2-Cyclopenten-1-one, a New Inducer of Heat Shock Protein 70 with Antiviral Activity
1996; Elsevier BV; Volume: 271; Issue: 50 Linguagem: Inglês
10.1074/jbc.271.50.32192
ISSN1083-351X
AutoresAntonio Rossi, Giuliano Elia, Massimo Santoro,
Tópico(s)interferon and immune responses
ResumoThe cytoprotective role of heat shock proteins (HSP) described in variety of human diseases, including ischemia, inflammation, and infection, suggests new therapeutic strategies relying upon the development of drugs that selectively turn on heat shock genes. Cyclopentenone prostaglandins, which contain an α,β-unsaturated carbonyl group in the cyclopentane ring and possess antiviral activity against several RNA and DNA viruses, were shown to function as signal for HSP synthesis in a nonstressful situation in a variety of mammalian cells. We now report that 2-cyclopenten-1-one selectively induces the expression of the 70-kDa HSP (HSP70) in human cells, through cycloheximide-sensitive activation of heat shock transcription factor 1 (HSF1). The α,β-unsaturated carbonyl group is the key structure triggering HSF1 activation. Induction is associated with antiviral activity during infection with vesicular stomatitis virus. These results identify the molecular structure of natural prostaglandins responsible for HSF1 activation and open new perspectives in the search for novel antiviral and cytoprotective drugs. The cytoprotective role of heat shock proteins (HSP) described in variety of human diseases, including ischemia, inflammation, and infection, suggests new therapeutic strategies relying upon the development of drugs that selectively turn on heat shock genes. Cyclopentenone prostaglandins, which contain an α,β-unsaturated carbonyl group in the cyclopentane ring and possess antiviral activity against several RNA and DNA viruses, were shown to function as signal for HSP synthesis in a nonstressful situation in a variety of mammalian cells. We now report that 2-cyclopenten-1-one selectively induces the expression of the 70-kDa HSP (HSP70) in human cells, through cycloheximide-sensitive activation of heat shock transcription factor 1 (HSF1). The α,β-unsaturated carbonyl group is the key structure triggering HSF1 activation. Induction is associated with antiviral activity during infection with vesicular stomatitis virus. These results identify the molecular structure of natural prostaglandins responsible for HSF1 activation and open new perspectives in the search for novel antiviral and cytoprotective drugs. INTRODUCTIONHeat shock proteins (HSP) 1The abbreviations used are: HSPheat shock proteinHSFheat shock transcription factorHSEheat shock elementPBSphosphate-buffered salineEMSAelectrophoretic mobility shift assayPAGEpolyacrylamide gel electrophoresisCHXcycloheximide. and molecular chaperones have been known for several years to protect cells against a wide variety of toxic conditions, including extreme temperatures, oxidative stress, virus infection, and the exposure to heavy metals or cytotoxic drugs (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4360) Google Scholar). HSP induction is not only a signal for detection of physiological stress, but is utilized by the cells in the repair process following different types of injury, to prevent damage resulting from the accumulation of non-native proteins (2Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Plainview, NY1990Google Scholar). In mammalian cells, several HSP are expressed during normal growth conditions and can be induced by biologically active molecules such as hemin (3Sistonen L. Sarge K.D. Phillips B. Abravaya K. Morimoto R.I. Mol. Cell. Biol. 1992; 12: 4104-4111Crossref PubMed Scopus (236) Google Scholar) and prostaglandins (4Santoro M.G. Garaci E. Amici C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8407-8411Crossref PubMed Scopus (151) Google Scholar, 5Amici C. Sistonen L. Santoro M.G. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6227-6231Crossref PubMed Scopus (153) Google Scholar), whereas others are expressed upon stress-activated regulation of transcriptional and translational switches. Induction requires the activation and translocation to the nucleus of a transregulatory protein, the heat shock transcription factor (HSF), which converts from a monomeric non-DNA-binding form to an oligomeric form that binds to specific promoter elements (HSE) located upstream of heat shock genes (6Morimoto R.I. Sarge K.D. Abravaya K. J. Biol. Chem. 1992; 267: 21987-21990Abstract Full Text PDF PubMed Google Scholar). Several HSFs have been identified in mammalian cells (6Morimoto R.I. Sarge K.D. Abravaya K. J. Biol. Chem. 1992; 267: 21987-21990Abstract Full Text PDF PubMed Google Scholar).We have shown previously that prostaglandins of the A and J type (PGAs and PGJs) were able to activate HSF and induce the synthesis of the 70-kDa HSP (HSP70) in a wide variety of mammalian cells (4Santoro M.G. Garaci E. Amici C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8407-8411Crossref PubMed Scopus (151) Google Scholar, 7Santoro M.G. Garaci E. Amici C. Stress Proteins: Induction and Function. Springer-Verlag, Berlin1990: 27-44Crossref Google Scholar). Induction of HSP70 by prostaglandins requires the presence of a reactive α,β-unsaturated carbonyl group in the cyclopentane ring (cyclopentenone), which renders this portion of the molecule able to form Michael's adducts with cellular nucleophilics and to covalently bind to cysteine residues of proteins via a thioether bond (8Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar, 9Khan S.H. Sorof S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9401-9405Crossref PubMed Scopus (67) Google Scholar). We have also shown that cyclopentenone prostaglandins possess a potent antiviral activity against RNA and DNA viruses in a wide variety of experimental models in vitro and in vivo (reviewed in 7Santoro M.G. Garaci E. Amici C. Stress Proteins: Induction and Function. Springer-Verlag, Berlin1990: 27-44Crossref Google Scholar and 10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar). In negative-strand RNA viruses the antiviral activity has been associated with the ability of cyclopentenone prostaglandins to induce HSP70 expression, and a role of this protein in the control of virus replication has been hypothesized (10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar, 11Amici C. Giorgi C. Rossi A. Santoro M.G. J. Virol. 1994; 68: 6890-6899Crossref PubMed Google Scholar).We now report that the cyclopentenone ring structure itself, 2-cyclopenten-1-one, is able to activate HSF and selectively induce HSP70 expression and show that the presence of an α,β-unsaturated carbonyl group is the key structure for triggering HSF activation. We also show that induction of HSP70 is associated with inhibition of infectious virus production and viral protein synthesis in monkey kidney epithelial cells infected with vesicular stomatitis virus (VSV).RESULTS AND DISCUSSIONHuman K562 erythroleukemic cells were exposed to varying concentrations of 2-cyclopenten-1-one or were subjected to heat shock. Whole-cell extracts were analyzed by EMSA to determine HSF activation. 2-Cyclopenten-1-one induced HSF DNA binding activity in a dose-dependent manner (Fig. 1A). Activation comparable with severe heat shock was achieved with concentration of 250-500 μM 2-cyclopenten-1-one. As in the case of heat shock, HSF type 1 (HSF1) is the primary component of the cyclopentenone-induced HSE binding activity, as determined by EMSA after preincubation of whole-cell extracts with polyclonal anti-HSF1 or anti-HSF2 antibodies (Fig. 1B).Heat shock-induced HSF activation is known to be dependent on de novo protein synthesis in the case of a mild (42°C) heat treatment, while HSF activation after severe (45°C) heat shock is independent of cellular protein synthesis (18Baler R. Welch W.J. Voellmy R. J. Cell Biol. 1992; 117: 1151-1159Crossref PubMed Scopus (273) Google Scholar). In order to determine whether HSF1 activation by 2-cyclopenten-1-one was dependent on de novo protein synthesis, K562 cells were either treated with 500 μM 2-cyclopenten-1-one or were stressed at 42 or 45°C for 20 min, in the presence or in the absence of 100 μM cycloheximide (CHX). Ninety or 180 min after 2-cyclopenten-1-one treatment, or 30 min after heat shock, whole-cell extracts were prepared and subjected to EMSA. As shown in Fig. 1C, 2-cyclopenten-1-one-induced HSF1 activation was found to be dependent on de novo protein synthesis, since it was inhibited by cycloheximide treatment.To investigate the kinetics of HSF activation by 2-cyclopenten-1-one, whole-cell extracts prepared at different times after treatment with 500 μM 2-cyclopenten-1-one or 3 h after heat shock (45°C for 20 min) were subjected to EMSA. The levels of HSF DNA binding activity in 2-cyclopenten-1-one-treated cells were quantitated by Molecular Dynamics PhosphoImager analysis. As shown in Fig. 2A, activation of HSF by 2-cyclopenten-1-one was detected 1.5 h after treatment, and maximal levels of HSF·HSE complexes were detected 9 h after addition of the drug. In the same experiment, the effect of 2-cyclopenten-1-one on HSP70 mRNA transcription was examined by nuclear run-on analysis. 2-Cyclopenten-1-one specifically induced HSP70 mRNA transcription, while it did not affect the transcription rates of messengers for other stress proteins, including HSC70, the glucose-regulated GRP78/BiP, or heme-oxygenase (Fig. 2B). HSP70 mRNA transcription started as soon as 1.5 h after 2-cyclopenten-1-one addition, and transcription rates were maximal by 6-9 h, to decline 24 h after treatment (Fig. 2B).Fig. 2Effect of 2-cyclopenten-1-one treatment on HSF activation, heat shock gene transcription, and protein synthesis in K562 cells. A, kinetics of HSF activation by 2-cyclopenten-1-one. Whole-cell extracts prepared at different times after treatment with 500 μM 2-cyclopenten-1-one or 3 h after heat shock (45°C for 20 min) were subjected to EMSA (right panel). Positions of HSF·DNA binding complex (HSF), constitutive HSE binding activity (CHBA), and nonspecific protein-DNA interactions (NS) are indicated, as in Fig. 1. The levels of HSF DNA-binding activity in 2-cyclopenten-1-one-treated cells were quantitated by Molecular Dynamics PhosphoImager analysis (left panel). HSF values were normalized to the level of HSF DNA binding activity at 9 h after treatment, which was given a value of 100%. B, transcription rates, measured by nuclear run-on assay. 32P-Labeled RNA was hybridized to nitrocellulose filters containing plasmids for the following human genes: HSP70 (pH2.3), GRP78/BiP (pHG23.1), HSC70 (pHA7.6), HO (clone 2/10), or gapdh. The radioactivity quantitated by Molecular Dynamics PhosphoImager analysis is shown in the left panel. Values are expressed as arbitrary units obtained by comparing transcription rates with control levels. C, effect of 2-cyclopenten-1-one on protein synthesis. K562 cells labeled with [35S]methionine at different times after treatment with 500 μM 2-cyclopenten-1-one were analyzed by SDS-PAGE and autoradiography (right, upper panel) or by immunoprecipitation with anti-HSP70 antibodies (right, lower panel). HSP70 synthesis (∘) was determined by densitometric analysis of the autoradiograms (left panel). Total protein synthesis (•) was determined as [35S]methionine incorporation into trichloroacetic acid-insoluble material.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the 2-cyclopenten-1-one-induced HSP70 mRNA was efficiently translated, K562 cells were labeled with [35S]methionine (10 μCi/106 cells, 1-h pulse) at different times after treatment with 500 μM 2-cyclopenten-1-one. Total protein synthesis, determined as [35S]methionine incorporation into trichloroacetic acid-insoluble material, was not inhibited by the drug under these conditions (Fig. 2C). Samples containing an equal amount of protein were analyzed by SDS-PAGE and autoradiography, and HSP70 synthesis was determined by densitometric analysis of the autoradiograms. As shown in Fig. 2C, 2-cyclopenten-1-one-induced HSP70 mRNA was efficiently translated in K562 cells. Synthesis of HSP70 was sustained for at least 9 h after treatment. Increased expression of this protein was also detected by immunoprecipitation (Fig. 2C, lower panel).To investigate whether the α,β-unsaturated carbonyl group present in 2-cyclopenten-1-one was the molecular structure responsible for HSF activation, K562 cells were treated with different concentrations of 2-cyclopenten-1-one, cyclopentanone (a similar molecule with a saturated carbonyl), or with cyclopentene, which contains a double bond, but not a carbonyl group. Neither cyclopentanone nor cyclopentene were able to induce HSF DNA binding activity (Fig. 3A), indicating that the presence of an α,β-unsaturated carbonyl is essential for HSF activation. Since the cyclopentenone prostaglandin A1 (PGA1) functions as signal for HSF activation in many types of mammalian cells (7Santoro M.G. Garaci E. Amici C. Stress Proteins: Induction and Function. Springer-Verlag, Berlin1990: 27-44Crossref Google Scholar, 10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar), we compared the effect of PGA1 with either 2-cyclopenten-1-one, or with 1-octen-3-ol and oenanthic acid, whose structure resembles the two aliphatic side chains of PGA1. Neither 1-octen-3-ol nor oenanthic acid were able to induce HSF activation, confirming that the key structural component for HSF activation by prostaglandins is the cyclopentenone moiety (Fig. 3B). However, PGA1 is able to activate HSF at concentrations much lower (approximately 25 times) than 2-cyclopenten-1-one (Fig. 3C). The presence of the aliphatic side chains could be functional by facilitating either entry into cells or detection of the molecular target.Fig. 3The α,β-unsaturated carbonyl group is responsible for HSF activation. A, K562 cells were treated with different concentrations of 2-cyclopenten-1-one (lanes 3-5), cyclopentanone (lanes 6 to 8), cyclopentene (lanes 9 to 11), or control diluent (lane 1) at 37°C or were stressed at 45°C for 20 min (HS; lane 2). After 3 h, whole-cell extracts were prepared and subjected to EMSA. A section of fluorograms from native gels is shown, and positions of HSF·DNA binding complex (HSF), constitutive HSE binding activity (CHBA), and nonspecific protein-DNA interactions (NS) are indicated, as in Fig. 1. B, K562 cells were treated with different concentrations of 2-cyclopenten-1-one (lanes 3-5), 1-octen-3-ol (lanes 6 to 8), oenanthic acid (lanes 9 to 11), or control diluent (lane 1) at 37°C or were heat shocked at 45°C for 20 min (HS; lane 2). EMSA was performed on whole-cell extracts 3 h after treatment. C, comparison of the effect of 2-cyclopenten-1-one (lanes 3-6) and PGA1 (lanes 7-10) on HSF activation. EMSA was performed on whole-cell extracts 3 h after treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Starting from the early observation that prostaglandins of the A type inhibit Sendai virus replication and prevent the establishment of persistent infections in cultured cells (19Santoro M.G. Benedetto A. Carruba G. Garaci E. Jaffe B.M. Science. 1980; 209: 1032-1034Crossref PubMed Scopus (87) Google Scholar), it is now well established that cyclopentenone prostaglandins possess a potent antiviral activity against several DNA and RNA viruses, including the human immunodeficiency virus type 1 (HIV-1) (20Rozera C. Carattoli A. De Marco A. Amici C. Giorgi C. Santoro M.G. J. Clin. Invest. 1996; 97: 1795-1803Crossref PubMed Scopus (70) Google Scholar), in vitro and in vivo (reviewed in 10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar and 21Santoro M.G. Garaci E. Amici C. Samuelsson B. Ramwell P.W. Paoletti R. Folco G. Granström E. Advances in Prostaglandins Thromboxane and Leukotriene Research. Vol. 21. Raven Press, New York1991: 867-874Google Scholar). The antiviral activity of a long acting synthetic analogue of PGA2 (di-M-PGA2) has also been shown in vivo, in a mouse model infected with influenza A virus (22Santoro M.G. Favalli C. Mastino A. Jaffe B.M. Esteban M. Garaci E. Arch. Virol. 1988; 99: 89-100Crossref PubMed Scopus (33) Google Scholar). In negative-strand RNA virus models prostaglandins of the A and J type provoke a selective and dramatic block of virus protein synthesis (11Amici C. Giorgi C. Rossi A. Santoro M.G. J. Virol. 1994; 68: 6890-6899Crossref PubMed Google Scholar, 12Pica F. Rossi A. Santirocco N. Palamara A. Garaci E. Santoro M.G. Antivir. Res. 1996; 29: 187-198Crossref PubMed Scopus (9) Google Scholar). This block is exerted at the translational level and is dependent on HSP70 expression in infected cells (reviewed in 10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar). A possible model has been hypothesized, in which HSP and virus messages, both of which can be translated in conditions where cellular protein synthesis is impaired, could possess similar mechanisms for preferential translation and could then compete with each other (23Santoro M.G. Feige U. Morimoto R.I. Yahara I. Polla B. Stress-Inducible Cellular Responses. Birkhauser-Verlag, Basel, Switzerland1996: 335-355Google Scholar).To investigate whether 2-cyclopenten-1-one could mimic the PG-induced block of virus protein synthesis, epithelial monkey kidney MA104 cells were infected with vesicular stomatitis virus and then treated with 2-cyclopenten-1-one, at concentrations which induced HSP70 synthesis. VSV titers were determined 24 h post-infection by cytopathic effect 50% assay. As shown in Fig. 4A, 2-cyclopenten-1-one was found to inhibit the production of VSV infectious particles in a dose-dependent manner and with a selective index of 36.Fig. 4Effect of 2-cyclopenten-1-one on VSV replication and protein synthesis. Confluent monolayers of MA104 cells were infected with VSV (1 plaque-forming unit/cell) and treated with different concentrations of 2-cyclopenten-1-one or control diluent soon after the 1-h adsorption period for the next 24 h. A, VSV titers were determined 24 h post-infection. B, uninfected (U) or VSV-infected (VSV) MA104 cells were treated with 500 μM (lanes 2and 4) 2-cyclopenten-1-one or with control diluent (lanes 1 and 3) soon after VSV infection and labeled with [35S]methionine (1-h pulse starting 5 h post-infection). Equal amounts of protein were analyzed by SDS-PAGE and autoradiography. The position of HSP70, identified by Western blot analysis using anti-human HSP70 antibodies (data not shown), is indicated by the arrow. VSV proteins L, G, N, NS, and M (12Pica F. Rossi A. Santirocco N. Palamara A. Garaci E. Santoro M.G. Antivir. Res. 1996; 29: 187-198Crossref PubMed Scopus (9) Google Scholar) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As compared with PGA1, which dramatically inhibits VSV replication in MA104 cells at concentrations lower than 50 μM (12Pica F. Rossi A. Santirocco N. Palamara A. Garaci E. Santoro M.G. Antivir. Res. 1996; 29: 187-198Crossref PubMed Scopus (9) Google Scholar, 24Pica F. De Marco A. De Cesare F. Santoro M.G. Antivir. Res. 1993; 20: 193-208Crossref PubMed Scopus (30) Google Scholar), higher concentrations of 2-cyclopenten-1-one were needed to inhibit virus replication. The relative potency of PGA1 and 2-cyclopenten-1-one in activating HSF (Fig. 3) correlates with the antiviral activity, reinforcing the hypothesis that the heat shock response is involved in the control of virus replication (10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar, 23Santoro M.G. Feige U. Morimoto R.I. Yahara I. Polla B. Stress-Inducible Cellular Responses. Birkhauser-Verlag, Basel, Switzerland1996: 335-355Google Scholar).To determine the effect of 2-cyclopenten-1-one on HSP70 induction and virus protein synthesis in MA104 cells, uninfected or VSV-infected cells were treated with 500 μM 2-cyclopenten-1-one, or with control diluent soon after VSV infection, and labeled with [35S]methionine (8 μCi/2 × 105 cells, 1-h pulse starting 5 h post-infection). Equal amounts of protein were analyzed on 10% SDS-PAGE gels and processed for autoradiography. As shown previously for PGA and PGJ compounds (10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar, 11Amici C. Giorgi C. Rossi A. Santoro M.G. J. Virol. 1994; 68: 6890-6899Crossref PubMed Google Scholar, 12Pica F. Rossi A. Santirocco N. Palamara A. Garaci E. Santoro M.G. Antivir. Res. 1996; 29: 187-198Crossref PubMed Scopus (9) Google Scholar), 2-cyclopenten-1-one treatment causes a selective and dramatic inhibition of viral protein synthesis, associated with HSP70 induction (Fig. 4B).The activation of heat shock genes and a cytoprotective role of HSP70 has been described in a wide variety of human diseases, including ischemia, metabolic disorders, inflammation, and infection (23Santoro M.G. Feige U. Morimoto R.I. Yahara I. Polla B. Stress-Inducible Cellular Responses. Birkhauser-Verlag, Basel, Switzerland1996: 335-355Google Scholar, 25Nowak T.S. Bound U. Schlesinger M.J. J. Neurochem. 1990; 54: 451-458Crossref PubMed Scopus (179) Google Scholar, 26Mestril R. Chi S. Sayen R. O'Reilly K. Dillmann W.H. J. Clin. Invest. 1994; 93: 759-767Crossref PubMed Scopus (281) Google Scholar, 27Williams R.S. Thomas J.A. Fina M. German Z. Benjamin I.J. J. Clin. Invest. 1993; 92: 503-508Crossref PubMed Scopus (152) Google Scholar, 28Uney J.B. Leigh P.N. Marsden C.D. Lees A. Anderton B.H. FEBS Lett. 1988; 235: 215-218Crossref PubMed Scopus (50) Google Scholar, 29Iaattela M. Wissing D. Bauer P.A. Li G.C. EMBO J. 1992; 11: 3507-3512Crossref PubMed Scopus (354) Google Scholar, 30Polla B.S. Kantengwa S. Curr. Top. Microbiol. Immunol. 1991; 167: 93-105Crossref PubMed Scopus (48) Google Scholar, 31Morris S.D. Cumming D.V.E. Latchman D.S. Yellon D.M. J. Clin. Invest. 1996; 97: 706-712Crossref PubMed Scopus (76) Google Scholar). HSP70 has been shown recently to be critically involved in myocardial protection by ischemia-induced injury also in animals (32Marber M.S. Walker J.M. Latchman D.S. Yellon D.M. J. Clin. Invest. 1994; 93: 1087-1094Crossref PubMed Scopus (124) Google Scholar). These observations justify the attempt to characterize new HSP inducers, which could be used therapeutically. The results described identify 2-cyclopenten-1-one as a new inducer of HSP70 with antiviral activity and indicate that the presence of an α,β-unsaturated carbonyl group in the cyclopentane ring is essential for induction. As shown previously for other HSP inducers, including antiviral prostaglandins, sodium arsenite, cadmium, and hyperthermia itself (10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar, 11Amici C. Giorgi C. Rossi A. Santoro M.G. J. Virol. 1994; 68: 6890-6899Crossref PubMed Google Scholar, 12Pica F. Rossi A. Santirocco N. Palamara A. Garaci E. Santoro M.G. Antivir. Res. 1996; 29: 187-198Crossref PubMed Scopus (9) Google Scholar), 2-cyclopenten-1-one-induced HSP70 synthesis is associated with a selective inhibition of virus protein synthesis, suggesting a cytoprotective role of this protein during viral infection. These results open new perspectives in the search for novel HSP inducers, which could be utilized as cytoprotective and antiviral drugs.In the present report we have also identified the molecular structure of natural prostaglandins responsible for HSF1 activation. Cyclopentenone prostaglandins are physiologically present in body fluids and, apart from the antiviral activity, are known to affect cell proliferation and differentiation (reviewed in 8Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar and 21Santoro M.G. Garaci E. Amici C. Samuelsson B. Ramwell P.W. Paoletti R. Folco G. Granström E. Advances in Prostaglandins Thromboxane and Leukotriene Research. Vol. 21. Raven Press, New York1991: 867-874Google Scholar). Recently the PGJ metabolite 15-deoxy Δ12,14PGJ2, which is characterized by the presence of a cyclopentenone ring and activates HSF (33Rossi A. Elia G. Santoro M.G. Proc. Natl. Acad Sci. U. S. A. 1996; (in press)Google Scholar), has been shown to be the natural ligand for the adipocyte determination factor PPARγ, inducing adipocyte differentiation (34Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2712) Google Scholar, 35Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). The fact that cyclopentenone prostaglandins activate HSF suggests the possibility that the heat shock response could play a role in physiological processes controlled by prostaglandins. INTRODUCTIONHeat shock proteins (HSP) 1The abbreviations used are: HSPheat shock proteinHSFheat shock transcription factorHSEheat shock elementPBSphosphate-buffered salineEMSAelectrophoretic mobility shift assayPAGEpolyacrylamide gel electrophoresisCHXcycloheximide. and molecular chaperones have been known for several years to protect cells against a wide variety of toxic conditions, including extreme temperatures, oxidative stress, virus infection, and the exposure to heavy metals or cytotoxic drugs (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4360) Google Scholar). HSP induction is not only a signal for detection of physiological stress, but is utilized by the cells in the repair process following different types of injury, to prevent damage resulting from the accumulation of non-native proteins (2Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Plainview, NY1990Google Scholar). In mammalian cells, several HSP are expressed during normal growth conditions and can be induced by biologically active molecules such as hemin (3Sistonen L. Sarge K.D. Phillips B. Abravaya K. Morimoto R.I. Mol. Cell. Biol. 1992; 12: 4104-4111Crossref PubMed Scopus (236) Google Scholar) and prostaglandins (4Santoro M.G. Garaci E. Amici C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8407-8411Crossref PubMed Scopus (151) Google Scholar, 5Amici C. Sistonen L. Santoro M.G. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6227-6231Crossref PubMed Scopus (153) Google Scholar), whereas others are expressed upon stress-activated regulation of transcriptional and translational switches. Induction requires the activation and translocation to the nucleus of a transregulatory protein, the heat shock transcription factor (HSF), which converts from a monomeric non-DNA-binding form to an oligomeric form that binds to specific promoter elements (HSE) located upstream of heat shock genes (6Morimoto R.I. Sarge K.D. Abravaya K. J. Biol. Chem. 1992; 267: 21987-21990Abstract Full Text PDF PubMed Google Scholar). Several HSFs have been identified in mammalian cells (6Morimoto R.I. Sarge K.D. Abravaya K. J. Biol. Chem. 1992; 267: 21987-21990Abstract Full Text PDF PubMed Google Scholar).We have shown previously that prostaglandins of the A and J type (PGAs and PGJs) were able to activate HSF and induce the synthesis of the 70-kDa HSP (HSP70) in a wide variety of mammalian cells (4Santoro M.G. Garaci E. Amici C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8407-8411Crossref PubMed Scopus (151) Google Scholar, 7Santoro M.G. Garaci E. Amici C. Stress Proteins: Induction and Function. Springer-Verlag, Berlin1990: 27-44Crossref Google Scholar). Induction of HSP70 by prostaglandins requires the presence of a reactive α,β-unsaturated carbonyl group in the cyclopentane ring (cyclopentenone), which renders this portion of the molecule able to form Michael's adducts with cellular nucleophilics and to covalently bind to cysteine residues of proteins via a thioether bond (8Fukushima M. Eicosanoids. 1990; 3: 189-199PubMed Google Scholar, 9Khan S.H. Sorof S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9401-9405Crossref PubMed Scopus (67) Google Scholar). We have also shown that cyclopentenone prostaglandins possess a potent antiviral activity against RNA and DNA viruses in a wide variety of experimental models in vitro and in vivo (reviewed in 7Santoro M.G. Garaci E. Amici C. Stress Proteins: Induction and Function. Springer-Verlag, Berlin1990: 27-44Crossref Google Scholar and 10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar). In negative-strand RNA viruses the antiviral activity has been associated with the ability of cyclopentenone prostaglandins to induce HSP70 expression, and a role of this protein in the control of virus replication has been hypothesized (10Santoro M.G. Experientia (Basel). 1994; 50: 1039-1047Crossref PubMed Scopus (66) Google Scholar, 11Amici C. Giorgi C. Rossi A. Santoro M.G. J. Virol. 1994; 68: 6890-6899Crossref PubMed Google Scholar).We now report that the cyclopentenone ring structure itself, 2-cyclopenten-1-one, is able to activate HSF and selectively induce HSP70 expression and show that the presence of an α,β-unsaturated carbonyl group is the key structure for triggering HSF activation. We also show that induction of HSP70 is associated with inhibition of infectious virus production and viral protein synthesis in monkey kidney epithelial cells infected with vesicular stomatitis virus (VSV).
Referência(s)