Heme oxygenase-1 (HO-1) is a stress-inducible protein present in many mammalian cell types. Various stimulants can induce upregulation of HO-1 protein expression, including hemolysis, inflammatory cytokines, and oxidative stress.1,2 HO-1 is the rate-limiting enzyme of heme degradation to free iron, carbon monoxide and biliverdin, which is readily converted into bilirubin.1,3 Induction of HO-1 upregulation has been reported to reduce tissue damage in organs such as the brain,4 lung,5 and liver.6 Caco-2 intestinal cells can transform normal intestinal epithelium cells, and Caco-2 cell monolayers have been well established as an in vitro model for the study of the normal intestinal epithelium as an intestinal barrier.7 As the intestinal epithelium displays rapid cell proliferation and differentiation and is an important immune regulatory organ,8 we previously investigated the effect of HO-1 activity on hypoxia inducible factor-1 (HIF-1) in a rat liver transplantation model and showed that the activity of HO-1 could be induced by transplant operation. HO-1 increases the survival rate after liver transplantation, and is related to the reduction of apoptotic ratio of hepatocyte and improves hepatic function; upregulation of HO-1 can promote the expression of HIF-1, and in turn, suppression of the expression of HO-1 can reduce the activity of HIF-1.9,10 We hypothesized that the HO-1 gene (heme oxygenase 1, HMOX1) may play an important role in Caco-2 cells. In the present study, to better understand the molecular mechanisms by which HMOX1 is altered in Caco-2 cells, we constructed a recombinant adeno-associated virus plasmid containing HMOX1, and transfected it into Caco-2 cells. We evaluated the effects of HO-1 upregulation in Caco-2 cells and demonstrated that HO-1 upregulation can significantly promote cellular proliferation and migration by targeting the CTNND1 gene.
The Caco-2 cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and was maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Invitrogen Ltd., Carlsbad, CA, USA) containing 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 10% (v/v) fetal bovine serum (FBS; GIBCO) that had been heat-inactivated for 30 minutes at 56°C, 100 U/ml penicillin, and 50 μg/ml streptomycin, and incubated at 37°C under 5% CO2-95% air atmosphere. Cells between passages 17 and 25 were used. All experiments were performed in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee of the Provincial Hospital Affiliated to Shandong University.
Analysis of gene expression using quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted using Trizol solution. Reverse transcription (RT) was performed in a 20 μl reaction system according to the manufacturer's recommendation. Gene transcript expression levels were determined by quantifying the intensity of the PCR product normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Quantitative measurement of gene mRNA levels was performed using an ABI Prism 7000 (Applied Biosystems, Foster City, CA, USA). RT-PCR primers are summarized in Table 1. Data were analyzed with the comparative CT method.
PCR for amplification of the HO-1 gene
A pair of PCR primers was designed using Primer 5.0 software according to the published sequence of human genome in GenBank (NM_002133). Primers are summarized in Supplementary Table 1. The primers contained BgLII and HindIII restriction sites at their 5'-termini, respectively. Total human genomic DNA was extracted from intestinal tissue. The amplification was performed in a 50 μl reaction mixture containing 5 μl 10×PCR buffer, 4 μl dNTP (each 2.5 mmol/L), 50 pmol of each primer, 0.25 μl Pyrobest DNA polymerase (5 U/μl) (Japan) and 6 μl extracted DNA. Reactions were run in a thermocycler (Techgene, UK) with the following program: denaturation at 95°C for 5 minutes, 35 cycles composed of denaturation at 95°C for 1.5 minutes, annealing at 54 °C for 1 minute, and extension at 72°C for 2 minutes. Finally, extension was carried out at 72°C for 10 minutes.
Recombinant adenovirus plasmid construction
The product of PCR amplification of the HO-1 gene was inserted into pMD18-simple T vector (TaKaRa, Japan) and then cloned into the transfer vector pShuttle-CMV (Qbiogene) using BgLII and HindIII (BioLabs, UK); the resulting vector was designated pShuttle-CMV-HO. The recombinant plasmid was identified by PCR, BgLII, and HindIII restriction enzyme digestion and sequencing. The plasmid was then linearized with PmeI (BioLabs) and transformed into Escherichia coli strain BJ5183 (Stratagene, CA, USA) competent cells using the pAdEasy-1 skeleton vector (Stratagene) with a Bio-Rad Gene Pulser at 200, 2.5 kV, and 25 F conditions for recombination; the plasmid was designated pAd-HO. The transformed bacteria were inoculated onto LB plates with kanamycin at 50 μg/ml. The recombinant plasmid was obtained by selecting blue colonies and identified by PCR and PacI (BioLabs) digestion.
Transfection and isolation of the recombinant adenovirus
To obtain recombinant adenovirus, human embryonic kidney 293 (HEK293) cells were cultured in DMEM (Invitrogen) supplemented with 3.7 g sodium bicarbonate/1 and 10% FBS (Hyclone, USA). The recombinant adenovirus plasmids were linearized with PacI and then mixed with Lipofectamine 2000 transfection reagent (Invitrogen). The recombinant adenovirus plasmids were inoculated into HEK293 cells for casing; the cells were incubated with 5% CO2 at 37°C for 3-7 days. The virus, designated rAd-HO, was propagated in HEK293 cells, collected at the appearance of a cytopathic effect, and then purified three times using the plaque test. The titer of the virus was determined by the method of 50% tissue culture infectious dose (TCID50) after passage in HEK293 cells.
For transduction, Caco-2 cells were infected with either control lentivirus or pShuttle-CMV-HO lentivirus. After 48 hours of transduction, the cells were harvested and prepared for subsequent studies. Cells were plated in 24-well plates (2×104 cells/well) overnight. The lentiviruses were diluted in 0.2 ml (108 TU/ml) complete medium containing polybrene (5 μg/ml) and added to the cells for 12 hours incubation at 37°C, followed by incubation in 0.3 ml of freshly prepared polybrene-RPMI 1640 for another 24 hours, which was then replaced with fresh RPMI 1640 medium and the cells were cultured for a further 48 hours period. The level of HO-1 expression in the cells was assayed by western blotting and qPCR 48 hours after transfection, as described above.
Subcellular localization of HO-1 expression in Caco-2 cells using fluorescence microscopy
Caco-2 cells were seeded in a 35-mm dish (Falcon) containing a coverslip coated with poly-D-lysine (Sigma). For permeabilized staining, 24 hours post-seeding cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with methanol at 4°C. The cells were blocked in 5% skimmed milk diluted in phosphate-buffered saline (PBS) and incubated in 5% skimmed milk/PBS containing a 1:100 dilution of mouse anti-HO-1 antibody (Abcam) at room temperature for 45 minutes. The cells were then washed in PBS three times followed by incubation with Cy3-conjugated donkey anti-mouse IgG (Jackson Immunologicals) at room temperature for 30 minutes. Then, the cells were washed in PBS three times followed by incubation with DAPI. The slides were mounted and observed under a fluorescent microscope (Olympus, Japan).
Cell proliferation assay
Caco-2 cells (3×103 cells/well) were incubated with 100 μl of culture medium in 96-multiwell plates, for 24 hours at 37°C in 5% CO2. The cells were transfected with pShuttle-CMV-HO or negative control for 0, 24, 48, 72, 96, or 120 hours. Cell numbers were assessed using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Briefly, 10 μl CCK-8 was added to each well. After 1 hour incubation at 37°C, absorbance at 450 nm was measured with an ARVO MX plate reader (PerkinElmer, MA, USA). Cell numbers were determined by relative absorbance at 450 nm. All experiments were performed in triplicates.
Scratch healing and migration assays
For the scratch assays, cells were treated with 10 mg/ml mitomycin C (Sigma) for 3 hours and then wounded with a pipette tip. Fresh, complete medium was added, and wound closure was observed for 48 hours. Photographs were taken every 6 hours. For migration assays, cell culture was performed in Transwell chambers (8 mm, 24-well format; Corning, NY, USA). Cells (2×105) were added to the upper chamber and cultured for 48 hours. For the migration assay, the insert membranes were not coated with Matrigel. Finally, the insert membranes were cut and stained with crystal violet (0.04% in 100 ml water) and permeating cells were counted and photographed under an inverted microscope. At least three independent experiments were performed for all conditions.
Cells and tissues were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) containing a cocktail of proteinase inhibitors (Bio-Rad). The lysed proteins were quantified using a bicinchoninic acid protein assay kit from Pierce. Subsequently, equal amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). Nonspecific binding sites were blocked by incubating with 5% nonfat milk in TBST buffer (TBS plus 0.1% Tween 20). The blots were washed with TBST and then incubated with a specific primary antibody overnight at 4 °C. The blots were again washed with TBST and then incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours at room temperature. Proteins were visualized with an enhanced chemiluminescence detection system (Amersham, Freiburg, Germany). Autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad). As a loading control, GAPDH-specific antibody (Sigma, St. Louis, MO, USA) was used. Relative protein levels were calculated by referring them to the amount of GAPDH protein (Sigma). The mean values from three independent experiments were taken as results. Monoclonal antibody recognizing HO-1 was purchased from Abcam (Shanghai, China).
Global cDNA microarray analysis and targetCTNND1gene verification
A whole human genome oligo-microarray (Agilent, Santa Clara, CA, USA) was used. After hybridization and washing, the microarray slides were scanned with an Agilent DNA microarray scanner. The resulting text files extracted from Agilent Feature Extraction Software (version 9.5.3) were imported into the Agilent GeneSpring GX software (version 7.3) for further analysis. Differentially expressed genes were identified through fold-change screening. For target CTNND1 gene verification, we used qPCR and chromatin immunoprecipitation assay. The primers for the target genes are listed in Table 1.
Chromatin immunoprecipitation assay
The assay was performed using the EZ ChIPTM Chromatin Immunoprecipitation Kit (Upstate Biotech, Waltham, MA, USA) in accordance with the manufacturer's instructions. Briefly, the cells were fixed with 1% formaldehyde for 10 minutes at 37°C, and nuclear extracts were obtained and sonicated to produce DNA of about 300 bp. Protein-DNA complexes were immunoprecipitated with anti-HO-1 antibody or normal rabbit IgG (negative control). The immunoprecipitated DNA was then purified and eluted with 50 μl of elution buffer. PCR amplification was performed using 2 μl of DNA sample with different sets of primers detailed in Table 1. Amplification of soluble chromatin prior to immunoprecipitation was used as an input positive control. qRT-PCR was carried out on eluted DNA with the same primer sets using Power SYBR Green PCR Master Mix (Applied Biosystems, USA) according to the manufacturer's instructions, and analyzed with an ABI Prism 7000 Biosystem machine (Applied Biosystems).
Statistical analysis was performed using SPSS16.0 software (SPSS Inc., USA). Data are expressed as the mean ± standard deviation from at least three separate experiments. Differences between groups were analyzed with Student's t test and χ2 test. A value of P <0.05 was considered statistically significant.
Construction and analysis of HO-1 gene recombinant adenovirus plasmid
To address the role of HO-1 in intestinal ischemia/reperfusion injury, the PCR amplification product of the HO-1 gene was inserted into pMD18-simple T vector and then cloned into the transfer vector pShuttle-CMV (Figure 1A, B). Then we transfected Caco-2 cells as confirmed by Cy3 fluorescence in the cytoplasm and as visualized by fluorescence microscopy (Figure 1C). After transfection, we examined HO-1 expression in Caco-2 cells using qPCR and western blotting. In HO-1 plasmid-transfected Caco-2 cells, HO-1 expression was significantly increased compared with pS-control transfectants (P <0.05; Figure 1D).
Ectopic expression of HO-1 promotes proliferation and migration of Caco-2 cellsin vitro
To study the effects of HO-1 upregulation on Caco-2 cell proliferation and migration, we transfected Caco-2 cells with HO-1 expression plasmids. Cell proliferation was significantly promoted compared with pS-control transfectants (P <0.05; Figure 2A). We also examined cell migration ability using a scratch healing assay, and migration using a Transwell invasion assay. HO-1 overexpressing transfected cells had closed the wound after 48 hours, whereas control-transfected cells were virtually unable to heal the wound. At 48 hours, mean wound distances of the experimental sample ((17.25±7.31) μm) and the control ((126.37±11.14) μm) were significantly different (P <0.01, Figure 2B). The number of migrating HO-1-overexpressing transfected cells (362.16±4.16) was significantly greater than controls (57.14±2.69; P <0.01, Figure 2C).
Identification of target genes after HO-1 gene transfection
We analyzed the genome-wide transcriptome profile of pS and pS/HO cells by Agilent oligo-microarray. The integrity of RNA was assessed by electrophoresis on a denaturing agarose gel (Figure 3A). A correlation matrix describes correlation among replicate experiments. A scatter-plot is useful for assessing the reproducibility between chips (Figure 3B). According to fold-change (≥2.0) screening between pS and pS/HO cells, we found 227 upregulated genes and 146 downregulated genes (not shown). Based on these differentially expressed genes, a tree with a clear distinction between the pS and pS/HO cells with Caco-2 cells was generated by cluster analysis (Figure 3C).
CTNND1gene is a target of the HO-1 gene
We chose to downregulate the CTNND1 gene for qPCR verification and to confirm our microarray findings (Figure 1A). To determine whether CTNND1 is a direct target gene of the HO-1, we conducted chromatin immunoprecipitation assays using HO-1 antibody and then analyzed the pulled-down DNA. We identified a downregulated gene, CTNND1 (NM_001085461), as a direct HO-1 target (Figure 1B).
HO-1 is a cytoprotective enzyme that breaks down heme to produce carbon monoxide, iron, and biliverdin.11,12 HO-1 is induced by multiple stimuli including oxidative stress, and pro-inflammatory cytokines, and has been shown to be upregulated in the lungs following mycobacterial infection.11,13 HO-1 overexpression exerts cytoprotection and ameliorates cellular damage in various experimental models.14-16 However, the precise mechanisms by which HO-1 may induce its cytoprotective effects have not been fully clarified. The beneficial effects of HO-1 are likely dependent on the end-products of heme degradation.17,18
In this study, we first constructed an HO-1 recombinant adenovirus plasmid. To evaluate the potential role of the HO-1 gene, we chose Caco-2 intestinal cells for gain-of-function assays, which can transform normal intestinal epithelium cells. Caco-2 cells are well established as an in vitro model for the study of the normal intestinal epithelium as an intestinal barrier. Our results showed that ectopic expression of HO-1 promotes Caco-2 cell proliferation and migration in vitro.
Microarray is a power technology that is able to perform genome-wide analysis in one experiment. Gene expressing profiles can characterize genes that are differentially regulated in different experimental conditions.8,19 In this study, we analyzed the genome-wide transcriptome profile of Caco-2 cells by Agilent oligo-microarray, and compared changes in gene expression profiles with or without HO-1 gene transfection to identify HO-1 target genes. According to fold-change (≥2.0) screening between pS and pS/HO cells, we found 227 upregulated genes and 146 downregulated genes. Based on these differentially expressed genes, a tree with a clear distinction between the pS and pS/HO cells was generated by cluster analysis. Subsequently, we identified a direct HO-1 target gene using qPCR and chromatin immunoprecipitation analysis. We identified a downregulated gene, CTNND1 (NM_001085461), as a direct HO-1 target; this gene encodes a member of the Armadillo protein family, which functions in cell adhesion and signal transduction.20,21 Wang et al22 noted that FRMD5 may play a role in CTNND1-based cell-cell contact and is involved in the regulation of tumor progression. Alcaide et al23 found that CTNND1 overexpression inhibited neutrophil leukocyte transendothelial migration independently of an effect on RhoA or Rac and instead blocks leukocyte transendothelial migration by preventing VE-cad tyrosine phosphorylation and association of active Src with the VE-cad complex. However, HO-1 is a well-conserved stress protein that protects organs, tissues, and cells from harm resulting from stimulating factors and pathological processes.24-27 HO-1 expression and activity are closely related to growth, apoptosis, angiogenesis, invasiveness, and metastasis of solid tumors.28 Induction of HO-1 by CoPP administration before intestinal ischemia/reperfusion injury resulted in a significant reduction of intestinal tissue injury.29,30 Development of strategies to induce HO-1 upregulation will be important to reduce intestinal ischemia/reperfusion injury in a clinical setting.16,31 Our results showed that HO-1 promotes proliferation and migration in Caco-2 cells by targeting the CTNND1 gene.
In summary, using gain-of-function assays, we found upregulated HO-1 expression promoted cell proliferation and migration in vitro. We identified a number of target genes by global microarray analysis combined with qPCR and chromatin immunoprecipitation assay after HO-1 transfection. CTNND1 gene, a member of the Armadillo protein family, was identified as a direct HO-1 target gene. Therefore, further understanding of the molecular mechanisms by which HO-1 upregulation exerts its effects might allow for the implementation of targeted clinical strategies.
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Keywords:© 2013 Chinese Medical Association
heme oxygenase-1; Caco-2 cells; microarray; CTNND1