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Gastrointestinal (GI) symptoms, such as diarrhea, abdominal pain, vomiting, and anorexia, are frequently observed in patients with coronavirus disease 2019 (COVID-19). However, the pathophysiological mechanisms connecting these GI symptoms to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections remain elusive. Previous studies indicate that the entry of SARS-CoV-2 into intestinal cells leads to downregulation of angiotensin converting enzyme 2 (ACE2) receptors resulting in impaired barrier function. While intestinal ACE2 functions as a chaperone for the amino acid transporter B0AT1, the B0AT1/ACE2 complex within the intestinal epithelium acts as a regulator of gut microbiota composition and function. Alternations to the B0AT1/ACE2 complex lead to microbial dysbiosis through increased local and systemic immune responses. Previous studies have also suggested that altered serotonin metabolism may be the underlying cause of GI disorders involving diarrhea. The findings of elevated plasma serotonin levels and high fecal calprotectin in COVID-19 patients with diarrhea indicate that the viral infection evokes a systemic inflammatory response that specifically involves the GI. Interestingly, the elevated proinflammatory cytokines correlate with elevated serotonin and fecal calprotectin levels further supporting the evidence of GI inflammation, a hallmark of functional GI disorders. Moreover, the finding that rectal swabs of COVID-19 patients remain positive for SARS-CoV-2 even after the nasopharynx clears the virus, suggests that viral replication and shedding from the GI tract may be more robust than that of the respiratory tract, further indicating fecal-oral transmission as another important route of viral spread. This review summarized the evidence for pathophysiological mechanisms (impaired barrier function, gut inflammation, altered serotonin metabolism and gut microbiota dysbiosis) underlying the GI symptoms in patients with COVID-19.

Functional gastrointestinal disorders (FGIDs), currently known as disorders of gut–brain interaction, are emerging microbiota–gut–brain abnormalities that are prevalent worldwide. The pathogenesis of FGIDs is heterogeneous and is intertwined with gut microbiota and its derived molecule‐modulated mechanisms, including gut dysmotility, visceral hypersensitivity, gut immune abnormalities, abnormal secretion, and impaired barrier function. There has been phenomenal progress in understanding the role of gut microbiota in FGIDs by underpinning the species alternations between healthy and pathological conditions such as FGIDs. However, the precise gut microbiota‐directed cellular and molecular pathogeneses of FGIDs are yet enigmatic. Determining the mechanistic link between the gut microbiota and gastrointestinal (GI) diseases has been difficult due to (i) the lack of robust animal models imitating the various aspects of human FGID pathophysiology; (ii) the absence of longitudinal human and/or animal studies to unveil the interaction of the gut microbiota with FGID‐relevant pathogenesis; (iii) uncertainty about connections between human and animal studies; and (iv) insufficient data supporting a holistic view of disease‐specific pathophysiological changes in FGID patients. These unidentified gaps open possibilities to explore pathological mechanisms directed through gut microbiota dysbiosis in FGIDs. The current treatment options for dysbiotic gut microbiota are limited; dietary interventions, antibiotics, probiotics, and fecal microbiota transplantation are the front‐line clinical options. Here, we review the contribution of gut microbiota and its derived molecules in gut homeostasis and explore the possible pathophysiological mechanisms involved in FGIDs leading to potential therapeutics options., This paper reviews the contribution of gut microbiota and its derived molecules in gut homeostasis and explore the possible pathophysiological mechanisms involved in FGIDs leading to potential therapeutics options. The current knowledge gaps warrant a modern approach for a holistic view to characterize patients based on the multiomic data from the gut microbiome, metabolome, transcriptome, host epigenome, and dietary profiles on longitudinal studies. The integration of these factors with physiological changes will enable us to develop targeted approaches to treat the patients, not only relieving symptoms but also restoring gut homeostasis.

Of all microorganisms in the human body, the largest and most complex population resides in the gastrointestinal (GI) tract. The gut microbiota continuously adapts to the host environment and serves multiple critical functions for their hosts, including regulating host immunity, procuring energy from food, and preventing the colonization of pathogens. Mounting evidence has suggested gut microbial imbalance (dysbiosis) as a core pathophysiology in the development of GI motility and metabolic disorders, such as irritable bowel syndrome and diabetes. Current research has focused on discovering associations between these disorders and gut microbial dysbiosis; however, whether these associations are a consequence or cause is still mostly unexplored. State-of-the-art studies have investigated how gut microbes communicate with our body systems through microbiota-derived metabolites and how they are able to modulate host physiology. There is now mounting evidence that alterations in the composition of small intestinal microbes have an association with GI dysmotility and metabolic disorders. Although treatment options for gut microbial dysbiosis are currently limited, antibiotics, fecal microbiota transplantation, probiotics, and dietary interventions are currently the best options. However, treatment with broad-spectrum antibiotics has been viewed with skepticism due to the risk of developing antibiotic resistant bacteria. Studies are warranted to elucidate the cellular and molecular pathways underlying gut microbiota-host crosstalk and for the development of a powerful platform for future therapeutic approaches. Here, we review recent literature on gut microbial alterations and/or interactions involved in the pathophysiology of GI dysmotility and metabolic disorders.

We read with great interest the recent publication by Lin et al ,1 which reported diarrhoea as the most common GI symptom in patients with COVID-19. Diarrhoea as a symptom of COVID-19 has been commonly observed,2 3 but the cause remains unknown. Diarrhoea occurs from excessive production of serotonin (5-hydroxytryptamine, 5-HT) in the GI tract.4 Ninety-five per cent of 5-HT is produced and released from the enterochromaffin cells within the epithelium of the GI tract. 5-HT localised to the GI tract is a key modulator of GI peristalsis.5 We hypothesised that plasma 5-HT levels are increased in patients with COVID-19 with diarrhoea. Table 1 summarises the COVID-19 severity, interleukin 6 (IL-6), IgM/IgG antibodies, fever, GI symptoms, plasma 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), underlying diabetes, body mass index, gender and age measured in 80 patients with COVID-19 (10 patients with diarrhoea) and 18 healthy donors (see online supplemental material). Correlation analysis identified 5-HT, 5-HIAA and IL-6 as the most noticeable features in the COVID-19 cohort with diarrhoea. Patients with COVID-19 had significantly increased plasma 5-HT and 5-HIAA levels compared with healthy donors (figure 1A,D). When patients were categorised by disease severity, 5-HT and 5-HIAA levels increased with higher severity of symptoms (figure 1B,E). Patients with COVID-19 with diarrhoea had substantially higher 5-HT and 5-HIAA levels compared with patients without diarrhoea and the healthy group (table 1 and figure …

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 cellular microenvironment composition and changes therein play an extremely important role in cancer development. Changes in the extracellular matrix (ECM), which constitutes a majority of the tumor stroma, significantly contribute to the development of the tumor microenvironment. These alterations within the ECM and formation of the tumor microenvironment ultimately lead to tumor development, invasion, and metastasis. The ECM is composed of various molecules such as collagen, elastin, laminin, fibronectin, and the MMPs that cleave these protein fibers and play a central role in tissue remodeling. When healthy cells undergo an insult like DNA damage and become cancerous, if the ECM does not support these neoplastic cells, further development, invasion, and metastasis fail to occur. Therefore, ECM-related cancer research is indispensable, and ECM components can be useful biomarkers as well as therapeutic targets. Colorectal cancer specifically, is also affected by the ECM and many studies have been conducted to unravel the complex association between the two. Here we summarize the importance of several ECM components in colorectal cancer as well as their potential roles as biomarkers.

Background & Aims Interstitial cells of Cajal (ICCs) and pancreatic β cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. Methods We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. Results miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of β cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Kruppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. Conclusions miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

Colorectal cancer is a significant cause of death since it frequently metastasizes to several organs such as the lung or liver. Tumor development is affected by various factors, including a tumor microenvironment, which may be an essential factor that leads to tumor growth, proliferation, invasion, and metastasis. In the tumor microenvironment, abnormal changes in various growth factors, enzymes, and cytokines can wield a strong influence on cancer. Thrombospondin-4 (THBS4), which is an extracellular matrix protein, also plays essential roles in the tumor microenvironment and mediates angiogenesis by transforming growth factor-&beta, (TGF&beta, ) signaling. Platelet-derived growth factor receptor &beta, (PDGFR&beta, ), which is a receptor tyrosine kinase and is also a downstream signal of TGF&beta, is associated with invasion and metastasis in colorectal cancer. We identified that PDGFR&beta, and THBS4 are overexpressed in tumor tissues of colorectal cancer patients, and that PDGF-D expression increased after TGF&beta, treatment in the colon cancer cell line DLD-1. TGF&beta, and PDGF-D increased cellular THBS4 protein levels and secretion but did not increase THBS4 mRNA levels. This response was further confirmed by the inositol 1,4,5-triphosphate receptor (IP3R) and stromal interaction molecule 1 (STIM1) blockade as well as the PDGFR&beta, blockade. We propose that the PDGFR&beta, signal leads to a modification of the incomplete form of THBS4 to its complete form through IP3R, STIM1, and Ca2+-signal proteins, which further induces THBS4 secretion. Additionally, we identified that DLD-1 cell-conditioned medium stimulated with PDGF-D promotes adhesion, migration, and proliferation of colon myofibroblast CCD-18co cells, and this effect was intensified in the presence of thrombin. These findings suggest that excessive PDGFR&beta, signaling due to increased TGF&beta, and PDGF-D in colorectal tumors leads to over-secretion of THBS4 and proliferative tumor development.

Autophagy is a major intracellular degradation process that plays an important role in cell survival, stress responses, nutrient sensing and development. Our previous studies have shown that Rg2, a triterpenoid saponin contained in ginseng, protects cells against UVB-induced genotoxicity by increasing DNA repair, in possible association with modulation of protein levels involved in p53 pathway. In this study, we determined an upregulation of autophagy by Rg2. Rg2 treatment for 24 h in MCF-7, a breast cancer cell, did not show cytotoxicity up to 200 μM. Rg2 also upregulated the level of p-p53, p-AMPK, p-ACC, Atg-7 and LC3-II and decreased the level of p62 in concentration-dependent manners. We also determined the level of p53, AMPK, p62, Atg-7 and LC3 after UVB exposure and subsequent incubation in growth medium for 24 h. UVB increased the level of p-p53, p-AMPK, p-ACC and decreased the levels of p62, Atg-7 and LC3-II. Interestingly, Rg2 treatment for 24 h after UVB exposure increased the levels of p-p53, p-AMPK, p-ACC, Atg-7 and LC3-II and decreased the level of cyclobutane pyrimidine dimer, a UVB-induced DNA damage in concentration-dependent manners. All these results suggest that Rg2 increased autophagy and decreased UVB-induced DNA damage, in possible association with the modulation of protein levels in p53- and autophagic pathways.

Ultraviolet B (UVB) radiation induces skin damage, skin matrix degradation, and wrinkle formation through photochemical reaction and oxidative stress. Therefore, protecting the skin from UVB can prevent skin aging. In this study, we investigated the effects of a mixture (RA) of Rg2, a ginsenoside, and astaxanthin, an antioxidant, on the responses of HaCaT cells exposed to UVB (700 J/m2). The cells were incubated for 24 h after UVB exposure and cell viability was determined by MTT assay. UVB decreased cell viability by 60% compared to that of untreated control cells, whereas RA increased cell viability in a concentration-dependent manner, and this increase was significantly higher than that in the single treatment groups. Further, UVB increased the levels of DNA lesions such as cyclobutane pyrimidine dimer (CPD) and 8-hydroxyguanine (8-OHdG). Conversely, RA decreased both CPD and 8-OHdG levels in a concentration-dependent manner. UVB exposure also increased phosphorylation of ataxia-telangiectasia mutated (ATM) protein kinase and p53 and subsequently increased the levels of GADD45α, p21, and matrix metalloproteinases (MMPs)-3, -9, and -13. Additionally, UVB exposure decreased the level of COL1A1. However, RA treatment decreased the levels of p-ATM, p-p53, GADD45α, p21, MMP-3, -9, and -13 and increased the level of COL1A1 in a concentration-dependent manner. These results suggest that RA reduces UVB-induced cytotoxicity and genotoxicity through up-regulation of DNA repair via the combined effects of Rg2 and astaxanthin.

Although electrical stimulation is therapeutically applied for neural regeneration in patients, it remains unclear how electrical stimulation exerts its effects at the molecular level on spinal cord injury (SCI). To identify the signaling pathway involved in electrical stimulation improving the function of injured spinal cord, 21 female Sprague-Dawley rats were randomly assigned to three groups: control (no surgical intervention, n = 6), SCI (SCI only, n = 5), and electrical simulation (ES; SCI induction followed by ES treatment, n = 10). A complete spinal cord transection was performed at the 10th thoracic level. Electrical stimulation of the injured spinal cord region was applied for 4 hours per day for 7 days. On days 2 and 7 post SCI, the Touch-Test Sensory Evaluators and the Basso-Beattie-Bresnahan locomotor scale were used to evaluate rat sensory and motor function. Somatosensory-evoked potentials of the tibial nerve of a hind paw of the rat were measured to evaluate the electrophysiological function of injured spinal cord. Western blot analysis was performed to measure p38-RhoA and ERK1/2-Bcl-2 pathways related protein levels in the injured spinal cord. Rat sensory and motor functions were similar between SCI and ES groups. Compared with the SCI group, in the ES group, the latencies of the somatosensory-evoked potential of the tibial nerve of rats were significantly shortened, the amplitudes were significantly increased, RhoA protein level was significantly decreased, protein gene product 9.5 expression, ERK1/2, p38, and Bcl-2 protein levels in the spinal cord were significantly increased. These data suggest that ES can promote the recovery of electrophysiological function of the injured spinal cord through regulating p38-RhoA and ERK1/2-Bcl-2 pathway-related protein levels in the injured spinal cord.

Disorders of gut–brain interactions (DGBIs) are heterogeneous in nature and intertwine with diverse pathophysiological mechanisms. Regular functioning of the gut requires complex coordinated interplay between a variety of gastrointestinal (GI) cell types and their functions are regulated by multiple mechanisms at the transcriptional, post-transcriptional, translational, and post-translational levels. MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate gene expression by binding to specific mRNA targets to repress their translation and/or promote the target mRNA degradation. Dysregulation of miRNAs might impair gut physiological functions leading to DGBIs and gut motility disorders. Studies have shown miRNAs regulate gut functions such as visceral sensation, gut immune response, GI barrier function, enteric neuronal development, and GI motility. These biological processes are highly relevant to the gut where neuroimmune interactions are key contributors in controlling gut homeostasis and functional defects lead to DGBIs. Although extensive research has explored the pathophysiology of DGBIs, further research is warranted to bolster the molecular mechanisms behind these disorders. The therapeutic targeting of miRNAs represents an attractive approach for the treatment of DGBIs because they offer new insights into disease mechanisms and have great potential to be used in the clinic as diagnostic markers and therapeutic targets. Here, we review recent advances regarding the regulation of miRNAs in GI pacemaking cells, immune cells, and enteric neurons modulating pathophysiological mechanisms of DGBIs. This review aims to assess the impacts of miRNAs on the pathophysiological mechanisms of DGBIs, including GI dysmotility, impaired intestinal barrier function, gut immune dysfunction, and visceral hypersensitivity. We also summarize the therapeutic alternatives for gut microbial dysbiosis in DGBIs, highlighting the clinical insights and areas for further exploration. We further discuss the challenges in miRNA therapeutics and promising emerging approaches.

Ulva lactuca (U. lactuca) is a green alga distributed worldwide and used as a food and cosmetic material. In our previous study, we determined the effects of U. lactuca methanol extracts on the UVB-induced DNA repair. In the present study, we fractionated U. lactuca methanol extracts to identify the effective compound for the DNA repair. MTT assay demonstrated that (+)-epiloliolide showed no cytotoxicity up to 100 μM in BJ-5ta human dermal fibroblast. Upon no treatment, exposure to UVB 400 J/m2 decreased cell viability by 45%, whereas (+)-epiloliolide treatment for 24 h after UVB exposure significantly increased the cell viability. In GO and GESA analysis, a number of differentially expressed genes were uniquely expressed in (+)-epiloliolide treated cells, which were enriched in the p53 signaling pathway and excision repair. Immunofluorescence demonstrated that (+)-epiloliolide increased the nuclear localization of p53. Comet assay demonstrated that (+)-epiloliolide decreased tail moment increased by UVB. Western blot analysis demonstrated that (+)-epiloliolide decreased the levels of p-p53, p21, Bax, and Bim, but increased that of Bcl-2. Reverse transcription PCR (RT-PCR) demonstrated that (+)-epiloliolide decreased the levels of MMP 1, 9, and 13, but increased that of COL1A1. These results suggest that (+)-epiloliolide regulates p53 activity and has protective effects against UVB.

Background & Aims Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. Methods We developed an inducible, EC cell–specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell–depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell–depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). Results Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. Conclusions The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.

Serum response factor (SRF) is a master transcription factor of the actin cytoskeleton that binds to highly conserved CArG boxes located within the majority of smooth muscle cell (SMC)-restricted promoters/enhancers. Although most studies of SRF focus on skeletal muscle, cardiac muscle, and vascular SMCs, SRF research has recently expanded into the gastrointestinal (GI) system. Genome scale analyses of GI SMC transcriptome and CArG boxes (CArGome) have identified new SRF target genes. In addition to circular and longitudinal smooth muscle layers, SRF is also expressed in GI mucosa and cancers. In the GI tract, SRF is the central regulator of genes involved in apoptosis, dedifferentiation, proliferation, and migration of cells. Since SRF is the cell phenotypic modulator, it may play an essential role in the development of myopathy, hypertrophy, ulcers, gastric and colon cancers within the GI tract. Given the multi-functional role displayed by SRF in the digestive system, SRF has received more attention emerging as a potential therapeutic target. This review summarizes the findings in SRF research pertaining to the GI tract and provides valuable insight into future directions.

The objective of this review is to provide recent evidence for the oral–gut axis connection and to discuss gastrointestinal (GI) immune response, inflammatory bowel disease (IBD) pathogenesis, and potential salivary biomarkers for determining GI health. IBD affects an estimated 1.3% of the US adult population. While genetic predisposition and environment play a role, abnormal immune activity and microbiota dysbiosis within the gastrointestinal tract are also linked in IBD pathogenesis. It has been inferred that a reduced overall richness of bacterial species as well as colonization of opportunistic bacteria induce systemic inflammation in the GI tract. Currently, there is supporting evidence that both oral and gut microbiota may be related to the development of IBD. Despite this, there are currently no curative therapies for IBD, and diagnosis requires samples of blood, stool, and invasive diagnostic imaging techniques. Considering the relative ease of collection, emerging evidence of association with non-oral diseases may imply that saliva microbiome research may have the potential for gut diagnostic or prognostic value. This review demonstrates a link between saliva and intestinal profiles in IBD patients, suggesting that saliva sampling has the potential to serve as a non-invasive biomarker for gut diseases such as IBD in the oral–gut axis.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFRα(+), and neuronal cells during the development of intestinal obstruction.

Background/Aims Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. Methods We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. Results Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRαlow) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRαlow cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. Conclusions SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRαlow cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn's disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.

Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K+, suggesting that it is regulated not only by K+ conductances but by inward conductances such as Na+ and/or Ca2+. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na+ with equimolar tetraethylammonium or Ca2+ with Mn2+ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na+ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (b I NSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of b I NSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle.

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized ‘slow wave’ current. Reversal of tail current analysis showed this current was due to a Cl− selective conductance. ICC express ANO1, a Ca2+-activated Cl− channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the slow wave current. Single Ca2+-activated Cl− channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC50= 4.8 μm). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca2+-activated Cl− conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.

Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.

Specific functions of interstitial cells of Cajal (ICC) have been linked to distinct classes that differ by morphology and distribution. In the small intestine, slow wave-generating ICC are located in the myenteric region (ICC-MY), whereas ICC that mediate neuromuscular neurotransmission occur either throughout the circular muscle layer (intramuscular ICC, ICC-IM) or in association with the deep muscular plexus (ICC-DMP). Selective isolation of ICC to characterize specific properties has been difficult. Recently, neurokinin-1 receptors have been detected in murine ICC-DMP and neurons but not in ICC-MY. Here we identified and isolated ICC-DMP/IM by receptor-mediated internalization of fluorescent substance P and Kit immunofluorescence. Specificity of labeling was verified by confocal microscopy. Mouse and human ICC-DMP/IM were detected in suspension by fluorescent microscopy and harvested for RT-PCR with micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle, or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M2 and M3 muscarinic receptors, P2Y1 and P2Y4 purinergic receptors, VIP receptor 2, soluble guanylate cyclase-1 subunits, and protein kinase G. L- or T-type Ca2+ channels were not detected in these cells. ICC-MY and ICC-DMP were simultaneously detected and enumerated by flow cytometry and sorted to purity by fluorescence-activated cell sorting. In summary, functional classes of ICC have distinct molecular identities that can be used to selectively identify and harvest these cells with, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that are consistent with their role in neuromuscular neurotransmission.

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERGUSO) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3– S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10−6 M), atropine (10−6M). and l-nitroarginine (10−4 M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10−8 M) and MK-499 (3 × 10−8 M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10−6 M) produced a 10–20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10−6 M) did not affect [14C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.

1. Two components of voltage-gated, inward currents were observed from murine colonic myocytes. One component had properties of L-type Ca(2+) currents and was inhibited by nicardipine (5 x 10(-7) M). A second component did not 'run down' during dialysis and was resistant to nicardipine (up to 10(-6) M). The nicardipine-insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. 2. This low-voltage-activated current (I(LVA)) was resolved with step depolarizations positive to -60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half-inactivation was -65 mV. Inactivation and activation time constants at -45 mV were 86 and 15 ms, respectively. The half-recovery time from inactivation was 98 ms at -45 mV. I(LVA) peaked at -40 mV and the current reversed at 0 mV. 3. I(LVA) was inhibited by Ni(2+) (IC(50) = 1.4 x 10(-5) M), mibefradil (10(-6) to 10(-5) M), and extracellular Ba(2+). Replacement of extracellular Na(+) with N-methyl-D-glucamine inhibited I(LVA) and shifted the reversal potential to -7 mV. Increasing extracellular Ca(2+) (5 x 10(-3) M) increased the amplitude of I(LVA) and shifted the reversal potential to +22 mV. I(LVA) was also blocked by extracellular Cs(+) (10(-4) M) and Gd(3+) (10(-6) M). 4. Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. 5. We conclude that the second component of voltage-dependent inward current in murine colonic myocytes is not a 'T-type' Ca(2+) current but rather a novel, voltage-gated non-selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.

Gastrointestinal motility results from coordinated contractions of the tunica muscularis, the muscular layers of the alimentary canal. Throughout most of the gastrointestinal tract, smooth muscles are organized into two layers of circularly or longitudinally oriented muscle bundles. Smooth muscle cells form electrical and mechanical junctions between cells that facilitate coordination of contractions. Excitation-contraction coupling occurs by Ca(2+) entry via ion channels in the plasma membrane, leading to a rise in intracellular Ca(2+). Ca(2+) binding to calmodulin activates myosin light chain kinase; subsequent phosphorylation of myosin initiates cross-bridge cycling. Myosin phosphatase dephosphorylates myosin to relax muscles, and a process known as Ca(2+) sensitization regulates the activity of the phosphatase. Gastrointestinal smooth muscles are 'autonomous' and generate spontaneous electrical activity (slow waves) that does not depend upon input from nerves. Intrinsic pacemaker activity comes from interstitial cells of Cajal, which are electrically coupled to smooth muscle cells. Patterns of contractile activity in gastrointestinal muscles are determined by inputs from enteric motor neurons that innervate smooth muscle cells and interstitial cells. Here we provide an overview of the cells and mechanisms that generate smooth muscle contractile behaviour and gastrointestinal motility.

Background & Aims Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. Methods Kit +/copGFP mice harboring a copepod super green fluorescent protein ( copGFP ) complementary DNA, inserted at the Kit locus, were generated. copGFP + ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP + ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit +/copGFP mice were crossbred with diabetic Lep +/ob mice to generate compound Kit +/copGFP ;Lep ob/ob mutant mice. copGFP + ICC from compound transgenic mice were analyzed by confocal microscopy. Results copGFP in Kit +/copGFP mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP + cells from jejunal muscles were Kit + and free of contaminating cell-specific markers. Kit +/copGFP ; Lep ob/ob mice displayed ICC networks that were dramatically disrupted during the development of diabetes. Conclusions Kit +/copGFP mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

International audience; Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

Serum response factor (SRF) is a transcription factor known to mediate phenotypic plasticity in smooth muscle cells (SMCs). Despite the critical role of this protein in mediating intestinal injury response, little is known about the mechanism through which SRF alters SMC behavior. Here, we provide compelling evidence for the involvement of SRF-dependent microRNAs (miRNAs) in the regulation of SMC apoptosis. We generated SMC-restricted Srf inducible knockout (KO) mice and observed both severe degeneration of SMCs and a significant decrease in the expression of apoptosis-associated miRNAs. The absence of these miRNAs was associated with overexpression of apoptotic proteins, and we observed a high level of SMC death and myopathy in the intestinal muscle layers. These data provide a compelling new model that implicates SMC degeneration via anti-apoptotic miRNA deficiency caused by lack of SRF in gastrointestinal motility disorders.

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Small-conductance Ca2+-activated K+ (SK) channels are important participants in inhibitory neurotransmission in gastrointestinal smooth muscles. Three isoforms of an SK channel family were cloned from murine proximal colon smooth muscle. The transcripts encoding these subunits (SK1, SK2, and SK3) were detected in murine proximal colon and other peripheral tissues. The mRNA of each subunit was expressed at different levels in murine and canine colonic smooth muscles. The mRNA quantitative ratio of SK transcriptional expression in murine proximal colon is SK2 > SK3 > SK1; transcriptional expression of SK isoforms in canine proximal colon is minimal. SK3 immunohistochemical localization in murine small intestine (jejunum) and proximal colon showed immunoreactivity in circular and longitudinal muscularis. In transversely sectioned muscularis, staining was localized at the cell membrane in smooth muscle cells. Immunoreactivity was more intense in myenteric ganglia between longitudinal and circular muscularis and neuronal processes in circular and longitudinal muscularis. Transient expression of mSK1, mSK2, and mSK3 in COS cells resulted in Ca2+-activated voltage-independent channels. mSK1 is less sensitive to apamin compared with SK2 and showed intracellular Ca2+ sensitivity (10−8 to 10−6 M) in asymmetrical K+ (5/140 mM K+) gradients. Our results suggest that SK channel expression varies in colonic myocytes from different species and may contribute differentially to inhibitory junction potentials.

Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.