Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid

Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A₂, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

Induction of HSP27 phosphorylation and thermoresistance in Chinese hamster cells by arsenite, cycloheximide, A23187, and EGTA

Incorporation of [3H]leucine, immunochemical analyses with a specific hamster HSP27 rabbit immunoserum, and [32P]orthophosphate labeling were used to monitor synthesis, accumulation, and phosphorylation of HSP27 in Chinese hamster cells after induction of thermoresistance by arsenite, cycloheximide, A23187, and EGTA. In contrast to arsenite-induced thermotolerance, which develops in parallel to synthesis and accumulation of HSP27, enhanced thermoresistance observed immediately after incubating cells in the presence of cycloheximide, A23187, or EGTA is independent of HSP27 or other HSP accumulation. All these treatments, however, result in a rapid phosphorylation of preexisting HSP27. In view of previous results which indicated that HSP27 is involved in cell protection from thermal killing (J. Landry, P. Chrétien, H. Lambert, E. Hickey, and L. A. Weber, J. Cell Biol. 109, 7-15, 1989), it is proposed that activation of HSP27 through phosphorylation may be a key determinant in the regulation of cell thermosensitivity.     

Contrasting effects of midazolam on induction of heat shock protein 27 by vasopressin and heat in aortic smooth muscle cells.

We previously showed that vasopressin stimulates the induction of heat shock protein (HSP) 27, a low molecular-weight HSP, through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined the effects of midazolam, an intravenous anesthetic, on the HSP27 induction stimulated by vasopressin, heat, or sodium arsenite (arsenite) in A10 cells. Midazolam inhibited the accumulation of HSP27 induced by vasopressin or 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of protein kinase C. Midazolam also reduced the vasopressin-induced level of the mRNA for HSP27. In contrast, midazolam enhanced the HSP27-accumulation induced by heat or arsenite. Midazolam also enhanced the heat-increased level of the mRNA for HSP27. However, midazolam had no effect on the dissociation of the aggregated form of HSP27 following stimulation by vasopressin, heat, or arsenite. These results suggest that midazolam suppresses vasopressin-stimulated HSP27 induction in vascular smooth muscle cells, and that this inhibitory effect is exerted at a point downstream from protein kinase C. In contrast, midazolam enhanced heat- or arsenite-stimulated HSP27 induction. Thus, midazolam has dual effects on the HSP27 induction stimulated by various stresses in vascular smooth muscle cells.     

Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27)

Exposure to ultraviolet (UV) light poses a health risk for eye disease, and solar ultraviolet in the B range (UVB, 280-320 nm) is known to be related to various corneal disorders. In this study, we investigated whether pre-conditioning of cells with arsenite (AsO2(-1)) can reduce UVB-induced apoptosis in human corneal epithelial cells, and whether the anti-apoptotic activity of 27 kDa heat shock protein (HSP27), a small heat shock protein, plays a role in this protection. UVB at levels comparable to physiologic solar exposure induces apoptosis of corneal epithelial cells in culture, demonstrated by activation of caspase 9 and caspase 3, and DNA fragmentation. When cells were pre-conditioned with arsenite prior to UVB exposure, the UVB-induced cell death was reduced, and UVB-induced activation of caspases and DNA fragmentation was inhibited. When cells were pre-treated with SB 203580, which inhibits HSP27 phosphorylation through inhibition of p38 MAP kinase activation, the arsenite-induced reduction of UVB-induced apoptosis was partially reversed. Arsenite pre-conditioning inhibited UVB-induced apoptosis in a two-phase pattern, which was temporally correlated with arsenite-induced HSP27 expression and phosphorylation. Neutralization of intracellular HSP27 with its antibody reduced arsenite's inhibition of UVB-induced caspase3 activation. Our results suggest that forms of stress that upregulate HSP27 and its phosphorylation may be useful as novel approaches to prevent adverse ocular effects arising from UV exposure in humans.     

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 microM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K(+)-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K(-)-MN). The increase in K(-)-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K(+)-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.     

Independent regulation of prostaglandin production and the stress response in human fibroblasts.

The stress, or heat shock response of eukaryotic cells is characterized by dramatic changes in the metabolism of responding cells, most notably the increased synthesis of a group of proteins known as heat shock proteins. In this study, we examined the relationship of prostaglandin synthesis/release to the stress response. Stress protein synthesis was induced with sodium arsenite, and prostaglandin E2 and prostacyclin (measured as 6-keto PGF1 alpha) levels were determined by enzyme immunoassay. The stress response was monitored by the incorporation of [35S]methionine and separation of protein by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prostaglandin synthesis and the stress response were both induced by sodium arsenite. However, aspirin, a cyclooxygenase inhibitor, inhibited arsenite-induced prostaglandin synthesis but did not inhibit stress protein synthesis. Conversely, the calcium ionophore A23187 also stimulated prostaglandin synthesis, but did not induce the stress response. The results of this study indicate that sodium arsenite, a stress response inducer, stimulates prostaglandin production, but this appears to be a correlative rather than causative occurrence in the stress response. Determination of the cytotoxicity of arsenite indicated a high correlation of stimulation of prostaglandin release with cytotoxicity.     

Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae.

The growth response of Saccharomyces cerevisiae to arsenite and arsenate and the relationship between the enhancement of heat shock protein (hsp) synthesis caused by these arsenic oxides and thermotolerance are reported. Arsenite and arsenate transiently inhibited cell growth and overall protein synthesis; arsenate enhanced the synthesis of the 42-, 74-, 84-, and 100-kilodalton hsps, whereas arsenite enhanced synthesis of only the 74-kilodalton hsp. The induction of these hsps reached a maximum 45 min following metal oxide treatment and then declined. A delayed thermotolerance peaked 4 h after metal oxide addition, at which time cell growth and protein synthesis were recovering. These data show that the arsenate- and arsenite-induced thermotolerance in S. cerevisiae cells does not appear to be causally related to either hsp synthesis or cell cycle arrest.     

Stress-induced glucose uptake in bovine chromaffin cells: a comparison of the effect of arsenite and anisomycin

The effect of the toxic chemical Na-arsenite and the protein synthesis inhibitor anisomycin on glucose transport in primary cultures of bovine chromaffin cells was compared using the effect of insulin-like growth factor I (IGF-I) as a reference. The enhanced uptake of glucose obtained in response to arsenite and anisomycin reached maximum after 60 min, with the response to anisomycin being delayed in onset relative to that of arsenite. At maximal doses the arsenite effect was consistently higher than that of anisomycin and comparable to the approximately 2-fold effect produced by IGF-I. The selective inhibitor of stress-activated protein kinase 2 (SAPK2), SB 203580, inhibited completely anisomycin-induced glucose uptake but only partly suppressed uptake stimulated by arsenite. Both substances, in concentrations producing maximal effects on glucose transport, led to a strong phosphorylation of SAPK2. In contrast to the effect on glucose transport, the arsenite-induced phosphorylation of SAPK2 was relatively slow compared to the anisomycin-induced activation. The results indicate that glucose uptake induced by the two types of cellular stress are mediated by at least two different signaling pathways, which also differ from that activated by IGF-I.     

Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo

When submitted to a heat-shock, mouse embryonal carcinoma (EC) and fibroblast cells show very different behavior. All the EC cells so far analyzed express very high levels of several heat-shock proteins (HSP) in the absence of stress and independent of their origin and culture conditions. In such cells, the 89-kd, 70-kd and 59-kd HSP are the most prominent proteins after actin. In addition, the 89-kd and 59-kd HSP are not stimulated by an arsenite shock in contrast to what is observed with fibroblasts or cells of the parietal yolk sac type. Arsenite induces the synthesis of a 105-kd polypeptide in fibroblasts but not in EC cells. In vitro differentiation of F9 cells induced by retinoic acid and dibutyryl cAMP is accompanied by a decrease in the spontaneous relative abundance of HSP and restores the arsenite-induced synthesis of the 105-kd polypeptide. EC cells are usually believed to be similar to inner cell mass cells of mouse blastocyst. Furthermore, data in the literature together with our own results suggest that the same three HSP are also spontaneously expressed in high amounts in the early mouse embryo.     

Arsenite induces apoptosis in chinese hamster ovary cells by generation of reactive oxygen species

Arsenic, a human carcinogen, possesses a serious environmental threat but the mechanism of its toxicity remains unclear. Knowledge of how arsenic induces cell death and how cells escape the death path may help to understand arsenic carcinogenesis. We have investigated the nature of sodium arsenite-induced cell death in Chinese hamster ovary K1 cells. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytometry. Immediately after 4 h of 40 μM arsenite treatment, no appreciable fraction of cells with sub-G1 DNA content was detected; however, the sub-G1 cell fraction increased with postarsenite incubation time, and detectable increase started at 8 h of incubation, whereas the intracellular peroxide level as measured by the fluorescent intensity of 2′,7′-dichlorofluorescein increased immediately following a 4-h arsenite treatment. Simultaneous treatment with arsenite plus antioxidant (N-acetyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); protein kinase inhibitor (H-7) or protein synthesis inhibitor (cycloheximide) reduced the fraction of sub-G1 cell and internucleosomal DNA degradation. Trolox, neocuproine, or cycloheximide given after arsenite treatment also effectively reduced apoptosis. These results lead to a working hypothesis that arsenite-induced apoptosis in CHO-K1 cells is triggered by the generation of hydrogen peroxide, followed by a copper-mediated Fenton reaction that catalyzes the production of hydroxyl radicals, which selectively activates protein kinase through de novo synthesis of macromolecules. © 1996 Wiley-Liss, Inc.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Stress granule assembly is mediated by prion-like aggregation of TIA-1

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.     

Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.

It is well-known that p38 mitogen-activated protein kinase (p38MAPK) participates in cellular responses to mitogenic stimuli, environmental and genotoxic stresses, and apoptotic agents. Although there are several reports on p38MAPK in relation to cell growth and apoptosis, the exact mechanism of p38MAPK-mediated cell growth regulation remains obscure. Here, we examined possible roles of p38MAPK in the sodium arsenite-induced cell growth inhibition in NIH3T3 cells. Sodium arsenite induced transient cell growth delay with marked activation of p38MAPK. In addition, arsenite induced CDK inhibitor p21(CIP1/WAF1) and enhanced its binding to the CDK2, which resulted in inhibition of CDK2 activity. The levels of cyclin D1 expression and the CDK4 kinase activity were also significantly reduced. pRB was hypophosphorylated by sodium arsenite. SB203580, a specific inhibitor of p38MAPK, blocked arsenite-induced growth inhibition as well as the arsenite-induced p21(CIP1/WAF1) expression. Expression of dominant negative p38MAPK also blocked arsenite-induced p21(CIP1/WAF1) expression. Inhibited-CDK2 activity was also completely reversed by SB203580 or expression of dominant negative p38MAPK, while the decreased-cyclin D1 protein by the compound was not restored. These data demonstrate a possible link between the activation of p38MAPK and induction of p21(CIP1/WAF1), suggesting that the activation of p38MAPK is, at least in part, related to the cell growth inhibition by sodium arsenite.     

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.     

Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in chinese hamster ovary cells

Arsenic is the first metal to be identified as a human carcinogen. Arsenite, one inorganic form of arsenic, has been found to induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety of in vitro systems. In this study of arsenite-induced genotoxicity represented as micronuclei production in Chinese hamster ovary cells (CHO-K1), we found that the calcium channel blocker, verapamil, can potentiate arsenite-induced micronuclei. And after arsenite treatment, the elevation of intracellular calcium was observed. When extracellular calcium was depleted during arsenite treatment, the arsenite-induced micronuclei formation was significantly suppressed. These data indicated that a calcium ion plays an essential role in arsenite-induced genotoxicity. Further, it was found that the cotreatment of arsenite and a calcium ionophore, A23187, can increase the micronuclei induction. In contrast, pretreatment of the intracellular calcium chelator, quin 2, significantly inhibited micronuclei production of arsenite administration. In addition, we measured the activity of calcium-and phospholipid-dependent protein kinase C (PKC) and found that arsenite can activate PKC activity in a dose-dependent manner. Subsequently, some PKC activators and inhibitors were applied to investigate the involvement of PKC on arsenite-induced micronuclei formation. It was found that H7, a PKC inhibitor, can depress but TPA, a PKC activator, can enhance arsenite-induced micronuclei significantly. These data indicated that arsenite exposure perturbs intracellular calcium homeostasis and activates PKC activity. As a result, the activation of PKC activity may play an important role in arsenite-induced genotoxicity. J. Cell. Biochem. 64:423–433. © 1997 Wiley-Liss, Inc.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.