Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and ß2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Hsp27 suppresses the formation of inclusion bodies induced by expression of R120G alpha B-crystallin, a cause of desmin-related myopathy.

The R120G mutation in the small heat shock protein (sHSP) alpha B-crystallin has been identified in a family suffering from desmin-related myopathy. In this study, we characterized the features of transiently expressed R120G alpha B-crystallin in mammalian cells. In addition, we examined interactions of this mutant alpha B-crystallin with Hsp27, another representative sHSP. In HeLa cells, transiently expressed R120G alpha B-crystallin was mainly fractionated in the insoluble fraction, although wild-type alpha B-crystallin was predominantly found in the soluble fraction. In immunofluorescence studies, we found 15-25% of R120G alpha B-crystallin-expressing cells to contain multiple cytosolic inclusion bodies, in which Hsp27 was also localized. When R120G alpha B-crystallin and Hsp27 were transiently co-expressed in HeLa cells, the amount of R120G alpha B-crystallin in the soluble fraction was greater than with expression of R120G alpha B-crystallin alone. Moreover, co-expression resulted in reduced formation of inclusion bodies, suggesting that Hsp27 acts as a molecular chaperone for R120G alpha B-crystallin.

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27.

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCI), or anisomycin, an activator of p38 mitogen-activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)-induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase-activated protein (MAPKAP) kinase-2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells.

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.

Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus.

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels.

A high level of hippocampal brain-derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (gamma-hexachlorocyclohexane); and L-type voltage-dependent calcium channel agonist, BAY-K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY-K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12--24 h, 8--16 h and 6 h following administration of kainic acid, PTZ and BAY-K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY-K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY-K 8644, but not PTZ, effects. omega-Conotoxin GVIA and DCG-IV partially blocked kainic acid-induced enhancement of BDNF, indicating involvement of L-type and N-type voltage-dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in D-myo-inositol-1,4,5-triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY-K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant-dependent stimulation of BDNF production and involvement of region-specific voltage-dependent calcium channels.

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.

Phosphorylation-induced change of the oligomerization state of alpha B-crystallin.

alphaB-crystallin in cells can be phosphorylated at three serine residues in response to stress or during mitosis (Ito, H., Okamoto, K., Nakayama, H., Isobe, T., and Kato, K. (1997) J. Biol. Chem. 272, 29934-29941 and Kato, K., Ito, H., Kamei, K., Inaguma, Y., Iwamoto, I., and Saga, S. (1998) J. Biol. Chem. 273, 28346-28354). In the present study, we determined effects of phosphorylation of alphaB-crystallin on its oligomerization state, mainly by using site-directed mutagenesis, in which all three phosphorylation sites were substituted with aspartate to mimic the phosphorylation state (3D-alphaB). From results of sucrose density gradient centrifugation, we found that wild type alphaB-crystallin (wt-alphaB) and 3D-alphaB sedimented in fractions corresponding to apparent molecular masses of about 500 and 300 kDa, respectively. Chaperone-like activity of 3D-alphaB was significantly weaker than that of wt-alphaB. When wt-alphaB and 3D-alphaB were expressed in COS-m6 cells, they sedimented at positions corresponding to apparent molecular masses of about 500 and 100 kDa, respectively. In U373 MG human glioma cells, alphaB-crystallin was observed as large oligomers with apparent molecular masses about 500 kDa and the oligomerization size was reduced after phosphorylation induced by phorbol 12-myristate 13-acetate and okadaic acid. Coexpression of luciferase and wt-alphaB or 3D-alphaB in Chinese hamster ovary cells caused protection of the enzyme from heat inactivation although the degree of protection with 3D-alphaB was less than that with wt-alphaB. From these observations, it is suggested that phosphorylation of alphaB-crystallin causes dissociation of large oligomers to smaller sizes molecules and reduction of chaperone-like activity, like in the case of HSP27.

AlphaB-crystallin phosphorylated at Ser-59 is localized in centrosomes and midbodies during mitosis.

We have reported that the three serine residues in alphaB-crystallin are phosphorylated under various stress conditions. We prepared affinity-purified antibodies recognizing each of the phosphorylated serine residues (Ser-19, Ser-45, and Ser-59, respectively) in alphaB-crystallin with peptides (p19S, p45S, or p59S) that contained the corresponding phosphorylated serine residue. Immunocytochemically anti-p45S antibodies stained the cytoplasm of mitotic cells (J. Biol. Chem. 273, 28,346-28,354). We have now found that the anti-p59S antibodies recognize centrosomes and midbodies of dividing cells. alphaB-Crystallin was the only protein recognized by the anti-p59S antibodies in Western blot analyses of isolated centrosome fractions. alphaB-Crystallin phosphorylated at Ser-59 was localized at the microtubule organizing centers by means of double staining with anti-beta-tubulin antibody in aster formation analysis and was co-localized with gamma-tubulin in centrosomes. Gamma-Tubulin was co-immunoprecipitated with alphaB-crystallin in U373 glioma cell extracts. On the other hand, the location of the phosphorylated alphaB-crystallin deviated from that of alpha-tubulin or gamma-tubulin in the midbody region. Taken together with the evidences that several chaperones are distributed to centrosomes, these results suggest that alphaB-crystallin as a chaperone might be also involved in the quality control of proteins.

The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization.

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1-17 and an unknown number of residues between 17-28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8-13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 of Drosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.

Brain-derived neurotrophic factor, nerve growth and neurotrophin-3 selected regions of the rat brain following kainic acid-induced seizure activity.

Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12-24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (approximately 10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (approximately 10-fold) and 8.2 ng/g in the olfactory bulb (approximately 16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.

AlphaB-crystallin in the rat lens is phosphorylated at an early post-natal age.

We determined the developmental changes in the phosphorylation state of alphaB-crystallin in lenses from rats at various post-natal ages by isoelectric focusing gel electrophoresis or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of lenses using antibodies that recognized the carboxy-terminal sequence or each of the three phosphorylated serine residues (Ser-19, Ser-45 and Ser-59) in alphaB-crystallin. Phosphorylated forms of alphaB-crystallin were barely detected at birth but they became detectable at 3 weeks of age and reached plateau levels at 8 weeks of age. The phosphorylation of alphaB-crystallin at Ser-45 was observed preferentially. The active form of p44/42 MAP kinase, which is responsible for the phosphorylation of Ser-45 in alphaB-crystallin, also increased in a development-dependent manner. Thus we found that the developmental increase of the phosphorylation at Ser-45 of alphaB-crystallin in the rat lens was due to the developmental activation of p44/42 MAP kinase.

Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation.

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.

Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small heat shock proteins in Drosophila melanogaster.

In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23. Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27. In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns. The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9. Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S. cerevisiae ubc9ts mutant. Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27. The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development. The significance of the sHsp-Ubc9 interaction is discussed.

Synthesis and accumulation of alphaB crystallin in C6 glioma cells is induced by agents that promote the disassembly of microtubules.

When C6 cells in culture were exposed at 37 degrees C to 1 microM colchicine or to 1 microM colcemid, a tubulin-binding antimitotic alkaloid, levels of alphaB crystallin in cells began to increase after about 10 h, reaching a maximum of more than 1 microg/mg protein after 24 h. The level of alphaB crystallin returned to near the control level within two subsequent days of culture in the normal medium. Northern blot analysis showed that the accumulation of alphaB crystallin was preceded by an increase in the level of the mRNA for alphaB crystallin. Nuclear run-off transcription assays showed that colchicine induced new synthesis of mRNA for alphaB crystallin. Immunofluorescence staining revealed that alphaB crystallin accumulated in the peripheral areas of cells, as did the depolymerized tubulin, after several hours of treatment with colcemid, and then it gradually became more conspicuous in the cytoplasm. Vinblastine and nocodazole, which also promote the disassembly of microtubules by binding to tubulins, also induced the synthesis of alphaB crystallin. Furthermore, induction of alphaB crystallin by these drugs was observed in quiescent cells that had been cultured in serum-free medium. However, taxol, a microtubule-stabilizing antimitotic agent, did not stimulate the synthesis of alphaB crystallin, but rather, it suppressed the induction of synthesis of alphaB crystallin by the microtubule-disrupting drugs. Induction of alphaB crystallin by colchicine or by other drugs that promote the disassembly of microtubules was sensitive to staurosporine, an inhibitor of protein kinases, and the induction was completely suppressed in the presence of 10 nM staurosporine. These results suggest that the expression of alphaB crystallin is stimulated, via phosphorylation reactions that are sensitive to staurosporine, when the depolymerization of microtubules is enhanced.

cDNA cloning of a 20-kDa protein (p20) highly homologous to small heat shock proteins: developmental and physiological changes in rat hindlimb muscles.

A cDNA clone encoding p20, a novel member of the small heat-shock protein family in mammals, was isolated from a rat soleus cDNA library. The clone contained an insert of 1.3 kb with an open reading frame specifying a polypeptide of 162 amino-acid residues. Southern blot analysis suggested that the p20 gene is a single gene in rat genome. Developmental changes and a sciatic nerve denervation experiment suggested that the expression of p20 in rat hindlimb muscle is related to muscle contraction, and specifically in slow-twitch muscles.

Modulation of the stress-induced synthesis of hsp27 and alpha B-crystallin by cyclic AMP in C6 rat glioma cells.

The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and alpha B-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 microM for 1 h) or heat (42 degrees C for 30 min), expression of hsp27 and alpha B-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 microM) or epinephrine (20 microM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of beta-adrenergic receptors. Cholera toxin (2 micrograms/ml), forskolin (20 microM), 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 alpha B-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 alpha B-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 alpha B-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 alpha B-crystallin is suppressed.

Enhancement of stress-induced synthesis of hsp27 and alpha B crystallin by modulators of the arachidonic acid cascade.

The regulation by intrinsic factors of responses to stress of two small stress proteins, hsp27 and alpha B crystallin, was examined in C6 rat glioma cells. Levels of hsp27 and alpha B crystallin were low in C6 glioma cells in confluent cultures. However, levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50 microM for 1 h) stress. When cells were exposed to arsenite or hear in the presence of indomethacin (50 microM), an inhibitor of cyclooxygenase, or in the presence of nordihydroguaiaretic acid (NDGA; 50 microM), an inhibitor of lipoxygenase, induction of hsp27 and alpha B crystallin was markedly stimulated as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. The presence of melittin (1 microM), an activator of phospholipase A2, during the stress period also stimulated the induction of the two proteins. The expression of hsp70 to each stress was also enhanced in the presence of indomethacin, NDGA, or melittin. The gel mobility shift assay revealed that these chemicals prolonged the arsenite-induced activation of heat shock element (HSE)-binding activity of heat shock transcriptional factor (HSF) in cells. Induction of hsp27 and alpha B crystallin in adrenal glands of heat-stressed (42 degrees C for 15 min) rats was also enhanced by prior injection of aspirin, another inhibitor of cyclooxygenase. These results indicate that the responses to stress of hsp27 and alpha B crystallin, as well as the response of hsp70, are coupled with the metabolic activity of the arachidonic acid cascade and the mechanism for regulation of stress responses observed in C6 cells is operative in tissues and organs in vivo.

[Mammalian small stress proteins and responses to stress].

When cells are exposed to heat stress or chemical stress, expression of genes for heat shock proteins or stress proteins (HSPs) is enhanced and the proteins are accumulated in cells. The cells with increased HSPs exhibit tolerance against the additional stress. HSPs are expressed also in unstressed tissues or cells for essential biochemical cellular processes including growth and differentiation. Since the responses of HSPs in tissues to stress loaded to a whole living body are much more sensitive compared to those in cultured cells, it is suggested that endogenous factors modulate the stress-induced expression of HSPs. Here we summarize the responses of small HSPs (alpha crystallins, HSP27 and p20) to stress and their modifications by various factors.