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Cellular and Molecular Mechanisms of Heat Stress-Induced Up-Regulation of Occludin Protein Expression

2008; Elsevier BV; Volume: 172; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2008.070522

1525-2191

Karol Dokładny, Dongmei Ye, John C. Kennedy, Pope L. Moseley, Y. Thomas,

Salivary Gland Disorders and Functions

The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39°C or 41°C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein. The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39°C or 41°C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein. The intestinal epithelial barrier consists of apical plasma membrane of the enterocytes that acts as a transcellular barrier and intercellular tight junctions (TJs) that act as a paracellular barrier against intercellular penetration of toxic luminal substances, including bacterial endotoxins, bacterial by-products, digestive enzymes, and food-degradation products.1Anderson JM Van Itallie CM Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol. 1995; 269: G467-G475PubMed Google Scholar, 2Baker JW Deitch EA Li M Berg RD Specian RD Hemorrhagic shock induces bacterial translocation from the gut.J Trauma. 1988; 28: 896-906Crossref PubMed Scopus (365) Google Scholar, 3Hollander D Crohn's disease—a permeability disorder of the tight junction?.Gut. 1988; 29: 1621-1624Crossref PubMed Scopus (223) Google Scholar, 4Ma TY Intestinal epithelial barrier dysfunction in Crohn's disease.Proc Soc Exp Biol Med. 1997; 214: 318-327Crossref PubMed Scopus (95) Google Scholar, 5Madara JL Loosening tight junctions. Lessons from the intestine.J Clin Invest. 1989; 83: 1089-1094Crossref PubMed Scopus (282) Google Scholar, 6Farquhar MG Palade GE Junctional complexes in various epithelia.J Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2132) Google Scholar The TJ complex consists of cytoplasmic and transmembrane proteins. The transmembrane proteins, which include occludin, claudin family of proteins, and junctional adhesion molecules, extend from cytoplasmic compartment across the plasma membrane into extracellular compartment to participate in the formation of an extracellular TJ seal.7Fanning AS Mitic LL Anderson JM Transmembrane proteins in the tight junction barrier.J Am Soc Nephrol. 1999; 10: 1337-1345PubMed Google Scholar, 8Anderson JM Molecular structure of tight junctions and their role in epithelial transport.News Physiol Sci. 2001; 16: 126-130Crossref PubMed Scopus (304) Google Scholar, 9Stevenson BR Siliciano JD Mooseker MS Goodenough DA Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.J Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1285) Google Scholar, 10Balda MS Matter K Transmembrane proteins of tight junctions.Semin Cell Dev Biol. 2000; 11: 281-289Crossref PubMed Scopus (121) Google Scholar The critical role of transmembrane proteins in the formation and maintenance of the TJ barrier is well established; however, the precise protein structure and components and molecular determinants of TJ barrier remain unclear.11Turner JR Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application.Am J Pathol. 2006; 169: 1901-1909Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar Occludin is an integral transmembrane TJ protein that has been shown to play a crucial role in TJ barrier function and TJ signaling process. Previous studies have shown that overexpression of occludin protein in MDCK cells leads to an enhancement of TJ barrier function.12McCarthy KM Skare IB Stankewich MC Furuse M Tsukita S Rogers RA Lynch RD Schneeberger EE: Occludin is a functional component of the tight junction.J Cell Sci. 1996; 109: 2287-2298Crossref PubMed Google Scholar Conversely, siRNA knock-down of occludin leads to an increase in TJ permeability to selected paracellular markers.13Yu AS McCarthy KM Francis SA McCormack JM Lai J Rogers RA Lynch RD Schneeberger EE Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells.Am J Physiol. 2005; 288: C1231-C1241Crossref Scopus (265) Google Scholar Molecular studies have shown that COOH-terminal end of occludin plays a crucial role in the maintenance of paracellular barrier function.14Chen Y Merzdorf C Paul DL Goodenough DA COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos.J Cell Biol. 1997; 138: 891-899Crossref PubMed Scopus (254) Google Scholar Additionally, biochemical alteration of occludin phosphorylation has been shown to be an important determinant of TJ localization of occludin protein and enhancement of TJ barrier function.15Clarke H Soler AP Mullin JM Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.J Cell Sci. 2000; 113: 3187-3196Crossref PubMed Google Scholar, 16Farshori P Kachar B Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells.J Membr Biol. 1999; 170: 147-156Crossref PubMed Scopus (143) Google Scholar, 17Rao RK Basuroy S Rao VU Karnaky Jr, KJ Gupta A Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the cytoskeleton by oxidative stress.Biochem J. 2002; 368: 471-481Crossref PubMed Scopus (345) Google Scholar The “pivotal role of occludin in maintenance of TJ barrier function” has also been demonstrated in gene transfection studies after Raf-1-induced depletion of occludin in Pa-4 epithelial cells.18Li D Mrsny RJ Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.J Cell Biol. 2000; 148: 791-800Crossref PubMed Scopus (196) Google Scholar However, the molecular and cellular mechanisms that regulate occludin gene activation and protein synthesis remain primarily unknown. Heat stress (HS) causes an increase in intestinal epithelial permeability to luminal antigens including endotoxins.19Gathiram P Gaffin SL Brock-Utne JG Wells MT Time course of endotoxemia and cardiovascular changes in heat-stressed primates.Aviat Space Environ Med. 1987; 58: 1071-1074PubMed Google Scholar, 20Gathiram P Wells MT Raidoo D Brock-Utne JG Gaffin SL Portal and systemic plasma lipopolysaccharide concentrations in heat-stressed primates.Circ Shock. 1988; 25: 223-230PubMed Google Scholar, 21Hall DM Buettner GR Oberley LW Xu L Matthes RD Gisolfi CV Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia.Am J Physiol. 2001; 280: H509-H521Google Scholar, 22Shapiro Y Alkan M Epstein Y Newman F Magazanik A Increase in rat intestinal permeability to endotoxin during hyperthermia.Eur J Appl Physiol Occup Physiol. 1986; 55: 410-412Crossref PubMed Scopus (69) Google Scholar Both human and animal studies have shown that HS-induced disruption of intestinal TJ barrier leading to systemic endotoxemia19Gathiram P Gaffin SL Brock-Utne JG Wells MT Time course of endotoxemia and cardiovascular changes in heat-stressed primates.Aviat Space Environ Med. 1987; 58: 1071-1074PubMed Google Scholar, 20Gathiram P Wells MT Raidoo D Brock-Utne JG Gaffin SL Portal and systemic plasma lipopolysaccharide concentrations in heat-stressed primates.Circ Shock. 1988; 25: 223-230PubMed Google Scholar, 21Hall DM Buettner GR Oberley LW Xu L Matthes RD Gisolfi CV Mechanisms of circulatory and intestinal barrier dysfunction during whole body hyperthermia.Am J Physiol. 2001; 280: H509-H521Google Scholar, 22Shapiro Y Alkan M Epstein Y Newman F Magazanik A Increase in rat intestinal permeability to endotoxin during hyperthermia.Eur J Appl Physiol Occup Physiol. 1986; 55: 410-412Crossref PubMed Scopus (69) Google Scholar, 23Brock-Utne JG Gaffin SL Wells MT Gathiram P Sohar E James MF Morrell DF Norman RJ Endotoxaemia in exhausted runners after a long-distance race.S Afr Med J. 1988; 73: 533-536PubMed Google Scholar is an important pathogenic factor contributing to fatality related to heat stroke.24Gathiram P Wells MT Brock-Utne JG Gaffin SL Antilipopolysaccharide improves survival in primates subjected to heat stroke.Circ Shock. 1987; 23: 157-164PubMed Google Scholar, 25Gathiram P Wells MT Brock-Utne JG Gaffin SL Prophylactic corticosteroid increases survival in experimental heat stroke in primates.Aviat Space Environ Med. 1988; 59: 352-355PubMed Google Scholar It had been shown that blood circulating endotoxin levels are greater than 1000-fold higher in heat stroke patients compared to normal healthy individuals, and that the degree of endotoxemia is predictive of fatal outcome.26Bouchama A Parhar RS el-Yazigi A Sheth K al-Sedairy S Endotoxemia and release of tumor necrosis factor and interleukin 1 alpha in acute heatstroke.J Appl Physiol. 1991; 70: 2640-2644PubMed Google Scholar Therapeutic strategies that eliminate luminal bacteria27Gathiram P Wells MT Brock-Utne JG Wessels BC Gaffin SL Prevention of endotoxaemia by non-absorbable antibiotics in heat stress.J Clin Pathol. 1987; 40: 1364-1368Crossref PubMed Scopus (38) Google Scholar and treatment with anti-endotoxin antibodies before the onset of heat shock24Gathiram P Wells MT Brock-Utne JG Gaffin SL Antilipopolysaccharide improves survival in primates subjected to heat stroke.Circ Shock. 1987; 23: 157-164PubMed Google Scholar have been shown to prevent fatality related to heat shock. Thus, therapeutic strategies that maintain intestinal TJ barrier function during HS are being actively pursued as an important therapeutic option in heat stroke.24Gathiram P Wells MT Brock-Utne JG Gaffin SL Antilipopolysaccharide improves survival in primates subjected to heat stroke.Circ Shock. 1987; 23: 157-164PubMed Google Scholar, 25Gathiram P Wells MT Brock-Utne JG Gaffin SL Prophylactic corticosteroid increases survival in experimental heat stroke in primates.Aviat Space Environ Med. 1988; 59: 352-355PubMed Google Scholar, 28Singleton KD Wischmeyer PE Oral glutamine enhances heat shock protein expression and improves survival following hyperthermia.Shock. 2006; 25: 295-299Crossref PubMed Scopus (99) Google Scholar Previous studies from our laboratory indicated that a physiologically relevant increase in temperature (39°C or 41°C) causes an increase in occludin protein expression and an increase in junctional localization.29Dokladny K Moseley PL Ma TY Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability.Am J Physiol. 2006; 290: G204-G212Google Scholar, 30Dokladny K Wharton W Lobb R Ma TY Moseley PL Induction of physiological thermotolerance in MDCK monolayers: contribution of heat shock protein 70.Cell Stress Chaperones. 2006; 11: 268-275Crossref PubMed Scopus (21) Google Scholar The increase in junctional localization of occludin has been postulated to be an important protective mechanism against HS-induced disruption of TJ barrier in intestinal epithelial monolayers. The inhibition of HS-induced occludin expression was associated with a marked increase in TJ barrier disruption.29Dokladny K Moseley PL Ma TY Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability.Am J Physiol. 2006; 290: G204-G212Google Scholar The intracellular and molecular mechanisms that mediate occludin expression remain unresolved. The major aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced increase in occludin protein expression, using Caco-2 intestinal epithelial monolayers as an in vitro intestinal epithelial model system. In this study, we used modest heat exposure as a physiologically relevant inducer of occludin protein expression to gain insight into cellular and molecular mechanisms that regulate occludin protein expression. Our data show that the HS-induced increase in occludin protein expression was regulated by activation of HSF-1. In addition, our studies provide insight into the cellular and molecular mechanisms that mediate HSF-1-induced up-regulation of occludin protein expression during HS. Cell culture media (Dulbecco's modified Eagle's medium, DMEM), trypsin, fetal bovine serum (FBS), and related reagents were purchased from Life Technologies (Gaithersburg, MD). Glutamine, penicillin, streptomycin, and phosphate-buffered saline (PBS) were purchased from Life Technologies, Inc. (Grand Island, NY). Anti-occludin antibody was obtained from Zymed Laboratories (South San Francisco, CA). Quercetin, Triton X-100, bovine serum albumin, normal donkey serum, and anti-β-actin antibody were purchased from Sigma (St. Louis, MO). Horseradish peroxidase-conjugated secondary antibodies for Western blot analysis were purchased from Zymed Laboratories. Cy-3 antibodies for immunostaining were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-HSP1 antibodies were purchased from Stressgen Biotechnologies (Victoria, Canada). Tween 20 and nonfat dry milk were purchased from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of reagent grade and were purchased from Sigma, VWR (West Chester, PA), or Fisher Scientific (Pittsburgh, PA). Caco-2 cells (passage 18) were purchased from the American Type Culture Collection (Rockville, MD) and maintained at 37°C in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mmol/L glutamine, and 25 mmol/L HEPES, and supplemented with heat-inactivated 10% FBS.31Hidalgo IJ Raub TJ Borchardt RT Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability.Gastroenterology. 1989; 96: 736-749Abstract Full Text PDF PubMed Scopus (1952) Google Scholar Culture medium was changed every 2 days. After partial digestion with 0.25% trypsin and 0.9 mmol/L ethylenediaminetetraacetic acid (EDTA) in Ca2+- and Mg2+-free PBS, Caco-2 cells were subcultured on tissue culture plates (Corning, Acton, MA). To study the time-course effect of HS on occludin protein expression, Caco-2 monolayers were exposed to elevated temperatures for varying time periods and analysis of protein expression was performed by Western blot analysis as previously described.29Dokladny K Moseley PL Ma TY Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability.Am J Physiol. 2006; 290: G204-G212Google Scholar At the end of the experimental period, Caco-2 monolayers were immediately rinsed with ice-cold PBS, and cells were lysed with lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 500 μmol/L NaF, 2 mmol/L EDTA, 100 μmol/L vanadate, 100 μmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 40 mmol/L paranitrophenyl phosphate, 1 μg/ml aprotinin, and 1% Triton X-100) and scraped, and the cell lysates were placed in microfuge tubes. Cell lysates were centrifuged to yield a clear lysate. Supernatant was collected, and protein measurement was performed using a Bio-Rad protein assay kit. Laemmli gel loading buffer was added to the lysate containing 5 to 10 μg of protein and boiled for 7 minutes, after which proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Proteins from the gel were transferred to the membrane (Trans-Blot transfer medium, nitrocellulose membrane; Bio-Rad Laboratories) overnight. The membrane was incubated for 2 hours in a blocking solution [5% dry milk in Tris-buffered saline (TBS)-Tween 20 buffer] followed by an incubation (1 to 2 hours) with appropriate primary antibodies in a blocking solution. After being washed in TBS-Tween buffer, the membrane was incubated (1 hour) in appropriate secondary antibodies and developed using the Western blotting luminol reagents (Santa Cruz Biotechnology, Santa Cruz, CA) on the Kodak BioMax MS film (Fisher Scientific). Caco-2 cells were pulse-labeled with [35S]methionine as previously described.32Wick DA Seetharam B Dahms NM Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells.Am J Physiol. 1999; 277: G506-G514PubMed Google Scholar Caco-2 monolayers were incubated in methionine-free DMEM medium supplemented with 10% (dialyzed) FBS at 37°C for 60 minutes. Subsequently, Caco-2 cells were pulsed overnight with [35S]methionine by incubation in DMEM medium containing 200 μCi/ml of [35S]methionine at 37°C. The radioactive media was removed, and Caco-2 cells were washed three times with DMEM. The [35S]methionine-labeled Caco-2 cells were then chased by incubation in DMEM media containing 10-fold excess of cold methionine. Caco-2 cells were exposed to 37°C or 41°C temperatures for various time periods. At the end of the chase period, Caco-2 cells were washed three times with cold PBS. Total protein degradation was assessed by counting the radioactivity of [35S]methionine in the sample. Subsequently, occludin protein degradation was assessed by immunoprecipitation of occludin followed by autoradiography. For immunoprecipitation of occludin protein, Caco-2 cell lysate was prepared as described above. In a 1.5-ml microcentrifuge tube, PBS-washed Protein G-Sepharose 4 Fast Flow (Amersham Biosciences Corp, Piscataway, NJ) was combined with occludin antibody and mixed end-over-end for 1 hour at 4°C in a tube rotator. After the incubation time, the beads were washed four times in a wash buffer to remove the unbound antibody. One hundred μg of the protein and 10% bovine serum albumin were added to the tube containing occludin antibody bound to protein G-Sepharose beads and incubated for 2 hours at 4°C while mixing end-over-end in a tube rotator. After the incubation time, the beads were washed four times in a wash buffer to remove the unbound proteins. Twenty μl of Laemmli gel-loading buffer was added to the beads and boiled for 7 minutes, after which proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel followed by gel drying in a Gel Dryer Model (Bio-Rad Laboratories) connected to the Universal vacuum system (Savant Instruments, Holbrook, NY). Dried gel was exposed to Kodak BioMax MS film. Cellular localization of HSF-1 was assessed by immunofluorescent antibody labeling. Caco-2 monolayers grown on coverslips were exposed to HS for 1 hour. At the end of the experimental period, Caco-2 monolayers were washed twice in cold PBS (4°C) and fixed with 2% paraformaldehyde for 20 minutes. After being permeabilized with 0.1% Triton X-100 in PBS at room temperature for 20 minutes, Caco-2 monolayers were then incubated in blocking solution composed of bovine serum albumin and normal donkey serum in PBS for 1 hour. Cells were then labeled with primary antibodies in blocking solution overnight at 4°C. After being washed with PBS, the cells were incubated in Cy-3-conjugated secondary antibody for 1 hour at room temperature. Before being mounted on microscope slides (Erie Scientific, Portsmouth, NH), cells were incubated in 4′,6-diamidino-2-phenyindole, dilactate (DAPI) (Sigma). Immunolocalizations of HSF-1 protein were visualized using a Nikon fluorescence microscope (Nikon, Garden City, NY) equipped with a Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were processed with Wasabi software (Hamamatsu Photonics Deutschland, Herrsching, Germany). A 2023-bp occludin promoter region was identified from the human genome database and the occludin promoter region was cloned using the GenomeWalker system (Clontech, Palo Alto, CA). Two gene-specific primers, OCCSEAP-1F (5′-GGGGTACCCGACCCCAAAGGAGAAACAACCC-3′) and OCCSEAP-1R (5′-GATCGCAGATCTCGAGCTGCGTCCTAGACCGGCTC-3′), were designed from human genome sequences upstream of the translation start site of occludin. A 2023-bp DNA fragment (GenBank accession no. DQ264390) was amplified by polymerase chain reaction (PCR). The amplification condition was 1 cycle at 94°C for 2 minutes, followed by 43 cycles at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes, 1 cycle at 72°C for 5 minutes. The resultant PCR product was digested with KpnI and XhoI, and inserted into pSEAP2-basic reporter vector (Clontech). The sequence was confirmed by the DNA services at the University of New Mexico. DNA construct of occludin promoter (pSEAP2-basic promoter vector, Clontech) were transiently transfected into Caco-2 cells using GeneJuice transfection reagent (EMD Biosciences, San Diego, CA). In brief, Caco-2 cells (5 × 105) were seeded into a six-well plate and grown to confluency. Caco-2 monolayers were then washed with PBS twice and 1.0 ml of Opti-MEM medium was added to each well. One μg of plasmid construct and 2 μl of transfection reagent were preincubated in 250 μl of Opti-MEM in two separate tubes (Invitrogen Corp., Carlsbad, CA). After 5 minutes of incubation, two solutions were mixed and incubated for another 20 minutes, and the mixture was added to each well. After incubation for 3 hours at 37°C, 500 μl of DMEM containing 10% FBS were added to each well to reach a 2.5% final concentration of FBS. Subsequently, media were replaced with normal Caco-2 growth media 16 hours after transfection. HS experiments were performed 48 hours after transfection. Secreted alkaline phosphatase (SEAP) activities were assessed by using the Great EscAPe SEAP chemiluminescence detection kit (BD Biosciences, Palo Alto, CA). Supernatants of the cell culture medium (15 μl) were placed in a separate well of a 96-well plate. Subsequently, the dilution buffer was added and the 96-well plate containing experimental samples was sealed with the adhesive aluminum foil and placed in the incubator (65°C) for 1 hour. Samples were cooled to room temperature. Assay buffer was added to each sample followed by 1.25 mmol/L Chemiluminescence Substrate Phosphatase Detection substrate working dilution. Samples were then incubated at room temperature for 10 minutes. The chemiluminescent signal was detected using a Veritas microplate luminometer (Turner BioSystems, Sunnyvale, CA). SEAP being a heat stable protein33Cullen BR Malim MH Secreted placental alkaline phosphatase as a eukaryotic reporter gene.Methods Enzymol. 1992; 216: 362-368Crossref PubMed Scopus (164) Google Scholar was used as the reporter protein instead of luciferase, which is heat-sensitive. The experimental values of reporter SEAP activities were normalized to the baseline values that were obtained before the start of the experiments to account for any differences in transfection efficiency between experimental samples. Caco-2 cells were exposed to heat (41°C) for 1 hour and nuclear extracts were prepared according to the manufacturer's instruction manual (Active Motif, Carlsbad, CA) with minor modifications. Cells were washed with 2 ml of ice-cold PBS, scraped, and centrifuged at 14,000 rpm for 2 minutes. The cell pellets were resuspended in 1 ml of hypotonic buffer (20 mmol/L HEPES, 5 mmol/L NaF, 10 μmol/L Na2MoO4, 0.1 mmol/L EDTA, pH = 7.5), and incubated on ice for 15 minutes. After the incubation period, 50 μl of 10% Nonidet P-40 was added followed by centrifugation at 14,000 rpm for 30 seconds, pelleted nuclei were resuspended in 40 μl of complete lysis buffer (20 mmol/L HEPES, 20% glycerol, 400 mmol/L NaCl, 10 mmol/L NaF, 0.1 mmol/L EDTA, 10 μmol/L Na2MoO4, 1 mmol/L NaVO3, 10 mmol/L p-nitrophenyl phosphate, and 10 mmol/L β-glycerophosphate, pH = 7.5). Before use 1 μl of 1 mol/L dithiothreitol and 10 μl of protease inhibitor cocktail were added per 1 ml of lysis buffer. After incubation on ice for 30 minutes, the lysates were centrifuged at 14,000 rpm for 10 minutes. The supernatants were stored at −70°C. Protein concentrations were determined using the Bradford method. To demonstrate the HSF-1 binding to the binding motif or heat shock element (HSE) on the occludin promoter, a double-stranded 50-bp oligonucleotide probe (Integrated DNA Technologies, Coralville, IA) encoding the occludin promoter region from −1046 to −997 was synthesized. The oligonucleotide binding reactions was performed according to the Flexi kit instruction manual (Active Motif) with modifications. The binding reactions contained 3 μg of proteins, 1 pmol/μl of biotinylated probe (Integrated DNA Technologies, Inc.) in a total volume of 55 μl of complete binding buffer. After incubation at room temperature for 30 minutes, the reaction mixtures were transferred to an individual well on the plate and incubated for 1 hour. Rabbit HSF-1 antibody was diluted in a total volume of 100 μl of antibody binding buffer (1:2000) and was added to the well to bind HSF-1 from the nuclear extract. After incubation for 1 hour, HSF-1 antibody was removed and 100 μl of anti-rabbit horseradish peroxidase-conjugated IgG (1:5000) were added to the well and incubated for 1 hour. Subsequently, 100 μl of developing solution were added for 2 to 10 minutes, and 100 μl of stop solution were added. The absorbance at 450 nm was determined using the SpectraMax 190 (Molecular Devices, Sunnyvale, CA). To silence HSF-1, ON-TARGETplus SMARTpool (Dharmacon, Inc. Chicago, IL) was used. The sequences for HSF-1 small interfering RNA (siRNA) were: 5′-PUACUUGGGCAUGGAAUGUGUU-3′; 5′-PGUCCAUAGCAUCCAAGUGGUU-3′; 5′-PUAUGUCUUCACUCUUCAGGUU-3′; 5′-PUGAAUCCGGGCUGCUGUUCUU-3′. Caco-2 monolayers were transiently transfected using DharmaFect transfection reagent (Dharmacon, Lafayette, CO). Caco-2 cells were seeded into a six-well plate and grown to confluency. Caco-2 monolayers were then washed with PBS and Opti-MEM medium was added to the well. The plasmid vector containing the siRNA of HSF-1 and DharmaFect reagent were preincubated in Opti-MEM. After 5 minutes of incubation, two solutions were mixed and incubated for another 20 minutes, and the mixture was added to each well. After incubation for 3 hours at 37°C, 500 μl of DMEM containing 10% FBS and no antibiotics were added to cell culture media to reach a 2.5% final concentration of FBS. Heat exposure was performed 6 days after transfection. The siRNA-induced silencing of HSF-1 was confirmed by immunoblot of HSF-1. Caco-2 cells/filter (5 × 105) were seeded into six-well transwell permeable inserts and grown to confluency. Filter-grown Caco-2 cells were exposed to HS for desired time periods. At the end of the experimental period, cells were washed twice with ice-cold PBS. Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Total RNA concentration was determined by absorbance at 260/280 nm using SpectrraMax 190 (Molecular Devices). The reverse transcription (RT) was performed using the GeneAmp Gold RNA PCR core kit (Applied Biosystems, Foster city, CA). Two μg of total RNA from each sample were reverse-transcribed into cDNA in a 40-μl reaction containing 1× real-time PCR buffer, 2.5 mmol/L MgCl2, 250 μmol/L of each dNTP, 20 U RNase inhibitor, 10 mmol/L dithiothreitol, 1.25 μmol/L random hexamer, and 30 U multiscribe RT. The RT reactions were performed in a thermocycler (PTC-100; MJ Research, Waltham, MA) at 25°C for 10 minutes, 42°C for 30 minutes, and 95°C for 5 minutes. The real-time PCRs were performed using an ABI Prism 7900 sequence detection system and TaqMan universal PCR master mix kit (Applied Biosystems, Branchburg, NJ) as previously described.34Ye D Ma I Ma TY Molec