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Patients with inflammatory bowel disease who are in remission and those that developed inflammatory bowel syndrome after enteric infection continue to have symptoms of diarrhea or constipation in the absence of overt inflammation, indicating motility dysfunction. We investigated whether oxidative stress during inflammation impairs integrity of the promoter of Cacna1c, which encodes the pore-forming α1C subunit of Cav1.2b calcium channels.
We used long-extension PCR (LX-PCR) to evaluate DNA integrity in tissues from distal colons of rats; trinitrobenzene sulfonic acid (TNBS) was used to induce inflammation.
H2O2 increased in the muscularis externa 1 to 7 days after inflammation was induced with TNBS. The oxidative stress significantly impaired DNA integrity in 2 specific segments of the Cacna1c promoter: −506 to −260 and −2,193 to −1,542. The impairment peaked at day 3 and recovered partially by day 7 after induction of inflammation; expression of the products of Cacna1c followed a similar time course. Oxidative stress suppressed the expression of Nrf2, an important regulator of anti-oxidant proteins. Intra-peritoneal administration of sulforaphane significantly reversed the suppression of Nrf2, oxidative damage in the promoter of Cacna1c, and suppression of Cacna1c on day 7 of inflammation. The inflammation subsided completely by 56 days after inflammation was induced; however, impairment of DNA integrity, expression of Nrf2 and Cacna1c, and smooth muscle reactivity to acetylcholine remained suppressed at this timepoint.
Oxidative stress during inflammation impairs the integrity of the promoter of Cacna1c; impairment persists partially after inflammation has subsided. Reduced transcription of Cacna1c contributes to smooth muscle dysfunction in the absence of inflammation.
Oxidative stress induced by aerobic metabolism continually causes about 100,000 to 1,000,000 DNA lesions in cells of an organism per day1. DNA repair enzymes detect and fix these lesions (primarily base excision) to maintain homeostasis2. In addition, pathogenic DNA damage occurs when reactive oxygen and reactive nitrogen species generated due to inflammation, UV radiation, pollutants, dietary components, overwhelm the endogenous repair mechanisms and impair genomic integrity. The susceptibility to these lesions and the capability of repair mechanisms to fix them are cell-type and DNA sequence-specific3, 4. The accumulation of DNA lesions is deleterious to cellular functions and organism’s survivability. Depending upon the location of the lesions in the genome, the loss of DNA integrity might result in the production of mutant/inactive proteins, causing senescence, apoptosis, unregulated cell division, or aberrant transcription of genes5. The failure of cells to maintain DNA integrity is a major contributory factor to diseases, such as cancer, Alzheimer’s disease and cystic fibrosis6, 7.
Numerous studies show that persistent or recurring inflammation in the gut- H. pylori infection, ulcerative colitis and reflux esophagitis- leads to cancer in the rapidly dividing epithelial cells8, 9. However, we know very little about the effects of inflammation on DNA integrity in terminally differentiated cells, such as smooth muscle cells in the gut. There is evidence of long-term impairment of their function after inflammation has subsided in at least two prominent conditions. 1) A sizable percent of IBD patients during remission have the classic symptoms of irritable bowel syndrome (IBS) (diarrhea/constipation and intermittent abdominal cramping)10–14. 2) Severe enteric infection, individually or in a community setting accompanied, with comorbid psychological conditions, results in IBS-like symptoms in a subset of the affected subjects (post-infectious IBS; PI-IBS)15–20. In both conditions, the classic inflammatory response is absent when the patients have these symptoms.
Some reports indicate, however, that the colonic mucosal biopsies in these patients show a low-grade inflammation comprised of an increase in T lymphocyte count in the lamina propria and/or epithelium16, 21 and increase in IL-1β mRNA in rectal mucosa22. These studies proposed that the low-grade mucosal inflammation is the cause of IBS-like symptoms – abnormal bowel function. However, there is no known mechanism by which inflammation in mucosal cells could alter the function of smooth muscle and enteric neuronal cells located at the other end of the colon wall. Significant inflammatory infiltrate in the microenvironment of the cells is required to alter their function.
Clinical and experimental studies show that oxidative stress is transmural in IBD23, 24. In this study, we tested a novel hypothesis that oxidative stress during active inflammation in the muscularis externa impairs the integrity of DNA in colonic smooth muscle cells. The impairment of DNA integrity persists after resolution of oxidative stress/inflammation, which causes persistent smooth muscle dysfunction. We tested this hypothesis by inducing inflammation in the rat colon with TNBS and examining the expression of Cacna1c, which encodes the pore-forming α1C-subunit of Cav1.2b (L-type) calcium channels in smooth muscle cells. Previous studies found that alterations in the expression of these channels play a significant role in smooth muscle dysfunction24–26. We found that oxidative stress generated by the inflammatory response causes DNA damage, which is not fully repaired for at least 56 days after the insult, resulting in smooth muscle dysfunction. The DNA damage persists in the absence of any overt inflammation/oxidative stress at that time.
Six to eight week old male adult rats were fasted for 24 hour before the induction of inflammation. During this period, regular water was replaced with Colyte® to cleanse the colon. Colonic inflammation was induced by intraluminal administration of TNBS (68 mg/kg in 250μl), dissolved in 40% ethanol. Age matched control rats received 0.9% saline. The rats were euthanized 1, 3, 7, 28, and 56 days after induction of inflammation.
Tables 1 and 2 in supplement materials show all primers. The primers for LX-PCR were designed to amplify the promoter of Cacna1c from −3,053 to +301 and promoter of β-actin gene from −2,480 to +288. Primers for rolling LX-PCR were designed to amplify 6 partially overlapping segments in Cacna1c promoter. The LX-PCR and rolling LX-PCR were performed with 15 ng genomic DNA, 400 nM primers, 300 μM dNTPs, and 5 units of LongAmp DNA polymerase (New England BioLabs, Ipswich, MA) in total volume of 50 ul. The PCR amplification was carried out in 30 cycles as follows; denaturation for 15 seconds at 94°C, 30 seconds at 58°C, and 2 minutes at 68°C. After completion of the cycles, the reaction mixtures were incubated for 7 minutes at 72°C. The PCR products were separated by electrophoresis on 0.8% agarose gels with ethidium bromode and visualized on a UV-transluminator.
The level of mRNA for each gene was measured with SYBR-Green– based real-time PCR. The cDNAs were diluted 1:10 with RNase/DNase free water to avoid saturation effect of template during PCR amplification. Each cDNA sample (7 ul of diluted cDNA) was amplified by using SYBR-Green PCR Master Mix according to the manufacturer’s instructions. PCR amplification was performed with Step-One Plus Real-time PCR system (Applied Biosystems, Austin, TX). The level of the housekeeping gene Rpl32 (L32) in each sample was used as an internal control.
Immunofluorescence staining was performed using 4 micron thick sections of formalin-fixed parrafin embedded tissues. After deparaffinization by sequential incubation in xylene and 100%, 95% and 85% ethanol, the samples were cooked with antigen retrieval buffer in a steamer for antigen retrieval. The samples were blocked with 10% donkey serum for 1 hour at room temperature. For co-immunofluorescence staining, the sections were incubated with anti-γH2AX antibody and Cy3™-conjugated smooth muscle α-actin overnight at 4°C in the dark, following incubation with Alexa488 anti-rabbit IgG for 1 h at room temperature in the dark. The sections were examined under fluorescence microscope.
For MPO assay, 100 mg of muscularis externae were homogenized in 20 mM phosphate buffer (pH 7.4) and centrifuged at 4°C for 10 minutes. The pellet was sonicated in 50 mM phosphate buffer (pH 6) containing 0.5% hexadecyl trimethyl ammonium bromide (HTAB) and centrifuged at 4°C for 5 minutes. The supernatant was used for MPO assay by incubation of 100 μl supernatant with 16 mM tetramethyl benzidine (TMB) in 50% ethanol, 0.3 mM H2O2 and 8 mM sodium phosphate buffer (pH 5.4) for 3 minutes. The level of MPO was measured by reading the absorbance at 655 nm in plate reader. Abasic site quantification in genomic DNA was assayed with DNA Damage Quantification Kit -AP site Counting- (Dojindo Molecular Tech, Gainthersburg, ND) by following manufacturer’s instructions. The level of hydrogen peroxide was measured with BIOXYTECH H2O2-560 Quantitative Hydrogen Peroxide Assay Kit (Cell biolabs Inc, Portland, OR) by following manufacturer’s instructions.
TNBS insult significantly increased the expression of GADD45G, a marker for DNA damage, on days 1, 3 and 7 of inflammation (Fig. 1A). LX-PCR showed time-dependent impairment of integrity of the promoter region (−3053/+301) of Cacna1c. The DNA damage (strand breaks) reached the maximum on day-3 of inflammation – and recovered partially by day-7 (Fig.1B). The integrity of the full Cacna1c promoter on day 56 post-inflammation was not different from that of age-matched controls. We used age-matched controls to avoid the possibility of any age-related changes in DNA integrity. Inflammation did not impair integrity of the housekeeping β-actin gene (Actb) (Fig.1C). In addition, TNBS insult did not cause DNA damage in the promoters of genes encoding CPI-17 and MLCK genes on day 1, 3 or 7 post-TNBS insult, suggesting specific damage to the Cacna1c (Fig. S2, supplement). We validated the LX-PCR method by using it to determine integrity of the mitochondrial genomic DNA in response to treatment of muscularis externae strips with 200 μM H2O2 for 30 minutes27. H2O2 decreased the DNA integrity of mitochondrial DNA to 43 ± 0.5 % of control, p<0.05 (Fig. 1D).
We then investigated whether inflammation impaired integrity of the entire promoter randomly or targeted specific segments, by dividing the ~3 kB (−3,053/+301) Cacna1c promoter into six slightly overlapping segments (Fig. 2A). We could not achieve uniform overlaps due to the presence of significant GC-rich sequences in segments 4 and 5. LX-PCR of individual segments on day-3 of inflammation showed damage of only segments three (−2,193/−1,542) and five (−506/−42) (Figs. 2B to 2D: data not shown for segments 2, 4, and 6). Segment six (−260/+301), which overlaps with segment 5 (−506/−42), showed no drop in the efficiency of PCR amplification. Therefore, we narrowed down the susceptible portion of DNA segment five to −506/−260 bps. Segment 3 had no overlap with its adjacent segments.
Loss of integrity of DNA in segment 5 was more rapid than in segment 3 (Figs. 2C and 2D). On the other hand, restoration of DNA integrity in segment 5 was slower than in segment 3, suggesting greater vulnerability of segment 5 than segment 3. Specifically, segment 3 DNA was fully repaired by day-56 post-inflammation, while the integrity of segment 5 was significantly less than in age matched controls (Figs. 2C and 2D).
The impairment of mRNA and protein expression of the Cacna1c followed similar time-course as the promoter (Figs. 1E and 1F). We noted that protein and mRNA levels of Cacna1c in the older control rats on day-56 post-inflammation were significantly greater than in younger control rats at the time of induction of inflammation. However, the protein and mRNA expressions of Cacna1c remained significantly suppressed on day-56 post-inflammation, compared with age-matched controls. In accord with this, we found that contractile response to ACh on day-56 was significantly suppressed, compared with age-matched controls (Fig. 3). In addition, the contractile response to 60 mM KCl, which depolarizes the colonic smooth muscle membrane to induce calcium influx through the Cav1.2b channels, was also smaller than that in age-matched controls.
The global level of apurinic/apyrimidinic (AP) sites, an indicator of base excision repair intermediate and DNA strand breaks was determined in genomic DNA extracted from the muscularis externa of the distal colon of TNBS rats on day-3 of inflammation and was found to be significantly greater than that in control rats (Fig. 4A). Immunofluorescence and Western blotting revealed significantly greater phosphorylated γ-H2AX level, an indicator of genomic double -strand breaks, in smooth muscle cells (Figs. 4B and 4C). We investigated whether there were temporal differences in the expression of genes involved in base excision and double strand break repair mechanisms (Supplement Fig. 1). The expression of genes for both repair mechanisms increased similarly from day 1 to day 7 of inflammation.
Hydrogen peroxide and myeloperoxidase activities in the muscularis externa peaked on day-1 of inflammation and declined thereafter. However, both activities remained significantly greater than in naïve controls on day-7 of inflammation. These activities did not differ between animals subjected to TNBS inflammation and their age-matched controls on day-56 post inflammation (Figs. 5A and 5B).
The nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a critical role in the manifestation of oxidative stress in inflammation28. We found that TNBS inflammation significantly suppresses the expression of Nrf2 from day-3 and it does not recover fully at least until day-56 post-inflammation (Fig. 5C). We investigated whether oxidative stress caused impairment of DNA in the promoter sequence of Cacna1c by administering (i.p.) 5 mg/kg sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane], an activator and inducer of Nrf2, daily for 3 days. In one series of experiments, sulforaphane treatment started 3 days prior to the TNBS insult and tissues were collected on day-3 post inflammation. In the second series, the daily administration of sulforaphane started on day 3 after the insult and tissues were collected on day-7 post-inflammation. In both cases, sulforaphane treatment significantly reversed the suppression of Nrf2, the instability of DNA in the promoter of Cacna1c and the suppression of Cacna1c on daty-7 of inflammation (Fig 6; data shown only for treatment starting 3 days prior to inflammatory insult). Prophylactic treatment with sulforaphane prevented the suppression of contractility by TNBS inflammation on day-7 post-TNBS insult (Fig. 7).
Inflammatory bowel disease (IBD) is recurring inflammation of the gut. It is a complex disease with multiple potential factors contributing to the spontaneous relapse of inflammation. Studies on human tissues and in experimental models of inflammation show an intense immune response comprised of oxidative and nitrosative stresses as well as release of myriad cytokines, chemokines and cell adhesion molecules at various levels and time courses. One of the adverse effects of these inflammatory mediators is impairment of the function of non-immune cells in the gut wall – smooth muscle cells, enteric neurons and epithelial cells, which regulate the motility and epithelial transport. Impairment of smooth muscle function significantly contributes to the morbid symptoms of diarrhea and constipation. Studies on human and animal tissues show that the inflammatory mediators inhibit smooth muscle reactivity to acetylcholine by transcriptional suppression of specific proteins of excitation-contraction coupling in smooth muscle cells24, 29. The impaired contractility of smooth muscle cells suppresses the generation of spontaneous rhythmic phasic contractions, which facilitates the rapid propulsion of fecal material in the colon by giant migrating contractions causing frequent bowel movements30, 31. However, the suppression of smooth muscle rhythmic phasic contractions in the absence of giant migrating contractions results in slower transit and constipation-like conditions32.
Our findings show that colonic inflammation induced by TNBS causes damage in the promoter region of Cacna1c starting at Day 1 of inflammation - reaching the maximum on day 3 and recovers partially by day 7. Further analysis with rolling LX-PCR showed that the DNA damage during active inflammation is confined to two segments of the promoter sequence, one located close to the core promoter (−506/−260) and the other upstream to it (−2,193/−1,542). The DNA damage in the upstream segment is repaired completely by 56 days after inflammation. However, the damage in the segment close to the promoter region persists at this time.
Most studies on DNA damage and repair mechanisms to date have focused in the coding regions of genes in proliferating cells because of their potential in initiating and promoting carcinogenesis. Our understanding of DNA damage and repair mechanisms in terminally differentiated cells is in its infancy. The investigation of these mechanisms is outside the scope of this work. However, one study in the literature points to a link between DNA instability in the promoter sequences and gene transcription. This study reported that the addition of OH· to the C-8 position of guanine within the recognition sequences of transcription factors, resulting in the formation of 8-oxydeoxy-guanosine (8-oxodG), might impair the binding of transcription factor to DNA33. The formation of 8-oxodG impaired the binding of AP-1 and Sp1 transcription factors to their cis-elements. However, 8-oxodG modifications in the NF-κB recognition sequence had no effect on its binding to the transcription factor.
DNA methylation of CpG islands can also suppress gene expression. The screening of Cacna1c promoter (−2910/+263 bp) with MethPath software (http://www.urogene.org/methprimer/index1.html) identified 4CpG islands. However, methylation specific PCR (MS-PCR) showed no change in the methylation status of any of the CpG Islands on the Cacna1c promoter (data not shown).
Our findings show that DNA damage and repair processes are sequence-specific. Oxidative stress induced by TNBS inflammation impaired DNA integrity in two specific segments of the promoter. In addition, the repair mechanisms effectively overcame the damage in one segment after inflammation subsided, but not in the other. We know very little about the mechanisms of this sensitivity. However, folate-sensitive (CGG)n sequences show susceptibility to 8-oxodG formation resulting in DNA instability34. Sequence analysis showed that segment five that is sensitive to DNA damage has the largest numbers of CGG sequences as a percent of total nucleotides (4% vs 0.2, 0.2, 0.9, 1.3, and 2.7% in segments 1, 2, 3, 4, and 6). It is noteworthy that the cis-elements of transcription factor Sp1 known as GC boxes [5′-(GT)GGGCGG(G/A)(GT)-3′] are rich in folate-sensitive sequences. This segment contains Sp1 binding motifs. Sp1 plays a critical role in the transcription of several genes35, 36. We speculate that impairment in the Sp1 cis-elements might contribute to the attenuated transcription of Cacna1c gene.
The suppression of mRNA and protein expressions of the pore-forming α1C subunit of Cav1.2b channels37 during and after inflammation followed a time course similar to that of DNA instability, which suggests that it contributes to impaired transcription of Cacna1c. Specifically, the DNA instability of segment 5 persisted for at least 56 days after induction of inflammation. The mRNA and protein expressions of the α1C subunit remained suppressed at this time, compared with those in age-matched controls. There was no evidence of inflammation at this time.
Other studies show that the density of voltage-dependent Ca2+ channels in hippocampal neurons increases with aging38, 39. We found that mRNA and protein expressions of the pore-forming α1C subunit increased significantly in control rats 56 days after inflammation, at which time they were 8 weeks older than at the time of inflammatory insult. However, in the presence of persistent DNA damage after the inflammatory insult the normal increase in the expression of α1C subunit was blunted. We do not know the effect of aging on the expression of Cav1.2b channels in human colonic myocytes. However, it is noteworthy that the IBS-like symptoms during remission in IBD patients and those in PI-IBS patients persist for several years after the onset of disease13, 40. Therefore, it is likely that changes in the age-related expression of Cacna1c as well as DNA instability due to prior inflammatory episodes together account for persistent smooth muscle dysfunction following inflammatory episodes.
Nrf2 is a basic leucine zipper (bZIP) transcription factor that serves as a central regulator of genes encoding antioxidant proteins and electrophile enzymes. In resting cells, Kelch like-ECH-associated protein 1 (Keap1) sequesters Nrf2 in the cytoplasm as an inactive complex41. The interaction of Nrf2 with Keap1 presents Nrf2 for ubiquitination and subsequent proteasomal degradation42, 43. Oxidative or nitrosative stress modifies this complex by several potential mechanisms, including thiol modification of Keap1 and phosphorylation of serine or threonine residues on Nrf2. Both mechanisms result in dissociation of the complex, accumulation of Nrf2 in the cytoplasm and eventual translocation to the nucleus44–49. Nrf2 forms heterodimers with a group of small musculoaponeurotic fibrosarcoma (Maf) proteins that lack transactivation domain28. Heterodimerization of Nrf2 with these proteins enhances the binding to a cis-acting enhancer ARE/EpRE located in the promoters of several genes encoding antioxidant proteins50–53.
We found that oxidative stress due to TNBS inflammation suppresses the expression of Nrf2 in the muscularis externa, which bottoms out on Day 3 and then recovers partially by day 7- the period during which integrity of the DNA comprising the Cacna1c promoter is impaired. Systemic administration of sulforaphane, an inducer and activator of Nrf2, prior to the induction of TNBS inflammation (prophylaxis) or after its induction (curative) significantly reduced the DNA instability as well as its biological effects on Cacna1c. Sulforaphane, present in cruciferous vegetables, exerts its chemopreventive and cytoprotective effects by the induction of phase 2 detoxifying and antioxidant enzymes through the induction of Nrf2 signaling. These findings suggest that DNA damage results due to the suppression of Nrf2.
Clinical findings show that the expression of antioxidant proteins in the inflamed segments of Crohn’s disease and ulcerative colitis patients is impaired54. Our findings suggest that this is likely due to the suppression of Nrf2. A notable finding in our study is that the partial, but significant, suppression of Nrf2 persists after inflammation has subsided. We speculate that persistent suppression of Nrf2 might exaggerate the inflammatory response to a subsequent insult resulting in greater DNA damage and organ dysfunction. It is noteworthy that sulfasalazine/masalazine, containing 5-ASA, used as a maintenance drug in IBD patients are radical scavengers that might restore balance between oxidants and antioxidants in the face of suppression of Nrf2 during remission.
The persistence of impaired motility function during remission in IBD patients (IBD-IBS) or that following an episode of severe inflammation (PI-IBS) remains an enigma. Some reports found a low-grade inflammatory response, comprised of increase in T lymphocytes in the lamia propria and epithelial layer in the mucosal biopsies of the IBD patients in remission12, 55 or in PI-IBS patients16, 21,22, 56, 57. However, the increase in T lymphocytes is not significantly different between patients who develop the symptoms of IBS and those who do not58. In addition, prednisone treatment significantly reduces lamina propria T-lymphocytes in PI-IBS patients, but it has no effect on the symptoms of diarrhea/constipation indicative of smooth muscle dysfunction21. Therefore, the low-grade mucosal inflammation is not the cause of smooth muscle dysfunction resulting in the symptoms of diarrhea/constipation in these patients. Our findings show that a single episode of inflammation causes persistent impairment of smooth muscle dysfunction due to persistent DNA instability in the Cacna1c promoter. In addition, the persistent low-grade inflammatory response after severe inflammation might be due to the persistent suppression on Nrf2 resulting in an imbalance in the oxidant-antioxidant response. The data on Nrf2 expression in the mucosa of IBD patients in remission or in PI-IBS patients are not available.
Human diseases are complex and multi-factorial. Animal models mimic only specific features of a human disease. Our model of TNBS inflammation shows that a single episode of robust colonic inflammation24 impairs the stability of specific segments of DNA comprising the promoter region of the Cacna1c gene. The stability of DNA recovers partially during active inflammation but remains significantly suppressed in long-term after inflammation has subsided. The impairment of DNA stability contributes to suppression of the pore-forming α1C subunit of Cav1.2b channels and hence smooth muscle contractility. This model does not mimic conditions under which the smooth muscle contractility is enhanced, resulting in diarrhea-like conditions59. Human patients are subject to environmental factors, such as chronic stress and medications that are absent in this model, but they might alter the outcomes.
Supported in part by NIDDK Grants DK 032346 and DK 072414 (SKS)
Study concept and design (SKS); acquisition of data (KC and JC); analysis and interpretation of data KC, SKS, JC and SM); drafting of the manuscript (SKS, KC); critical revision of the manuscript for important intellectual content (SKS, KC, SM) statistical analysis (KC); obtained funding (SKS); technical, or material support (SM)
There are no conflicts of interest to disclose for all authors.
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