the control group, where the control group was set to 100%. All values are relative to the respective β-actin band density. Data are expressed as mean values ± S.E.M.3 15; KCl 4.6; MgCl2 1.2; NaH2PO4 1.2; CaCl2 1.5 and glucose 5.6, while the potassium-rich bicarbonate buffer composition was as follows (mM): NaCl 59.5; NaHCO3 15; KCl 63.5; MgCl2 1.2; NaH2PO4 1.2; CaCl2 1.5 and glucose 5.6.The bicarbonate buffer had the following composition (mM): NaCl 119; NaHCOThe DMEM supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and amphotericin B (25 μg/ml) was purchased from Gibco .Bis I, Ro-32-0432, chelerythrine chloride and PKCi 20–28 were purchased from Calbiochem  and dissolved in dimethyl-sulfoxide. ET-1 and S6c  were dissolved in 0.9% saline with 0.1% bovine serum albumin.FastRNA Pro Green Kit was purchased from BIO 101 .® Green kit were purchased from Applied Biosystems .GeneAmp RNA PCR kit and GeneAmp SYBRB receptor antibody was purchased from IBL , goat-antimouse ETA receptor antibody from Santa Cruz Biotechnologies  and mouse-antirat CD31 antibody from Serotec . The secondary antibodies, donkey-antimouse Cy™5 conjugated, donkey-antirabbit Cy™3 conjugated and donkey-antigoat Cy™2 conjugated, were purchased from JacksonImmunoResearch .For the immunohistochemistry, Tissue TEK was purchased from Gibco . Rabbit-antihuman ET3VO4, 0.5% Deoxycholate, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton X-100, 1 mM DTT, 20 μM Pepstatin, 20 μM Leupeptin, 0.1 U/ml Aprotinin, 1 nM Calyculin and 1 mM PMSF.The lysis-buffer for Western blot had the following composition: 10 mM Tris pH 7.4, 50 mM β-Glycerophosphate, 100 μM NaThe primary antibodies for the Western blot were rabbit polyclonal antibodies with following specificity: anti-PKCα phosphospecific (phosphorylated at threonine 638), anti-PKCδ phosphospecific (phosphorylated at serine 664), anti-PKCε phosphospecific (phosphorylated at serine 739), anti-PKCβI phosphospecific (phosphorylated at threonine 642) and anti-PKCγ phosphospecific (phosphorylated at threonine 655) were purchased from Biosource . The mouse polyclonal β-actin antibody was purchased from Sigma . The secondary antibodies; goat anti-rabbit IgG-horseradish peroxidase and goat anti-mouse IgG-horseradish peroxidase were purchased from Pierce .MH participated in the design of the study, carried out the artery preparations, organ culture, myograph experiments, analyzed the data and wrote most of the manuscript.PV carried out the immunohistochemistry.ES participated in the design of the study, participated in the myographs experiments and revised the manuscript.SB carried out the Western blots and contributed to the writing and revision of the manuscript.LE participated in the design of the study and revised the manuscript.All authors read and approved the final manuscript.


The effects of tumour necrosis factor-alpha  on the growth and DNA synthesis of the human breast cell line, T47D, were studied. A dose-dependent, reversible inhibition of thymidine incorporation and cell growth was observed in the range of 0.1 ng ml-1 to 100 ng ml-1 of TNF alpha. Cell viability was not impaired in any of the experiments. Flow-cytometric DNA analysis demonstrated that after 24 h exposure to TNF alpha, T47D cells accumulated in the G1 phase of the cell cycle, and were depleted in the G2/M and S phases, suggesting a block in the progression of the G1/S transition. The involvement of protein kinases (PK) and protein phosphatases in TNF alpha-induced signal transduction was also investigated. A transient and rapid 2-fold increase in total cellular protein kinase C (PKC) activity was detected after 10 min exposure to TNF alpha. To study the role of the observed PKC activation in the cytostatic effect of TNF alpha, T47D cells were exposed to the cytokine in the presence of the potent PKC inhibitor, H7. The inhibitory effect of TNF alpha on thymidine incorporation was not affected by exposure to H7 at concentrations sufficient to block the stimulation of thymidine up-take induced by the PKC agonist, phorbol-12-myristate-13-acetate (PMA). The involvement of other signalling pathways was addressed using the cyclic nucleotide-dependent PK inhibitor, H8; the calmodulin-dependent PK inhibitor, W7; and the inhibitor of protein phosphatases PP1 and PP2B, okadaic acid. Exposure of T47D cells to these enzyme inhibitors failed to antagonise the inhibition of thymidine incorporation by TNF alpha. Taken together, these results indicate that the cytostatic effect of TNF alpha on T47D cells occurs at the G1/S transition of the cell cycle, and is mediated by an intracellular pathway which does not involve the activity of protein kinases C and A, nor protein phosphatases PP1, PP2B.


Pfiesteria are reported to have been responsible for massive fish kills in the southeastern United States. Some reports suggest that exposure to waters having Pfiesteria blooms or occupation-related exposure might result in Pfiesteria-induced dermal irritation and inflammation. Although the toxin has not been isolated and purified, the original data suggested both hydrophilic and hydrophobic toxic components. Some investigators propose that dermonecrotic properties are associated with a hydrophobic fraction.Members of the estuarine dinoflagellate genus Pfiesteria-laden aquarium water during active fish-killing conditions was examined in the present study to evaluate its potential to produce inflammation and dermal sensitization and to determine whether the inflammation and dermatitis reported in early human exposure studies were allergic or irritant in nature.A bioactive C18-bound putative toxin (CPE) extracted from This fraction was cytotoxic to mouse Neuro-2A cells and primary human epidermal keratinocytes (NHEK) at a concentration of 1 mg/mL. Balb/C mice exposed to 50–200% CPE by skin painting exhibited a 6–10% increase in ear swelling relative to vehicle-treated mice in a primary irritancy assay. There was no increase in lymph node cell proliferation as measured using the local lymph node assay. Exposure to CPE in culture up-regulated interleukin-8 in NHEK, whereas granulocyte macrophage–colony-stimulating factor and tumor necrosis factor α were only minimally altered.This study suggests that CPE is cytotoxic to keratinocytes in culture at high concentrations and that it induces mild, localized irritation but not dermal sensitization. Pfiesteria are reported to have been responsible for massive fish kills between 1991 and 1998, primarily in the estuaries of North Carolina and Maryland. The classic signs of Pfiesteria poisoning in fish are skin ulcerations and altered, erratic swimming patterns (a) that Pfiesteria complex organisms produce a potent exotoxin that destroys the integrity of the skin directly (b) that the dinoflagellate directly attaches to and feeds on fish epidermis  pidermis . Anotherresponse  and indiresponse .Pfiesteria-related illness in a multiple system syndrome known as possible estuary-associated syndrome (PEAS)  includes e (PEAS) . Under td waters , intense lesions . Initialammation , 1998. Tammation . In thesammation . These iammation . More relth risk . Evaluatnditions . In two nditions . The nonnditions .Pfiesteria toxicity is still unknown, although numerous efforts have been made to isolate the toxin and characterize the chemical structure and mechanism of action  is preferentially released from the cells  as an allergen and contact sensitizer and to distinguish a possible local irritancy response from dermal sensitization.Earlier studies have associated intense exposure to ailments , 1998. Tytokines . The goaad libitum and were monitored daily for acute toxicity and weighed weekly. Treatment had no effect on the body weight of the mice (data not shown). We conducted all experiments under an approved National Institute of Environmental Health Sciences’ (NIEHS) Animal Care and Use Committee protocol. The mice were pathogen free, treated humanely, and housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility. All experiments using laboratory animals were conducted with regard for alleviation of pain and distress.Female Balb/C mice were obtained from Taconic Farms, Inc.  at 6 weeks of age, maintained on a 12-hr light/dark cycle at 20–22°C, and acclimated for 1 week before treatment. The mice received NIH-31 diet  and water Pfisteria piscicida and Pseudo-fisteria shumwayae and represents the natural estuarine environment. The CPE was prepared as described by Orechromis niloticus) fingerlings to dinoflagellate cultures in marine aquaria. The fish were maintained in 20-L aquaria at room temperature in 20 ppt artificial seawater  on a 12-hr light:dark cycle in an approved biocontainment facility with four conditioned box filters for aeration and nitrification. Water quality was ammonia < 0.002 mg/L, pH 8.0–8.2, salinity 20 ppt, and dissolved oxygen at saturation. The aquarium was inoculated with the algal culture, and 8–10Tilapia nilotica 2.5– 5 cm in length were added. As fish died, more were added to the aquarium until the bloom had reached a level sufficient to kill 90–100% of all fish within 48 hr. Time to death of the fish was used as the indicator of sample toxicity because it proved to be a better predictor than algal counts. The bioactive material was extracted from the seawater at the peak of the fish-killing event by adsorption to C18 silica gel. The aquarium water was pumped into a separate vessel and 1 g C18 resin  was added for every liter of aquarium water. The resin and aquarium water were reacted together for 40–60 min while the magnetic stir bar slowly mixed the solution to keep the resin suspended. The resin was allowed to settle; the aquarium water was aspirated from the vessel; and the resin was washed with a small volume of clean 20 ppt seawater and transferred to polypropylene tubes. One volume of 40% acetonitrile (ACN) was added to the resin , and the resin was stored at 4°C in the dark under nitrogen until the bioactive fraction was eluted. A 2-mL–packed aliquot of the C18 resin with bound test article was poured into a 15-mL Bio-Rad Econo Pac disposable column ; the liquid was drained; and the flowthrough was passed over the resin 5 times. The resin was washed with 20 mL 20% ACN, and the CPE was eluted with 20 mL 100% ACN and dried under nitrogen. The resulting product from 2 mL of packed resin resuspended in 3 mL dimethyl sulfoxide (DMSO) was defined as 100% CPE for in vivo assays the same quantity of product resuspended in 1.5 mL DMSO was defined as 200%. To preserve maximal potency, aliquots of CPE were dried, stored at −80°C, and prepared fresh in DMSO or the appropriate dilution solvent for each experiment.A CPE was produced at North Carolina State University (NCSU) from the isolate referenced in To establish the minimal irritating and maximal nonirritating concentrations of the CPE for use in sensitization experiments and to ensure that the mice would tolerate multiple days of topical exposure to the ear, we conducted a primary irritancy assay following a modification of the method described by methyl-3H]-thymidine  in 250 μL sterile phosphate-buffered saline (PBS). Five hours later the mice were euthanized via carbon dioxide asphyxiation and the cervical (auricular) lymph nodes (3H]-thymidine was measured by β-scintillation counting as disintegrations per minute (dpm) for each mouse.We performed the LLNA according to Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) guidelines . The micph nodes  excised;4 cells/well at 37°C and 5% CO2. Neuro-2A cells  were cultured in modified minimal essential medium with Earle’s salts and nonessential amino acids , 10% fetal calf serum, 2 mM glutamine, and (100 U/ 100 μg)/mL penicillin–streptomycin in 96-well plates at 2 × 104 cells/well at 37°C and 5% CO2. Keratinocytes and Neuro-2A cells were allowed to grow in culture for 24 hr, then the medium was removed and appropriate dilutions of CPE in fresh medium were added (n = 6). After 24 hr of exposure to CPE, the medium was removed and frozen at −80°C for use in cytokine assays. Cell proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay according to the manufacturer’s instructions . Twenty microliters of the reagent 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium salt (MTS) in 100 μL fresh medium was added to each well; the cultures were returned to the incubator; and the absorbance at 490 nm was measured 1, 2, and 3 hr later using a microplate reader and SoftMax Pro analysis software . Cell viability was measured in NHEK by neutral red dye uptake. Cells were incubated with 50 μg/mL neutral red dye in 200 μL fresh medium for 3 hr. The medium-containing dye was removed and the cells were fixed for 1 min in 200 μL of fixing solution . The fixing solution was removed, and the dye taken up by viable cells was extracted with 200 μL 50% ethanol/1% acetic acid before absorbance determination at 540 nm, as previously described , obtained from reduction mammoplasty , were cultured in serum-free, low calcium (0.15 mM) keratinocyte basal medium  supplemented with epidermal growth factor (0.1 ng/mL), bovine pituitary extract (0.4%), insulin (5 μg/mL), hydrocortisone (0.5 μg/mL), gentamicin (50 μg/mL), and amphotericin (50 ng/mL) in 96-well plates at 1 × 10escribed .n = 6). With the first cytokine kit, each culture was assayed independently, that is, three experiments with six replicates per group per experiment. Expression levels for IL-8, IL-1β, and IL-6 were all above the limit of detection (per the manufacturer’s specifications) in the first assay. IL-1β, and IL-6 data are presented from this assay. Because TNF-α and GM-CSF expression levels were generally below the limit of detection for the assay, two replicate cultures were pooled and concentrated to increase the possibility of detection, giving three samples per group, that is, five experiments with three replicates per group per experiment. All samples from the first assay were repeated in the second assay. Each experiment was maintained separately and there were matched controls for each experiment. After the assay was completed, the data were pooled for statistical analysis. Data for TNF-α, GM-CSF, IL-8, MIP-1α, and MIP-1β are presented from the second assay.The levels of selected cytokines in cell culture supernatants from NHEK cultures before the addition of MTS or neutral red reagent (described above) were measured using the Luminex xMAP Protein Immunoassay according to the manufacturer’s instructions . Briefly, 50 μL of sample was incubated with 50 μL of assay diluent and antibody-coated beads for 2 hr at room temperature in the dark with vigorous shaking. After washing, the samples were incubated with 100 μL biotinylated detection antibody for 1 hr, then with 100 μL streptavidin–R-phycoerythrin (RPE) for 30 min. Cytokine bead flourescence was measured and analyzed using the Luminex LabMAPscanner and software (Biosource). Two xMAP kits were used. One kit measured tumor necrosis factor (TNF)-α, granulocyte macrophage–colony-stimulating factor (GM-CSF), interleukin (IL)-8, IL-1β, and IL-6 in supernatants from three different cell culture experiments; the other measured TNF-α, GM-CSF, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β from five cell culture experiments. In each experiment, six culture wells were independently treated for each group , Fisher’s least significant difference (LSD) test, and Dunnett’s test to determine the significance of the results; we selected a maximal value of Pfiesteria extracts . The MTS compound is reduced via NADPH or NADH into a soluble, colored formazan product in metabolically active cells; nonproliferating cells cannot affect this reaction. CPE induced cytotoxicity in Neuro-2A cells at 1 × 103 μg/mL , keratinocytes consistently secreted elevated levels of IL-8. Secretion of MIP-1α and MIP-1β was also increased at 1 × 103 μg/mL. Synthesis of TNF-α was statistically elevated at 1 × 103 μg/mL, although the average absorbance value (8.8 pg/mL) was below the minimum limit of detection for the assay (10 pg/mL). IL-6, GM-CSF, and IL-1β expression were not significantly altered by exposure to CPE.Keratinocyte-derived cytokines mediate inflammatory responses in the skin, regardless of whether the stimulus is a result of allergen-specific or irritant effects, therefore, we evaluated cytokine profiles in NHEK following exposure to CPE . At cytoPfiesteria has been reported to be an extremely toxic marine microbe , whereas contact irritant dermatitis is a localized, reversible, inflammatory response induced by primary contact with a chemical or mechanical irritant . Cytokinemotaxis , were setization , were miPfiesteria extract preparation altered the permeability of rat pituitary cells.Cumulative irritant dermatitis, a common nonspecific skin disease, is characterized by erythema, dryness and scaling that are usually observed with repeated exposure to a mixture of weak chemicals . SeveralP. piscicida and P. shumwayae potentiated the learning, memory, attention, and behavior alterations induced by the anticholinergic drug scopolamine in rats (Pfeisteria-laden water might exacerbate the response to a true sensitizer, enhancing the dermatitis, inflammation, and lesions caused by other microorganisms, allergens, and occupational activities, the CPE tested is not capable of inducing dermal sensitization at the concentrations evaluated. These findings are consistent with recent reviews and the CDC-sponsored epidemiology studies (Pfiesteria-containing estuarine environments does not pose a significant health risk.Both human and animal studies have demonstrated that irritancy can act as an adjuvant to skin sensitization . At low- in rats . Our res studies  that att


Nitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells.Nitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1.The transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (±)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)α and PKCδ from the cytosol to the membrane. Furthermore, the PKC-α,βinhibitors, GÖ6976 (10 nM) and PKCδ inhibitors, rottlerin (4 μM) inhibited the NOR-1 induced migration of PKCα and PKCδ respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCα and PKCδ with ERK1/2 pathways.Exogenous NO induced the MUC5AC mucin gene and protein through the PKCα and PKCδ – ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways. Production of NO is generally increased during inflammatory airway diseases such as asthma or bronchiectasis, or after exposure to irritant gases such as ozone . NO is pGoblet cell hyperplasia and metaplasia are well established hallmarks of the airways of cigarette smokers, with and without chronic obstructive pulmonary disease (COPD). Enhanced epithelial mucin expression is believed to be the rate limiting step for goblet cell metaplasia . Four geNO donation by isosorbide dinitrate increased MUC5AC mucin secretion in the goblet cell line HT29-MTX  but suppIn this study, we examined effects of NO on MUC5AC mucin synthesis and PKC-mediated second messenger pathways that may be involved in physiological functions of airway epithelium. Our results suggest that the PKC inhibitors inhibit the MUC5AC mRNA expression and mucin synthesis through inhibiting the PKCα and PKCδ-ERK1/2-MUC5AC promoter pathways during donation of NO to the A549 cells.2. The cells were replenished with fresh media every 2–3 days. The cell viability was periodically determined by trypan blue exclusion method.Human lung adenocarcinoma-derived A549 cells were cultured in Roswell Park Memorial Institute (RPMI1640) media supplemented with 10% fetal bovine serum (FBS), penicillin 100 U/ml and streptomycin 100μg/ml. Cells were maintained in a humidified incubator at 37°C with 95% air (vol/vol) and 5% (vol/vol) COG-nitro-L-arginine methyl ester (L-NAME) was used as a nitric oxide synthase inhibitor. Phorbol 12-myristate 13-acetate (PMA) was used as a protein kinase C (PKC) activator and inhibitors of PKC isoforms were used such as GÖ6976 (PKCα/β inhibitor), rottlerin (PKCδ inhibitor) and calphostin C (a ubiquitous PKC inhibitor) which were purchased from Calbiochem .NOR-1  was used as a NO donor. For control experiment, NMUC5AC protein was measured as described previously . Briefly5 of A549 cells were seeded on 100 mm dish and incubated until 80–90% confluency. After adapted in serum-free medium for 24 h, cells were stimulated by NOR-1 for 3 h and supernatant was collected for Greiss assay. For Nitrate, 200 μl of culture media and 200 μl of nitrate reductase buffer that contained 50 μM NADPH, 40 mM KH2PO4 and 50 mU nitrate reductase were mixed and incubated at room temperature for 2 h. 200 μl of 0.8% N-1-naphthyl-ethylene diamine was added to same amounts of 2% sulfanilamide in 0.2 N HCl. After incubation at room temperature for 10 minutes, the absorbance was measured on a spectrophotometry at 540 nm. Nitrite of cell supernatant was determined using a mixture of 50 μl of 2% sulfanilamide in 0.2 N HCl and 50 μl of 0.8% N-1-naphthyl-ethylene diamine. Sodium nitrite was used as the standard.Nitrate and nitrite were measured via the Greiss assay in the culture media. 1 × 105cells/well) and incubated for 48 h in serum free medium. Before transfection, the pGL3-MUC5AC-3752pro luciferase reporter plasmid and control pGL3-Basic vector were adjusted to 200 ng/μl, and β-galactosidase was adjusted to 100 ng/μl. The tube designated 'A' contained 300 μl of serum media, 5 μl of pGL3-MUC5AC-3752pro luciferase reporter plasmid, 5 μl of Plus reagent (GIBCO BRL), and 3 μl of β-galactosidase, while 'B' tube contained 300 μl of serum free media and 4 μl of LIPOFECTAMINEβ REAGENT (GIBCO BRL). Each tube was mixed well in room temperature and 200 μl of the mixture was added to the wells containing A549 cells. After 5h, 1 ml of 20% FBS was added to the wells and further incubated for 24 h.In size of 1.3 Kb fragment MUC5AC promoter which was cloned into the pGL3-Basic luciferase vector was generously provided by Carol Basbaum . A549 cells were seeded on 6-well plates . β-galactosidase activity was measured in the luminometer  in accordance with the manufacturer's instructions. All transfections were performed in triplicate wells; results were reported as emitted light per well (mean ± SD).® reagent  and chloroform from A549 cells. The RNA was incubated with 10 mM dNTP, 0.1 M DTT, 1 μl random hexamer (1 pmole) and 1 μl SuperScript II  at 42°C for 50 min, and then heat-inactivated at 70°C for 15 min. After reverse transcription, PCR was performed with specific primer pairs for the MUC5AC and β-actin genes in a thermocycler  with an initial denaturation step of 94°C for 4 min, followed by 28 cycles of 1 min at 94°C, 1 min at 60°C, 1 min at 72°C, with a final extension at 72°C for 7 min. The following primer pairs were used for the PCR: MUC5AC, 5-TCC GGC CTC ATC TTC TCC-3 (forward) and 5-ACT TGG GCA CTG GTG CTG-3 (reverse); β-actin, 5-CAA GAG ATG GCC ACG GCT GCT TCC-3 (forward) and 5-TCC TTC TGC ATC CTG TCG GCA ATG-3 (reverse). The amplified PCR products were visualized on a 1% agarose gel by ethidium bromide staining.Total RNA was isolated using TRIzol5) were seeded on 100 mm dishes and cultured in 10 ml until 80–90% confluency. After PKC inhibitors were treated for 30 min, cells were washed and incubated with NOR-1 for 3 h. Cells were harvested by centrifugation  and pumped by 1 ml syringe for destruction. For cytosol and membrane fraction, destroyed cells were centrifuged at 50,000 rpm  for 1 h at 4°C, and then supernatant (cytosol fraction) was collected. After RIPA buffer  was added into the pellet (membrane fraction), it was sonicated about 5 s. Both fractions were quantitated by Bradford method and equal amount of protein (20 §P) were resolved separately on 7.5% of SDS polyacrylamide gradient gels and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, membranes were incubated with anti-PKC antibodies  followed by horseradish peroxidase (HRP)-conjugated antibodies. The detection was performed using a chemiluminescence method (Amersham Life Science). The density of signals was quantified using a densitometer.A549 cells . Cells were destroyed by RIPA buffer on ice for 20 minutes. After destroyed cells were centrifuged, proteins were collected from supernatant and determined by Bradford method. 50 §P of protein were separated on a discontinuous 10 % and 4% PAGE gel and then the proteins were transferred to a PVDF membrane at 80 V for 1 h. The membrane was blocked with 5 % skim milk in TBS buffer  for 1 h, and then incubated with the mouse anti-human p-ERK antibody  or rabbit anti-human p-p38MAPK antibody  at 4°C overnight. The membrane was washed 3 times with TBST buffer (TBS + 0.1% Tween20) and incubated with HRP-conjugated secondary antibody (1:2000) at room temperature for 1 h. The target protein was detected by ECL Kit  using X-ray film.All data are presented as means ± SE. Data obtained from all the experiments was analyzed by Kruskal-Wallis one-way non-parametric analysis of variance with post hoc evaluations by Mann-Whitney's rank sum test . A level of significance was considered at p < 0.05.The concentrations of NO in the culture medium of A549 cells after incubation with the synthetic NO donors, NOR-1 for 3 hours were well correlated the concentrations of NOR-1  inhibited NOR-1 induced MUC5AC mRNA expression Figure .The present study clearly demonstrates a potent stimulatory effects of NO donor on MUC5AC mucin secretion from A549 cells. Activation of the PKCα and PKCδ with ERK1/2 mediated NO donor induced MUC5AC mucin gene expression and mucin synthesis. We used NOR-1 as a NO donor which releases NO with a more rapid kinetics . NO dono1, β2, γ), novel , and atypical . The classical isoforms are calcium and phorbol ester-activated, the novel are calcium-insensitive but activated by phorbol esters, and the atypical isoforms are both calcium and phorbol ester-insensitive, with all isofoms activated by phosphatidyl serine[Protein kinase C (PKC) is a family of serine/threonine-specific protein kinases with at least 10 different isoforms . The PKCyl serine.The interaction between NO and PKC has been the subject of many studies, with most focused on the role of PKC in the regulation of NO production ,28. WithIn this study, NOR-1 directly increased the transcriptional activity of transfected MUC5AC promoter, indicating that NO-induced upregulation of MUC5AC mRNA occurs at the transcriptional level. NOR-1 also moved the PKCα and PKCδ from the cytosol to the membrane and this intracellular activation of PKC was inhibited by PKCα inhibitor and PKCδ inhibitor.Involvement of PKC in secretion of airway mucin in response to various stimuli has been indicated previously -38. The Phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), are important inflammatory stimuli that have been shown to modulate diverse cellular events through PKC activation . PMA ind2-terminal kinases  , and p38 kinases [The mitogen-activated protein kinase (MAPK) cascades consist of serine threonine kinases that are sequentially phosphorylated by upstream kinases  and subdivided into three major pathways: ERKs, c-Jun-NH kinases ,46. MAPK kinases . Recent  kinases ,49. p38  kinases ,51.In this study, we found that NO donation by NOR-1 activated ERK1/2 but not p38 and this ERK1/2 activation was inhibited by several types of PKC inhibitors and by MEK inhibitor, PD98059 figure . These fToday, it is widely accepted that NO plays an important role in airway function. NO is an important mediator in the lung and has been shown to be associated with inflammatory lung diseases such as asthma and chronic bronchitis -58. In aNOR1 = (±)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide; NO = nitric oxide; PKC = protein kinase C; ELISA = enzyme linked immunosorbent assay; TNF-α = tumor necrosis factor α; EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinase; PMA = Phorbol 12-myristate 13-acetate


Hyaluronan (HA), an important component of connective tissues, is highly metabolically active, but the mechanisms involved in its catabolism are still largely unknown. We hypothesized that a protein similar to sperm PH-20, the only mammalian hyaluronidase known to be active at neutral pH, could be expressed in connective tissue cells. An mRNA transcript similar to that of PH-20 was found in chondrocytes, synoviocytes, and dermal fibroblasts, and its levels were enhanced upon stimulation with IL-1. In cell layers extracted with Triton X-100 – but not with octylglucoside – and in culture media, a polyclonal antipeptide anti-PH-20 antibody identified protein bands with a molecular weight similar to that of sperm PH-20 (60 to 65 kDa) and exhibiting a hyaluronidase activity at neutral pH. Further, upon stimulation with IL-1, the amounts of the neutral-active hyaluronidase increased in both cell layers and culture media. These findings contribute potential important new insights into the biology of connective tissues. It is likely that PH-20 facilitates cell-receptor-mediated uptake of HA, while overexpression or uncontrolled expression of the enzyme can cause great havoc to connective tissues: not only does HA fragmentation compromise the structural integrity of tissues, but also the HA fragments generated are highly angiogenic and are potent inducers of proinflammatory cytokines. On the other hand, the enzyme activity may account for the progressive depletion of HA seen in osteoarthritis cartilage, a depletion that is believed to play an important role in the apparent irreversibility of this disease process. Hyaluronan (HA), a linear, megadalton glycosaminoglycan, has important biological and structural functions . First, in vitro have also indicated that HA fragments similar in size to that of fragmented HA molecules found in vivo in inflammatory sites induce the expression of inflammatory genes in dendritic cells, macrophages, eosinophils, and certain epithelial cells dCTP by random priming (Megaprime system) and used as a PH-20 probe.As the 1,000-bp RT-PCR product amplified by PH-20 primers B does not contain any + membranes prior to fixation by UV cross-linking. After a prehybridization step for 4 hours in 6 × SSC , 5 × Denhardt's solution, 0.5% SDS, and 20 μg/ml denatured Salmon sperm DNA , the membranes were hybridized overnight at 64°C in a similar solution containing either the PH-20 or the actin probe. At the end of the hybridization procedure, membranes were washed at 64°C , dried, and then exposed to x-ray film for 12 hours (β-actin) or 48 hours (PH-20).Intact RNA samples (25 μg) were subjected to electrophoresis in 1% agarose/formaldehyde gels and transferred by capillarity to Hybond-N2 each) containing cells at near confluence were used for each condition. Data represent the means ± standard deviations obtained for five different sets of experiments.In separate experiments, 20 μg of total RNA from cells either nonstimulated or stimulated with IL-1 at a concentration of 5 or 10 ng/ml were subjected to northern blotting as stated above and the dried membranes were exposed to a PhosphorImager screen  for the detection and quantification of the radioactive signals. As the 1.4-kb transcript represented approximately 10% of the total radioactivity, quantification was restricted to the 2.4-kb transcript. Results obtained for stimulated cells were normalized with values obtained for the corresponding unstimulated control cells, to which a value of 1 arbitrary unit was assigned. In each set of experiments, three Petri dishes (79 cm2 (amino acids 155 to 169) was chosen. The sequence of its first 10 amino acids is also present in bovine PH-20. Further comparison between the sequence of the immunizing peptide and that of other known human proteins  also reveals that both hyaluronidases 2 glycine) SDS–PAGE in accordance with the procedure of Schagger and von Jagow [After being concentrated by ultrafiltration, proteins eluted from the immunoaffinity gel were characterized by Tricine  per milligram). To generate this substrate, preconfluent synoviocytes were incubated in the presence of 2-acetamido-2-deoxy-D-glucurono-1,5-lactone (50 mM) and tritium-labelled glucosamine (50 μCi/ml); after digestion with proteinase K and precipitation in ethanol, the glycosaminoglycans were passed through a DEAESepharose column equilibrated in 50 mM pyridine, pH 5.5 (buffer A), and the high-molecular-weight tritium-labelled HA molecules were eluted at a NaCl concentration of 0.4 M. Assays were conducted in duplicate as follows: specimens and known relative turbidity-reducing units (rTRUs) of testicular hyaluronidase were incubated with purified tritium-labelled HA molecules  in either 0.1 M Hepes (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid) (pH 7 to 8), 0.1 M Mops (3-(N-morpholino)propanesulfonic acid) (pH 6.5 to 7.5), 0.1 M Mes (pH 5.5 to 6.5), or 0.1 M acetate (pH 4.5 to 5.5) containing a final concentration of 0.15 M NaCl, 7 mM 1,4-saccharolactone, and 1% Triton X-100. After incubations, specimens were heated at 95°C for 5 min, diluted with 10 volumes of buffer A, and applied on 1 ml DEAESepharose gel equilibrated in the same buffer. Gels were then washed with 10 volumes of buffer A before being eluted step by step with increasing amounts of NaCl dissolved in the same buffer: 10 volumes of 0.2 M NaCl for step 1; 10 volumes of 0.3 M NaCl for step 2; 10 volumes of 0.4 M NaCl for step 3, and 3 volumes of 0.8 M NaCl for step 4. More than 95% of the radioactivity applied onto the gel was recovered in steps 1 to 3. HA disaccharides produced by chondroitinase ABC elute at 0.2 M NaCl, the HA tetrasaccharides and hexasaccharides produced by testicular hyaluronidase elute at 0.2 and 0.3 M NaCl, and the intact HA molecules elute at 0.4 M NaCl. For amounts of testicular hyaluronidase ranging between 0.005 and 0.05 rTRUs, there is a highly statistically significant linear correlation (r = 0.9) between, on the one hand, the enzyme activity and, on the other hand, the % of radioactivity recovered in steps 2 and 3 (range 10 to 45%). The intra-assay and interassay variations are less than 5 and 10%, respectively.Hyaluronidase activity was also assessed by using high-molecular-weight radiolabelled HA  at 5 ng/ml IL-1 and 2.4 ± 0.3  at 10 ng/ml IL-1. Concentration of 10 ng/ml.Because HA depolymerization is consistently observed in inflammatory sites , and as Because, thus far, chondrocyte and fibroblast cell layers extracted with octylglucoside have been reported to contain a hyaluronidase activity that cannot be detected above pH 5 ,28, a prBecause obviously fibroblasts expressed PH-20 at the mRNA level, we used the anti-PH-20 antibodies to test whether these cells expressed the neutral-active hyaluronidase at the protein level. After having extracted fibroblast cell layers with either octylglucoside or Triton X-100, a detergent used to solubilize sperm PH-20 , specimeIn parallel experiments, chondrocytes were cultured in the absence and in the presence of IL-1 (5 ng/ml). Cell layers extracted with Triton X-100 as well as their corresponding culture media were allowed to interact with the immunoaffinity gel, and proteins eluted with the glycine–HCl buffer were subjected to SDS-PAGE Fig. . Specime® Gentle elution buffer were subjected to HA substrate gel electrophoresis at pH 6.5  to open up tissue spaces and facilitate penetration , spermatSeveral factors could explain why the enzyme has not been detected before in connective tissue cell lines. Northern blot analysis and the more sensitive RT-PCR technique detected the PH-20 transcript in testis, normal breast tissue, metastatic cancer cell lines, fetal and placental cDNA libraries, and murine kidneys, as well as in trace amounts in the prostate, but those studies never investigated the RNA from articular cartilage, synovium, or skin -21. Two HA is not an inert constituent of the extracellular matrix, but, rather, is highly metabolically active, with a half-life ranging from less than a day in the skin to about 3 weeks in articular cartilage ,38. As t2, and matrix metalloproteinase-3 by arthritic synovium [PH-20 generates a mixture of HA oligosaccharides and fragments that may interact with various cells and produce distinct and important biological effects quite different from those induced by the native, high-molecular-weight polymer. Thus, by stimulating the proliferation and migration of vascular endothelial cells via multiple signaling pathways, HA fragments, but not high-molecular-weight HA molecules, are angiogenic ,9. Obviosynovium -12, fragsynovium  but alsosynovium .The finding that human dermis contains a neutral-active hyaluronidase suggests that depolymerization of HA can occur locally within the dermis. Accumulation of dermal HA with its associated water of hydration, as seen in urticaria and bullous skin lesions, can arise from both increased synthesis and local catabolic failures. On the other hand, PH-20-induced depolymerization of HA may contribute to the scar formation that occurs in adult wounds: fetal wounds have an extracellular matrix rich in HA of high molecular weight and heal without fibrosis, whereas the addition of hyaluronidase to the wound fluid enhances wound fibrosis ,46. By bUncontrolled and/or up-regulated PH-20 activity may be very deleterious to cartilage matrix, both directly and indirectly, since even a couple of cleavages along the filamentous backbone of aggrecan aggregates dramatically reduce the viscoelastic properties of articular cartilage as well as the size of these aggregates, which become no longer effectively immobilized within the collagen network of the articular tissue. Further, the oligosaccharides produced by PH-20 have been shown to induce a dose-dependent chondrocytic chondrolysis as well as up-regulation of aggrecan synthesis and HA synthase 2 mRNA . In bothThe observation that IL-1 up-regulates the production of PH-20 by both chondrocytes and synoviocytes is worth noting, because enhanced expression of the enzyme may contribute to the degradation of cartilage matrix in arthritides such as rheumatoid arthritis; this contention is strengthened by the observation that proinflammatory cytokines increase the loss of HA fragments from cartilage explants . On the Furthermore, because the pH of synovial fluid is usually above 6 , PH-20 cAs PH-20 has a HA-binding domain that is distinct from its hyaluronidase domain, the molecule may also act as a HA receptor at the cell surface. Indeed, binding of HA to this distinct domain of sperm PH-20 results in thyrosine phosphorylation and an increase in intracellular calcium ). GlycosOur study provides strong evidence that connective tissue cells express PH-20 and that the production of this neutral-active hyaluronidase is up-regulated by IL-1. Since, besides having unique physicochemical properties, HA can modify cell behavior and plays a key role in the organization of the extracellular matrix of connective tissues, this finding may contribute new insights into the pathophysiology of several disorders including skin and joint diseases. Although the overall hyaluronidase activity detected at neutral pH was relatively low, remodeling of the extracellular matrix during wound healing, inflammatory processes, and cancer growth and metastasis are slow processes. In this context, even very slow enzymatic activities at physiological pH, operating on a time scale of hours to days rather than minutes, may suffice to create great havoc.bp = base pairs; BSA = bovine serum albumin; DMEM = Dulbecco's modified Eagle's medium; dpm = distintegrations per minute; FCS = fetal calf serum ; HA = hyaluronan; IL-1 = interleukin-1; kb = kilobases; PBS = phosphate-buffered saline; PH-20 = sperm hyaluronidase or sperm adhesion molecule 1; RT-PCR = reverse transcriptase polymerase chain reaction; rTRUs = relative turbidity-reducing units; SSC = 0.15 M Na Cl, 0.015 M sodium citrate, pH 7.0.The author(s) declare that they have no competing interests.in situ hybridization and histological studies. DHM designed the research protocols, supervised the studies, and drafted the manuscript. All authors read and approved the final manuscript.HEL played a leading role in the coordination of the study, contributed to the research protocols, participated in the interpretation of results, and prepared the manuscript. AAC conducted the


In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation.Proliferation of primary HASMC was measured by [4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation.We demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF-R through the intervention of PI3K and ROS. While PI3K and ROS involvement is an early event, the activation of Src occurs downstream of EGF-R activation and is followed by the classical Ras-ERK1/2 signaling pathway to control G1 progression and cell proliferation.Collectively, our data demonstrate that in HASMC LTD Reagents and films for chemiluminescence and [3H]Thymidine were from Amersham Bioscience . All reagents and supplies for electrophoresis and DC™ Protein assay were from Bio-Rad Laboratories .Cell culture supplies, EGF, 2 in Medium 199, additioned with 20% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in 25 cm2 culture flask. The primary isolates were positively stained with an anti-smooth muscle α-actin antibody to assess the identity of the cultures. Thereafter, cells were routinely grown in monolayers in MEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, passaged at a 1:3 ratio in 75 cm2 culture flask and used between the 3rd and 8th passage (our isolates) or between the 3rd and the 10th passage (purchased cells).Smooth muscle cells from human bronchi were purchased from Cambrex  or isolated in our laboratory as previously described . Briefly® Reagent", according to the manufacturer's instructions. After denaturation , 1–2 μg of total purified RNA was retrotranscribed in the presence of MMLV-Reverse Transcriptase (5 U/μl) in optimized reaction conditions . Specific amino- and carboxyl-terminal primers for hCysLT1 receptor  and for CysLT2 receptor  have been selected on the basis of the sequences previously published Thymidine incorporation assay was performed as previously described [4  and EGF (single pulse) at the indicated concentration for 48 hours and incubated with [3H]thymidine for the last 4 hours at a final concentration of 1 μCi ml-1. Cells were then washed twice with ice-cold phosphate buffered saline to rinse loosely associated radioactive tracer. Acid-soluble radioactivity was removed by 20 minutes treatment with 5% trichloroacetic acid at 4°C followed by a two-step washing with 95% ethanol. The acid-insoluble portion was recovered by 60 minutes digestion with 2% Na2CO3 in 0.1 M NaOH. The radioactivity was then measured by liquid scintillation counting., where I represents the intensity of fluorescence at the specified time points.HASMC were loaded with 10 μM DCFH-DA in saline buffer supplemented with 0.1% of bovine serum albumin for 1 hat 37°C (stained). At the end of the incubation, cells were washed in PBS and oxidative activity was assessed as follows. The production of reactive oxygen species (ROS) was measured by the intensity of DCF emission at 525 nm (excitation 503 nm – Perkin-Elmer LS 50B) in both stained and unstained cells. Results are expressed as the difference in fluorescence  calculated as AU = thymidine incorporation , as well as its potentiating effect of the EGF-induced DNA synthesis  .Because we were interested in investigating whether the EGF-R can function as a downstream signaling partner of cysteinyl-LTs, therefore acting as a point of convergence for heterogeneous signaling pathways, we used immunoblot analysis to assess the phosphorylation state of this receptor following the treatment of the cells with LTDM., Fig. , n = 20.M., Fig. , LTD4 alion Fig. . In addily) Fig. , a resul4 is able to activate ERK [4-induced [3H]thymidine incorporation. Indeed, 1 hour pretreatment with 20 μM PD98059 fully prevented LTD4-induced DNA synthesis  produced a maximal ERK1/2 phosphorylation of 162% ± 16 S.E.M. , if efficient cell proliferation is to be achieved.The time frame of EGF-R activation (3 min) is fully compatible with that of MAPK activation (5 min). Despite there is disagreement in the literature on the necessity for prolonged activation of the MAPK cascade to produce a significant mitogenic effect ,42, we r4-induced [3H]thymidine incorporation, we pretreated HASMC with 100 ng/ml PTX for 20 hours. Preincubation of HASMC with PTX fully abolished LTD4-induced DNA synthesis  also fully inhibited LTD4-induced EGF-R and ERK1/2 phosphorylation, demonstrating that not only EGF-R transactivation, but also ERK1/2 phosphorylation is totally dependent upon a Gi/o protein activation , or by costimulation of EGF and LTD4 (> 70%). Furthermore, PP1 and to a lesser extent genistein were able to inhibit the effect induced by EGF alone.It is known that GPCRs might trigger activation of a protein tyrosine kinase (PTK) such as Src in order to activate MAPKs. Thus, we tested the effect of genistein, a broad spectrum PTK inhibitor, and of PP1, a specific Src kinase inhibitor, on LTDi coupled receptors requires Src kinase activity. However, Figure 4-stimulated EGF-R transactivation as well as EGF-induced autophosphorylation (data not shown). At variance, both compounds inhibited LTD4-  or EGF-stimulated  ERK1/2 phosphorylation in HASMC in a similar way , confirming that also in these cells LTD4 is able to activate the small G protein Ras at a concentration relevant for its pathophysiological role in HASMC proliferation , and stimulated with either LTD4 or PDGF .Production of ROS has been noted upon growth factor stimulation of arterial smooth muscle cells  and boviion Fig. . Similarion Fig. . Thus, t4 at the concentration of 1 μM was observed to induce almost a 4-fold increase of fluorescence over baseline, within 5 min . This effect was specifically inhibited by a 30 min pretreatment with 1 μM of the specific CysLT1-R antagonist montelukast  in another natural expressing systems such as the human monocyte/macrophage-like U937 cells [Interestingly, PTX pretreatment totally inhibited LTD systems , is alsose cells , and tha37 cells .1-induced EGF-R transactivation in HASMC did not seem to involve Src, as demonstrated by the inability of two different Src kinase inhibitors to significantly affect LTD4-induced EGF-R phosphorylation. We also excluded that EGF-R transactivation might require metalloproteinase cleavage of proHB-EGF as well as PKC activity [4-induced EGF-R phosphorylation (data not shown). Conversely, EGF-R transactivation seems to require the involvement of a PI3K, likely activated by the βγ complex of Gi, which, in turn, is known to activate a number of downstream signaling molecules, including different PTKs [CysLTactivity . In factent PTKs .4-stimulated DNA synthesis was sensitive to Src inhibition, which suggests the involvement of Src kinase downstream of EGF-R phosphorylation [4-induced DNA synthesis, and inhibited the potentiating effect of LTD4 on EGF-stimulated thymidine incorporation, indicative of the involvement of the MAPK cascade. These data have been confirmed by demonstrating that LTD4 was able to increase ERK1/2 phosphorylation and to augment EGF-induced MAPK activation, again with a pharmacological profile characteristic of a CysLT1-R, and that both effects were sensitive to Src inhibition. Furthermore, LTD4 was observed to increase the amount of active Ras (Ras-GTP), a capacity that we already demonstrated in U937 cells [However, LTDrylation , and, in37 cells .4-stimulated ERK1/2 phosphorylation. Furthermore, while wortmannin and LY294002 had only a negligible effect on EGF-stimulated MAPK activation, they produced a significant inhibition of LTD4-induced ERK1/2 phosphorylation. These data, therefore, confirm that PI3K activation is an early event upstream of EGF-R phosphorylation in the signaling cascade linking CysLT1-R to thymidine incorporation. Usually, the mitogenic effect caused by EGF involves the activation of a PI3K, but this pathway is parallel to and independent from ERK1/2 phosphorylation. Accordingly, the MEK1 inhibitor was only partially able to inhibit EGF-induced thymidine incorporation. These observations are in agreement with the paradigm that Gi-coupled receptor- and Gβγ-stimulated MAPK activation is attenuated by inhibition of PI3K acting at a point upstream of Ras, and that this pathway requires a tyrosine kinase and Raf [Consistent with a predominant role of EGF-R transactivation, AG1478 and PTX strongly reduced LTD and Raf .1-R activation and EGF-R phosphorylation, intriguingly we observed that LTD4 rapidly increased ROS formation in HASMC, an effect specifically inhibited by the antagonist montelukast. Indeed, different GPCRs have been suggested to increase ROS, which, in turn, inactivate PTP that negatively control RTK activity [4 has been proposed to increase production of superoxide anion [4-induced EGF-R transactivation, MAPK activation and thymidine incorporation.Looking for a mechanism linking CysLTactivity , and theactivity . While iactivity ,52. In tactivity . In agrede anion , while, de anion ,56. Acco4, the most potent bronchoconstrictor yet identified, might potentiate EGF-induced mitogenesis in HASMC, activating a CysLT1-R coupled to a Gi protein to cause EGF-R transactivation through the intervention of PI3K and ROS. This EGF-R induced activation triggers, downstream, the classical Src-Ras-ERK1/2 pathway to control G1 progression and cell proliferation. Furthermore, because interaction between GPCR and RTK might be exceedingly complex and potentially occurs at multiple levels, we may not completely rule out the hypothesis that a second cooperative Gq/11 pathway might contribute to the LTD4-induced effects in HASMC [In conclusion, our data demonstrate that LTDin HASMC . This poTaken together, these data corroborate a second, and potentially more important, role for cysteinyl-LTs in modulating cell physiology, from epithelial ,58 to meThe author(s) declare that they have no competing interests.in vitro cell proliferation as well MAPK phosphorylation studies.SR helped in study design and data analysis, was involved in cell culture and carried out RT-PCR, binding, in vitro cell proliferation, EGF-R and MAPK phosphorylation studies.SC was involved in cell culture and performed in vitro studies on ROS generation and subsequent data analysis.BV was the expert for ROS and was involved in the VC conceived and designed the study and participated to the manuscript preparation.GER participated in the design of the experiments, coordination and manuscript preparation.


It has recently become possible to generate human osteoclasts in vitro, by incubation of peripheral blood mononuclear cells (PBMCs) in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). However, the assays at present available do not distinguish clearly between the distinct effects of agents on differentiation and function.Very little is known of the regulation of the function of human osteoclasts, largely due to the virtual impossibility of obtaining human osteoclasts vs after incubation for 1–24 h in test agent.We developed a novel assay for resorptive function of human osteoclasts that minimizes inter-assay variability by using each culture as its own baseline, and that minimizes the confounding effects of agents on differentiation by assessing resorptive function over a short test period. In this assay, the development of resorptive activity is monitored in sample cultures. When resorption is underway, bone resorption  into the supernatant) is compared before Using this assay, we found that changes in bone resorption could be detected using substantially fewer cultures per variable. Moreover, we could detect effects of agents on resorption within 1 h of addition, a time sufficiently short that a change in release is likely to reflect an effect on function rather than on differentiation.The assay makes it possible to distinguish the effects of agents on osteoclastic function, independent of their effects on differentiation. The maintenance of skeletal integrity depends on continual resorption of bone by osteoclasts and its replacement by osteoblasts. Recently, there have been considerable advances in our understanding of the mechanisms through which osteoclast formation is regulated -3. In coex vivo with which to address this question. In their absence, human osteoclastic cells can be generated in vitro by incubation of peripheral blood mononuclear cells (PBMCs) in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) on plastic culture surfaces or on bone/dentine slices [It is virtually impossible to obtain human osteoclasts e slices ,6. Howeve slices -10. If aRecently, it has become possible to measure bone resorption as the release into culture supernatants of products of bone solubilization, the concentration of which has been shown to correlate with bone resorption . This apThe difficulties in the interpretation of resorption data mentioned above are compounded by the length of time it normally takes for osteoclastic differentiation to occur: the long incubation times magnify small initial differences between cultures, and in our experience can lead to substantial inter-culture variability. This variability increases the number of cultures required per variable, and because relatively small numbers of monocytes are available from a given donor, the number of variables that can be studied in each experiment is in practice severely limited.vs after incubation for 1–24 h with test agent. Using this approach, we found that changes in bone resorption could be detected using substantially fewer cultures per variable; and that effects of agents on resorption could be detected within 1 h of addition, a time sufficiently short that a change in release is likely to reflect an effect on function rather than on differentiation.We therefore developed a novel assay that minimizes the confounding effects of agents on differentiation by measuring resorption over a shorter period; and that minimizes inter-culture variability by using each culture as its own baseline. In this assay, sample cultures are inspected to monitor the development of actively resorbing osteoclasts. When resorption is underway, release of products of bone resorption is compared before Cells were incubated in MEM with Earle's salts , supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU/ml benzylpenicillin, and 100 μg streptomycin  (MEM/FCS). Recombinant human M-CSF and soluble recombinant murine RANKL were from Insight Biotechnology . The specific cathepsin K inhibitors MV061194, MV061748, MV061940, MV061645 and MSX-081 were provided by Medivir UK . Cortical bovine bone slices (4 × 4 × 0.1 mm) were prepared as previously described . Salmon 5 cells were added per well to a 96-well plate, each well of which contained a bone slice. The cultures were incubated in a total volume of 200 μl MEM/FCS containing M-CSF (50 ng/ml) and RANKL (30 ng/ml) for 24 h at 37°C in 5% CO2 in humidified air. Bone slices were then removed from the wells and placed in the wells of a 24-well plate, in 1 ml of MEM/FCS containing M-CSF and RANKL at the above concentrations, before continued incubation. Cultures were fed three times per week by replacing 60% of the medium with fresh medium and cytokines.Heparinized blood was obtained from healthy human male or female volunteers (aged 22 – 57) with the consent of St George's Ethical Committee. The blood was layered over Histopaque-1077 and centrifuged for 30 min at 400 g. The opaque interface containing mononuclear cells (PBMCs) was collected with a pasteur pipette, washed in PBS, then resuspended in MEM/FCS. 5 × 10To monitor cultures for the development of resorptive function, sample bone slices were removed at intervals, fixed in 10% formalin and stained using toluidine blue (0.1% for 1 min). Bone slices were then inspected by light microscopy for the presence of osteoclasts and excavations , bone slices were removed from the 24-well plates, washed in PBS, placed in the wells of a 96-well plate, and incubated for 2 or 24 h in 100 μl of fresh MEM/FCS containing M-CSF and RANKL. All the culture medium was removed after this period of incubation and stored frozen. Bone slices were washed again in PBS and transferred to new 96-well plates, the wells of which contained 100 μl of fresh MEM/FCS containing M-CSF and RANKL, together with test agent or vehicle control unless otherwise stated. Supernatants were collected and stored frozen after a further 1–24 h of incubation.®)  in the culture supernatants. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions.The resorption activity of the cultures was determined by quantifying the C-terminal telopeptide degradation product of type I collagen  cytochemistry and reflected light or scanning electron microscopy. In other experiments, the total quantity of TRAP present in the cells at the end of the culture was assessed by enzyme assay of TRAP in cell lysates.After incubation, bone slices were immersed in 10% (v/v) sodium hypochlorite for 10 min to remove cells. The bone slices were then washed in water, air-dried, mounted and sputter-coated with gold. Bone slices on stubs were inspected in a scanning electron microscope . The extent of bone resorption on bone slices mounted onto microscope slides was quantified by counting the number of grid intersections in an eyepiece graticule that overlay an area of bone resorption using reflected light microscopy.p-nitrophenyl phosphate to p-nitrophenol in the presence of sodium tartrate. 80 μl of each lysate, diluted appropriately, was added to 96-well plate wells containing 80 μl 0.09 M citrate buffer (pH 4.8) with 20 mM phosphatase substrate and 80 mM tartaric acid and incubated at room temperature for 40 min. The reaction was stopped by addition of 40 μl of 0.5 M sodium hydroxide. Optical absorbance was measured at 405 nm on an Opsys MR plate reader  against a standard curve of p-nitrophenol.After removal of supernatant, bone slices were washed in PBS, transferred to fresh wells of a 96-well plate and incubated in 100 μl 0.1% Triton in water (v/v) for 10 min. TRAP enzyme activity was measured by the conversion of p < 0.05All data are expressed as mean ± standard error of mean, of four replicate cultures, unless otherwise stated. The significance of differences between control and experimental groups was assessed by Student's t test or ANOVA (Dunnett's Multiple Comparison Test). Differences were considered significant if Previous assays have used an incubation time of three days to measure CTX-I release ,13. To dWe noted that there was frequently considerable variation in the extent of bone resorption on different bone slices incubated under identical conditions in the same experiment Fig. . This vaWe hypothesized that the effect of inter-culture variability could be minimized by a longitudinal rather than cross-sectional assay design. These alternative approaches were compared in an assay using an inhibitor of cathepsin K, an enzyme crucial to the degradation of collagen during bone resorption by osteoclasts Fig. . The intvs the cross-sectional approach, we used data from the control groups of assays similar to those shown in Fig. To assess the relative statistical power of the longitudinal -7 M – 10-5 M). We found a strong correlation between initial CTX-I and TRAP in cultures incubated in control medium (r2 = 0.96) (n = 24), but the correlation was less clear for cultures incubated in E64 (r2 = 0.52) (n = 24). This conforms to our previous experience, that the TRAP content of cells is influenced by agents that modulate bone resorption [The high cost of CTX-I assays led us to test alternative approaches that might provide a measure of the osteoclastic content of cultures that would substitute for the first CTX-I reading. It might be that the TRAP content of lysates reflects the resorptive capacity of cultures. We therefore tested the relationship between the first of the pair of CTX-I readings, and the TRAP in the lysate of the corresponding culture after incubation in either control medium or the cysteine protease inhibitor E64 (10sorption : TRAP is-7 M), a cathepsin K inhibitor, and within 2 h of incubation in salmon CT (10 ng/ml). This result additionally suggests that inhibition of bone resorption by the cathepsin K inhibitor has a more rapid onset than inhibition by CT. This might be because intracellular degradation of endocytosed matrix fragments continues to completion despite cell-inhibition, while the effects of the (membrane-permeant) enzyme inhibitor are essentially immediate.We tested the ability of the assay to detect changes in bone resorption over shorter time periods Fig. . In thesin vivo of changes in functional activity that correspond to changes in plasma calcium concentration [Very shortly after administration of hormones such as CT or PTH, osteoclasts show morphological evidence ntration . Only muntration ,3,17. Inex vivo. In their absence, osteoclasts can be generated in vitro, but osteoclast formation from human PBMCs typically takes 14–21 days [This is because human osteoclasts are essentially unavailable –21 days -10. ThusThis problem is partly circumvented in assays that measure bone resorption as the release into culture supernatants of products of bone solubilization ,13,18. TA second difficulty is the substantial inter-culture variability in osteoclast formation, which might be accentuated by the prolonged incubation required to form osteoclasts from PBMCs: long incubation times might magnify small initial differences between cultures, especially since cells capable of forming osteoclasts represent only a very small proportion (<1%) of PBMCs , so thatWe therefore developed a novel assay that, by addressing these difficulties, facilitates the measurement of osteoclast function independent of differentiation. First, we optimized bone resorption, since if there is more bone resorption it can be measured over a shorter period. Short measurement periods minimize the confounding effects of agents on differentiation. We optimized bone resorption by optimizing combinations of serum batches and PBMC-donors. This led to substantially greater and earlier release of CTX-I antigen, to levels an order of magnitude greater than previously reported ,12,13. TIn view of the significant expense of longitudinal measurements of CTX-I, we tested whether measurement of the quantity of TRAP in the cell lysates at the termination of the experiment could substitute for the initial CTX-I assay. We found that, while TRAP correlated well with the initial CTX-I reading in control cultures, the correlation in experimental groups in which bone resorption was modulated was unreliable. We have previously noted that agents that modulate resorption also modulate lysate TRAP levels . HoweverThe short incubation times and the longitudinal sampling makes the assay we have described a powerful tool with which to detect and quantify the effects of agents that activate or modulate the resorptive activity of osteoclasts, while minimizing the potentially confounding effects of the same agent on osteoclastic differentiation. The assay design also reduces the inter-culture variability of resorption data, compared to previous approaches.UG and MS are employed by Medivir UK; BS is employed by Medivir AB. TJC receives consultancy fees from Medivir UKBK and KF performed all the experimental studies. KF prepared the figures. UG, BS, MS and TJC conceived the assay approach. UG, BS and MS created the novel inhibitors of cathepsin K. TJC supervised the experimental work and wrote the manuscript. All authors read and approved the final manuscript.


The microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimer's disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization.We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau.The results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies. Although many studies have been done on methanol and formaldehyde intoxication ,2, none Methanol ingestion is an important public health concern because of the selective actions of its toxic metabolites, formaldehyde and formic acid, on the retina, optic nerve and central nervous system . Severe et al. have classified formaldehyde as "probably neurotoxic" [Formaldehyde is a common environmental contaminant found in paint, clothes, medicinal and industrial products, and is a component of diesel and gasoline exhaust ,23. Recerotoxic" , becauserotoxic" , causingrotoxic" -31. TherHere, we examine the role of formaldehyde in induction of protein misfolding. In particular, we investigate the effect of formaldehyde on the aggregation of human neuronal tau in vitro and the toxicity of tau aggregates in mammalian neuronal cells. The results imply that low concentrations of formaldehyde are sufficient to induce formation of amyloid-like tau aggregates, which in turn induce apoptosis of both human neuroblastoma cells (SH-SY5Y) and rat hippocampal cells.In order to investigate the potential of formaldehyde to induce protein misfolding leading to neurodegenerative disease, we studied the effect of low concentrations of formaldehyde on the biochemical and biophysical properties of human neuronal tau. Incubation with increasing concentrations of formaldehyde from 0.01–0.5% was observed to result in formation of increasing amounts of SDS-insoluble aggregates of tau, as detected by SDS-PAGE Fig. , curve 1We then compared the time course of the aggregation reaction in the presence Fig. , curve 1-3s-1) was greater than that in the presence of formaldehyde (~3.66 × 10-4s-1). This demonstrates that formaldehyde reacts with protein tau and blocks amino groups.OPT is commonly used as a fluorescent probe to detect both α- and ε-amino groups of a protein . As showThe time course of tau aggregation in the presence and absence of formaldehyde was also monitored using the fluorescent dye ThT, which was considered to be highly specific for amyloid-like structure . Under tConsequentially, we measured the activity of the different types of tau aggregates in tubulin assembly. While the spontaneously formed aggregates and those formed in the presence of acetaldehyde, maintained a relatively high level of residual activity, the formaldehyde-induced aggregates of tau were practically inactive in promoting tubulin assembly Table . TogetheThe ability of tau aggregates to bind the dyes ThT and Congo Red suggests that the aggregated tau contains relatively more β-sheet structure compared with the natively unfolded protein. To prove this prediction, htau 40 was incubated in the presence or absence of formaldehyde and then examined by circular dichroism (CD) spectroscopy. In the absence of formaldehyde, both native tau and acetaldehyde-treated tau showed CD spectra typical for an unfolded protein as reported ,38, withIn addition to its secondary structure signature, the premolten globule state is characterized by a partially collapsed structure with a loosely packed hydrophobic core. To confirm that the aggregates had partially folded character, htau40 was incubated with formaldehyde and then examined for the ability to bind ANS, a fluorescent probe of surface-exposed hydrophobic patches , which wIn order to investigate whether amyloid-like tau has an effect on neurons, amyloid-like tau was added to neurotypic SH-SY5Y cells after the residual formaldehyde had been removed completely from the protein-deposits by ultrafiltration (see Methods). Figure The effect of amyloid-like tau on cell viability was estimated using the MTT assay . As showIn order to further investigate the neurotoxicity of amyloid-like tau, we used flow cytometry analysis to examine the effect of formaldehyde-aggregated tau on rat hippocampal cells Fig. . Cells wMethanol is an ocular toxicant that causes visual dysfunction often leading to blindness after acute exposure. The physiological and biochemical changes responsible for this toxicity are poorly understood . AccordiIn recent years, however, formaldehyde has been found to be a neurotoxic molecule and to damage the prefrontal cortex of rats including the hippocampus ,47. ThesThe microtubule associated protein tau plays an important role in maintenance of the cytoskeleton. It promotes and maintains assembly of microtubules, which are required for axonal morphogenesis and transport . In receRecently, Kuret and colleagues described a tau assembly pathway in which anionic inducers, for instance arachidonic acid (AA), favor a shift in the equilibrium between unfolded and filamentous tau species. The microtubule binding function of tau is lost and tau protein accumulates in a partially folded, ThT-positive intermediate which then self-aggregates into a hydrophobic nucleus (as detected by fluorescent ANS), before the filament nucleus elongates to form full fibrils ,39. In c2) groups and to increase the net negativity of a protein [et al. (1994) [As shown above, formaldehyde reacted with the amino groups of tau, as demonstrated by the OPT test. Reaction with formaldehyde is known to eliminate positive  , the con. (1994) ,32. On tKhlistunova and colleagues found that the repeat domains of intracelluar tau could aggregate and were toxic to neuronal cells. The degree of tau aggregation and toxicity depends on the propensity to form β-structure ,38,50,51Here we investigate the effect of low concentrations of formaldehyde on protein misfolding and aggregation. We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of 0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. After removal of the formaldehyde, the amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic SH-SY5Y cells and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Control cells incubated with formaldehyde alone, or with tau aggregates formed in the presence of acetaldehyde or in the absence of additives (and which did not show appreciable binding of thioflavin T or Congo Red), did not show signs of apoptosis. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau. The results suggest that low concentrations of formaldehyde may play a role in induction of tauopathies.The clone of recombinant human tau-40 was kindly provided by Dr. Goedert  [E. coli, was purified as described previously [et al. [2) in a final volume of 500 μl. Once mixed, the samples were pipetted into quartz microcuvettes (1 ml), equilibrated at 37°C, and the absorbance at 350 nm was measured on a Hitachi U-2010 spectrophotometer. The specific activity of tau was the same as that described by Goedert et al. (1990) [Neuronal tau, overexpressed in eviously -56. Brieeviously  and the  [et al. . Tau (4 . (1990) .For protein aggregation experiments, tau (1.2 μM for light scattering or 20 μM for electrophoresis) was incubated at 37°C with different concentrations of formaldehyde in 100 mM phosphate buffer (pH 7.2) for 24 h to allow the reaction to reach completion. Acetaldehyde and BSA were used as controls. For time course analysis, aliquots were taken at different time intervals during incubation of tau with or without formaldehyde. Light scattering was measured in a Hitachi F-4500 fluorescence spectrophotometer (slits: Em = 5.0 nm and Ex = 5.0 nm) with excitation at 480 nm. Kinetic data were analyzed according to Tsou (1965) . ElectroTo test conformational changes, ANS was employed to detect whether the aggregates of formaldehyde-treated tau had exposed hydrophobic surface area. The fluorescence at 480 nm of ANS after excitation at 350 nm for different concentrations of tau was determined at room temperature using a Hitachi F-4500 spectrofluorometer. CD spectra were recorded using a Jasco J-720 CD spectrometer. The spectra were measured in 1-mm pathlength quartz cuvettes, and data were collected from 195 nm to 250 nm at 0.5-nm intervals. The samples were all in 100 mM sodium phosphate buffer (pH 7.2). The bandwidth was set at 1.5 nm (37°C). The spectrum baselines were corrected using the spectrum for the buffer measured under identical conditions.Neuronal tau  was resuspended in 50 mM phosphate buffer containing OPT (molar ratio: reagent/protein = 20/1) in the presence of formaldehyde at different concentrations at 37°C for 120 min. The fluorescence (Ex340 nm/Em455 nm) was then measured (slits: Em = 5.0 nm and Ex = 5.0 nm). Under the same conditions, tau was resuspended in 0.005% formaldehyde and 2 μM OPT; and aliquots were taken to measure the fluorescence at different time intervals. The data were analyzed according to Tsou (1965) .The assay  was perfFor electron microscopy, tau (40 μM) was incubated with 0.1% formaldehyde at 37°C for 24 h. The incubated samples were loaded on a carbon-coated grid for 2 min, stained with 2% (w/v) uranyl acetate for 1 min, and then dehydrated through a graded water-ethanol series. Samples were visualized under a JEOL JEM-100CX electron microscope. Tau alone (40 μM) was incubated under the same conditions in the presence of heparin (1 mg/ml) as a control.Neuronal tau  was incubated at 37°C with formaldehyde (0.05%) in 25 mM phosphate buffer (pH 7.2) over night. Then protein solution was diluted using phosphate buffer and 3 μl of the sample (10 ng tau protein) was dropped onto the mica surface and left for 5 min at room temperature before drying with nitrogen gas. The mica diaphragm was rinsed with ultra-purified water 20 times and dried down with nitrogen gas before observation under the atomic force microscope . The horizontal diameter at half height of a particle (globular protein) was measured and the data were analyzed using the Nanoscope 6.11r1 software.2 incubator, as described [6 cells/ml) or glass coverslips . Cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 10% fetal horse serum. For most experiments, the culture medium was replaced with serum-free medium before the addition of formaldehyde-treated tau. Native tau and acetaldehyde-treated tau were used as controls. Cells were incubated with the samples for 72 h. As an additional control, cells were treated with an equivalent amount of 100 mM phosphate butter (pH 7.2) in the absence of tau, and no effect was detected. To inhibit apoptosis, Congo Red was incubated with the cells for 5–10 min before adding the protein of interest. Photos were taken using an Olympus IX-71 inverted contrast microscope.SH-SY5Y human neuroblastoma cells were cultured in DMEM medium supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% COescribed . The medescribed . Brieflyet al. [As described by Mayo et al. , viabiliet al. . After aAfter incubation with tau, the formaldehyde was removed from the protein sample by a series of dilution steps in an Amicon Microcon-10 column. In order to verify the absence of formaldehyde before use, the presence of the aldehyde group was detected as described , and no 2) at a concentration of 1 × 106 cells/ml. Then the cells were incubated with AnnexinV- FITC and propidium iodide (PI) for 15 min (22–25°C) in dark. The stained cells were immediately analyzed by flow cytometry . Annexin V-FITC selectively passed through the plasma membranes of apoptotic cells and stained them with green fluorescence. Apoptosis was considered to have taken place in cells positive for Annexin V-FITC and negative for PI. All data were analyzed with Cell Quest software (BD). Each measurement was carried out at least in triplicate.Cells undergoing apoptosis were detected with the use of double staining with Annexin V-FITC/PI in dark according to the manufacturer's instructions . BrieflyCLN was responsible for the experiments, data analysis and drafted the manuscript. XSW conducted the electron microscopy assay, and contributed throughout the experimental process. YL and SP drafted portions of the text. RQH participated as a supervisor in the study design, analyses and writing. All authors read and approved the final manuscript.