Heat Shock-sensitive Expression of Calreticulin.
1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês
10.1074/jbc.270.28.17011
ISSN1083-351X
AutoresEdward M. Conway, Lili Liu, B Nowakowski, Marta Steiner‐Mosonyi, Sérgio Pinto Ribeiro, Marek Michalak,
Tópico(s)ATP Synthase and ATPases Research
ResumoCalreticulin (CRT) is an ubiquitous, highly conserved, Ca2+-binding protein of the sarcoplasmic and endoplasmic reticulum. The precise function(s) of CRT is unknown. However, based on sequence analyses and observations that it may bind to steroid receptors and integrins and store Ca2+ within the cell, it has been postulated to play a "housekeeping" role. To determine whether the level of expression of CRT is affected by stress, we examined the heat shock response of CRT from a variety of cultured cells, including vascular endothelial, lung epithelial, and lung fibroblasts. Following exposure of the cells to 42°C, CRT mRNA transiently accumulated 2.5-4.2-fold at 1-6 h. Nuclear run-on studies and mRNA stability experiments confirmed that the predominant mechanism of augmentation was transcriptional. Chloramphenicol acetyltransferase assays further indicated that the promoter region, containing a putative heat shock element between −172 and −158 of the human CRT gene, is heat shock-sensitive. Finally, we demonstrated the in vivo significance of these findings by exposing rats to hyperthermia. This resulted in accumulation of CRT mRNA and an augmentation of CRT protein in lung tissue. We hypothesize that this stress-induced up-regulation of CRT contributes to the mechanism(s) by which the vascular endothelium and lung tissue, and possibly other organ systems, maintain homeostasis when exposed to a variety of pathophysiological conditions. Calreticulin (CRT) is an ubiquitous, highly conserved, Ca2+-binding protein of the sarcoplasmic and endoplasmic reticulum. The precise function(s) of CRT is unknown. However, based on sequence analyses and observations that it may bind to steroid receptors and integrins and store Ca2+ within the cell, it has been postulated to play a "housekeeping" role. To determine whether the level of expression of CRT is affected by stress, we examined the heat shock response of CRT from a variety of cultured cells, including vascular endothelial, lung epithelial, and lung fibroblasts. Following exposure of the cells to 42°C, CRT mRNA transiently accumulated 2.5-4.2-fold at 1-6 h. Nuclear run-on studies and mRNA stability experiments confirmed that the predominant mechanism of augmentation was transcriptional. Chloramphenicol acetyltransferase assays further indicated that the promoter region, containing a putative heat shock element between −172 and −158 of the human CRT gene, is heat shock-sensitive. Finally, we demonstrated the in vivo significance of these findings by exposing rats to hyperthermia. This resulted in accumulation of CRT mRNA and an augmentation of CRT protein in lung tissue. We hypothesize that this stress-induced up-regulation of CRT contributes to the mechanism(s) by which the vascular endothelium and lung tissue, and possibly other organ systems, maintain homeostasis when exposed to a variety of pathophysiological conditions. Calreticulin (CRT)1 1The abbreviations used are: CRTcalreticulinHSEheat shock elementHUVEChuman umbilical vein endothelial cellsHSPheat shock proteinCATchloramphenicol acetyltransferaseHMEChuman microvascular endothelial cells. 1The abbreviations used are: CRTcalreticulinHSEheat shock elementHUVEChuman umbilical vein endothelial cellsHSPheat shock proteinCATchloramphenicol acetyltransferaseHMEChuman microvascular endothelial cells. is a Ca+-binding protein that was first described in the sarcoplasmic reticulum of skeletal muscle (1MacLennan D. Yip C. Iles G. Seeman P. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: 469-477Crossref Google Scholar, 2Ostwald T. MacLennan D. J. Biol. Chem. 1974; 249: 974-979Abstract Full Text PDF PubMed Google Scholar) and subsequently found to be a major Ca2+-binding protein of the lumen of endoplasmic reticulum (ER) membranes(3Milner R. Baksh S. Shemanko C. Carpenter M. Smillie L. Vance J. Opas M. Michalak M. J. Biol. Chem. 1991; 266: 7155-7165Abstract Full Text PDF PubMed Google Scholar). The nucleotide sequences of cDNAs encoding CRT from several diverse species have since been determined(4Treves S. Zorzato F. Pozzan T. Biochem. J. 1992; 287: 579-581Crossref PubMed Scopus (21) Google Scholar, 5Hawn T. Tom T. Strand M. J. Biol. Chem. 1993; 268: 7692-7698Abstract Full Text PDF PubMed Google Scholar, 6Kennedy T. Kuhl D. Barzilai A. Sweatt J. Kandel E. Neuron. 1992; 9: 1013-1024Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 7Liu N. Fine R. Johnson R. Biochim. Biophys. Acta. 1993; 1202: 70-76Crossref PubMed Scopus (15) Google Scholar, 8Fliegel L. Burns K. MacLennan D. Reithmeier R. Michalak M. J. Biol. Chem. 1989; 264: 21522-21528Abstract Full Text PDF PubMed Google Scholar, 9Michalak M. Milner R. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (404) Google Scholar, 10McCauliffe D. Zappi E. Lieu T.-S. Michalak M. Sontheimer R. Capra J. J. Clin. Invest. 1990; 86: 332-335Crossref PubMed Scopus (81) Google Scholar, 11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar, 12Nakamura M. Oshio S. Tamura A. Okinaga S. Arai K. Biochem. Biophys. Res. Commun. 1992; 186: 984-990Crossref PubMed Scopus (19) Google Scholar), and the deduced amino acid sequences reveal a remarkably high degree of conservation. CRT is an ubiquitous protein, synthesized by all mammalian cells tested except erthrocytes(9Michalak M. Milner R. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (404) Google Scholar, 13Baksh S. Michalak M. J. Biol. Chem. 1991; 266: 21458-21465Abstract Full Text PDF PubMed Google Scholar, 14Khanna N. Tokuda M. Waisman D. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar). In spite of considerable advances in elucidating the structure and tissue distribution of CRT, less is known about its precise function or regulation. calreticulin heat shock element human umbilical vein endothelial cells heat shock protein chloramphenicol acetyltransferase human microvascular endothelial cells. calreticulin heat shock element human umbilical vein endothelial cells heat shock protein chloramphenicol acetyltransferase human microvascular endothelial cells. Based on Ca2+-binding properties of the protein, CRT has been proposed by several laboratories to be a major Ca2+-binding/storage protein of the endoplasmic reticulum (9). The protein has two distinct Ca2+-binding sites (high affinity/low capacity and low affinity/high capacity)(13Baksh S. Michalak M. J. Biol. Chem. 1991; 266: 21458-21465Abstract Full Text PDF PubMed Google Scholar). Recent investigations have indicated that CRT might also be important in protein-protein interactions within the cell. Guan et al. (15) reported a tight association of flavin-containing lung monooxygenases with CRT, while Rojiani and co-workers (16Rojiani M. Finlay B. Gray V. Dedhar S. Biochemistry. 1991; 30: 9859-9866Crossref PubMed Scopus (184) Google Scholar) demonstrated interactions between the integrin α subunit and CRT. A set of endoplasmic reticulum proteins has also been identified that interact with CRT(17Burns K. Michalak M. FEBS Lett. 1993; 318: 181-185Crossref PubMed Scopus (23) Google Scholar). Furthermore, Burns et al.(18Burns K. Duggan B. Atkinson E. Famulski K. Nemer M. Bleackley R. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (323) Google Scholar) and Dedhar et al.(19Dedhar S. Rennie P. Shago M. Hagesteijn C.-Y. Yang H. Filmus J. Hawley R. Bruchovsky N. Cheng H. Matusik R. Giguere V. Nature. 1994; 367: 480-483Crossref PubMed Scopus (304) Google Scholar) have provided evidence that CRT may interact with steroid hormone receptors leading to the modulation of their transcriptional activity. Finally, CRT has also been implicated to play a role in the pathogenesis of autoimmune disorders(10McCauliffe D. Zappi E. Lieu T.-S. Michalak M. Sontheimer R. Capra J. J. Clin. Invest. 1990; 86: 332-335Crossref PubMed Scopus (81) Google Scholar, 20Rokeach L. Haselby J. Meilof J. Smeenk R. Unnasch T. Greene B. Hoch S. J. Immunol. 1991; 147: 3031-3039PubMed Google Scholar). Although analysis of the promoter region of the human CRT gene (11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar) has revealed several potential sites that may play an important role in its transcriptional regulation, none of these have been functionally defined. CRT is reported to be augmented in response to cellular proliferation(21Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (127) Google Scholar, 22Gersten D. Bijwaard K. Law L. Hearing V. Biochim. Biophys. Acta. 1991; 1096: 20-25Crossref Scopus (14) Google Scholar), mitogenic stimulation(23Burns K. Helgason C. Bleackley R. Michalak M. J. Biol. Chem. 1992; 267: 19039-19042Abstract Full Text PDF PubMed Google Scholar), and viral transfection (24O'Banion M. Winn V. Settleman J. Young D. J. Virol. 1993; 67: 3427-3434Crossref PubMed Google Scholar) and may be released from activated neutrophils(25Eggleton P. Lieu T. Zappi E. Sastry K. Coburn J. Zaner K. Sontheimer R. Capra J. Ghebrehiwet B. Tauber A. Clin. Immunol. Immunopath. 1994; 72: 405-409Crossref PubMed Scopus (91) Google Scholar). Other investigations have indicated that CRT is a major prompt glycosylation protein in Chinese hamster ovary cells in response to heat shock(26Jethmalani S. Henle K. Kaushal G. J. Biol. Chem. 1994; 269: 23603-23609Abstract Full Text PDF PubMed Google Scholar). In this report, we demonstrate, using Northern analyses, mRNA stability measurements, and nuclear run-on assays, that heat shock also up-regulates CRT expression in cultured cells predominantly via transcriptional mechanisms. Furthermore, CAT assays support our hypothesis that the promoter region of the CRT gene containing a putative heat shock element (HSE) is sensitive to heat shock stress. Finally, utilizing rats exposed to heat shock stress, we demonstrate that both the transcriptional and post-translational glycosylation responses are active in vivo. These results, and recent observations indicating the in vivo protective role of heat shock in sepsis-induced lung injury(27Ribeiro S. Villar J. Downey G. Edelson J. Slutsky A. Crit. Care Med. 1994; 22: 922-929Crossref PubMed Scopus (158) Google Scholar, 28Villar J. Ribeiro S. Brendan J. Mullen J. Kulixzewski M. Post M. Slutsky A. Crit. Care Med. 1994; 22: 914-921Crossref PubMed Scopus (313) Google Scholar), suggest that stress-induced CRT up-regulation may be important in a variety of biological systems and pathophysiologic conditions. Goat-anti-rabbit CRT antibodies have been fully characterized (3Milner R. Baksh S. Shemanko C. Carpenter M. Smillie L. Vance J. Opas M. Michalak M. J. Biol. Chem. 1991; 266: 7155-7165Abstract Full Text PDF PubMed Google Scholar, 8Fliegel L. Burns K. MacLennan D. Reithmeier R. Michalak M. J. Biol. Chem. 1989; 264: 21522-21528Abstract Full Text PDF PubMed Google Scholar) and shown not to cross-react with calsequestrin (3). Restriction enzymes were from Boehringer Mannheim Canada (Dorval, Quebec). 32P-Labeled nucleotides were purchased from ICN Biomedicals (Canada). RNase T1, yeast tRNA, and DNase 1 were from Life Technologies, Inc. (Burlington, Ontario, Canada); NTPs were from Pharmacia Biotech. Inc. (Baie d'Urfe, Quebec, Canada); and endothelial cell mitogen was from Biomedical Technologies Inc. (Stoughton, MA). HUVEC were harvested by the method of Jaffe et al.(29Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2751Crossref PubMed Scopus (5968) Google Scholar), grown, and characterized as reported previously (30). Experiments were performed on cells between passages 2 and 5. The immortalized human microvascular endothelial cell line HMEC-1 were kindly provided by the Biological Products Branch of the Centers for Disease Control (Atlanta, GA) and cultured as reported(31Ades E. Candal F. Swerlick R. George V. Summers S. Bosse D. Lawley T. J. Invest. Dermatol. 1992; 99: 683-690Abstract Full Text PDF PubMed Google Scholar, 32Hooper W. Phillips D. Ribeiro M. Benson J. George V. Ades E. Evatt B. Blood. 1994; 84: 483-489Crossref PubMed Google Scholar). A549 human lung adenocarcinoma cells, SKLU-1 human epithelial lung cancer cells, and MRC-5 human diploid fibroblasts (passage 25-30) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Media was changed 24 h prior to all experiments. For the purposes of heat shock, the cell monolayers were first grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C and were transferred to another humidified, water-jacketed incubator preset at 42°C. The incubator was regulated to stay within 0.2°C of the target temperature. At the appropriate time points, the cells were immediately placed on ice, washed with ice-cold phosphate-buffered saline, lysed, and used for membrane preparations, RNA isolations, or nuclear extractions for run-on assays. The full-length 1.9-kilobase cDNA encoding human CRT (33McCauliffe D. Lux F. Lieu T.-S. Sanz I. Hanke J. Newkirk M. Bachinski L. Itoh Y. Siciliano M. Reichlin M. Sontheimer R. Capra J. J. Clin. Invest. 1990; 85: 1379-1391Crossref PubMed Scopus (140) Google Scholar) was a gift of Dr. R. Sontheimer (South West Medical Center, Dallas, Texas). The plasmid pGAD28 containing the cDNA for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was provided by Dr. Robert J. Schwartz, Houston, TX(34Dugaiczyk A. Haron J. Stone E. Dennison O. Rothblum K. Schwartz R. Biochemistry. 1983; 22: 1605-1613Crossref PubMed Scopus (376) Google Scholar), and the cDNA encoding the human heat shock protein HSP70 was obtained from Dr. Jack Hensold, Case Western Reserve University, Cleveland, OH. Plasmids were purified by alkaline lysis and CsCl gradient centrifugation(35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar). Following purification from low melting point agarose gels, the entire 1.9-kilobase cDNA of CRT, the 547-base pair HindIII-XbaI fragment from the coding region of glyceraldehyde-3-phosphate dehydrogenase, and a 2.3-kilobase BamHI-HindIII fragment encoding HSP70 were used as probes for Northern blots after labeling with [α-32P]dCTP using the random primer synthesis method (36). The specific activity of the probes was approximately 2 × 106 cpm/ng of DNA. For nuclear run-on experiments, the above cDNA inserts were subcloned into the plasmid Bluescript (pBS) (Stratagene), and 10 μg of each were dot-blotted onto nitrocellulose for each condition (see below). Total RNA was isolated from confluent cell monolayers by the method of Chomczynski et al.(37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). Northern analyses were performed as described previously(38Conway E. Nowakowski B. Blood. 1993; 81: 726-733Crossref PubMed Google Scholar). To determine the t of a specific mRNA, postconfluent cells were exposed to actinomycin D, 25 μg/ml, in culture media for varying periods of time, after which the cells were lysed for Northern analysis. Actinomycin D at this concentration totally inhibited transcription as evaluated by nuclear run-on experiments (data not shown). Densitometry was performed on scanned autoradiographs (MacIntosh Color OneScanner) using the NIH Image Analysis Version 1.52 computer program for the MacIntosh. To obtain the CRT promoter region with the putative heat shock element included, genomic DNA was prepared from approximately 4 × 106 second passage HUVEC. The following oligonucleotide primers were synthesized to span the region from −223 to 22(11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar) : 1) 5′-gcgtggtcgaccatcat; 2) 5′-cgatctagagcggctctgcagt. Primer 1 is the upstream sense strand, while primer 2 is the anti-parallel strand between the transcription and translation start sites and has an XbaI site added to provide for easier subcloning. To 1 μg of each of the primers, polymerase chain reaction buffer was added to a final concentration of 10 mM Tris, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.01% gelatin. After heating to 94°C, Taq polymerase, 2.5 units (Perkin Elmer Cetus, Mississauga, Canada), was added to initiate the reaction, and amplification was performed for 30 cycles, with 1-min primer extensions at 72°C, denaturation for 30 s at 94°C, and primer annealings for 30 s at 45°C. The resultant 261-base pair fragment was digested with SalI and XbaI and subcloned into pBS, and the nucleotide sequence was confirmed by DNA sequencing. The CRT promoter region was then subcloned into pCAT-Basic vector (pCAT-BV) (Promega), the latter, which contains the CAT gene, in the appropriate orientation, to yield pCRT-CAT for CAT assays. pCAT-BV lacks eukaryotic promoter and enhancer sequences, and expression of functional CAT activity in transfected cells depends on the insertion of a functional promoter within the multiple cloning sequence. Preconfluent cells in 100-mm dishes were washed twice with serum-free medium, OPTI-MEM (Life Technologies, Inc.). Transfection was accomplished by overnight incubation at 37°C of a mixture containing 20 μg of the CAT vector, 20 μg of pSVTKGH(39Selden R. Rowe M. Goodman H. Moore D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (470) Google Scholar), and 25 μg of lipofectin in a total volume of 1.5 ml of OPTI-MEM. pSVTKGH contains the thymidine kinase promoter, an SV40 enhancer, and the human growth hormone gene. The cells were then fed with 5 ml of fresh growth medium, 48 h after which heat shock experiments were started. Following heat shock stress, the cells were immediately put on ice, and the medium was carefully removed for quantitation of growth hormone levels by a nonisotopic immunoassay (system 9670SA from Life Technologies, Inc.). The cell monolayers were washed 3 times with cold phosphate-buffered saline, scraped, and pelleted, and lysates were prepared by four freeze-thaw cycles, after which they were heated at 60°C for 10 min. CAT reactions using [3H]chloramphenicol were performed overnight at 37°C in duplicate according to the manufacturer's instructions (Promega). Transfections with pCAT-BV or without any CAT vector yielded similar (± 5%) background tritium counts. Experiments were performed 3 times, and after subtracting the background counts as determined from parallel transfections with pCAT-BV, the results were corrected for transfection efficiency by dividing by the respective growth hormone levels as determined by the immunoassay. Following heat shock, nuclear pellets from approximately 5 × 107 cells/sample were prepared, and nuclear run-on assays were performed as previously reported(40Conway E. Liu L. Nowakowski B. Steiner-Mosonyi M. Jackman R. J. Biol. Chem. 1994; 269: 22804-22810Abstract Full Text PDF PubMed Google Scholar). The 32P-labeled RNA specifically bound to the filters was visualized by autoradiography. Nonspecific hybridization was determined to be negligible as assessed by using filters dot-blotted with 10 μg of pBS. All animal experiments and treatments were approved by the Institutional Animal Care and Use Committee of the University of Toronto. Sprague-Dawley rats, 250-300 g (Harlan, Indianapolis, IN), were anesthetized with pentobarbital 35 mg/kg intraperitoneally and placed into a prewarmed 41 ± 0.5°C ambient temperature, humidified neonatal incubator (Isolette infant incubator, Air-Shields Inc., Harboro, PA). The temperature of each animal, which was continuously monitored by rectal thermometer, persistently increased until it reached 41°C (1-1.5 h). Fifteen minutes later, while maintaining a temperature between 41 and 42.5°C, the animals were transferred to room temperature (21°C), from which point the post-heat shock duration was determined. Sham-treated rats were also anesthetized but were not exposed to hyperthermia. The temperatures of these rats dropped from a mean of 37.5-36.1 ± 0.5°C 1 h after anesthesia administration and returned to baseline within 4-6 h. At the end of the post-heat shock period, the rats were sacrificed by sodium pentobarbital overdose. Thoracotomy was performed, and the lungs were resected and immediately inserted into liquid nitrogen and stored at −80°C for further processing. Samples were subsequently homogenized in phosphate-buffered saline for SDS-polyacrylamide gel electrophoresis and Western immunoblotting or lysed in 4 M guanidium isothiocyanate solution (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar) for total RNA preparation and Northern blot analyses. Protein concentration was determined using the Bio-Rad Protein Assay (Richmond, CA) according to the manufacturer's instructions. SDS-polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose membranes were carried out as described earlier(38Conway E. Nowakowski B. Blood. 1993; 81: 726-733Crossref PubMed Google Scholar). CRT was identified with the goat anti-CRT antibodies by overnight incubation at room temperature in a buffer containing 20 mM Tris, pH 8.0, 0.15 M NaCl, 5% Carnation milk and detected with alkaline phosphatase-conjugated rabbit anti-goat antibody. Statistical analyses were conducted by standard techniques with the aid of StatView computer program for the MacIntosh (Abacus Concepts Inc., CA). The means are provided with associated standard deviations (S.D.). Fig. 1 shows the Northern blot analysis of CRT mRNA in heat-shocked HUVEC. Following continuous growth of the cells at 37°C to a postconfluent state, the HUVEC were transferred to an incubator preset at 42°C, while control cell monolayers were maintained at 37°C. The specific mRNA for CRT had risen approximately 4.2-fold (see Fig. 3) following 6 h of heat shock as quantitated by densitometry, and, although some attenuation was noted, an elevation was maintained for at least 36 h during continuous heat stress. The pattern of rise of CRT mRNA was similar to that seen with HSP70 mRNA expression under the same stress conditions (Fig. 1). HSP70 mRNA levels were undetectable under nonstress conditions, but increased over 100-fold from 1.5 to 6 h following heat shock. mRNA levels of the "housekeeping" gene, glyceraldehyde-phosphate-3′-dehydrogenase, were unaltered by the stress, indicating the specific nature of the heat shock response (Fig. 1) and equal loading of total RNA/lane.Figure 3:mRNA levels of CRT from different cells exposed to 42°C heat shock. Postconfluent cells (HMEC-1, HUVEC, A549, SKLU-1, MRC-5) were exposed to continuous heat shock for 0-24 h, and 10 μg of total RNA from each sample were separated on a denaturing formaldehyde-agarose. Northern blot analyses were performed, detecting with radiolabeled cDNA probes for CRT and glyceraldehyde-3-phosphate dehydrogenase. Densitometry was performed as detailed under "Experimental Procedures," and CRT mRNA levels were quantitated relative to the non-heat shocked levels (100%) and corrected for glyceraldehyde-3-phosphate dehydrogenase levels at each time point. The results reflect the mean of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We examined the specificity of the heat shock response by exposing other cultured cells to hyperthermia. These included human lung epithelial carcinoma A549 cells, human lung adenocarcinoma SKLU-1 cells, an immortalized line of human microvascular endothelial cells (HMEC-1)(31Ades E. Candal F. Swerlick R. George V. Summers S. Bosse D. Lawley T. J. Invest. Dermatol. 1992; 99: 683-690Abstract Full Text PDF PubMed Google Scholar, 32Hooper W. Phillips D. Ribeiro M. Benson J. George V. Ades E. Evatt B. Blood. 1994; 84: 483-489Crossref PubMed Google Scholar), and human diploid lung fibroblasts. Although variabilities in the rate and degree of response were observed, specific CRT mRNA levels in each of these cell lines as evaluated by Northern blot analyses (Fig. 2) and densitometry (Fig. 3) rose 2.5-3.6-fold following 1.5-6 h of exposure to hyperthermia and, in all but the A549 cells, remained elevated at 24 h. These results suggested that the factors active in up-regulating transcription of the CRT gene are not specific to a single cell type but rather are probably functional in most cell biologic systems. Given the above results, we determined whether alterations in CRT mRNA stability were contributing to the heat shock-induced augmentation in expression. This was done by evaluating the effect of hyperthermia on actinomycin D-induced, time-dependent decrease in CRT mRNA. Postconfluent HUVEC were incubated at either 37 or 42°C in the presence or absence of the RNA polymerase inhibitor actinomycin D, 25 μg/ml, at which dose the incorporation of [3H]thymidine was suppressed by over 95% within 30 min of exposure, and in vitro transcription was abrogated. In a representative experiment, densitometry analyses of the Northern blots shown in Fig. 4 indicated that the t of CRT mRNA when the cells were maintained at 37°C was 8 h, while the t when the cells were exposed to heat shock was 7.5 h. The lack of a significant alteration in the stability of the CRT mRNA by heat shock, suggested that the major mechanism of up-regulation of the message was via transcription. We directly evaluated the in vitro transcriptional response of CRT to heat shock stress by performing nuclear run-on studies. HSP70 transcripts from HUVEC maintained at 37°C were almost undetectable. Transcription of the HSP70 gene was augmented over 100-fold by the 42°C heat shock (Fig. 5) in a similar kinetic pattern of expression as its specific mRNA (Fig. 1). CRT gene transcription was also up-regulated 3.5-fold by the 42°C heat shock stress within 90 min as quantitated by densitometry (Fig. 5) and relative to glyceraldehyde-3-phosphate dehydrogenase gene transcription. This was followed by gradual attenuation over the ensuing 24 h. These findings were consistent with the Northern blot analysis (Fig. 1-3) and also supportive of a predominantly transcriptional response of the CRT gene to the stress. On examination of the promoter region of the human CRT gene between −172 and −158(11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar), we have identified a putative HSE composed of three inverted repeats of the consensus pentad nGAAn (Fig. 6). This region may provide a site of interaction with heat shock transcription factors (41Pelham H. Cell. 1982; 30: 517-528Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 42Mirault M. Southgate R. Delwart E. EMBO J. 1982; 1: 1279-1285Crossref PubMed Scopus (80) Google Scholar, 43Xiao H. Perisic O. Lis J. Cell. 1991; 64: 585-593Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 44Perisic O. Xiao H. Lis J. Cell. 1989; 59: 797-806Abstract Full Text PDF PubMed Scopus (322) Google Scholar) to mediate the observed heat shock sensitivity of CRT. The putative HSE within the CRT gene was therefore examined for response to heat shock by inserting the region from −223 to 22 in front of a CAT construct (pCRT-CAT). A549 cells were transfected with the pCRT-CAT or pCAT-BV, the latter as a negative control, and exposed to 37 or 42°C for varying periods of time. Growth hormone assays and transfections with pCAT-BV were performed to correct the CAT activity results for transfection efficiency and nonspecific background as detailed in the methods. As seen in Table I, pCRT-CAT expression was augmented by over 3-fold following 5 h of heat shock stress, indicating that the promoter region containing the putative HSE is important for heat shock sensitivity of the CRT gene.Table I:CAT activity of CRT promoter in A549 cells Open table in a new tab In order to confirm that the CRT gene is up-regulated in vivo, we exposed anesthetized rats normally maintained at room temperature (21°C) to heat shock. Immediately following anesthesia, rats were exposed to an ambient temperature of 41°C for 60-90 min until the rectal temperature reached 41-42.5°C for 15 min. The heat-shocked rats (n = 5) were then returned to room temperature during which time the rectal temperature dropped to 38.6 ± 0.3 and 37.8 ± 0.6°C at 30 and 60 min post-heat shock, respectively. The rectal temperatures all returned to base line within 90 min, and no episodes of hypothermia were documented for 18 h post-heat shock. Consequently, the rats were hyperthermic for a total duration of approximately 2-3.5 h. 12 and 18 h post-heat shock exposure, the rats were sacrificed for lung tissue analyses. Control sham-treated animals were not exposed to heat stress but did develop transient hypothermia to a mean minimum temperature of 36.1°C 1 h following the anesthesia. Rectal temperatures of these animals were also normal by 4-6 h after administration of anesthesia. Fig. 7(top) is a Western immunoblot depicting total CRT expression within the lung tissue as a result of heat shock. In this representative experiment, lung-derived CRT was notably increased 12 and 18 h following heat shock, as compared with that from the sham-treated control animal or animals not exposed to heat shock or anesthesia (not shown). Furthermore, a second larger band detected by the specific anti-CRT antibodies was also evident in the immunoblot, consistent with heat shock-induced glycosylation of CRT observed in vitro by other investigators(26Jethmalani S. Henle K. Kaushal G. J. Biol. Chem. 1994; 269: 23603-23609Abstract Full Text PDF PubMed Google Scholar). The increase in expression of CRT protein was paralleled by a persistent increase by 3-fold (by densitometry) in specific CRT mRNA (Fig. 7) from the same tissue samples. We can conclude, therefore, that heat shock stress induces transcription and translation of CRT that is not only manifested in cell culture but that is also evident in vivo. In this report, we have shown that heat shock results in increased expression of CRT predominantly at the level of transcription. The physiological significance of this finding was confirmed by in vivo experiments; CRT mRNA and protein are elevated in the lung tissue of heat-shocked rats. Additionally, the previously observed early glycosylation response of CRT to hyperthermia (26Jethmalani S. Henle K. Kaushal G. J. Biol. Chem. 1994; 269: 23603-23609Abstract Full Text PDF PubMed Google Scholar) was also evident in our in vivo experiments. In response to heat shock, CRT transcription transiently increased 3-4-fold followed by a recovery phase in which gene transcription attenuated and eventually returned toward pre-heat shock levels. The ubiquitous nature of this CRT response was evident from our observation that different cells (HUVEC, A549, SKLU-1, MRC-5, and HMEC) responded to the stress by enhancing transcription of the CRT gene and accumulation of its mRNA. Our data further suggested that a major mechanism of CRT mRNA accumulation as depicted in the Northern blots, was via enhanced transcription, with little or no contribution from alterations in message stability. Opas et al.(21Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (127) Google Scholar) previously reported that in a cultured rat myogenic cell line, heat shock of 45°C for 3 h followed by return to 37°C for 2-48 h did not result in an increase in the levels of CRT protein. Indeed, a transient reduction in CRT was observed during the recovery phase between 6 and 8 h. The apparent discordance of our results with those of Opas et al.(21Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (127) Google Scholar) might be explained by the use of different cultured cells or alternatively by the high temperature used in their experiments, which may have abrogated the transcriptional response. This possibility is consistent with observations that the transcriptional response of some "classic" heat shock proteins may be suppressed, and indeed diminished, by particularly large increases in temperature(45Lindquist S. Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar). The human CRT gene has recently been cloned(11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar). Several putative regulatory sequences were identified using human HeLa cell nuclear extracts, gel mobility shift analyses, and DNA footprint localization studies. The possibility that CRT might provide a housekeeping function similar to the role of other "chaperones" was clearly recognized by these investigators(11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar). Our analysis of the region between −172 and −158 of the human CRT gene revealed the presence of a putative HSE. Strong biochemical evidence indicates that this sequence motif, i.e. arrays of two or more inverted repeats of the five-base pair sequence nGAAn, provides a site of interaction with heat shock transcription factors. The arrays may function even with imperfect five-base pair units where there are substitutions for some of the consensus GAA bases (41Pelham H. Cell. 1982; 30: 517-528Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 42Mirault M. Southgate R. Delwart E. EMBO J. 1982; 1: 1279-1285Crossref PubMed Scopus (80) Google Scholar, 43Xiao H. Perisic O. Lis J. Cell. 1991; 64: 585-593Abstract Full Text PDF PubMed Scopus (194) Google Scholar) or if the repeats are variably oriented (44). As noted in Fig. 6, the identified consensus sequence, gGAAccCAGcgTTCc, within the 5′-flanking region of the CRT gene, has two perfect inverted repeats with an intervening imperfect pentad. We propose that this putative HSE in the CRT gene may be responsible for heat shock-dependent expression of CRT via transcriptional mechanisms. Our CAT assays support this hypothesis. However, further CAT studies using genetically altered DNA sequences within the putative HSE are indicated for confirmation as to the significance of this region. Furthermore, although the HSE may confer stress inducibility, the multiple putative cis- and/or trans-regulatory elements (11McCauliffe D. Yang Y. Wilson J. Sontheimer R. Capra J. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar) may also confer a range of transcriptional responses, and consequently the kinetics and magnitude of DNA binding of heat shock transcription factors to the HSE may be regulated(46Morimoto R. Sarge K. Abravaya K. J. Biol. Chem. 1992; 267: 21987-21990Abstract Full Text PDF PubMed Google Scholar). HSPs, often referred to as molecular chaperones, are currently believed to function as protectors of cells from harmful effects of a variety of stresses and represent one of the most conserved families of proteins in evolution (see Welch et al.(47Welch W. Philos. Trans. R. Soc. Lond. 1993; 339: 327-333Crossref PubMed Scopus (155) Google Scholar) and Morimoto et al.(48Morimoto R. Science. 1993; 259: 1409-1410Crossref PubMed Scopus (1205) Google Scholar) for reviews). Following exposure of rats to heat stress for 2-3.5 h, we observed a persistent increase in lung CRT mRNA and protein, the latter characterized by the presence of an apparently alternatively glycosylated form(26Jethmalani S. Henle K. Kaushal G. J. Biol. Chem. 1994; 269: 23603-23609Abstract Full Text PDF PubMed Google Scholar). Recent in vivo studies have demonstrated the protective property of hyperthermia and heat shock proteins in sepsis-induced lung injury in rats (27Ribeiro S. Villar J. Downey G. Edelson J. Slutsky A. Crit. Care Med. 1994; 22: 922-929Crossref PubMed Scopus (158) Google Scholar, 28Villar J. Ribeiro S. Brendan J. Mullen J. Kulixzewski M. Post M. Slutsky A. Crit. Care Med. 1994; 22: 914-921Crossref PubMed Scopus (313) Google Scholar) and endotoxin-mediated mortality in mice(49Hotchkiss R. Nunnally I. Lindquist S. Taulien J. Perdrizet G. Karl I. Am. J. Physiol. 1993; 265: R1447-R1457Crossref PubMed Google Scholar). Our findings of an increase in CRT mRNA and protein in lung tissue following heat shock, and the persistence of this response several hours after the removal of the stress, suggests that CRT also may play a protective role during and following fever or a variety of other physiological stresses. Although CRT is ubiquitous to all cells except erythrocytes, our studies were restricted to vascular endothelial and lung-derived tissue and cells. We and others have recently suggested that the stress response may protect the vasculature by transcriptionally up-regulating important vascular endothelial cell proteins, including thrombomodulin (40), thrombospondin(50Ketis N. Lawler J. Hoover R. Karnovsky M. J. Cell Biol. 1988; 106: 893-904Crossref PubMed Scopus (20) Google Scholar), and HSP70(51Udelsman R. Blake M. Stagg C. Li D. Putney D. Holbrook N. J. Clin. Invest. 1993; 91: 465-473Crossref PubMed Scopus (212) Google Scholar). It is therefore reasonable to hypothesize that CRT may also contribute to this process in the vasculature. However, it should be noted that considerable variation in organ expression of HSP72 has been observed in studies by Hotchkiss et al.(49Hotchkiss R. Nunnally I. Lindquist S. Taulien J. Perdrizet G. Karl I. Am. J. Physiol. 1993; 265: R1447-R1457Crossref PubMed Google Scholar) when mice were exposed to heat shock and endotoxin. Consequently, further studies will be required to evaluate the organ and tissue specificity of the response of CRT to heat shock stress. The mechanism(s) by which CRT may play a physiologically protective role is currently unknown. In addition to its Ca2+-binding properties, CRT also interacts with the DNA-binding domain of the glucocorticoid receptor and other nuclear hormone receptors(18Burns K. Duggan B. Atkinson E. Famulski K. Nemer M. Bleackley R. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (323) Google Scholar, 19Dedhar S. Rennie P. Shago M. Hagesteijn C.-Y. Yang H. Filmus J. Hawley R. Bruchovsky N. Cheng H. Matusik R. Giguere V. Nature. 1994; 367: 480-483Crossref PubMed Scopus (304) Google Scholar), inhibiting glucocorticoid-sensitive gene expression and retinoic acid-dependent differentiation. Consequently, stress-induced up-regulation of CRT could modulate the steroid sensitivity of different cells, thereby protecting cells/organisms from different stresses. This regulatory mechanism may therefore have far-reaching effects during a variety of physiological processes, including differentiation, development, and homeostasis. A549 cells were transiently co-transfected with pCRT-CAT and pSVTKGH as described in the methods, and exposed to either 37°C or 42°C for the noted times. Results with associated standard deviations reflect the average of 3 experiments each performed in duplicate, corrected for transfection efficiency, and relative to activity when cells were maintained at 37°C for the same period of time.
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