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
The suppression of mRNA and protein expressions of the pore-forming α1C
subunit of Cav
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 Cav
1.2b 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 Cav
1.2b 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.