Gene Expression In Intestinal Cells Research Articles

The Impact of Physical Activity on Gut Microbiota: An In-Depth Literature Review

Introduction. The human body is home to a diverse community of microorganisms known as the microbiota. These microorganisms, including bacteria, fungi, and viruses, play crucial roles in maintaining overall health. They colonize the digestive tract shortly after birth, and the composition of the gut microbiota changes throughout an individual's life, largely influenced by diet and physical activity. Breastfeeding has long-term beneficial effects on the gut microbiota and gene expression in intestinal cells. Regular exercise not only improves cardiovascular and respiratory health but also affects metabolic, immunological, neural, and microbial pathways. This review explores how diet and physical activity impact the composition and diversity of gut microbiota, highlighting important findings and their implications for health. Aim of study. This study aims to review how physical activity influences gut microbiota, investigating the effects of different types and intensities of exercise on microbial composition and diversity, and their implications for overall health. Materials and methods. A non-systematic examination of the scientific literature was conducted using specific keywords such as gut microbiota, physical activity, sport, and microbiome. The examination took place on PubMed, with scrutiny of a total of 31 references published up to 2023. Conclusions. This review highlights the crucial role of gut microbiota in maintaining good health and its complex interaction with diet and physical activity. Regular exercise has a positive impact on the diversity of microbiota, especially when started at a young age. Imbalance in gut bacteria is linked to various diseases, underscoring the significance of a healthy microbiome. Encouraging physical activity and a nutritious diet can improve gut health, and more research is needed to understand the mechanisms and potential health indicators involved.

Intestinal regulation of suppression of tumorigenicity 14 (ST14) and serine peptidase inhibitor, Kunitz type -1 (SPINT1) by transcription factor CDX2

The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.

Identifying the location of epidermal growth factor-responsive element involved in the regulation of type IIb sodium-phosphate cotransporter expression in porcine intestinal epithelial cells.

Phosphate is an important mineral nutrient for both human and animals in growth and physiological functions; thus, much effort in the past has been made to clarify the mechanisms governing its absorption. Previous studies have found that epidermal growth factor (EGF) inhibits phosphate absorption in human intestinal cells via modulating the interaction of transcriptional factor c-myb with sodium-phosphate cotransporter (NaPi-IIb) gene promoter. This finding provoked our interest in determining the effect of EGF on NaPi-IIb gene expression in intestinal cells of pigs and the location of EGF-responsive element in the gene promoter. Using quantitative PCR, it was observed that EGF significantly reduced NaPi-IIb gene expression in porcine intestinal epithelial IPEC-J2 cells. Transfection with a series of constructs that contain different lengths of the 5'-flanking promoter region of the NaPi-IIb gene manifested that EGF-responsive element is located in the -1200 to -800 region. Further, c-myb was extracted from the cell nucleus of IPEC cells that were exposed to EGF or not via immunoprecipitation. The electrophoretic mobility shift assay showed a specific binding of transcription factor c-myb to labelled probes encompassing DNA sequence from -1092 to -1085 (-TCCAGTTG-). This protein-DNA complex was decreased with cells exposed to EGF and abrogated when c-myb was pre-incubated with excessive unlabelled competitive probes. Results from mutagenesis studies demonstrated that the c-myb-binding site is the EGF-responsive element involved in the regulation of NaPi-IIb expression. Identifying the location of EGF-responsive element contributes to understanding mechanisms underlying EGF down-regulated NaPi-IIb gene expression and provides a foundation for further investigating EGF-regulatory functions in phosphate absorption in pig intestine.

Mitigation of whole-body gamma radiation–induced damages by Clerodendron infortunatum in mammalian organisms

Several phytoceuticals and extracts of medicinal plants are reported to mitigate deleterious effects of ionizing radiation. The potential of hydro-alcoholic extract of Clerodendron infortunatum (CIE) for providing protection to mice exposed to gamma radiation was investigated. Oral administration of CIE bestowed a survival advantage to mice exposed to lethal doses of gamma radiation. Radiation-induced depletion of the total blood count and bone marrow cellularity were prevented by treatment with CIE. Damage to the cellular DNA (as was evident from the comet assay and the micronucleus index) was also found to be decreased upon CIE administration. Radiation-induced damages to intestinal crypt cells was also reduced by CIE. Studies on gene expression in intestinal cells revealed that there was a marked increase in the Bax/Bcl-2 ratio in mice exposed to whole-body 4 Gy gamma radiation, and that administration of CIE resulted in significant lowering of this ratio, suggestive of reduction of radiation-induced apoptosis. Also, in the intestinal tissue of irradiated animals, following CIE treatment, levels of expression of the DNA repair gene Atm were found to be elevated, and there was reduction in the expression of the inflammatory Cox-2 gene. Thus, our results suggest a beneficial use of Clerodendron infortunatum for mitigating radiation toxicity.

Food-Derived Peptides Stimulate Mucin Secretion and Gene Expression in Intestinal Cells

In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells.

Chitin‐binding protein GbpA of Vibrio cholerae induces interleukin‐8 gene expression in intestinal cells through a TLR2/TLR1/CD14 complex

Vibrio cholerae N‐acetylglucosamine binding protein (GbpA) is a secretory protein which is essential for bacterial survival in environment as well as in human gut. Here we show that GbpA binds to toll‐like receptor 2 (TLR2) and initiates downstream cellular inflammatory signaling pathway by IL8 secretion. GbpA triggered formation of lipid raft microdomain on intestinal cell surface by interacting with TLR2 with a Kd of 76 nM. The microdomain contains TLR1, CD14 and flotilin in addition to TLR2. As a downstream effect, the lipid raft complex triggered IL8 secretion and the effect was specific in HT29 intestinal cells. The critical involvement of the GbpA‐induced lipid raft microdomain complex in IL8 secretion was indicated by the decrease in secretion caused by disruption of the raft by extraction of cholesterol by methyl‐β‐D‐cyclodextrin and by its restoration by extraneous addition of cholesterol. The GbpA‐mediated elevation in IL8 secretion involved activation of NFκB and formation of AP1 complex which eventually increased IL8 promoter activity. Taken together, the results suggest that GbpA is a key player in pathogenesis of cholera by triggering pro‐inflammatory response in infection by inducing downstream activation through clustering of GbpA‐TLR1/2‐CD14 complex in lipid raft microdomain. [Supported in part by grant from CSIR, Govt. of India No. 37(1410)/10/EMR‐II]

Hepatocyte nuclear factor-4α regulates human cellular retinol-binding protein type II gene expression in intestinal cells

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4alpha expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4alpha significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4alpha protein levels by HNF-4alpha small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4alpha protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4alpha protein levels, HNF-4alpha protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4alpha is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4alpha in the regulation of human intestinal vitamin A absorption and metabolism.

Repression of the Desmocollin 2 Gene Expression in Human Colon Cancer Cells Is Relieved by the Homeodomain Transcription Factors Cdx1 and Cdx2

Desmosomes are intracellular junctions that provide strong cell-cell adhesion in epithelia and cardiac muscle. Their disruption causes several human diseases and contributes to the epithelial-to-mesenchymal transition observed in cancer. Desmocollin 2 (DSC2) is a cadherin superfamily member and a critical component of desmosomes found in intestinal epithelium. However, the mechanism regulating DSC2 gene expression in intestinal cells is not known. Cdx1 and Cdx2 are homeodomain transcription factors that regulate intestine-specific gene expression. Cdx expression in the past has been associated with the induction of desmosomes. We now show that the DSC2 gene is a transcriptional target for Cdx1 and Cdx2. Colon cancer cell lines retaining Cdx2 expression typically express DSC2. Restoration of Cdx expression in Colo 205 cells induced DSC2 mRNA and protein and the formation of desmosomes. The 5'-flanking region of the DSC2 promoter contains two consensus Cdx-binding sites. Electrophoretic mobility shift assays show that Cdx1 and Cdx2 bind these sites in vitro, and chromatin immunoprecipitation confirmed Cdx2 binding in vivo. DSC2 promoter truncations established that these regions are Cdx responsive. The truncations also identify a region of the promoter in which potent transcriptional repressors act. This repressor activity is relieved by Cdx binding. We conclude that the homeodomain transcription factors Cdx1 and Cdx2 regulate DSC2 gene expression in intestinal epithelia by reversing the actions of a transcriptional repressor. The regulation of desmosomal junctions by Cdx contributes to normal intestinal epithelial columnar morphology and likely antagonizes the epithelial-to-mesenchymal transition necessary for the metastasis of colon cancer cells in humans.

Comparison of different vehicles to study the effect of tocopherols on gene expression in intestinal cells

Recent studies have focused on the ability of tocopherols to regulate gene expression. For such experiments, the methodology used to deliver molecules to the cells is crucial and could lead to different results depending on the vehicle used. The objective of the present study was to compare commonly used tocopherol vehicles (ethanol, BSA and mixed micelles) in terms of toxicity, stabilization of tocopherols, uptake efficiency of tocopherols by cells and effect on gene expression. Lactate dehydrogenase measurements did not reveal cytotoxicity of any of the tested vehicles. Tocopherol recovery measurements showed that ∼80% of the tocopherol was lost in ethanolic solutions, while only ∼30% and 10% were lost in BSA and mixed micelles, respectively. After 24 h incubation, Caco-2 cell monolayers treated with mixed micelles exhibited the highest α-tocopherol intracellular concentrations (5-times those measured with the two other vehicles). Similar results were obtained with γ-tocopherol. Vehicles, except mixed micelles that activate the FXR/bile acids signalling pathway, did not affect expression of nuclear receptors involved in lipid metabolism or their target genes. This study establishes mixed micelles as the best vehicle to deliver tocopherols to intestinal cell monolayers in culture.

Phytanic acid and docosahexaenoic acid increase the metabolism of all- trans-retinoic acid and CYP26 gene expression in intestinal cells

Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all- trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRα) and peroxisome proliferator activating receptor (PPARα) activator, also activated PPARδ, and to a lesser degree PPARγ, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all- trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all- trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all- trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all- trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all- trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all- trans-RA in intestinal cells.

Cdx1 promotes cellular growth of epithelial intestinal cells through induction of the secretory protein PAP I

Expression of the Cdx1 homeobox gene in epithelial intestinal cells promotes cellular growth and differentiation. Cdx1and the Pancreatitis Associated Protein I (PAP I) are concomitantly expressed in the epithelial cells of the lower part of the intestinal crypts. Because Cdx1 is a transcription factor and PAP I, in other tissues, is a proliferative factor, we looked for a relationship between these two proteins in the intestinal-derived IEC-6 cells. After stable transfection with a Cdx1 expression vector, they produce high levels of the PAP I transcript and protein indicating a functional link between the two genes. Demonstration of Cdx1 binding to the PAP I promoter region and suppression of PAP I induction after deletion of the corresponding sequence indicated that Cdx1 is a transcription factor controlling PAP I gene expression in intestinal cells. By infecting IEC-6 cells with adenoviruses expressing PAP I, we demonstrated that PAP I induces mitosis in these cells. On the other hand, inhibition of the PAP I expression in the IEC-6 Cdxl-expressing cells using an antisense strategy confirmed the requirement of this protein for the effect of Cdx1 on cell growth. Finally, addition of the immunopurified PAP I to the culture medium promotes cell growth of the IEC-6 cells in a dose-dependent manner. Maximal effect was obtained at 1 ng/ml. Taken together these results demonstrate that PAP I is a target of the Cdx1 homeobox gene in intestinal cells which participates in the regulation of intestinal cell growth via an autocrine and/or paracrine mechanism.

Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5'-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5'-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor kappaB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2alpha and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.