Epithelial Cell Homeostasis Research Articles

FRI161 An in Vitro Triple Screen Model for Human Mineralocorticoid Receptor Activity

Abstract Disclosure: H. Liu: None. S.M. Konzen: None. A. Coy: None. J. Rege: None. C.E. Gomez-Sanchez: None. W.E. Rainey: None. A.F. Turcu: None. Background: The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron. MR is expressed in a variety of other tissues, including gastrointestinal tract, exocrine glands, central nervous system, heart, and adipose tissue. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone. In humans, cortisol concentrations far exceed those of aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. Objective: To develop an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. Methods: The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussian luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). Cells were also treated with serum from patients with LRH (N=3) or primary hypertension (PH, N=3). Luminescence was measured in conditioned medium using coelenterazine. Results: In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45nM and 0.30nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6nM and 56.0nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20nM (CV1-MRLuc) to 850nM, while the EC50 for aldosterone remained unchanged. Serum from patients with both LRH and PH led to variable degrees of luminescence in the CV1-MRLuc cells, but not in the CV1-MRluc-HSD11B2 cells, suggesting a cortisol-mediated MR activation in these patients, possibly via glycyrrhetinic acid-like factors. Conclusions: Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts. Presentation Date: Friday, June 16, 2023

Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

Mucus secreted by goblet cells (GCs) may play an important role in intestinal transit function. Our previous study found that Piezo1 protein is essential for GC function; however, the effect of GC Piezo1 on intestinal transit function is unclear. Our study aimed to investigate the effect of Piezo1 in GCs on intestinal transit and the potential mechanism. We compared intestinal mucus, fecal form, intestinal transit time, intestinal epithelial cell composition, and stem cell function in WT and GC-specific Piezo1-deficient (Piezo1ΔGC) mice. Our results revealed a correlation between mucus and intestinal transit: the less mucus there was, the slower the intestinal transit. Piezo1 deficiency in GCs led to decreased mucus synthesis and also disrupted the ecological niche of colon stem cells (CSCs). Through organoid culture, we found that the capacity of proliferation and differentiation in Piezo1ΔGC mouse CSCs was significantly decreased, which also led to a reduced source of GCs. Further studies found that the reduced Wnt and Notch signals in colon crypts might be the potential mechanism. These results indicated the importance of GC Piezo1 in intestinal transit function, which acts by maintaining the homeostasis of intestinal epithelial cells and mucus.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Human milk-derived MANF, as an immuno‐nutritional factor, maintains the intestinal epithelial barrier and protects against necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Compared to formula milk, breastfeeding protects against NEC. However, the composition of breast milk is quite complicated, and many immunological compositions remain unknown. In this study, we aimed to investigate the concentration of a secreted protein, Mesencephalic astrocyte-derived neurotrophic factor (MANF), in breastmilk and evaluate its immune-regulatory function in protecting the intestinal epithelial barrier. Our data indicated that MANF was secreted in human milk but could not be detected in infant formulas. More importantly, the amount of MANF in colostrum was higher than that in mature milk. We also clarified that MANF was mainly expressed in intestinal macrophages and was capable of inducing apoptosis and decreasing the inflammation of pro-inflammatory macrophages in both NEC intestinal tissues and BMDMs. Mechanismly, MANF protein significantly inhibited the apoptosis of intestinal epithelial cells and protected epithelial tight junctions through downregulation of the NF-κB pathway in pro-inflammatory macrophages. These results reveal the crucial function of human milk-derived MANF in intestinal macrophages, which contributes to downregulating the intestinal inflammatory response and protecting the homeostasis of intestinal epithelial cells. Our study not only demonstrates a potential mechanism underlying breastfeeding protective effects in NEC but also, more importantly, enables clinical translation, facilitating new strategies for the development of nutritional interventions in the prevention of NEC.

The Pathogenicity of Fusobacterium nucleatum Modulated by Dietary Fibers-A Possible Missing Link between the Dietary Composition and the Risk of Colorectal Cancer.

The dietary composition has been approved to be strongly associated with the risk of colorectal cancer (CRC), one of the most serious malignancies worldwide, through regulating the gut microbiota structure, thereby influencing the homeostasis of colonic epithelial cells by producing carcinogens, i.e., ammonia or antitumor metabolites, like butyrate. Though butyrate-producing Fusobacterium nucleatum has been considered a potential tumor driver associated with chemotherapy resistance and poor prognosis in CRC, it was more frequently identified in the gut microbiota of healthy individuals rather than CRC tumor tissues. First, within the concentration range tested, the fermentation broth of F. nucleatum exhibited no significant effects on Caco-2 and NCM460 cells viability except for a notable up-regulation of the expression of TLR4 (30.70%, p < 0.0001) and Myc (47.67%, p = 0.021) and genes encoding proinflammatory cytokines including IL1B (197.57%, p < 0.0001), IL6 (1704.51%, p < 0.0001), and IL8 (897.05%, p < 0.0001) in Caco-2 cells exclusively. Although no marked effects of polydextrose or fibersol-2 on the growth of F. nucleatum, Caco-2 and NCM460 cells were observed, once culture media supplemented with polydextrose or fibersol-2, the corresponding fermentation broths of F. nucleatum significantly inhibited the growth of Caco-2 cells up to 48.90% (p = 0.0003, 72 h, 10%) and 52.96% (p = 0.0002, 72 h, 10%), respectively in a dose-dependent manner. These two kinds of fibers considerably promoted butyrate production of F. nucleatum up to 205.67% (p < 0.0001, 6% polydextrose at 24 h) and 153.46% (p = 0.0002, 6% fibersol-2 at 12 h), which explained why and how the fermentation broths of F. nucleatum cultured with fibers suppressing the growth of Caco-2 cells. Above findings indicated that dietary fiber determined F. nucleatum to be a carcinogenic or antitumor bacterium, and F. nucleatum played an important role in the association between the dietary composition, primarily the content of dietary fibers, and the risk of CRC.

FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/− mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6+/− mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6+/− mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERβ subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERβ attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERβ promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and β-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERβ were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERβ subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

Epithelial TIPE1 Protein Guards against Colitis by Inhibiting TNF-α-Mediated Inflammation.

Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response, and IEC dysfunction has been linked to multiple inflammatory diseases, including inflammatory bowel disease. In this study, we found that a member of the TNF-α-induced protein 8 (TNFAIP8 or TIPE) family called TIPE1 is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. TIPE1-deficient mice, or chimeric mice that were deficient in TIPE1 in their nonhematopoietic cells, were more sensitive to dextran sulfate sodium-induced experimental colitis; however, TIPE1 deficiency had no impact on the development of inflammation-associated and sporadic colorectal cancers. Mechanistically, TIPE1 prevented experimental colitis through modulation of TNF-α-dependent inflammatory response in IECs. Importantly, genetic deletion of both TIPE1 and its related protein TNFAIP8 in mice led to the development of spontaneous chronic colitis, indicating that both of these two TIPE family members play crucial roles in maintaining intestinal homeostasis. Collectively, our findings highlight an important mechanism by which TIPE family proteins maintain intestinal homeostasis and prevent inflammatory disorders in the gut.

Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata.

Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.

Molecular Insights on Shigellosis: How the Interaction Between Invasin IpaA and Vinculin Hijacks Cellular Mechanotransduction

With 270 million infections annually and nearly half a million death a year, shigellosis is a severe intestinal infection caused by bacteria of the Shigella family. Appearance and spread of drug-resistant strains renewed global concerns for public health and finding novel targets for treatment is fast becoming a priority. To this end, invasins are a potentially good candidate. Also called Ipa(s), which is the short for Invasion Plasmid Antigen, invasins play a key role in mediating bacterial invasion and infection of the host cell. Importantly, they have been reported to hijack inbuilt mechanical capability of the host cells such as cell adhesion and active processes mediated by the actin cytoskeleton to enable bacterial ingress into the host cells. IpaA is an invasin of particular interest as it presents three motifs that mimic vinculin binding sites and thus it allows IpaA to interact with vinculin, which is one of the most critical regulators of cellular and tissue mechanics. Using a mechanobiology point-of-view, we aim to provide an overview of Shigella´s infection mechanism, to highlight recently discovered molecular mechanisms of IpaA/vinculin interaction and to finally discuss their consequences for epithelial cell and tissue mechanical homeostasis that may result in the symptomatic outcomes seen in severe shigellosis.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

Inactivation of epithelial sodium ion channel molecules serves as effective diagnostic biomarkers in clear cell renal cell carcinoma.

Non-voltage-gated sodium channel, also known as the epithelial sodium channel (ENaC), formed by heteromeric complexes consisting of SCNN1A, SCNN1B, and SCNN1G, is responsible for maintaining sodium ion and body fluid homeostasis in epithelial cells. However, no systematic study of SCNN1 family members has been conducted in renal clear cell carcinoma (ccRCC) to date. To investigate the abnormal expression of SCNN1 family in ccRCC and its potential correlation with clinical parameters. The transcription and protein expression levels of SCNN1 family members in ccRCC were analyzed based on the TCGA database, and were confirmed by quantitative RT-PCR and immunohistochemical staining assays, respectively. The areaunder curve (AUC) was used to evaluate the diagnostic value of SCNN1 family members for ccRCC patients. The mRNA and protein expression of SCNN1 family members was significantly downregulated in ccRCC compared with normal kidney tissues, which might be due to DNA hypermethylation in the promoter region. It is worth noting that the AUC of SCNN1A, SCNN1B, and SCNN1G were 0.965, 0.979, and 0.988 based on the TCGA database (p < 0.0001), respectively. The diagnostic value was even higher when combing these three members together (AUC = 0.997, p < 0.0001). Intriguingly, the mRNA level of SCNN1A was significantly lower in females compared with males, while SCNN1B and SCNN1G were increased with the progression of ccRCC and remarkably associated with a worse outcome for patients. The aberrantly decrease of SCNN1 family members might serve as valuable biomarkers for the diagnosis of ccRCC.

Cell-autonomous complement C3 maintains prostate epithelial cell homeostasis and prevents malignant transformation

Abstract Intracellularly active complement C3 and C3a emerged as central regulators of normal immune cell function and proliferation via control of mTOR and glycolysis. Because increased glycolysis is a hallmark of many cancer cells, we hypothesized that augmented cell-intrinsic C3 activity may contribute to epithelial cell malignancy. However, while benign prostate epithelial cells (PECs) harbored expected intracellular C3/C3a storages, cancerous PECs, surprisingly, exhibited loss of C3/C3a. Further, C3/C3a reduction levels in PECs correlated inversely with worsening Gleason scores in patients, silencing of C3 in healthy PECs induced uncontrolled proliferation, and reconstitution of intracellular C3a in prostate cancer cell line DU145 normalized their hyper-proliferation. In line with an unexpected anti-proliferative role for C3a in PECs, C3ar−/−mice developed spontaneous hyperplasia of prostate tubular cells with age, treatment of DU145 tumors, implanted subcutaneously into the flanks of athymic mice, with C3a-expressing adenovirus significantly reduced tumor growth in vivo, and augmented C3 levels are associated with a better prognosis in patients with prostate and other types of epithelial cell cancers. RNA-sequencing of C3or C3aR-deficient human PECs identified intrinsic C3a as novel controller of several known PEC oncogenes, likely via up-stream impact on the c-MYC pathway. In sum, while intracellular C3 drives proliferation in immune cells, in (prostate) epithelial cells, cell-autonomous C3/C3a is a negative regulator of growths and, thus, represses oncogenic transformation. Such cell-specific intracellular C3/C3a activities should be taken into consideration when targeting this system therapeutically.

MRPL12-ANT3 interaction involves in acute kidney injury via regulating MPTP of tubular epithelial cells

Acute kidney injury (AKI) is a serious disease with no effective treatment. Abnormal opening of mitochondrial permeability transition pore (MPTP) is an important pathological process in ischemia reperfusion injury (IRI), the key factor of AKI. It is essential to elucidate MPTP regulation mechanism. Here, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) specifically binds to adenosine nucleotide translocase 3 (ANT3) under normal physiological conditions, stabilizes MPTP and maintains mitochondrial membrane homeostasis in renal tubular epithelial cells (TECs). During AKI, MRPL12 expression was significantly decreased in TECs, and MRPL12-ANT3 interaction was reduced, leading to ANT3 conformation change, MPTP abnormal opening, and cell apoptosis. Importantly, MRPL12 overexpression protected TECs from MPTP abnormal opening and apoptosis during hypoxia/reoxygenation (H/R). Our results suggest MRPL12-ANT3 axis involves in AKI by regulating MPTP, and MRPL12 could be potential intervention target for treatment of AKI.

Abstract 1424: Selective blockade of neuropeptide Y receptor 1 induces cell cycle arrest and promotes apoptosis in colon cancer cells

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer related deaths worldwide. The balance between cancer cell proliferation and apoptosis, which maintains intestinal epithelial cell homeostasis, is disturbed in CRC. Furthermore, deregulation of the apoptotic pathway contributes to therapeutic resistance. Therefore, determination of the mechanisms employed by CRC cells to evade apoptosis is important for development of new therapeutic strategies. Our group has recently reported that neuropeptide Y (NPY), which is a 36-amino acid neuropeptide, is associated with increased angiogenic response in colon cancer. However, the regulatory role of NPY in cellular homeostasis in CRC is largely unknown. NPY is secreted by colon cancer cells that express Y1 receptors (Y1R). The aim of the present study was to investigate the role of NPY in regulation of cell growth and survival in CRC and to determine the apoptosis inducing effects of Y1R antagonist, BIBO 3304 trifluoroacetate (BIBO), in CT26 colon carcinoma cells. Methods: Correlation between NPY expression and cell cycle and apoptosis-related genes were analyzed in cBioPortal for cancer genomics with a sample number of 594 CRC patients from the cancer genome atlas database (TCGA). Based on the TCGA database, 16 apoptosis-related genes (11 proapoptotic and 5 antiapoptotic) were checked. The effect of Y1R blocker (BIBO, Bio-Techne, MN) on CT26 (ATCC) cell proliferation, apoptosis and cell cycle progression was determined by performing in vitro cell proliferation assay and flow cytometry. Results and Conclusion: NPY expression in CRC shows a positive correlation with pro-survival/anti-apoptotic genes (BCL2, BCL2L1, BCL2L2, BCL2A1, MCL1) and negative correlation with key genes associated with the induction of cellular apoptosis (GZMB, TRIB3, TPX2, BAX, BAK1, BAD, BID, BIK, BOK, HRK) in CRC. Decreased cell proliferation and increased apoptosis was observed in CT26 mouse CRC cells upon inhibition of Y1R. An increased number of CT26 cells in early and mid-apoptotic phase was observed upon treatment with Y1R antagonist, BIBO, compared to untreated cells. In addition, cell cycle analysis indicated an increase in cell numbers in the G1/S cell cycle transition phase in the BIBO treated group. Our findings therefore indicate that treatment of CT26 CRC cells with Y1R blocker results in cell cycle arrest, which provides an opportunity for the cells to either undergo repair mechanisms or follow the apoptotic pathway. Blocking the actions of NPY by Y1R blockers thus may be a promising therapeutic approach in CRC patients. Citation Format: Sooraj Kakkat, Steven McClellan, Debanjan Chakroborty, Chandrani Sarkar. Selective blockade of neuropeptide Y receptor 1 induces cell cycle arrest and promotes apoptosis in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1424.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells

Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs invivo. We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed invitro. Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Simvastatin as a topical anti-inflammatory: Effect on airway epithelial cell homeostasis.

Despite the significant morbidity associated with chronic rhinosinusitis in individuals with asthma (CRSwA) there is a paucity of codified, evidence-based management strategies for CRS in this population. Using PubMed, EMBASE, and Cochrane Review Databases, a systematic review was performed covering management strategies for CRSwA. 5,903 articles were screened; 70 were included for full-text analysis. After application of exclusion criteria, 53 articles comprised the qualitative synthesis. The level of evidence was graded, and benefit-harm assessments, as well as value judgment and recommendations were provided RESULTS: : Strong evidence confirms the benefit of oral and topical medications on sinonasal-specific outcomes in individuals with CRSwA; there is low-grade evidence demonstrating that these agents improve lung function and/or asthma control. Moderate-to-strong evidence suggests that endoscopic sinus surgery (ESS) improves both sinonasal- and asthma-specific quality of life. Although there is insufficient-to-low evidence to indicate that ESS improves pulmonary function in this population, data indicate a positive impact of this intervention on asthma control. Biologic medications strongly improve both subjective and objective sinonasal- and asthma-specific outcomes. Evidence supports managing CRS in individuals with CRSwA in a stepwise fashion, starting with traditional non-biologic oral and topical medication, and escalating to second-line treatments, such as ESS and biologics. Optimal treatment of individuals who suffer from CRSwA often requires concurrent, directed management of asthma, as not all CRS interventions impact asthma status. This article is protected by copyright. All rights reserved.

RUNX3 in Stem Cell and Cancer Biology.

The runt-related transcription factors (RUNX) play prominent roles in cell cycle progression, differentiation, apoptosis, immunity and epithelial-mesenchymal transition. There are three members in the mammalian RUNX family, each with distinct tissue expression profiles. RUNX genes play unique and redundant roles during development and adult tissue homeostasis. The ability of RUNX proteins to influence signaling pathways, such as Wnt, TGFβ and Hippo-YAP, suggests that they integrate signals from the environment to dictate cell fate decisions. All RUNX genes hold master regulator roles, albeit in different tissues, and all have been implicated in cancer. Paradoxically, RUNX genes exert tumor suppressive and oncogenic functions, depending on tumor type and stage. Unlike RUNX1 and 2, the role of RUNX3 in stem cells is poorly understood. A recent study using cancer-derived RUNX3 mutation R122C revealed a gatekeeper role for RUNX3 in gastric epithelial stem cell homeostasis. The corpora of RUNX3R122C/R122C mice showed a dramatic increase in proliferating stem cells as well as inhibition of differentiation. Tellingly, RUNX3R122C/R122C mice also exhibited a precancerous phenotype. This review focuses on the impact of RUNX3 dysregulation on (1) stem cell fate and (2) the molecular mechanisms underpinning early carcinogenesis.

Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction.

Redox Homeostasis in Well-differentiated Primary Human Nasal Epithelial Cells.

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H2O2) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H2O2 exposure, cell integrity decreased (p<0.05), GSH/GSSG ratio reduced (p<0.05), and ATP production declined by 83% (p<0.05) in the sensitive and 55% (p<0.05) in the resistant group; mtROS production increased 3.4-fold (p<0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function.