The Role of HIF1 in Regulating Cobalt-Induced Cytokine Expression in Alveolar Type II Cells

Ricardo Rivera-Soto1, Steve P. Proper2 and John J. LaPres2

1

Department of Biology, University of Puerto Rico at Arecibo, Arecibo, Puerto Rico. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan.

Abstract
Asthma is a chronic inflammation of the airways that affects approximately 300 million of people worldwide and the symptoms include cough, chest tightness, and shortness of breath. This disease is characterized by an immunological response mediated by T helper 2 (TH2) cells and select cytokines associated with their recruitment. Recent studies have shown that mice deficient in the protein, hypoxia inducible factor 1 alpha (HIF1), and exposed to cobalt exhibit an asthma-like immune response. In contrast, control mice respond to cobalt by displaying symptoms similar to hard metal lung diseases. Previous research in the laboratory and literature review led us hypothesize that loss of HIF1 in alveolar type II (ATII) cells lead to a change in the cells cytokine expression profile that promotes the observable change in cobalt-induced inflammation. To address this hypothesis, A549 cells, a human ATII-like cell line, were used. To model what happens in the transgenic mouse lung, these cells were manipulated, using siRNA, to decrease the expression of HIF1Following knock down, these cells, and their respective controls were exposed to cobalt for 6 and 24 hours and the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), vascular endothelial growth factor (VEGF) and Interleukin (IL) 5 and 10 genes was assessed by quantitative RT-PCR. The preliminary results indicate that a longer exposure to cobalt increased the expression of cytokines by HIF1-deficient cells while in the others cells strains were decreased. These results were confounded by a mixed population within the cell strains used. Therefore, to reach a conclusion further studies are necessary. Keywords: Hypoxia Inducible Factor, siRNA, Asthma, Toxicogenomics

Introduction
Asthma is a chronic inflammation of the airways that affect approximately 300 millions of people worldwide (Masoli, Fabian, Holt, Beasley, & Global Initiative for Asthma, 2004). This disease is characterized by symptoms that include, cough, wheezing, chest tightness and shortness of breath. The immunological response to asthma is drive by T helper Type 2 (TH2) cells and includes eosinophilia, and the appearance of specific cytokines and chemokines, such as interleukin 5 (IL-5) and IL-13 (Holgate, 2012). Hypoxia is defined as a decrease in available oxygen reaching the cells or tissue of the body. The cellular response to hypoxia is regulated by a family of transcription regulators known as the hypoxia-inducible factors (HIFs). The most ubiquitously expressed and widely studied HIF is HIF1. HIF1 is a dimer, composed of an oxygen sensor, HIF1 and a constantly expressed HIF1, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT). When oxygen levels are normal (i.e.normoxia), HIF-1 is modified at specific proline residues by a family of enzymes known as prolyl hydroxylase domain containing enzymes (PHD). This hydroxylation leads to the ubiquitination of HIF1 by the vonHippel-Lindau tumor suppressor (VHL), followed by HIF1s rapid proteosomal degradation (Bruick & McKnight, 2001; Marxsen et al., 2004). When the levels of O2 decrease, the PHD becomes inhibited and the HIF1 translocate to the nucleus where it is free to interact with HIF1, forming the function transcription factor, HIF1. HIF1 binds specific genomic sequences known as hypoxia-response elements (HREs) within promoter regions of target genes. This binding can promote the regulation of more than 100 genes, including glycolytic enzymes, angiogenic factors, and inducers of apoptosis (Carmeliet et al., 1998; Pugh & Ratcliffe, 2003; Semenza, Roth, Fang, & Wang, 1994). Recent studies have shown that mice deficient of HIF-1 in the alveolar type II (ATII) and Club cells (previously known as Clara cells) and exposed to cobalt exhibit a TH2mediated inflammation resembling asthma. This includes eosinophilia, mucous cell metaplasia, and increased expression of chitinase-like proteins. In contrast, control mice display a TH1-mediated inflammation, including, neutrophilia, fibrosis, and tumor

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necrosis factor (TNF) expression (Saini, Greenwood, et al., 2010; Saini, Kim, et al., 2010). However, how loss of HIF1 mediates this change in inflammatory response remains unknown. Previous research and literature reviews has led us to hypothesize that this change in inflammatory response is due to changes in the cells cytokine expression profile due to loss of HIF1 in the ATII cells. To address this hypothesis, A549 cells, a human ATII-like cell line, were used. To model what happens in the transgenic mouse lung, these cells were manipulated, using siRNA, to express less HIF1. Following successful knock down, these cells, and their respective controls were exposed to cobalt and the expression of various inflammatory mediator genes was assessed using quantitative RT-PCR.

Materials and Methods

Cell lines and culture conditions The human alveolar adenocarcinoma A549 cell line was purchased from American Type Culture Collection (Manassas, VA). A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 g/ml streptomycin. Cells were grown at 37C in a humidified incubator under 5% CO2. HIF-1-siRNA constructions The HIF-1 siRNA lentivector set (Cat#000058) and the 2nd Generation Packaging Mix (Cat# LV003) were purchased from Applied Biological Materials (Richmond, BC, Canada). This vector set includes four unique HIF1 target sequences expressed in a lentiviral vector. The cells that were transfected will be named for the siRNA sequences they were infected with (i.e. 2357 thru 2360) for the rest of the paper. Additionally, a negative control (scrambled) was also used. Lentiviral packaging and infection were performed via manufactures protocols.

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Evaluation of efficiency of the transfection The plasmid used to knockdown the expression of HIF1 includes expression cassettes for the Green Fluorescence Protein (GFP) and Puromycin resistance genes. The expression of these genes can be used to determine the efficiency of the infection and selection of infected cells, respectively. First, the expression of the GFP was assessed using fluorescent microscopy. Second, cells that successfully incorporated the infected DNA were selected by adding puromycin (1.5 g/mL) to the media. Following puromycin selection, cells were maintained in puromycin containing media. Evaluating the level of HIF1 knockdown To evaluate the efficiency of HIF1 knockdown following infection, western blot was performed. Cells were exposed for six hours to hypoxia (1% O2) or normoxia (21% O2). Cells were washed three times with ice cold 1x PBS and manually removed from the culture dish in lysis buffer (20mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 420 mM KCl, 1 mM EDTA, 20% Glycerol and protease inhibitors). Cells were centrifuged at 2,000 RPM for 2 minutes at 4o C (Sorvall Biofuge Fresco; Thermo Scientific, Rockford, Illinois). Following centrifugation, the supernatant was collected and incubated at 4o C with rotation for 30 minutes. Insoluble material was removed by centrifugation at 13,000 RPM for 30 minutes. Protein concentrations of each sample were determined using BioRad assay and a bovine serum albumin standard curve. Equal amounts (100 g) of each sample were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membrane and the membrane was probed with a HIF1-specific antibody (1:500 dilution, NB-100-105, Novus Biologicals, Littleton, Colorado), and anti-Histone-H3 (1:5000 dilution, abcam, Cambridge, Massachusetts). Then, the proteins were exposed to secondary antibodies; goat-anti-mouse-IgG-HRP (1:1000) and goat-anti-rabbit-IgGHRP (1:500) respectively, (Santa Cruz Biotechnology, Santa Cruz, California). Detection was performed with the Pierce enhanced chemilumescent kit (Thermo Scientific) and exposed to film for 30 seconds, 1 and 5 minutes.

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Cobalt Exposure Each individual cell strain expressing the individual siRNA sequences and the parent control were plated into a 6-well dish. At 50-60 % confluence, half of the plate was treated with DMEM containing CoCl2 (114 M) and the other half received normal DMEM. Cells were exposed for and incubated for six or twenty four hours. RNA isolation and Quantitative Real Time-Polymerase Chain Reaction Following cobalt exposure, cells were manually removed from the dish in the presence of Trizol (Life Technologies, Grand Island, New York). RNA was isolated according to the manufacturers protocols. Total RNA quantification was performed

spectrophotometrically (Nano-Drop ND-1000 UV-Vis Spectrophotometer; Thermo Scientific). Total RNA (250 ng) was reverse transcribed using Primescript Reverse Transcriptase (Takara Bio, Mountain View, California) or Superscript III (Life Technologies). cDNA from each experimental group was used to analyze the gene expression through qRT-PCR. The array analysis reaction were carried out in ABI Prism 7000 96 well block using Power SYBR Green (Applied Biosystems, Foster, California). The changes in the gene expression were calculated using the 2-Ct method and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene as the reference.

Results
Green Fluorescent Protein Expression As can be seen by the positive GFP signal in figure 1, cells were successfully infected with the siRNA sequences. It is also apparent that all the cells are not expressing equal levels of GFP, suggesting that the cells represent a mixed population.
Figure 1. The Green Fluorescent Protein was
used as a marker gene to detect positively infected cells. Expression of this gene is related with the infection efficiency. (A) Phase contrast image (B) Fluorescent image. The exposure time for the image was 5 seconds

A
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Western Blot To determine if the siRNA sequences were capable of decreasing the level of HIF1 protein within the cells, a western blot was performed following the six hours of hypoxia. The parental cells, A549s, and scrambled controls displayed little HIF1 under normoxic conditions. The levels of HIF1 are dramatically increased following hypoxia exposure. Clones expressing sequences 2357 and 2359 closely resembled the parental and scrambled controls, suggesting these sequences were not functional. In contrast, sequences 2358 and 2360 show a decrease in the amount of HIF1 protein following hypoxia exposure (Figure 2).

+ - + - + - + HIF1 H3

+ -

+ -

Figure 2. HIF1 analysis from nuclear extracts of A549 control (1), Scramble (2), 2357 (3), 2358 (4), 2359 (5), and
2360 (6) was performed after six hours of exposure to hypoxia (+) or normoxia (-). The target protein was detected using a monoclonal anti-HIF1 antibody and anti-histone H3 was used for the loading control.

Genes Expression HIF1. To further confirm the HIF1 knockdown, the level of HIF1 mRNA was

assessed by qRT-PCR. Interestingly, each cell strain displayed equal levels of HIF1 mRNA (Figure 3). Notably the maximum expression was seen in the 2360 sequence.
mRNA Expression (arb. units)

HIF1

Figure 3. HIF1 mRNA was assessed by qRT-PCR.

Each strain was normalized to the parental cell line. The graph illustrates the mean of the samples and the standard deviation is shown in the error bars .

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BNIP3. To determine if the loss of HIF1 protein observed in Figure 2 translates into decreased HIF1 function, the expression of BNIP3, a known HIF1 target gene, was assessed by qRT-PCR following the 6 (Fig. 4A) or 24 (Fig. 4E) hours of cobalt exposure. At six hours the maximum expression was on the 2357 cell strain and

scrambled control. These results suggest that the loss of HIF1 observed in the western blot (Fig. 2) translates to a functional loss of cobalt-induced signaling. On the other hand, at 24 hours the only cell that is expressing the gene is the 2359. VEGF. Vascular endothelial growth factor is another HIF1 target gene and its expression was assessed by qRT-PCR. The results at six hours (Fig. 4B) show that the cells strains 2359 and 2360 decrease the expression in comparison with the others. This data support the BNIP3 expressions results. Interestingly, the cell strain 2358 was expressing the maximum quantity of VEGF mRNA following 6 and 24 hours (Fig. 4F) of cobalt exposure. Interleukin. The hypothesis suggests that loss of HIF1 will lead to changes in cytokine expression. To test this hypothesis, the expression of the genes that encode two cytokines important for the asthma phenotype, IL5 and IL10, were assessed in the various cell strains using qRT-PCR. IL5 expression mirrored that observed for BNIP3. After six hours (Fig. 4C) of cobalt exposure each of the cell strains displayed a decreased expression of IL5 following cobalt challenge in comparison with the scrambled control. In contrast, the expression of IL5 gene after the 24 hours (Fig. 4G) was only observed in the cell strains 2359 and 2360. Likewise, the expression of IL10 was consistent with the expression of IL5. After six hours (Fig. 4D) of exposure all the cell strains show a decrease in comparison with the scrambled control. Similarly to IL5 at 24 hours (Fig. 4H) of exposure only the cell strains 2359 and 2360 increased their expression of the gene, while the others strains exhibit a decrease.

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6 hours

24 hours
Control

BNIP3

E
Cobalt

Gene Expression (arb. units)

VEGF

IL-5

IL-10

Cell strains
Figure 4. Results of gene expressions. qRT-PCR was performed for the
BNIP3, VEGF, IL-5 and IL-10 mRNA. Cells were exposed to 6 hours (left) and 24 (right) to cobalt. The data presented is the mean of the samples and the standard deviation is shown in the error bars.

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Discussion
Our research was designed to test that hypothesis that loss of HIF1 in ATII cells will alter the cells ability to express cytokines following cobalt exposure. To this aim, the level of HIF1 was decreased in A549 cells using siRNA. The successful knock down in these ATII-like cells would partially model what happened in the transgenic mice used previously in our laboratory by Saini et al. The expression of GFP in the figure 1 suggest that the infection of the plasmid was successful, however, as can be visualized in the same figure, not all the cells express equal levels of GFP and this could be one of the reason why our results were not consistent. The western blot results (figure 2) indicate that sequences 2358 and 2360 were the most effective in decreasing the expression of the HIF1. Interestingly, the HIF1 mRNA expression level did not parallel the protein expression (figure 3). The results of the qRT-PCR show that all sequences where expressing similar levels of mRNA. Given the levels of HIF1 proteins levels observed in figure 2, it is assume that cell strains 2358 and 2360 were successfully decrease the expression of the HIF1 . These results were also supported by the fact that the expression of the BNIP3 mRNA was also decreased after six hours of cobalt treatment (Fig. 4A) in the same sequences compared to the scrambled control. The BNIP3 gene is a known HIF1 target gene and its expression is correlated with the level of HIF1 activation in the cells. These results suggest that the level of HIF1 knockdown did impact the cells ability to respond to cobalt-induced hypoxia signaling. However, when another target gene, VEGF, was assessed, the results show that strain 2358 was the most expressed in both 6 (Fig. 4C) and 24 (Fig. 4G) hours. These results do not parallel the BNIP3 expression. Interestingly, the expression of the VEGF in the 2359 and 2360 strains was highly increased after the 24 hours challenge while the others decreased. Interestingly, VEGF has been linked to asthma etiology and this increased expression in the cells expressing HIF1 might offer clues to what is happening within the transgenic mice. Though it does not follow classic hypoxic responses, it offers a possible explanation for the change in immunity observed in mice lacking HIF1 in their ATII cells. It is known

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that the exposure of A549 cells to cobalt for 24 hours promotes the activation of certain genes while highly suppress others (Li, Ke, & Costa, 2009). Taken together the results from the western blot (figure 2) and the target genes expression (figure 4), sequence 2360 seems to be the most effective in decreasing the HIF1 expression. Therefore, the cell strains expressing less HIF1 protein was then used to characterize the transcription factors role in modulating cytokine expression following cobalt exposure. Each cell strains and the control lines were exposed to cobalt for 6 or 24 hours. We evaluated by means of qRT-PCR the expression of the cytokines, interleukins 5 and 10, which play a role in asthma (Chung, 2001; Greenfeder, Umland, Cuss, Chapman, & Egan, 2001). Our results indicate that the cobalt-induced expression of these cytokines was altered upon changing HIF1 levels, however, their expression did not perfectly correlate with the level of HIF1 protein found. After the six hours (Fig. 4C and D) of cobalt challenge all the cell strains showed a decrease in comparison with the scrambled control. Importantly, the cell strains expressing sequences 2357 and 2359 did not display a similar increase in cytokines as the scrambled control, even though these strains express equal levels of HIF1 protein. This discrepancy makes us speculate that the mixed population in these cells affected the expression of the evaluated genes. Interestingly, as in the others genes expression assays, the strain 2360 increased their expression of both interleukins only after the 24 hours (Fig. 4G and H) challenge. Our preliminary results partially support our hypothesis. In particular, taken together, the data suggests that a longer exposure to cobalt is necessary for changes in cytokine expression profiles in HIF1-deficient ATII cells to become evident. However, the lack of inducibility in two of the cells strains expressing normal levels of HIF1 and the notable decrease of gene expression of some of the strain raise some concern. To be able to reach a definitive conclusion, further studies are necessary. To acquire more precise data, only the cells that express a decrease in the HIF1 should be test with different variables. As an example, exposing the cells to hypoxia could give concrete results. Additionally, to analyze their inflammation response, lipopolysaccharide can be used due to their capacities as an inflammation inducer. If the new results exhibit the

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expected change in cytokine profiles, co-culture the HIF1-deficient A549 cells with alveolar macrophage or dendritic cell lines to evaluate their immune response following challenge. There are a lot of questions regarding the HIF that needs to be answer. The purpose of our study was to add knowledge and to have a better understanding of the role that this protein could play in various diseases, especially in the asthma. To accomplish this goal there is a lot of work and research that have to be done.

Acknowledgments
Student support provided by NIH R25 HL103156 grant and the 2013 Undergraduate Student Summer Research Program in the Biomedical Sciences at Michigan State University.

References
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