Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl2. In the present study, CdCl2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl2-induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl2-induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures.

Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells.

Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.

The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates.

Characterization of cadmium chloride-induced BiP accumulation in Xenopus laevis A6 kidney epithelial cells.

Endoplasmic reticulum (ER) stress can result in the accumulation of unfolded/misfolded protein in the ER lumen, which can trigger the unfolded protein response (UPR) resulting in the activation of various genes including immunoglobulin-binding protein (BiP; also known as glucose-regulated protein 78 or HSPA5). BiP, an ER heat shock protein 70 (HSP70) family member, binds to unfolded protein, inhibits their aggregation and re-folds them in an ATP-dependent manner. While cadmium, an environmental contaminant, was shown to induce the accumulation of HSP70 in vertebrate cells, less information is available regarding the effect of this metal on BiP accumulation or function. In this study, cadmium chloride treatment of Xenopus laevis A6 kidney epithelial cells induced a dose- and time-dependent increase in BiP, HSP70 and heme oxygenase-1 (HO-1) accumulation. Exposure of cells to a relatively low cadmium concentration at a mild heat shock temperature of 30°C greatly enhanced BiP and HSP70 accumulation compared to cadmium at 22°C. Treatment of cells with the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced cadmium-induced BiP and HSP70 accumulation. Immunocytochemistry revealed that cadmium-induced BiP accumulation occurred in a punctate pattern in the perinuclear region. In some cells treated with cadmium chloride or the proteasomal inhibitor, MG132, large BiP complexes were observed that co-localized with aggregated protein or aggresome-like structures. These BiP/aggresome-like structures were also observed in cells treated simultaneously with cadmium at 30°C or in the presence of buthionine sulfoximine. In amphibians, the association of BiP with unfolded protein and its possible role in aggresome function may be vital in the maintenance of cellular proteostasis.

Expression and localization of the Xenopus laevis small heat shock protein, HSPB6 (HSP20), in A6 kidney epithelial cells.

Small heat shock proteins (sHSPs) are molecular chaperones that bind to unfolded protein, inhibit the formation of toxic aggregates and facilitate their refolding and/or degradation. Previously, the only sHSPs that have been studied in detail in the model frog system, Xenopus laevis, were members of the HSP30 family and HSPB1 (HSP27). We now report the analysis of X. laevis HSPB6, an ortholog of mammalian HSPB6. X. laevis HSPB6 cDNA encodes a 168 aa protein that contains an α-crystallin domain, a polar C-terminal extension and some possible phosphorylation sites. X. laevis HSPB6 shares 94% identity with a X. tropicalis HSPB6, 65% with turtle, 59% with humans, 49% with zebrafish and only 50% and 43% with X. laevis HSPB1 and HSP30C, respectively. Phylogenetic analysis revealed that X. laevis HSPB6 grouped more closely with mammalian and reptilian HSPB6s than with fish HSPB6. X. laevis recombinant HSPB6 displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of citrate synthase. Immunoblot analysis determined that HSPB6 was present constitutively in kidney epithelial cells and that heat shock treatment did not upregulate HSPB6 levels. While treatment with the proteasomal inhibitor, MG132, resulted in a 2-fold increase in HSPB6 levels, exposure to cadmium chloride produced a slight increase in HSPB6. These findings were in contrast to HSP70, which was enhanced in response to all three stressors. Finally, immunocytochemical analysis revealed that HSPB6 was present in the cytoplasm in the perinuclear region with some in the nucleus.

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport.

Fluoxetine-induced transactivation of the platelet-derived growth factor type ß receptor reveals a novel heterologous desensitization process.

Many G protein-coupled receptors (GPCRs), including serotonin (5-HT) receptors promote the activity of receptor tyrosine kinases (RTKs) via intracellular signaling pathways in a process termed transactivation. Although transactivation pathways are commonly initiated by a GPCR, a recent report demonstrated that serotonin-selective reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of the platelet-derived growth factor (PDGF) type ß receptor. We show that a 45 min pretreatment of SH-SY5Y cells with the SSRI fluoxetine indeed blocked 5-HT-induced transactivation of the PDGFß receptor. However, upon further examination, we discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFß receptor via 5-HT2 receptor activation. After 45min, the increase in PDGFß receptor phosphorylation induced by fluoxetine had returned to baseline, but a subsequent transactivating stimulus (5-HT) failed to "re-transactivate" the PDGFß receptor. We further demonstrate that 45min, but not 3h, 5-HT pretreatment blocks dopamine-induced PDGFß receptor transactivation. This did not involve changes in PDGF receptor function, since ligand (PDGF)-induced PDGFß receptor activation was not inhibited by 5-HT pretreatment. To our knowledge this is the first demonstration of the heterologous desensitization of an RTK transactivation pathway and reveals a previously unknown short-term "blackout" period where no additional transactivation signaling is possible.

Differential expression of the seven rRNA operon promoters from the plant growth-promoting bacterium Pseudomonas sp. UW4.

Bacteria often have multiple copies of ribosomal RNA (rrn) genes in their genomes. The presence of multiple rrn operons suggests an advantage to the organism, perhaps through adjustable control of protein expression in response to altered environmental conditions. In the work described here, the strengths of the seven rRNA promoters of Pseudomonas sp. UW4 were individually assessed by separately cloning each promoter region into an expression vector and monitoring the activity of the reporter protein, the Escherichia coli lacZ gene product. The lacZ expression was the highest for the rrnE promoter under all growth conditions, with the various promoters demonstrating a range of strengths. These findings indicate that these promoters are not functionally identical. This observation suggests that the differential expression of rrn operons under various physiological conditions and growth stages allows better regulation of rRNA, conferring an advantage to P. sp. UW4 through a more fine-tuned control of protein expression in a wide range of environmental situations.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Distinct patterns of HSP30 and HSP70 degradation in Xenopus laevis A6 cells recovering from thermal stress.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. In this study, we examined the pattern of accumulation of HSP30 and HSP70 in Xenopus laevis A6 kidney epithelial cells recovering from heat shock. Immunoblot analysis revealed the presence of elevated levels of HSP30 after 72h of recovery. However, the relative levels of HSP70 declined to near control levels after 24h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. This study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation.

Reactive oxygen species are required for 5-HT-induced transactivation of neuronal platelet-derived growth factor and TrkB receptors, but not for ERK1/2 activation.

High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type ß receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H2O2 induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H2O2 failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFß receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFß receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFß and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFß and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring.

Enhanced HSP30 and HSP70 accumulation in Xenopus cells subjected to concurrent sodium arsenite and cadmium chloride stress.

Heat shock proteins (HSPs) are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in A6 kidney epithelial cells of the frog, Xenopus laevis. Immunoblot analysis revealed that HSP30 and HSP70 accumulation in concurrent stressor treatments were significantly higher than the sum of HSP30 or HSP70 accumulation in individual treatments. This finding suggested a synergistic action between sodium arsenite and cadmium chloride. KNK437 inhibitor studies indicated that the combined stressor-induced accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemistry revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined sodium arsenite and cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin proteasome degradation system. The addition of a mild heat shock further enhanced the accumulation of HSP30 and HSP70 in response to relatively low concentrations of sodium arsenite plus cadmium chloride.

Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) ß receptors which can protect neurons against NMDA-induced neurotoxicity. RESULTS: In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered. CONCLUSIONS: In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.

The complete genome sequence of the plant growth-promoting bacterium Pseudomonas sp. UW4.

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated "housekeeping" genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.

5-Hydroxytryptamine type 7 receptor neuroprotection against NMDA-induced excitotoxicity is PDGFß receptor dependent.

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors regulates growth factor receptor expression, including the expression of platelet-derived growth factor (PDGF) ß receptors. Direct activation of PDGFß receptors in primary hippocampal and cortical neurons inhibits NMDA receptor activity and attenuates NMDA receptor-induced neurotoxicity. Our objective was to investigate whether the 5-HT7 receptor-induced increase in PDGFß receptor expression would be similarly neuroprotective. We demonstrate that 5-HT7 receptor agonist treatment in primary hippocampal neurons also increases the expression of phospholipase C (PLC) γ, a downstream effector of PDGFß receptors associated with the inhibition of NMDA receptor activity. To determine if the up-regulation of PDGFß receptors is neuroprotective, primary hippocampal neurons were incubated with the 5-HT7 receptor agonist, LP 12, for 24 h. Indeed, LP 12 treatment prevented NMDA-induced neurotoxicity and this effect was dependent on PDGFß receptor kinase activity. Treatment of primary neurons with LP 12 also differentially altered NMDA receptor subunit expression, reducing the expression of NR1 and NR2B, but not NR2A. These findings demonstrate the potential for providing growth factor receptor-dependent neuroprotective effects using small-molecule ligands of G protein-coupled receptors.

5-HT(1A) receptors transactivate the platelet-derived growth factor receptor type beta in neuronal cells.

In the absence of ligand, certain growth factor receptors can be activated via G-protein coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate platelet-derived growth factor (PDGF) ß receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here we show that 5-HT can transiently increase the phosphorylation of PDGFß receptors through 5-HT(1A) receptors in a time- and dose-dependent manner in SH-SY5Y neuroblastoma cells. 5-HT also transactivates PDGFß receptors in primary cortical neurons. This transactivation pathway is pertussis-toxin sensitive and Src tyrosine kinase-dependent. This pathway is also dependent on phospholipase C activity and intracellular calcium signaling. Several studies involving PDGFß receptor transactivation by GPCRs have also demonstrated a PDGFß receptor-dependent increase in the phosphorylation of ERK1/2. Yet in SH-SY5Y cells, 5-HT treatment causes a PDGFß receptor-independent increase in ERK1/2 phosphorylation. This crosstalk between 5-HT and PDGFß receptors identifies a potentially important signaling link between the serotonergic system and growth factor signaling in neurons.

Withaferin A induces proteasome inhibition, endoplasmic reticulum stress, the heat shock response and acquisition of thermotolerance.

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.

Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor ß receptor expression.

Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)ß receptors. The increased PDGFß receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFß receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

Sodium arsenite and cadmium chloride induction of proteasomal inhibition and HSP accumulation in Xenopus laevis A6 kidney epithelial cells.

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 µM) or CdCl(2) (100-200 µM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Curcumin-induced inhibition of proteasomal activity, enhanced HSP accumulation and the acquisition of thermotolerance in Xenopus laevis A6 cells.

In the present study, curcumin, a phenolic compound with anti-inflammatory, anti-tumor and anti-amyloid properties, inhibited proteasomal activity and induced the accumulation of HSPs in the frog model system, Xenopus laevis. Treatment of A6 kidney epithelial cells with curcumin enhanced ubiquitinated protein levels and inhibited chymotrypsin-like activity. Furthermore, exposure of cells to 10-50 µM curcumin for 24h induced HSP30 and HSP70 accumulation. This phenomenon was controlled at the transcriptional level since pre-treatment of cells with KNK437, a heat shock factor 1 (HSF1) inhibitor, repressed HSP accumulation. Additionally, elevation of the incubation temperature from 22 to 30 °C greatly enhanced the curcumin-induced accumulation of HSP30 and HSP70. Immunocytochemical analysis revealed that curcumin-induced HSP30 was detectable primarily in the cytoplasm in a punctate pattern with minimal detrimental effects on the actin cytoskeleton. Finally, prior exposure of cells to curcumin conferred a state of thermotolerance since it protected them against a subsequent cytotoxic thermal challenge. These findings are of importance given the interest in identifying agents that can upregulate HSP levels with minimal effects on cell structure or function as a therapeutic treatment of protein folding diseases.