The mammalian intestine contains many different cell types but is comprised of 2 main cell types: epithelial cells and smooth muscle cells. Recent in vivo and in vitro evidence has revealed that various alterations to the DNA methylation apparatus within both of these cell types can result in a variety of cellular phenotypes including modified differentiation status, apoptosis, and uncontrolled growth. Methyl groups added to cytosines in regulatory genomic regions typically act to repress associated gene transcription. Aberrant DNA methylation patterns are often found in cells with abnormal growth/differentiation patterns, including those cells involved in burdensome intestinal pathologies including inflammatory bowel diseases and intestinal pseudo-obstructions. The altered methylation patterns being observed in various cell cultures and DNA methyltransferase knockout models indicate an influential connection between DNA methylation and gastrointestinal cells’ development and their response to environmental signaling. As these modified DNA methylation levels are found in a number of pathological gastrointestinal conditions, further investigations into uncovering the causative nature, and controlled regulation, of this epigenetic modification is of great interest.
The mammalian intestine is a complex organ containing of a diverse array of cells that have their genesis in all 3 germ layers1 and demand careful coordination between each unique cell type in order to accomplish the absorption of dietary nutrients and water, expulsion of waste through peristaltic contractions, as well as provide a physical barrier to pathogens.2 The 2 most prominent cellular populations of the intestine are the epithelial and smooth muscle populations. Epithelial cells begin their development as Wnt responsive Lgr5+ stem cells at the base of intestinal crypts3 and differentiate into mature, absorptive Alpi+, Lgr5− enterocytes (ME)4 as they move up the villus until they arrive at the villus tip where they undergo apoptosis and extrude themselves into the lumen while the protective barrier is maintained under physiological conditions.5–7 These epithelial cells have an incredibly fast turnover rate of 2 to 5 days.1,8 Due to the rapid turnover and differentiation observed in intestinal epithelial cells, they have become prime targets for the study of differential expression based on changes in modulable epigenetic mechanisms, especially DNA methylation, as both global and site-specific changes in DNA methylation levels are hallmarks of differentiating cells.9–13 Several recent in vivo studies have indeed found that DNA methylation levels change at various genomic sites during both development and differentiation of intestinal epithelium from Lgr5+ stem cells into fully mature ME.14–18 Many of these changes strongly correlate with DNA methylation levels found in human disease states such as inflammatory bowel diseases,19–22 and certain colorectal cancers.23–27 Variable and characteristic patterns of genomic DNA methylation that change under various phenotypic conditions are also found in another cell in the gastrointestinal (GI) tract: smooth muscle cells (SMC).
Surrounding the epithelium, and separated by a submucosal region, are 2 perpendicular layers of smooth muscle (circular and longitudinal) that produce peristaltic movement via calcium initiated, actin-myosin contractions28,29 when stimulated by neuronal input mediated through the pace making interstitial cells of Cajal.30,31 Unlike most terminally differentiated somatic cells, SMC have a unique plasticity in which they are able to transition between a mature, contractile state, and a synthetic, proliferative, more stem cell-like state.32,33 Of note, a similar type of dedifferentiation plasticity has recently been observed in villus epithelium.4 When comparing the 2 states in SMC, mature SMC express high levels of proteins necessary for contractility such as MYH11, ACTA2, and TAGLN and have low rates of proliferation while synthetic SMC have higher rates of proliferation, lower levels of contractile proteins, produce high levels of extracellular matrix33–35 and have lowered levels of the necessary microRNAs (miRNAs), miR-143/145.36 These synthetic SMC are no longer functionally contractile and proliferate in response to injury and begin to transition to a more differentiated state once the tissue repair has been accomplished.37 This plasticity and ability to proliferate is important for tissue repair but does carry with it the potential for SMC to improperly regulate the dynamic differentiation and growth process. Aberrant growth patterns of SMC in the GI tract is associated with burdensome GI diseases such as megacystis-microcolon-intestinal hypoperistalsis syndrome38–40 and intestinal pseudo-obstructions.41,42 The combination of plasticity and the potential for the dysregulation of growth/differentiation patterns make SMC a strong candidate for phenotypic alteration through epigenetic mechanism manipulation. In this vein, several research teams have been able to manipulate the phenotypic status of SMC through alteration of DNA methylation mechanisms and enzymes,43–48 similar to previously mentioned research into DNA methylation dynamics in intestinal epithelium.
Higher levels of genomic cytosine methylation are regularly associated with gene inactivation or silencing, especially when 5-methylcytosine (5-mc) nucleotides are within promoters.9,13 It is well established that the methylation of cytosines occurs through the transfer of a methyl group from
The GI tract begins its development at the gastrulation stage in early embryonic development. When DNA methylation is inhibited at this early developmental stage in zebrafish embryos through use of cytidine analogs (5-azacytidine, 5-aza and 5-aza-2′ deoxycytidine, 5-aza-dC), which non-selectively inhibit all DNMT isoforms and induce hypomethylation, gastrulation does not proceed and muscle progenitors do not organize as expected,59 indicating the shared importance of DNA methylation for both epithelial (endoderm) and muscle tissue (mesoderm). As it pertains to intestinal epithelium, there are 2 differentiation states of focus in relation to cellular identity: intestinal epithelial stem cells (IESC) and mature ME. IESC are found at the base of intestinal crypts in close contact to supportive Paneth cells, express
Inflammatory bowel disease (IBD) encompasses 2 related but different pathologies affecting the intestinal epithelium that both manifest in pro-inflammatory conditions: Crohn’s disease (CD) and ulcerative colitis (UC). In general, both IBD conditions arise in genetically susceptible individuals whose GI mucosa fails to maintain barrier integrity allowing for infiltration of microbiota and other environmental factors that initiate the recruitment of immune cells to the affected area causing the phenotypic inflammatory condition.80 UC is restricted to the colon while CD can occur anywhere along the GI tract. As both IBD diseases are of unknown etiology, most researchers in the field have turned their attention to establishing genetic, epigenetic, and environmental links to the disease states themselves through forward screening techniques.22,81–83 Both diseases states have commonalities in regards to symptom manifestation and genetic loci that are most often associated with immune regulation83,84 as well as a the dysbiosis of the gut microbiota.85 Many of the genes found to be commonly dysregulated, with correlating aberrant methylation patterns, in the genetics screens of UC affected tissue are associated with regulating apoptosis (
Much of the initial focus that has been given to DNA methylation in SMC has been centered on vascular (vSMC) or airway SMC (aSMC) as these cell types are associated with common, and costly, chronic conditions such as atherosclerosis and asthma, respectively. SMC have an uncommon ability, known as plasticity, to shift between a mature, contractile state and a more proliferative, synthetic condition that is dynamic based on environmental and genetic conditions.33 The more proliferative SMC state is phenotypically similar to that of the less-differentiated myofibroblasts through the production of extracellular matrix.37 This shift between states of differentiation has repeatedly been shown to be inextricably linked to, and often regulated by, changes in DNA methylation at various genomic elements.13,97,98 Early studies on cells at various developmental stages of SMC differentiation (fibroblasts, myofibroblasts, and SMC), made use of cytidine analogs (5-aza and 5-aza-dC) to inhibit DNMT activity under in vitro conditions.44,46 Regardless of differentiation status, cytidine analogs consistently interfered with proper differentiation. Hepatic stellate cells were not able to transdifferentiate into myofibroblasts when exposed to 5-aza-dC.78 Cultured aSMC were not able to alter their phenotypic status when subjected to 5-aza-dC treatment even when the cells were induced to phenotypically switch by the addition of PDGF.46 Phenotypic switching in vSMC was revealed to be regulated by the presence of miR-1298 whose expression is directly dependent on levels of DNA methylation.99 In contrast to mature SMC, when cultured fibroblasts were exposed to 5-aza-dC they began to express higher levels of α-SMA, indicating the maturing of fibroblasts towards mature SMC.44 Recently, high passage primary human intestinal SMC (iSMC), known for a lack of contractile protein expression compared to lower passage cells, were found to restore their contractile protein expression upon treatment of 5-aza.77 Collectively, these results emphasize the importance of DNA methylation to phenotypic switching in SMC of various origin. Unfortunately, the preponderance of these studies were done in vitro and not on iSMC, and thus, these conclusions needed to be tested in vivo for confirmation across experimental conditions. In order to rectify this, a murine model was employed to show the first in vivo evidence regarding the importance of DNA methylation in the development of iSMC.43 In a smooth muscle restricted (
While SMC and EC diverge in developmental lineages early in the embryo, they share the interdependent physiological goals of GI motility and absorption and thus the fate of one cell type will likely affect the other cell type’s homeostasis. Despite epithelial cells being endoderm derived and SMC mesoderm derived, they do share congruous characteristics. As previously mentioned, 5-aza will halt all gastrulation processes that would lead to the development of both mature SMC and epithelial cells in the absence of 5-aza,59 indicating both cell types require precise methylation patterns in order to properly develop. Additionally, when
The GI tract is a massively complex organ system that, in varying cell types, relies on careful regulation of DNA methylation patterns in order to develop or differentiate as needed. Careful, and conditionally dependent, coordination of DNA methylation patterns and locations is crucial to ensure necessary growth and differentiation as well as when to halt these same processes (Fig. 2). As aberrant DNA methylation patterns, or altered DNMT levels, have been useful in understanding and treating pathologies in the intestinal epithelium, similar strategies should be employed for SMC disease states in the GI tract.