Abstract
Background
As environmental and body temperatures vary, lung epithelial cells experience temperatures significantly different from normal core temperature. Our previous studies in human lung epithelium showed that: (1) heat shock accelerates wound healing and activates profibrotic gene expression through heat shock factor-1 (HSF1); (2) HSF1 is activated at febrile temperatures (38°−41°C); and (3) hypothermia (32°C) activates and hyperthermia (39.5°C) reduces expression of a subset of miRNAs that target protein kinase Cα (PKCα) and enhance proliferation.
Methods
We analyzed the effect of hypo- and hyperthermia exposure on Wnt signalling by exposing human small airway epithelial cells (SAECs) and HEK293T cells to 32°C, 37°C, or 39.5°C for 24h, then analyzing Wnt-3a-induced epithelial mesenchymal transition (EMT) gene expression by qRT-PCR and TOPFlash reporter plasmid activity. Effects of miRNA mimics and inhibitors and the HSF1 inhibitor, KNK437, were evaluated.
Results
Exposure to 39.5°C for 24h increased subsequent Wnt-3a-induced EMT gene expression in SAECs and Wnt-3a-induced TOPFlash activity in HEK293T cells. Increased Wnt responsiveness was associated with HSF1 activation and blocked by KNK437. Overexpressing temperature-responsive miRNAs mimics reduced Wnt responsiveness in 39.5°C-exposed HEK293T cells, but inhibitors of the same miRNAs failed to restore Wnt responsiveness in 32°C-exposed HEK293T cells.
Conclusions
Wnt responsiveness, including expression of genes associated with EMT, increases after exposure to febrile-range temperature through an HSF1-dependent mechanism that is independent of previously identified temperature-dependent miRNAs. This process may be relevant to febrile fibrosing lung diseases, including the fibroproliferative phase of acute respiratory distress syndrome (ARDS) and exacerbations of idiopathic pulmonary fibrosis (IPF).
Keywords: hyperthermia, Wnt, Heat Shock Factor-1, fibrosis, epithelial-mesenchymal transition
Introduction
Pulmonary epithelia perform multiple essential functions required for homeostasis and response to infection and injury, including secretion of mucins [1], antimicrobial peptide [2], and surfactant [3], gas exchange [3], and detoxification of xenobiotics [4]. Epithelial cells engage in bidirectional communication with lung fibroblasts, which is essential to lung development and lung healing, but may also contribute to fibrosis [5]. Epithelial dysfunction is associated with reversible and chronic obstructive lung disease [6], pulmonary fibrosis [7], recurrent infections [8], and acute lung injury [9], and most lung cancers are epithelial in origin [10]. Potential mechanisms linking epithelial injury, lung fibrosis, and neoplasia includes: loss of antifibrotic factors such as hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and prostaglandin (PG)-E2, epithelial to mesenchymal transition (EMT), and expression of profibrotic factors such as TGFβ [11]. Wnt signaling is central to many of these disease pathways [12].
We [13, 14, 15, 16] and others [17, 18] have shown that modest deviations in temperature within the clinically relevant range impact cell survival and function of lung epithelium, including regulation of the heat shock response. Specifically, exposure to febrile-range temperature (~39.5°C) sensitizes lung epithelium to Fas-associated protein with Death Domain (FADD)/caspase-8-dependent apoptosis [13, 17] and stimulates submaximal heat shock factor-1 (HSF1) activation and heat shock protein (HSP)-72 expression [16]. We have also shown that activation of HSF1 in human lung epithelial cells accelerates wound healing and modifies gene expression, including activation of several profibrotic genes [15]. We recently showed that expression of a small subset of microRNAs in human lung epithelium is temperature-dependent, induced by hypothermia (32°C) and reduced at febrile range temperature (39.5°C) [14]. Three of the hypothermia-induced miRNAs targeted protein kinase Cα (PKCα), reduced PKCα protein levels, and enhanced proliferation by releasing cells from a PKCα-mediated G1/S proliferation block. Since the lung epithelium is exposed to a temperature gradient between ambient and core temperatures [19], the epithelium may be exposed to temperatures well below or well above normal core temperature, depending on ambient and core temperature, minute ventilation, location within the bronchoalveolar tract, and deviations in core temperature during fever and hypothermia.
In this paper, we have extended our previous study by analyzing the effects of clinically relevant hypo- and hyperthermic exposures on Wnt signaling in primary cultured human small airway epithelial cells (SAECs) and HEK293T cells. We demonstrate that that exposure to 39.5°C increases responsiveness to Wnt ligand and subsequent expression of genes that drive EMT through a mechanism that is independent of our previously identified temperature-dependent miRNAs but requires activated HSF1.
Materials and Methods
Cell culture and treatment
Primary cultured human SAECs (lot no. 58704924) isolated from a 37-year old Caucasian male were purchased from the ATCC (Manassas, VA) and maintained in airway epithelial cell basal medium supplemented with the SAEC growth kit (ATCC) at 37°C and 5% CO2, in automatic CO2 incubators. BEAS2B cells were obtained from ATCC and cultured in DMEM with high glucose (Gibco Thermo Fisher) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, pH 7.3, and 5% heat-inactivated fetal bovine serum (FBS). HEK293T cells were obtained from ATCC and were maintained at 37°C in Dulbecco’s Minimal Essential Medium (DMEM) with 4.5 g/L glucose and supplemented with 5% FBS and 1% penicillin-streptomycin. Recombinant human Wnt-3a (Wnt-3a) was purchased from R&D Biosystems (cat# 5036-WN-010). KNK437 was purchased from EMD Millipore (Billerica, MA) and cells were pretreated for 60 min at 30 μM final concentration in 0.1% DMSO.
Immunoblotting
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane (Immobilon-FL; Millipore) blocked with Odyssey™ Blocking Buffer (LI-COR; Lincoln, NE), probed with primary mouse antibodies against β-Catenin (BD Biosciences; San Jose, CA; cat#610154), Transcription factor-7 (TCF7) (Novus Biologicals; Littleton, CO; cat#H00006932-M01 and H00006932-M02), and HSP27 (Cell Signaling Technology; Danvers, MA; Cat # 2402) and β-tubulin (Millipore; Cat#MAB3408), then secondary IRDye 680RD Goat anti-Mouse IgG (LI-COR). The immunoblots were imaged on a LI-COR Odyssey infrared imaging system and band densities were quantified using the LI-COR software.
TOPFlash reporter assay
HEK293T cells were seeded at 70,000 cells/well in 24-well plates and pairs of monolayers were transfected 24h later with 500ng/well reporter plasmid TOPFlash driven by TCF/Lymphoid enhancer-binding factor (LEF) response elements or the control FOPFlash reporter plasmid in which the TCF/LEF sequences have been mutated (Addgene; Cambridge, MA) and 5ng the renilla luciferase expression plasmid, pRL (Promega; Madison, WI) using lipofectamine 2000 (Life technologies) as per manufacturer instructions. The cells were allowed to recover at 37°C for 24h, then were incubated at 32°C, 37°C, or 39.5°C for an additional 24 h. The cells were then switched back to 37°C and treated with 200ng/ml Wnt-3a and lysed 24h later using passive lysis buffer (Promega). Dual luciferase assay was performed using the dual luciferase assay kit (Promega) as per standard protocol. The firefly luciferase activities of TOPFlash and FOPFlash constructs were corrected for transfection efficiency by normalizing to renilla luciferase activity. The normalized TOPFlash was expressed as a ratio to the normalized FOPFlash activity in corresponding monolayers.
Quantitative RT-PCR analysis of baseline and Wnt-3a responsive gene expression
Primary cultured SAECs monolayers were incubated at 32°C, 37°C, or 39.5°C for 24h, then switched back to 37°C and treated with 200ng/ml recombinant human Wnt-3a for 3h for early Wnt-responsive genes or 24h for EMT genes. Cells were lysed and total RNA was extracted using Trizol™ (Thermo Fisher) as per the manufacturer’s instructions. The purity and concentration of RNA samples were determined by absorbance at 260 and 280 nm (Nanodrop; Thermo-Fisher Scientific). cDNA was generated from 1ug of total RNA by reverse transcription using iScript™ cDNA Synthesis Kit (Bio-Rad; Hercules, CA; cat #1708891). qRT-PCR was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad cat #1725204) on a Biorad CFX96 or StepOnePlus™ Real-Time PCR System (Thermo Fisher) as per the manufacturer’s instructions and the primer sequences listed in Table 1. Ct values were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and fold-change vs. 37°C was calculated using the delta-delta method (16).
Table 1.
PCR primers used for quantitative RT-PCR.
| Gene primer name1 | Primer sequence |
|---|---|
| CDH1_FP | 5’-AGGCCAAGCAGCAGTACATT |
| CDH1_RP | 5’-ATTCACATCCAGCACATCCA |
| TWIST1_FP | 5’-GCCAATCAGCCACTGAAAGG |
| TWIST1_RP | 5’-TGTTCTTATAGTTCCTCTGATTGTTACCA |
| cMYC_FP | 5’-CGACGAGACCTTCATCAAAA |
| cMYC_RP | 5’-TGCTGTCGTTGAGAGGGTAG |
| CCDN2_FP | 5’-TGATGGGGAAGGAGTACAAA |
| CCDN2_RP | 5’-TTGGAAGAAGAAGCAGGATG |
| MMP7_FP | 5’-AGCCAAACTCAAGGAGATGC |
| MMP7_RP | 5’-GCCAATCATGATGTCAGCAG |
| MMP2_FP | 5’-TTGACGGTAAGGACGGACTC |
| MMP2_RP | 5’-ACTTGCAGTACTCCCCATCG |
| Axin2_FP | 5’-CTGGCTCCAGAAGATCACAAA |
| Axin2_RP | 5’-GTGACAACCAACTCACTCGC |
| CDH2_FP | 5’-AGCCAACCTTAACTGAGGAGT |
| CDH2_RP | 5’-GGCAAGTTGATTGGAGGGATG |
| COL1A1_FP | 5’-TGGCCTCGGAGGAAACTTT |
| COL1A1_RP | 5’-TCCGGTTGATTTCTCATCATAGC |
FP and RP denote forward and reverse primer sequence.
Transfection with miRNA mimics and inhibitors
HEK293T cells were transfected with miRNA mimics and inhibitors as we have previously described [14]. Briefly, cells were seeded in 6-well cell culture plates (BD Biosciences) and transfected 24h later. For experiments with miRNA mimics, the cells were transfected with a combination of synthetic miRNA mimics for hsa-miR-92a1–5p (50nM), hsa-miR-27b-5p (50nM) and hsa-miR-1260a (25nM) or 125 nM scrambled sequence control MISSION® microRNA mimics (Sigma-Aldrich; St. Louis, MO). For experiments with miRNA inhibitors, the cells were transfected with pEZX-AM03 plasmid-based miRNA inhibitors from Genecopoeia (Rockville, MD) against hsa-miR-92a-1–5p (cat# HmiR-AN0831-AM03-B), hsa-miR-27b-5p (cat # HmiR-AN0360-AM03-B) and hsa-miR-1260a (cat# HmiR-AN0100-AM03-B) or a control plasmid with scrambled sequence (cat# CmiR-AN0001-AM03-B). All transfections were performed using lipofectamine 2000 (Thermo Fisher). Cells were recovered at 37°C for 24h, then incubated for 24h at 32°C, 37°C, or 39.5°C, lysed in RIPA, and immunoblotted for TCF7, ß-catenin, and ß-tubulin.
Immunofluorescence microscopy
Human SAECs were seeded at 10,000 cells per well in Millicell EZ SLIDE 4-well glass slides (EMD Millipore) and incubated at 37°C for 24h, then switched to 32°C, 37°C, or 39.5°C for next 24h. The cells were then treated with 200ng/ml Wnt-3a and switched back to 37°C for 3h. Cells were washed twice with ice cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS. Cells were blocked for 1h at room temperature (RT) in blocking buffer (PBS, 0.03%Triton, 1% bovine serum albumin (BSA) and 1% donkey serum), incubated overnight at 4°C with mouse anti-β-Catenin (BD Biosciences cat#610154) in blocking buffer. Cells were washed twice with washing buffer (PBS, 0.03%Triton). Cells were incubated with donkey anti-mouse CY3-conjugate (Jackson Immunoresearch; West Grove, PA) for 1h at room temperature, washed twice with washing buffer, and stained with 4, 6-diamidine-2-phenylindole, dihydrochloride (DAPI) (Kirkegaard & Perry Laboratories; Gaithersburg, MD; Inc cat# 710301) for 1 min at RT. Cells were washed once with PBS and the slides were sealed with coverslips. To analyze HSF1 activation, triplicate SAEC monolayers were exposed to 39.5°C for 1, 3, or 24h or to 42°C for 2h. Some monolayers were recovered at 37°C for 3h after 24h exposure to 39.5°C. Cells were immunostained as described above using rabbit anti-HSF1 primary antibody (Santa Cruz Biotechnology; Santa Cruz, CA; cat# sc9144) and donkey anti-rabbit Cy3-conjugate (Jackson Immunoresearch). Cells were visualized using Olympus (Tokyo, Japan) confocal microscope and Olympus Fluo View software.
Statistics
For quantitative RT-PCR and immunoblot data, differences between treatment groups were analyzed by repeat Mann-Whitney test using JMP 9 (SAS Institute, Inc; Cary, N.C.). Differences among >2 treatment conditions were analyzed by one-way ANOVA. In some cases we applied a post-hoc Tukey’s Honestly Significant Difference test to compare pairs of treatments.
Results
Exposure to clinically relevant hypothermia and hyperthermia modifies canonical Wnt-3a dependent gene expression and transactivation
To assess the impact of hypo- and hyperthermia exposure on canonical Wnt signaling in human lung epithelium, we analyzed the responsiveness of SAECs to the soluble Wnt ligand, Wnt-3a, and measured expression of Wnt-dependent genes. After 24h pre-exposure to 32°C, 37°C, or 39.5°C, all cells were incubated with 200ng/ml Wnt-3a for 3h at 37°C and expression levels of four Wnt-dependent genes, axin-2, cyclin D2 (CCND2), matrix metallopeptidase (MMP)-2, and avian v-myc myelocytomatosis viral oncogene homolog (c-Myc), were analyzed by qRT-PCR (Fig. 1a–d). All four genes exhibited the same temperature-dependent expression pattern, 44–74% reduction in expression at 32°C and 1.5 to 2-fold increase at 39.5°C, consistent with reduced canonical Wnt/β-catenin signaling after exposure to hypothermia and enhanced signaling after exposure to hyperthermia. Importantly, none of these four genes are predicted to be direct targets of our previously identified temperature-sensitive miRNAs [14]. Wnt-induced expression of a fifth gene, MMP7, which is a predicted direct target of the temperature-responsive miRNA, has-miR-92a-5p, was reduced by 11% and increased by 18-fold in 32° and 39.5°C cells, respectively (Fig. 1e).
Figure 1. Effects of clinically relevant hypothermia and hyperthermia on canonical Wnt-3a signaling capacity.
A-E. SAECs were incubated at 32°C, 37°C or 39.5°C for 24 h, switched back to 37°C, treated with 200 ng/ml recombinant human Wnt-3a for 3 h, total RNA was collected and mRNA levels of AXIN2 (A) Cyclin D2 (CCND2) (B) c-Myc (C) MMP2 (D), and MMP7 (E) were measured by qRT-PCR and expressed as fold-change vs. levels in 37°C cells. F. HEK293T cells treated similarly and Axin2 mRNA levels were measured. G. HEK293T cells were co-transfected with TOPFlash or FOPFlash and pRL renila luciferase control plasmid, recovered for 24h at 37°C, then incubated at 32°C, 37°C, or 39.5°C for 24h with transactivation of 200 ng/ml Wnt-3a for 24h at 37°C, and firefly and renilla luciferase measured. Firefly luciferase activity was normalized to renilla activity and the ratio of TOPFlash:FOPFlash calculated and expressed as fold-change vs. 37°C. Mean±SE of 3 experiments for qRT-PCR and 6 experiments for TOPFlash. * denotes p< 0.05 vs. 37°C and † p<0.01 vs. 32°C.
Wnt-3a activates gene expression through the canonical β-catenin pathway by stimulating the nuclear translocation of β-catenin where it complexes with intranuclear TCF7/LEF1 to form a transcription factor that transactivates promoters containing TCF binding sites [20]. Activation of the canonical pathway was measured in HEK293T cells using the TOPFlash luciferase reporter plasmid, which contains multiple TCF binding sites in its promoter region. To confirm that HEK293T cells exhibit the same temperature-dependent gene expression as SAECs, we quantified axin-2 mRNA in HEK293T cells treated with same hypo- and hyperthermia exposure and Wnt-3a stimulation protocol as the SAECs. Levels of axin-2 mRNA were reduced by 42% in 32°C-exposed HEK293T cells and increased 2.2-fold in cells 39.5°C-exposed cells (Fig. 1f). In HEK293T cells transfected with the TOPFlash reporter plasmid, Wnt-3a-induced TOPFlash activity was reduced by 65% in 32°C-exposed cells and increased by 270% in 39.5°C-exposed cells compared with 37°C cells (Fig. 1g).
Effect of hypothermia and hyperthermia on levels and distribution of TCF7 and β-Catenin
Since Wnt-induced gene expression and TOPFlash activity is driven by increased intranuclear levels of TCF7 and ß-catenin, we compared whole cell levels of TCF7 and ß-catenin protein and Wnt-induced nuclear translocation of ß-catenin in SAECs incubated at 32°C, 37°C, and 39.5°C. Immunoblot analysis showed similar levels of the two proteins in cells lysates from SAECs incubated at the three temperatures (Fig. 2a). To assess whether pre-exposure to 32°C or 39.5°C altered Wnt-induced nuclear translocation of ß-catenin, we analyzed cellular distribution of ß-catenin in SAECs after 24h pre-exposure to 32°C, 37°C, or 39.5°C followed by 3h stimulation with 200ng/ml Wnt-3a at 37°C. Immunofluorscent confocal imaging of immunostained cells greater Wnt-induced nuclear ß-catenin translocation in 39.5°C pre-exposed cells and reduced ß-catenin translocation in 32°C pre-exposed cells compared with 37°C cells (Fig. 2b).
Figure 2. Effects of clinically relevant hypothermia and hyperthermia on cell levels and distribution of TCF7 and β-Catenin.
A. SAECs were incubated at 32°C, 37°C or 39.5°C for 24h and whole cell lysate was immunoblotted for β-Catenin, TCF7, and ß-tubulin. The band densities were quantified, normalized to ß-tubulin band density, and expressed as fold-change vs. 37°C. A representative immunoblot and a graph summarizing the mean±SE of 4 experiments is shown. B. SAECs grown on chamber slides were incubated at 32°C, 37°C or 39.5°C for 24h and either fixed (left panels) or switched back to 37°C and stimulated with 200 ng/ml Wnt-3a for 3h and fixed. The fixed monolayers were immunostained for β-Catenin (red) and DAPI (blue) and analyzed by confocal immunofluorescence microscopy. Representative fields from three similar experiments are shown. C. HEK293T cells were transfected with either inhibitors (inh) or mimics (mim) of the three temperature responsive miRNAs (miR x 3), scrambled sequence controls for each (cnt) or nothing (−), recovered for 24 h, incubated at the indicated temperature for 24 h, then stimulated with 200 ng/ml Wnt-3a at 37°C for 3h and Axin2 mRNA levels were measured by qRT-PCR and expressed as fold-change vs. unstimulated 37°C cells. Mean±SE of 3 experiments. * and † denote p<0.05 vs. unstimulated 39.5°C and mimic-pretreated cells, respectively.
Analysis of Potential Targets of Temperature-dependent miRNAs
An in silico search for predicted gene targets of our previously identified temperature-dependent miRNAs using miRTar algorithm [21] (Table 2) identified Wnt signaling as the pathway most likely to be modified by these miRNAs. Wnt signaling comprises three overlapping pathways, the canonical, planar cell polarity (PCP), and Wnt/Ca2+ pathways [20]. Of the 182 genes known to participate in Wnt signaling, 43 were targeted by at least one of the following temperature-sensitive miRNAs, hsa-miR-92a-1–5p, 24, hsa-miR-1260a, or hsa-miR-27b-5p. The 43 genes participate in one or more Wnt pathways, including 26 that participate in the canonical pathway, 17 in the PCP pathway, and 14 in the Wnt/Ca2+ pathway (Table 3). Since these miRNAs are induced at 32°C and reduced at 39.5°C and are predicted to target Wnt signaling elements, we reasoned that they may mediate the effects of hypo- and hyperthermia exposure on Wnt signaling capacity.
Table 2.
Pathways targeted by a representative Temperature Sensitive miRNA hsa-miR-92a-1–5p1.
| KEGG Pathway2 | p-value | Number pathway genes targeted | Percent of all genes in pathway3 |
|---|---|---|---|
| Wnt signaling pathway [hsa04310] | 1.66463E-33 | 22 | 12 |
| Melanogenesis [hsa04916] | 4.22712E-19 | 14 | 13 |
| Basal cell carcinoma [hsa05217] | 7.59544E-15 | 10 | 17 |
| Pathways in cancer [hsa05200] | 4.64796E-11 | 14 | 3 |
| Hedgehog signaling pathway [hsa04340] | 1.7015E-09 | 7 | 12 |
| Long-term potentiation [hsa04720] | 7.65295E-06 | 5 | 7 |
| Colorectal cancer [hsa05210] | 9.42127E-06 | 5 | 7 |
| Adherens junction [hsa04520] | 0.000101195 | 5 | 4 |
| Acute myeloid leukemia [hsa05221] | 0.000134987 | 4 | 6 |
| Thyroid cancer [hsa05216] | 0.000513415 | 3 | 8 |
Identified using miRTar.
Human pathway name and (KEGG number) listed.
Based on total number of pathway genes listed in KEGG database.
Table 3.
Wnt Signaling Genes Targeted by Temperature-sensitive miRNAs1.
| Gene | Pathways2 | 92a1–5p | 27b-5p | 1260a |
|---|---|---|---|---|
| WNT5B | 1,2,3 | . | ||
| WNT5A | 1,2,3 | . | ||
| WNT9B | 1,2,3 | . | ||
| WNT7B | 1,2,3 | . | ||
| WNT3A | 1,2,3 | . | ||
| WNT10B | 1,2,3 | . | ||
| WNT1 | 1,2,3 | . | ||
| NKD1 | 1,2 | . | ||
| PRKCG | 3 | . | ||
| PRKCA | 3 | . | . | . |
| PLCB4 | 3 | . | ||
| FZD4 | 3 | . | . | |
| CHP2 | 3 | . | ||
| CAMK2G | 3 | . | ||
| CAMK2A | 3 | . | ||
| RHOA | 2 | . | ||
| VANGL2 | 2 | . | ||
| RAC1 | 2 | . | ||
| PRICKLE2 | 2 | . | ||
| MAPK8 | 2 | . | ||
| MAPK10 | 2 | . | ||
| DVL1 | 2 | . | ||
| DAAM2 | 2 | . | ||
| DAAM1 | 2 | . | ||
| TCF7L2 | 1 | . | ||
| TCF7L1 | 1 | . | ||
| TCF7 | 1 | . | ||
| TBL1X | 1 | . | ||
| SMAD2 | 1 | . | ||
| SFRP1 | 1 | . | ||
| PRKACA | 1 | . | ||
| PPP2R5C | 1 | . | ||
| PPARD | 1 | . | ||
| FRAT2 | 1 | . | ||
| FBXW11 | 1 | . | ||
| DVL3 | 1 | . | ||
| CTNNB1 | 1 | . | ||
| CSNK2B | 1 | . | ||
| CSNK1E | 1 | . | ||
| CCND2 | 1 | . | ||
| BTRC | 1 | . | ||
| AXIN1 | 1 | . |
Gene targets identified by miRTar
Pathways 1, 2, and 3 are canonical, Planar Cell Polarity, and Wnt/Ca2+, respectively
To analyze the potential contribution of our previously identified temperature-dependent miRNAs to temperature-dependent Wnt signaling capacity, we measured Wnt-3a-induced axin-2 expression in HEK293T cells after transfection with inhibitors of hsa-miR-92a1–5p (50nM), hsa-miR-27b-5p (50nM) and hsa-miR-1260a and incubation at 32°C for 24h or in HEK293T cells after transfection with mimics of these three miRNAs and incubation at 39.5°C for 24h. Wnt-3a-induced axin-2 expression was increased in the 39.5°C pre-exposed cells and tended to be decreased in the 32°C pre-exposed cells compared with 37°C cells. Treating 39.5°C-exposed cells with miRNA mimics reduced Wnt-3a-induced axin-2, but treating 32°C-exposed cells with miRNA inhibitors failed to increase Wnt-3a-induced axin-2 expression (Fig. 2c).
Impact of temperature-dependent Wnt signaling on Genes Involved in EMT and Fibrosis
Wnt signaling stimulates EMT gene expression, including increasing levels of collagen type I (COL1A) [12] and twist family basic helix-loop-helix transcription factor 1 (TWIST1) [22], and increasing the ratio of N-cadherin (CDH2) to E-cadherin (CDH1) [23]. To determine whether the temperature-dependent changes in Wnt signaling are associated with EMT gene expression, we analyzed baseline and Wnt-3a-induced expression of these EMT markers in human SAECs after 24h pre-incubation at 32°C, 37°C, or 39.5°C. Wnt-induced EMT gene expression was measured after treating the cells with 200ng/ml Wnt-3a for 24h at 37°C. Incubating SAECs at 32°C for 24h had no significant effect on either baseline or Wnt-3a-stimulated expression of COL1A1 or TWIST1 or CDH2:1 ratio (Fig. 3a–d). Incubating SAECs at 39.5°C for 24h reduced baseline TWIST1 expression by 80% and increased Wnt-3a-stimulated expression of COL1A1 and TWIST1 by 2.8-fold and 9.2-fold, respectively and increased the CDH2:1 ratio by 3.6-fold compared with Wnt-3a-stimulated 37°C cells.
Figure 3. Effect of temperature-dependent Wnt signaling on genes involved in EMT and fibrosis.
SAECs were incubated at 32°C, 37°C or 39.5°C for 24h and total RNA was collected for analysis of basal EMT gene expression (A) or the cells were stimulated 200 ng/ml Wnt-3a for 24h at 37°C and total RNA collected for analysis of Wnt-3a-stimulated gene expression (B-D). Type I collagen alpha (COL1A1) (B), ratio of N-cadherin (CDH-2) to E-cadherin (CDH-1) (C) and TWIST1 (D) were quantified by qRT-PCR and expressed as fold-change vs. unstimulated 37°C cells. Mean±SE of 4 experiments are shown. * denotes p<0.005 vs. 37°C and † P<0.02 vs. 32°C.
Contribution of HSF1 to hyperthermia effects on Wnt-3a signaling
While hypothermia may reduce Wnt signaling capacity, in part through increased expression of one or more temperature-responsive miRNAs, the inability of miRNA inhibitors to increase Wnt responsiveness in the 32°C-exposed cells suggests the participation of other temperature-dependent mechanisms. The heat/stress-activated transcription factor, HSF1 is activated at 38°−39.5°C [16] and can regulate expression of many genes that are not classic heat shock proteins [24], including >500 genes in cancer and cancer stromal cells [25, 26]. Whether HSF1 modifies expression of any genes that participate in Wnt signaling is unknown. To assess whether HSF1 contributes to the increased Wnt-signaling capacity in 39.5°C pre-exposed SAECs, we analyzed HSF1 activation by measuring expression of a known HSF1-dependent gene, HSP27 (Fig. 4a), and by analyzing HSF1 nuclear translocation (Fig. 4b) in SAECs incubated at 37°C or 39.5°C. Levels of HSP27 protein increased after 6h exposure to 39.5°C. Nuclear translocation of HSF1 was evident in 39.5°C-exposed cells after 1h and persisted for 24h, but less so than occurred during 42°C exposure.. To evaluate whether HSF1 was required for the increased Wnt responsiveness in the 39.5°C-exposed cells, we used KNK-437, a pharmacologic inhibitor of HSF1-dependent transcription [27]. Pretreating SAECs with 30 μM KNK-437 prior to initiating the 39.5°C exposure blocked both HSP27 expression (Fig. 4c) and the increase in Wnt-3a-induced axin-2 expression (Fig. 4d).
Figure 4. Contribution of HSF1 to Hyperthermia-enhanced Wnt Signaling Capacity.
A. SAECs were incubated at 39.5°C for 6 or 24h, 39.5°C for 24h then 37°C for 3h, or 42°C for 2h then 37°C for 2h. Cells were lysed and immunoblotted for HSP27 and ß tubulin (representative of 3 similar blots). B. SAECs were grown on cover slips and incubated at 39.5°C for 0, 1, 3, or 24h, at 39.5°C for 24h then 37°C for 3h, or 42°C for 2h. Cells were fixed, immunostained for HSF1 and DAPI, and imaged by confocal immunofluorescence. Representative images for 0,3, and 24h at 39.5°C and 2h at 42°C. Percent of cells with nuclear HSF1 staining calculated and Mean±SE from 3 experiments shown. † denotes p<0.009 vs. time 0, * denotes p<0.0001 vs. all other treatments. C. SAECs were pretreated for 30 min with nothing (0), vehicle (1% DMSO) or 30 μM KNK437 then incubated at 39.5°C for 24h, lysed and immunoblotted for HSP27 and ß-tubulin (representative of 3 similar blots). D. SAECs were pretreated with DMSO or 30 μM KNK437 for 30 min, then at 37°C or 39.5°C for 24h, then with or without 200 ng/ml Wnt-3a for 3h. Cells were lysed, total RNA collected and axin mRNA quantified by qRT-PCR and expressed as percent untreated 37°C control cells. Mean±SE of 6 experiments. * denotes p<0.0001 vs. all unstimulated cells; † denotes p = 0.007 vs. DMSO-treated, Wnt-stimulated 39.5°C cells.
Discussion
We analyzed the effects of exposure to hypo- and hyperthermia on basal levels of Wnt-dependent genes and the capacity for canonical Wnt signaling, utilizing the canonical soluble Wnt ligand, Wnt-3a [28, 29]. Prior studies of how hypothermia [30] and hyperthermia [31] affect Wnt signaling focused on the temperature-dependent modification of ß-catenin levels, but at a lower hypothermic temperature (30°C) and higher hyperthermic temperatures (43°−48°C) than the clinically relevant temperatures used in our study. The temperatures and the exposure time used in the present study were based on our previous study [14] and are clinically relevant to accidental and therapeutic hypothermia exposures and febrile illnesses. Since the experiments were designed to determine how exposure to hypo- and hyperthermia modifies Wnt signaling capacity, all cells were stimulated with Wnt-3a at 37°C following hypo- or hyperthermia exposure. We used two measures of canonical Wnt signal activation, mRNA expression levels of five known early Wnt-dependent genes and Wnt-induced activation of the TOPFlash reporter plasmid. TOPFlash is a firefly luciferase reporter driven by an artificial promoter containing eight copies of the TCF binding sequence upstream of the thymidine kinase promoter and is activated when TCF7 and ß-catenin co-localize within the nucleus [32]. Although the magnitude of the temperature-dependent changes in expression levels varied among the five Wnt-dependent genes, the pattern of expression, lower in 32°C-exposed cells and higher in 39.5°C cells was consistent and paralleled the effects of hypo- and hyperthermia on Wnt-induced TOPFlash activity. Expression of one of the genes, MMP7, was increased by hyperthermia exposure much more than the other four genes. It is possible that since MMP7 is the only one of these Wnt-responsive genes to be directly targeted by the temperature-responsive miRNAs, it may be modified by multiple temperature-dependent mechanisms.
The increased Wnt-dependent gene expression and TOPFlash activity in HEK293T cells exposed to 39.5°C suggests higher Wnt-3a-induced intranuclear levels of TCF7 and ß-catenin after cells are exposed to 39.5°C. Although ß-catenin and TCF7 are predicted to be targets of the temperature-responsive miRNAs [14] (Table 2), immunoblotting failed to detect a significant effect of hypo- and hyperthermia exposure on whole cell levels of either protein. However, confocal immunofluorescence imaging showed that treatment with Wnt-3a induced greater ß-catenin nuclear translocation after 39.5°C exposure and less after 32°C exposure, suggesting that exposure to hypo- and hyperthermia modifies one or more elements in the canonical Wnt signaling pathway.
Consistent with the canonical Wnt signaling pathway containing 26 potential temperature-dependent miRNA targets, treatment with mimics of these miRNAs reduced Wnt responsiveness in 39.5°C-exposed cells. However, treating 32°C-exposed cells with inhibitors of the same miRNAs failed to increase Wnt-3a responsiveness to the levels in 37°C or 39.5°C cells. These data demonstrate that the temperature-responsive miRNAs can reduce Wnt signaling capacity, but suggest that other factors are responsible for the changes in Wnt responsiveness following exposure to clinically relevant hypo- and hyperthermia.
We have shown that exposure to temperature within the usual febrile range activates a submaximal heat shock response [16]. Chou et al. demonstrated that phosphorylation of HSF1 on serine 326 by mTOR stabilizes β-catenin expression through the RNA binding protein HuR [33], but we found that hyperthermia increased Wnt-stimulated ß-catenenin nuclear translocation rather than whole cell levels of ß-catenin. Zhang et al. [34] demonstrated that treating rat cardiac stem cells with low dose LPS increased total cell and nuclear levels of ß-catenin and the effect was blocked by the HSP90 inhibitor geldanamycin. Cordonnier et al. [35] demonstrated that HSP27 depletion in prostate cancer cells reduced EGF-stimulated interaction of ß-catenin with GSK3ß, which is required for its phosphorylation and nuclear translocation, and EMT gene expression. HSP27 overexpression had the opposite effects, demonstrating a potential mechanism for HSF1 activation to increase Wnt signaling capacity. Cytoplasmic HSF1 trimerizes and translocates to nucleus in response to stress [36], including, exposure to febrile range hyperthermia [16]. In this study, exposing SAECs to 39.5°C stimulated a submaximal nuclear translocation of HSF1 that occurred within 1h and persisted for 24h, and an increase in expression of HSP27. Pretreatment with the HSF1 transcriptional inhibitor, KNK437 [27], blocked both HSP27 expression and increased Wnt-induced axin2 expression. Although the SAECs used in this study were obtained from a single individual, the SAECs and HEK293T cells had similar responses to shifts in temperature, suggesting the effect of hypo- and hyperthermia on Wnt signaling capacity may be a common property of human cells. These results implicate gene expression driven by submaximal HSF1 activation in the increased Wnt signaling capacity following exposure to febrile range hyperthermia. Moreover, the data suggest that the hypo- and hyperthermia modify Wnt signaling capacity through distinct mechanisms.
Canonical Wnt-dependent changes in EMT gene regulation, including a switch from E-cadherin to N-cadherin expression [23], activation of the transcriptional repressor, TWIST1 [22], and increase in type I collagen expression [12], contributes to embryogenesis, tissue repair, fibrosis, and cancer metastasis in multiple organs, including lung [37, 38, 39, 40, 41]. Consistent with the effect of hyperthermia on Wnt responsiveness, Wnt-3a-induced expression of type I collagen and TWIST1 and switch from E-cadherin to N-cadherin expression in SAECs was increased after exposure to 39.5°C. These gene products and MMP7, which was also expressed at higher levels in 39.5°C-exposed SAECs, are associated with fibrosing disorders, including pulmonary fibrosis [42, 43, 44]. The association of Wnt signaling activation and human idiopathic pulmonary fibrosis [45] and the effectiveness of pharmacological Wnt signaling inhibitors in reducing fibrosis in the rodent bleomycin-induced lung injury [46] underscores the importance of understanding the modifiable factors that regulate Wnt signaling. To that end, we note that fever is often associated with certain fibrosing lung processes, including the Acute Respiratory Distress Syndrome (ARDS) [47] and exacerbations of idiopathic pulmonary fibrosis (IPF) [48].
Conclusion
We have shown that exposure to clinically relevant hypothermia reduces and fever-range hyperthermia increases Wnt responsiveness and Wnt-stimulated EMT gene expression in human lung epithelium. These results support the importance of clinical studies to determine whether fever is profibrotic in these clinical settings and whether hypothermia or fever prevention, or inhibition of HSF1-dependent transcription will be protective against post-ARDS fibrosis and irreversible loss of lung function following IPF exacerbations.
Acknowledgments
Funding: The work was supported by NHLBI grant no. R01HL69057 and VA Merit Review no. IBX002143A.
Footnotes
Declaration of interest: The authors alone are responsible for the content and writing of the paper.
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