Preserve Tissue Homeostasis Research Articles

The intricate interplay between ferroptosis and efferocytosis in cancer: unraveling novel insights and therapeutic opportunities.

The complex interplay between ferroptosis and efferocytosis in cancer has attracted significant interest recently. Efferocytosis, the process of eliminating apoptotic cells, is essential for preserving tissue homeostasis and reducing inflammation. However, dysregulation of efferocytosis can have profound effects on cancer. Apoptotic cells accumulate because of impaired efferocytosis, which triggers chronic inflammation and the release of pro-inflammatory chemicals. Surprisingly, accumulating evidence suggests that dysregulation of ferroptosis- a form of controlled cell death characterized by lipid peroxidation and the buildup iron-dependent reactive oxygen species (ROS)-can influence efferocytic activities within the tumor microenvironment. Dysfunctional iron metabolism and increased lipid peroxidation, are associated with ferroptosis, resulting in inadequate apoptotic cell clearance. Conversely, apoptotic cells can activate ferroptotic pathways, increasing oxidative stress and inducing cell death in cancer cells. This reciprocal interaction emphasizes the complex relationship between efferocytosis and ferroptosis in cancer biology. Understanding and managing the delicate balance between cell clearance and cell death pathways holds significant therapeutic potential in cancer treatment. Targeting the efferocytosis and ferroptosis pathways may offer new opportunities for improving tumor clearance, reducing inflammation, and sensitizing cancer cells to therapeutic interventions. Further research into the interaction between efferocytosis and ferroptosis in cancer will provide valuable insights for the development of novel therapies aimed at restoring tissue homeostasis and improving patient outcomes.

Detailed role of SR-A1 and SR-E3 in tumor biology, progression, and therapy.

The first host defense systems are the innate immune response and the inflammatory response. Among innate immune cells, macrophages, are crucial because they preserve tissue homeostasis and eradicate infections by phagocytosis, or the ingestion of particles. Macrophages exhibit phenotypic variability contingent on their stimulation state and tissue environment and may be detected in several tissues. Meanwhile, critical inflammatory functions are played by macrophage scavenger receptors, in particular, SR-A1 (CD204) and SR-E3 (CD206), in a variety of pathophysiologic events. Such receptors, which are mainly found on the surface of multiple types of macrophages, have different effects on processes, including atherosclerosis, innate and adaptive immunity, liver and lung diseases, and, more recently, cancer. Although macrophage scavenger receptors have been demonstrated to be active across the disease spectrum, conflicting experimental findings and insufficient signaling pathways have hindered our comprehension of the molecular processes underlying its array of roles. Herein, as SR-A1 and SR-E3 functions are often binary, either protecting the host or impairing the pathophysiology of cancers has been reviewed. We will look into their function in malignancies, with an emphasis on their recently discovered function in macrophages and the possible therapeutic benefits of SR-A1 and SR-E3 targeting.

β-Galactosidase-Triggered Photodynamic Elimination of Senescent Cells with a Boron Dipyrromethene-Based Photosensitizer.

Senescence is a cellular response having physiological and reparative functions to preserve tissue homeostasis and suppress tumor growth. However, the accumulation of senescent cells would cause deleterious effects that lead to age-related dysfunctions and cancer progression. Hence, selective detection and elimination of senescent cells are crucial yet remain a challenge. A β-galactosidase (β-gal)-activated boron dipyrromethene (BODIPY)-based photosensitizer (compound 1) is reported here that can selectively detect and eradicate senescent cells. It contains a galactose moiety connected to a pyridinium BODIPY via a self-immolative nitrophenylene linker, of which the photoactivity is effectively quenched. Upon interactions with the senescence-associated β-gal, it undergoes enzymatic hydrolysis followed by self-immolation, leading to the release of an activated BODIPY moiety by which the fluorescence emission and singlet oxygen generation are restored. The ability of 1 to detect and eliminate senescent cells is demonstrated in vitro and in vivo, using SK-Mel-103 tumor-bearing mice treated with senescence-inducing therapy. The results demonstrate that 1 can be selectively activated in senescent cells to trigger a robust senolytic effect upon irradiation. This study breaks new ground in the design and application of new senolytic agents based on photodynamic therapy.

Mesenchymal Stem Cell-Derived Exosomes in Various Chronic Liver Diseases: Hype or Hope?

Chronic liver conditions are associated with high mortality rates and have a large adverse effect on human well-being as well as a significant financial burden. Currently, the only effective treatment available for the effects of liver failure and cirrhosis resulting from the progression of several chronic liver diseases is liver transplantation carried out at the original location. This implies that developing novel and effective treatments is imperative. Regenerative medicine has long been associated with stem cell therapy. Mesenchymal stem cells (MSCs), a type of cell with great differentiation potential, have become the preferred source for stem cell therapy. According to recent studies, MSCs' paracrine products-rather than their capacity for differentiation-play a significant therapeutic effect. MSC exosomes, a type of extracellular vesicle (MSC-EV), came into view as the paracrine substances of MSCs. According to research, MSC exosomes can maintain tissue homeostasis, which is necessary for healthy tissue function. All tissues contain them, and they take part in a variety of biological activities that support cellular activity and tissue regeneration in order to preserve tissue homeostasis. The outcomes support the use of MSCs and the exosomes they produce as a therapeutic option for a range of diseases. This review provides a brief overview of the source of MSC-EVs and outlines their physiological roles and biochemical capabilities. The elucidation of the role of MSC-EVs in the recovery and repair of hepatic tissues, as well as their contribution to maintaining tissue homeostasis, is discussed in relation to different chronic liver diseases. This review aims to provide new insights into the unique roles that MSC-EVs play in the treatment of chronic liver diseases.

Effect of stretching on inflammation in a subcutaneous carrageenan mouse model analyzed at single-cell resolution.

Understanding the factors that influence the biological response to inflammation is crucial, due to its involvement in physiological and pathological processes, including tissue repair/healing, cancer, infections, and autoimmune diseases. We have previously demonstrated that in vivo stretching can reduce inflammation and increase local pro-resolving lipid mediators in rats, suggesting a direct mechanical effect on inflammation resolution. Here we aimed to explore further the effects of stretching at the cellular/molecular level in a mouse subcutaneous carrageenan-inflammation model. Stretching for 10 min twice a day reduced inflammation, increased the production of pro-resolving mediator pathway intermediate 17-HDHA at 48 h postcarrageenan injection, and decreased both pro-resolving and pro-inflammatory mediators (e.g., PGE2 and PGD2 ) at 96 h. Single-cell RNA sequencing analysis of inflammatory lesions at 96 h showed that stretching increased the expression of both pro-inflammatory (Nos2) and pro-resolution (Arg1) genes in M1 and M2 macrophages at 96 h. An intercellular communication analysis predicted specific ligand-receptor interactions orchestrated by neutrophils and M2a macrophages, suggesting a continuous neutrophil presence recruiting immune cells such as activated macrophages to contain the antigen while promoting resolution and preserving tissue homeostasis.

Liver Wound Healing Is Associated with An Increase in Mfge8 Expression in Hepatocytes

Being recognized as a typical pathological feature of liver diseases, apoptosis-induced hepatocyte injury leading to sterile inflammation drives a wound-healing path in vivo. We and other researchers reported that Milk fat globule-EGF factor 8 (MFGE8) preserves tissue homeostasis, protects against tissue injury and inflammation, and attenuates nonalcoholic fatty liver disease and fibrosis. However, it is unclear whether and how MFGE8 is involved in liver regeneration from hepatocyte-apoptosis-induced injury. Our aim is to study current knowledge to this extent, we created a novel triple-transgenic mouse model, an inducible hepatocyte-specific apoptosis phenotype (namely, 3XTg-iHAP), in which the inducible hepatocyte-specific FasL expression is under control of the combined LoxP-Albumin-Cre system and rtTA Tet-On regulatory element. The liver tissues from 3XTg-iHAP and their littermates were collected at 8 h, 24 h, 48 h, 72 h, and 5 D after doxycycline (Dox, 5mg/kg, s.c., single dose) treatment and subjected to histological examination, RNAScope in situ hybridization (ISH) and qRT-PCR analysis. We first confirmed that the 3XTg-iHAP mice developed normal, viable, and fertile. Upon Dox treatment, qRT-PCR confirmed FasL expression was specifically induced in the liver tissues of 3XTg-iHAP mice (male, 7 weeks) but not in their littermates. H&E sections show the induction of hepatocyte apoptosis as early as 24 h followed by apoptosis-induced sterile inflammation and injury, hepatocyte mitosis, and regeneration in the liver parenchyma of 3XTg-iHAP mice. In the ISH assay, we found that (1) In littermate control mice, Mfge8 transcripts were localized in the intrahepatic bile duct epithelial cells (i.e., cholangiocytes) within the portal area. (2) In Dox-treated 3XTg-iHAP mice, we observed a profound increase of Mfge8 gene expression in parenchymal hepatocytes of the liver featured with apoptosis-associated sterile injury. In summary, we validated a novel 3XTg-iHAP mouse liver wound-healing model. The Mfge8 gene is constitutively expressed in the bile duct epithelial cells under the normal physiological condition, while upregulated in hepatocytes in response to the apoptosis-induced injury and repair. Our results suggest that liver wound healing is associated with an increase in Mfge8 expression in hepatocytes. The underlying mechanisms of whether and how MFG-E8 promotes the liver wound healing process need to be further studied. Grants are supported by NIH and the U.S. Department of Veterans Affairs. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Age and Sex on Gene Expression-Based Radiation Biodosimetry Using Mouse Peripheral Blood

Blood-based gene expression profiles that can reconstruct radiation exposure are being developed as a practical approach to radiation biodosimetry. However, age and sex could potentially limit the accuracy of the approach. In this study, we determined the impact of age on the peripheral blood cell gene expression profile of female mice exposed to radiation and identified differences and similarities with a previously obtained transcriptomic signature of male mice. Young (2 months) and old (24 months) female mice were irradiated with 4 Gy X-rays, total RNA was isolated from blood 24 hours later and subjected to whole-genome microarray analysis. Dose reconstruction analyses using a gene signature trained on gene expression data from irradiated young male mice showed accurate reconstruction of 0 or 4 Gy doses with root mean square error of ±0.75 Gy (R2 = 0.90) in young female mice. Although dose reconstruction for irradiated old female mice was less accurate than young female mice, the deviation from the actual radiation dose was not statistically significant. Pathway analysis of differentially expressed genes revealed that after irradiation, apoptosis-related functions were overrepresented, whereas functions related to quantities of various immune cell subtypes were underrepresented, among differentially expressed genes from young female mice, but not older animals. Furthermore, young mice significantly upregulated genes involved in phagocytosis, a process that eliminates apoptotic cells and preserves tissue homeostasis. Both functions were also overrepresented in young, but not old, male mice following 4 Gy X-irradiation. Lastly, functions associated with neutrophil activation that is essential for killing invading pathogens and regulating the inflammatory response were predicted to be uniquely enriched in young but not old female mice. This work supports the concept that peripheral blood gene expression profiles can be identified in mice that accurately predict physical radiation dose exposure irrespective of age and sex.

Tight Junction Protein Signaling and Cancer Biology.

Tight junctions (TJs) are intercellular protein complexes that preserve tissue homeostasis and integrity through the control of paracellular permeability and cell polarity. Recent findings have revealed the functional role of TJ proteins outside TJs and beyond their classical cellular functions as selective gatekeepers. This is illustrated by the dysregulation in TJ protein expression levels in response to external and intracellular stimuli, notably during tumorigenesis. A large body of knowledge has uncovered the well-established functional role of TJ proteins in cancer pathogenesis. Mechanistically, TJ proteins act as bidirectional signaling hubs that connect the extracellular compartment to the intracellular compartment. By modulating key signaling pathways, TJ proteins are crucial players in the regulation of cell proliferation, migration, and differentiation, all of which being essential cancer hallmarks crucial for tumor growth and metastasis. TJ proteins also promote the acquisition of stem cell phenotypes in cancer cells. These findings highlight their contribution to carcinogenesis and therapeutic resistance. Moreover, recent preclinical and clinical studies have used TJ proteins as therapeutic targets or prognostic markers. This review summarizes the functional role of TJ proteins in cancer biology and their impact for novel strategies to prevent and treat cancer.

Imp interacts with Lin28 to regulate adult stem cell proliferation in the Drosophila intestine.

Stem cells are essential for the development and long-term maintenance of tissues and organisms. Preserving tissue homeostasis requires exquisite control of all aspects of stem cell function: cell potency, proliferation, fate decision and differentiation. RNA binding proteins (RBPs) are essential components of the regulatory network that control gene expression in stem cells to maintain self-renewal and long-term homeostasis in adult tissues. While the function of many RBPs may have been characterized in various stem cell populations, how these interact and are organized in genetic networks remains largely elusive. In this report, we show that the conserved RNA binding protein IGF2 mRNA binding protein (Imp) is expressed in intestinal stem cells (ISCs) and progenitors in the adult Drosophila midgut. We demonstrate that Imp is required cell autonomously to maintain stem cell proliferative activity under normal epithelial turnover and in response to tissue damage. Mechanistically, we show that Imp cooperates and directly interacts with Lin28, another highly conserved RBP, to regulate ISC proliferation. We found that both proteins bind to and control the InR mRNA, a critical regulator of ISC self-renewal. Altogether, our data suggests that Imp and Lin28 are part of a larger gene regulatory network controlling gene expression in ISCs and required to maintain epithelial homeostasis.

RIP2 Contributes to Expanded CD4+ T Cell IFN-γ Production during Efferocytosis of Streptococcus pneumoniae–Infected Apoptotic Cells

Apoptotic cell clearance by professional and nonprofessional phagocytes in the process of efferocytosis is critical to preserve tissue homeostasis. Uptake of apoptotic cells by dendritic cells generates regulatory T cells and induces immunologic tolerance against self-antigens. In contrast, ingestion of infected apoptotic cells promotes activation of TLR4/MyD88-dependent bone marrow-derived dendritic cells (BMDCs) and triggers Th17 cell differentiation. In this study, we evaluated the impact of Streptococcus pneumoniae-infected apoptotic cell efferocytosis by BMDCs derived from C57BL/6 mice on differentiation and expansion of CD4+ T cell subsets, as well as the role of TLR2/4 and receptor-interacting protein 2 (RIP2) receptors in recognizing intracellular pathogens during efferocytosis. We demonstrated that BMDC-mediated efferocytosis of S. pneumoniae-infected apoptotic cells induced Th1 cell differentiation and expansion. Although TLR2/4 and RIP2 deficiency in BMDCs did not affect Th1 cell differentiation during efferocytosis, the absence of RIP2 decreased IFN-γ production by CD4 T cells during the expansion phase. These findings suggest that RIP2-mediated IL-1β production during efferocytosis of S. pneumoniae-infected apoptotic cells partially supports a Th1-mediated IFN-γ production microenvironment.

MRNA Translation Is Dynamically Regulated to Instruct Stem Cell Fate.

Stem cells preserve tissue homeostasis by replacing the cells lost through damage or natural turnover. Thus, stem cells and their daughters can adopt two identities, characterized by different programs of gene expression and metabolic activity. The composition and regulation of these programs have been extensively studied, particularly by identifying transcription factor networks that define cellular identity and the epigenetic changes that underlie the progressive restriction in gene expression potential. However, there is increasing evidence that post-transcriptional mechanisms influence gene expression in stem cells and their progeny, in particular through the control of mRNA translation. Here, we review the described roles of translational regulation in controlling all aspects of stem cell biology, from the decision to enter or exit quiescence to maintaining self-renewal and promoting differentiation. We focus on mechanisms controlling global translation rates in cells, mTOR signaling, eIF2ɑ phosphorylation, and ribosome biogenesis and how they allow stem cells to rapidly change their gene expression in response to tissue needs or environmental changes. These studies emphasize that translation acts as an additional layer of control in regulating gene expression in stem cells and that understanding this regulation is critical to gaining a full understanding of the mechanisms that underlie fate decisions in stem cells.

Contribution of proteases to the hallmarks of aging and to age-related neurodegeneration.

Protein quality control ensures the degradation of damaged and misfolded proteins. Derangement of proteostasis is a primary cause of aging and age‐associated diseases. The ubiquitin–proteasome and autophagy‐lysosome play key roles in proteostasis but, in addition to these systems, the human genome encodes for ~600 proteases, also known as peptidases. Here, we examine the role of proteases in aging and age‐related neurodegeneration. Proteases are present across cell compartments, including the extracellular space, and their substrates encompass cellular constituents, proteins with signaling functions, and misfolded proteins. Proteolytic processing by proteases can lead to changes in the activity and localization of substrates or to their degradation. Proteases cooperate with the autophagy‐lysosome and ubiquitin–proteasome systems but also have independent proteolytic roles that impact all hallmarks of cellular aging. Specifically, proteases regulate mitochondrial function, DNA damage repair, cellular senescence, nutrient sensing, stem cell properties and regeneration, protein quality control and stress responses, and intercellular signaling. The capacity of proteases to regulate cellular functions translates into important roles in preserving tissue homeostasis during aging. Consequently, proteases influence the onset and progression of age‐related pathologies and are important determinants of health span. Specifically, we examine how certain proteases promote the progression of Alzheimer's, Huntington's, and/or Parkinson's disease whereas other proteases protect from neurodegeneration. Mechanistically, cleavage by proteases can lead to the degradation of a pathogenic protein and hence impede disease pathogenesis. Alternatively, proteases can generate substrate byproducts with increased toxicity, which promote disease progression. Altogether, these studies indicate the importance of proteases in aging and age‐related neurodegeneration.

Murine Ex Vivo Cultured Alveolar Macrophages Provide a Novel Tool to Study Tissue-Resident Macrophage Behavior and Function.

Tissue-resident macrophages are of vital importance as they preserve tissue homeostasis in all mammalian organs. Nevertheless, appropriate cell culture models are still limited. Here, we propose a novel culture model to study and expand murine primary alveolar macrophages (AMs), the tissue-resident macrophages of the lung, invitro over several months. By providing a combination of granulocyte-macrophage colony-stimulating factor, TGFβ, and the PPARγ activator rosiglitazone, we maintain and expand mouse ex vivo cultured AMs (mexAMs) over several months. MexAMs maintain typical morphologic features and stably express primary AM surface markers throughout invitro culture. They respond to microbial ligands and exhibit an AM-like transcriptional profile, including the expression of AM-specific transcription factors. Furthermore, when transferred into AM-deficient mice, mexAMs efficiently engraft in the lung and fulfill key macrophage functions, leading to a significantly reduced surfactant load in those mice. Altogether, mexAMs provide a novel, simple, and versatile tool to study AM behavior in homeostasis and disease settings.

Electricity auto-generating skin patch promotes wound healing process by activation of mechanosensitive ion channels

Electricity constitutes a natural biophysical component that preserves tissue homeostasis and modulates many biological processes, including the repair of damaged tissues. Wound healing involves intricate cellular events, such as inflammation, angiogenesis, matrix synthesis, and epithelialization whereby multiple cell types sense the environmental cues to rebuild the structure and functions. Here, we report that electricity auto-generating glucose-responsive enzymatic-biofuel-cell (EBC) skin patch stimulates the wound healing process. Rat wounded-skin model and in vitro cell cultures showed that EBC accelerated wound healing by modulating inflammation while stimulating angiogenesis, fibroblast fuctionality and matrix synthesis. Of note, EBC-activated cellular bahaviors were linked to the signalings involved with calcium influx, which predominantly dependent on the mechanosensitive ion channels, primarily Piezo1. Inhibition of Piezo1-receptor impaired the EBC-induced key functions of both fibroblasts and endothelial cells in the wound healing. This study highlights the significant roles of electricity played in wound healing through activated mechanosensitive ion channels and the calcium influx, and suggests the possibility of the electricity auto-generating EBC-based skin patch for use as a wound healing device.

Phenotypical peculiarities and species-specific differences of canine and murine satellite glial cells of spinal ganglia.

Satellite glial cells (SGCs) are located in the spinal ganglia (SG) of the peripheral nervous system and tightly envelop each neuron. They preserve tissue homeostasis, protect neurons and react in response to injury. This study comparatively characterizes the phenotype of murine (mSGCs) and canine SGCs (cSGCs). Immunohistochemistry and immunofluorescence as well as 2D and 3D imaging techniques were performed to describe a SGC‐specific marker panel, identify potential functional subsets and other phenotypical, species‐specific peculiarities. Glutamine synthetase (GS) and the potassium channel Kir 4.1 are SGC‐specific markers in murine and canine SG. Furthermore, a subset of mSGCs showed CD45 immunoreactivity and the majority of mSGCs were immunopositive for neural/glial antigen 2 (NG2), indicating an immune and a progenitor cell character. The majority of cSGCs were immunopositive for glial fibrillary acidic protein (GFAP), 2',3'‐cyclic‐nucleotide 3'‐phosphodiesterase (CNPase) and Sox2. Therefore, cSGCs resemble central nervous system glial cells and progenitor cells. SGCs lacked expression of macrophage markers CD107b, Iba1 and CD204. Double labelling with GS/Kir 4.1 highlights the unique anatomy of SGC‐neuron units and emphasizes the indispensability of further staining and imaging techniques for closer insights into the specific distribution of markers and potential colocalizations.

Impact of aging on gene expression response to x-ray irradiation using mouse blood

As a radiation biodosimetry tool, gene expression profiling is being developed using mouse and human peripheral blood models. The impact of dose, dose-rate, and radiation quality has been studied with the goal of predicting radiological tissue injury. In this study, we determined the impact of aging on the gene expression profile of blood from mice exposed to radiation. Young (2 mo) and old (21 mo) male mice were irradiated with 4 Gy x-rays, total RNA was isolated from whole blood 24 h later, and subjected to whole genome microarray analysis. Pathway analysis of differentially expressed genes revealed young mice responded to x-ray exposure by significantly upregulating pathways involved in apoptosis and phagocytosis, a process that eliminates apoptotic cells and preserves tissue homeostasis. In contrast, the functional annotation of senescence was overrepresented among differentially expressed genes from irradiated old mice without enrichment of phagocytosis pathways. Pathways associated with hematologic malignancies were enriched in irradiated old mice compared with irradiated young mice. The fibroblast growth factor signaling pathway was underrepresented in older mice under basal conditions. Similarly, brain-related functions were underrepresented in unirradiated old mice. Thus, age-dependent gene expression differences should be considered when developing gene signatures for use in radiation biodosimetry.

The G protein-coupled receptor 15 (GPR15) regulates cutaneous immunology by maintaining dendritic epidermal T cells and regulating the skin microbiome.

The G protein-coupled receptor 15 (GPR15) regulates homing of different T-cell populations into the gut, thus, preserving tissue homeostasis. Its potential role in the preservation of homeostasis on other body interfaces, including the skin, is less well understood. We addressed the impact of GPR15 on cutaneous T-cell populations and the skin microbiome under steady-state conditions. Genetic deficiency in GPR15 substantially altered the composition of skin-resident T-cell populations. Precisely, dendritic epidermal T cells were almost absent in the epidermis of Gpr15-/- mice. The niche of dendritic epidermal T cells in the epidermis was, instead, populated by αβ TCR+ T cells. These changes were associated with shifts in the skin microbiota in Gpr15-/- mice. Collectively, our results uncover a role of GPR15 in the regulation of the cutaneous immune system and, thus, highlight the receptor as important general regulator of tissue homeostasis of exterior body interfaces.

Specialized immune responses in the peritoneal cavity and omentum.

The peritoneal cavity is a fluid filled space that holds most of the abdominal organs, including the omentum, a visceral adipose tissue that contains milky spots or clusters of leukocytes that are organized similar to those in conventional lymphoid tissues. A unique assortment of leukocytes patrol the peritoneal cavity and migrate in and out of the milky spots, where they encounter Ags or pathogens from the peritoneal fluid and respond accordingly. The principal role of leukocytes in the peritoneal cavity is to preserve tissue homeostasis and secure tissue repair. However, when peritoneal homeostasis is disturbed by inflammation, infection, obesity, or tumor metastasis, specialized fibroblastic stromal cells and mesothelial cells in the omentum regulate the recruitment of peritoneal leukocytes and steer their activation in unique ways. In this review, the types of cells that reside in the peritoneal cavity, the role of the omentum in their maintenance and activation, and how these processes function in response to pathogens and malignancy will be discussed.

Age-Related Deterioration of Mitochondrial Function in the Intestine.

Aging is an important and inevitable biological process in human life, associated with the onset of chronic disease and death. The mechanisms behind aging remain unclear. However, changes in mitochondrial function and structure, including reduced activity of the mitochondrial respiratory chain and increased production of reactive oxygen species—thus oxidative damage—are believed to play a major role. Mitochondria are the main source of cellular energy, producing adenosine triphosphate (ATP) via oxidative phosphorylation. Accumulation of damaged cellular components reduces a body's capacity to preserve tissue homeostasis and affects biological aging and all age-related chronic conditions. This includes the onset and progression of classic degenerative diseases such as cardiovascular disease, kidney failure, neurodegenerative diseases, and cancer. Clinical manifestations of intestinal disorders, such as mucosal barrier dysfunction, intestinal dysmotility, and chronic obstipation, are highly prevalent in the elderly population and have been shown to be associated with an age-dependent decline of mitochondrial function. This review summarizes our current understanding of the role of mitochondrial dysfunction in intestinal aging.

Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer

BackgroundWnt signaling drives epithelial self-renewal and disease progression in human colonic epithelium and colorectal cancer (CRC). Characterization of Wnt effector pathways is key for our understanding of these processes and for developing therapeutic strategies that aim to preserve tissue homeostasis. O-glycosylated cell surface proteins, such as α-dystroglycan (α-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/α-DG signaling pathway which triggers this mode of colonic epithelial cell-to-matrix interaction in health and disease.MethodsNext generation sequencing upon shRNA-mediated silencing of adenomatous polyposis coli (APC), and quantitative chromatin immunoprecipitation (qChIP) combined with CRISPR/Cas9-mediated transcription factor binding site targeting characterized LARGE2 as a Wnt target gene. Quantitative mass spectrometry analysis on size-fractionated, glycoprotein-enriched samples revealed functional O-glycosylation of α-DG by LARGE2 in CRC. The biology of Wnt/LARGE2/α-DG signaling was assessed by affinity-based glycoprotein enrichment, laminin overlay, CRC-to-endothelial cell adhesion, and transwell migration assays. Experiments on primary tissue, human colonic (tumor) organoids, and bioinformatic analysis of CRC cohort data confirmed the biological relevance of our findings.ResultsNext generation sequencing identified the LARGE2 O-glycosyltransferase encoding gene as differentially expressed upon Wnt activation in CRC. Silencing of APC, conditional expression of oncogenic β-catenin and endogenous β-catenin-sequestration affected LARGE2 expression. The first intron of LARGE2 contained a CTTTGATC motif essential for Wnt-driven LARGE2 expression, showed occupation by the Wnt transcription factor TCF7L2, and Wnt activation triggered LARGE2-dependent α-DG O-glycosylation and laminin-adhesion in CRC cells. Colonic crypts and organoids expressed LARGE2 mainly in stem cell-enriched subpopulations. In human adenoma organoids, activity of the LARGE2/α-DG axis was Wnt-dose dependent. LARGE2 expression was elevated in CRC and correlated with the Wnt-driven molecular subtype and intestinal stem cell features. O-glycosylated α-DG represented a Wnt/LARGE2-dependent feature in CRC cell lines and patient-derived tumor organoids. Modulation of LARGE2/α-DG signaling affected CRC cell migration through laminin-coated membranes and adhesion to endothelial cells.ConclusionsWe conclude that the LARGE2 O-glycosyltransferase-encoding gene represents a direct target of canonical Wnt signaling and mediates functional O-glycosylation of α-dystroglycan (α-DG) in human colonic stem/progenitor cells and Wnt-driven CRC. Our work implies that aberrant Wnt activation augments CRC cell-matrix adhesion by increasing LARGE/α-DG-mediated laminin-adhesiveness.AtSC-WjwGanCvAL3hWEdJuVideo abstract.Graphical abstract