Goblet Cell Formation Research Articles

Full thickness 3D in vitro conjunctiva model enables goblet cell differentiation

In vitro culture and generation of highly specialized goblet cells is still a major challenge in conjunctival 3D in vitro equivalents. A model comprising all physiological factors, including mucus-secreting goblet cells has the potential to act as a new platform for studies on conjunctival diseases. We isolated primary conjunctival epithelial cells and fibroblasts from human biopsies. 3D models were generated from either epithelial layers or a combination of those with a connective tissue equivalent. Epithelial models were investigated for marker expression and barrier function. Full-thickness models were analyzed for goblet cell morphology and marker expression via immunofluorescence and quantitative real-time PCR. Simple epithelial models cultured at the air–liquid interface showed stratified multi-layer epithelia with pathologic keratinization and without goblet cell formation. The combination with a connective tissue equivalent to generate a full-thickness model led to the formation of a non-keratinized stratified multi-layer epithelium and induced goblet cell differentiation. In our model, a high resemblance to natural conjunctiva was achieved by the combination of conjunctival epithelial cells with fibroblasts embedded in a collagen-hydrogel as connective tissue equivalent. In the future, our conjunctival in vitro equivalent enables the investigation of goblet cell differentiation, conjunctival pathologies as well as drug testing.

Comparative valuation of the chlorpyrifos, acetamiprid, and lambda-cyhalothrin toxicity and their hematological and histopathological consequences in pigeons.

Globally agrochemicals are widely used in the agricultural sectors, posing potential eco-toxicological risks and disrupting various lifeforms including birds. Thus, the current work was conducted to compare the acute toxic impacts of pesticides (e.g., chlorpyrifos, acetamiprid, and lambda-cyhalothrin) on the pigeon's health. In total 50 adult pigeons were purchased from a local market where these pigeons were fed on pollution-free food. Post adaptation period (15 days), the pigeons were arbitrarily separated into five distinct groups after having been identified in this manner by chance (each group containing 10 pigeons). Control group (group 1) was not treated with any pesticide while the remaining groups (groups 2, 3, and 4) were treated with 0.25-mg/kg body weight of chlorpyrifos, acetamiprid, lambda-cyhalothrin, and a mixture of all three pesticides (group 5), respectively. After 36 days of exposure, the groups that had been exposed to the pesticide showed a significant (p < 0.05) increase in both the total number of platelets and the number of white blood cells (WBCs), in comparison to the control group. On the other hand, the groups that were exposed to the insecticides had significantly lower levels of red blood cells (RBCs), hemoglobin (Hb), and packed cell volume (PCV) (p < 0.05). The value of mean corpuscular volume (MCV) was significantly (p < 0.05) reduced in acetamiprid-exposed group, while a significant increase was observed in other pesticide-exposed groups. Obvious histopathological changes were observed in the tissues of control group and no such changes were reported by control group. Necrosis, pyknosis, lymphocyte infiltration, congestion of blood, dissolution of plasma membrane, and vacuolation were observed in the livers of pesticide-treated pigeons. The intestinal study showed the formation of goblet cells, villi rupturing, degeneration of serosa, necrosis, and pyknosis in treated groups. Renal alterations, dilation of renal tubules, reduction of glomerulus tissue, and edema were observed. This study manifests that the uncontrolled use of pesticides impairs ecosystems and poses a substantial health risk to wildlife and ultimately to human.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr−/− mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

Repeated exposure of house dust mite induces progressive airway inflammation in mice: Differential roles of CCL17 and IL-13.

We conducted a systematic evaluation of lung inflammation indued by repeated intranasal exposure (for 10 consecutive days) to a human aeroallergen, house dust mite (HDM) in BALB/c mice. Peak influx of neutrophils, monocytes/lymphocytes, and eosinophils was observed in bronchoalveolar lavage (BAL) on days 1, 7 and 11, respectively, and normalized to baseline by day 21. Peak elevations of Th2, myeloid‐derived cytokines/chemokines and serum IgE were seen both in BAL and lung tissue homogenates between days 7 and 11, and declined thereafter; however, IL‐33 levels remained elevated from day 7 to day 21. Airway hyperreactivity to inhaled methacholine was significantly increased by day 11 and decreased to baseline by day 21. The lung tissue showed perivascular and peribronchial cuffing, epithelial hypertrophy and hyperplasia and goblet cell formation in airways by day 11, and resolution by day 21. Levels of soluble collagen and tissue inhibitors of metalloproteinases (TIMP) also increased reflecting tissue remodeling in the lung. Microarray analysis demonstrated a significant time‐dependent up‐regulation of several genes including IL‐33, CLCA3, CCL17, CD4, CD10, CD27, IL‐13, Foxa3, IL‐4, IL‐10, and CD19, in BAL cells as well as the lung. Pre‐treatment of HDM challenged mice with CCL17 and IL‐13 antibodies reduced BAL cellularity, airway hyper‐responsiveness (AHR), and histopathological changes. Notably, anti‐IL‐13, but not anti‐CCL17 monoclonal antibodies (mAbs) reduced BAL neutrophilia while both mAbs attenuated eosinophilia. These results suggest that CCL17 has an overlapping, yet distinct profile versus IL‐13 in the HDM model of pulmonary inflammation and potential for CCL17‐based therapeutics in treating Th2 inflammation.

Human Bronchial Epithelial Cell Growth on Homologous Versus Heterologous Tissue Extracellular Matrix

BackgroundExtracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. Materials and methodsPorcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. ResultsBoth trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. ConclusionsECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.

Long noncoding RNA H19 disrupts the intestinal barrier by inhibiting Goblet cell function

The intestinal mucosal barrier consists of multiple elements including mucus, epithelial layer, and immune defense; nonetheless, barrier dysfunction occurs commonly in various disorders. Goblet cells secrete mucin proteins that form surface mucus layer to act as the first physical defense in the barrier, but the exact mechanism underlying the control of Goblet cell function remains largely unknown. Long noncoding RNAs (lncRNAs) modulate a variety of biological functions by controlling gene expression at different levels. The lncRNA H19 is transcribed from the conserved imprinted H19/igf2 gene cluster and implicated in many aspects of gut mucosal pathophysiology. In this study, we examined if H19 regulates Goblet cell function and further investigated its impact in intestinal barrier function.METHODSIntestinal mucosal tissues were collected from H19‐deficient (H19−/−) and control littermate mice. The function of Goblet cells was examined by alcian blue staining and mucin 2 immunohistochemical staining assays. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer FITC‐dextran assays. Intestinal organoids were isolated from H19−/− and littermate mice and treated with lipopolysaccharide (LPS).RESULTSH19 deletion in mice promoted Goblet cell function in vivo and ex vivo, as evidenced by increases in the numbers of alcian blue‐positive cells in the intestinal mucosa of H19−/− mice and mucin 2‐positive cells in the H19‐deficient intestinal organoids isolated from H19−/− mice, compared with those in control littermate mice. In contrast, ectopically expressed H19 prevented the increased numbers of Goblet cells in the H19‐deficient organoids from H19−/− mice, although it had only a minor inhibition on Goblet cells in the organoids from littermate mice. H19 knockout also elevated the levels of autophagy proteins LC3‐II, lysozyme and beclin in the intestinal epithelium. H19 deletion in mice protected the intestinal barrier function in response to septic stress by protecting Goblet cells. Exposure to CLP reduced the numbers of Goblet cells in littermate mice, but H19 deletion significantly prevented the CLP‐elicited loss of Goblet cells. H19−/− mice also displayed much minor increase in gut permeability than that observed in control littermates after CLP. In the ex vivo model, treatment with LPS suppressed the formation of Goblet cells as shown by a dramatic decrease in the number of mucin 2‐positive cells in intestinal organoids generated from control littermate mice, but this inhibition was alleviated in the organoids from H19−/− mice.CONCLUSIONSThese results indicate that 1) H19 deletion in mice enhances Goblet cell function in the intestinal mucosa; and 2) preventing the rise in H19 protects Goblet cells from septic stress. These results suggest that induced H19 disrupts intestinal mucosal barrier by inhibiting Goblet cell function.Support or Funding InformationUS Department of Veterans Affairs (JNR, J‐Y.W) and NIH (J‐Y.W)

The Effects of Halofuginone on Wound Healing in the Rat Nasal Mucosa.

Background Halofuginone is an alkaloid febrifugine analogue and bioactive molecule that was isolated incidentally from the Dichroa febrifuga plant. The therapeutic efficacy of halofuginone in parasitic infections, scleroderma, inflammation, and fibrosis-related diseases, as well as in some types of cancer, has been previously reported. The effects of halofuginone on nasal mucosal damage are not yet known. Objective The aim of this study was to investigate the potential effect of topically applied halofuginone on wound healing in the mechanically injured nasal mucosa of rats. Methods A unilateral mucosal wound was created in the nasal cavity of 32 rats (aged 4 weeks) using the brushing technique. These rats were randomly divided into 4 groups. Although the control group did not receive an intervention, a dry pad, a saline-impregnated pad, or a pad impregnated with halofuginone were placed in the rats of the other 3 groups and left for 5 minutes. Rats were sacrificed on the 14th day, and a histological examination was performed. The nasal mucosa was assessed via hematoxylin-eosin and Masson’s trichrome staining. Results There were no statistically significant differences in epithelial thickness, inflammation, goblet cell formation, and epithelial disarray values between the halofuginone group and the control group ( P &gt; .05). The subepithelial thickness was significantly decreased in the saline-treated group and the halofuginone-treated group ( P &lt; .05), but a significantly lower level of subepithelial fibrosis was only observed in the halofuginone group compared to the other groups ( P &lt; .05). Conclusions Topical halofuginone administration reduces the development of fibrosis and subepithelial edema after experimentally induced nasal mucosal injury, but it does not exert therapeutic or preventive effects on epithelial damage, inflammation, and goblet cell hyperplasia.

Inflammation, vascularization and goblet cell differences in LSCD: Validating animal models of corneal alkali burns

Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid–Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.

Clinicobacterial and histopathological study of chronic dacryocystitis

Introduction: Diseases of lacrimal drainage system account for nearly 3% of visits to eye clinic. Chronic dacryocystitis is a frequently encountered disorder among these patients. The aim of this study was to identify the organisms responsible and to determine the antibiotic susceptibility pattern of the bacterial isolates from conjunctiva and nasal mucosa in cases of chronic dacryocystitis and comparing with lacrimal sac specimen. Material and Methods: Prospective longitudinal study carried out in 40 cases suffering from chronic dacryocystitis. After complete history and local examination of the cases, the sac was excised and examined histopathologically in pathology department. Swabs of the smear were taken in all cases were cultured and antibiotic sensitivity was done. The results were analyzed by using mean, median and the Chi-square (χ2) test. Results: The maximum incidence of the disease has been in the 4th and 5th decade of life, females were more predominantly affected than the males. The epithelial hypertrophy was the most common histological finding i.e 77.5%, the second common was squamous metaplasia followed by goblet cell formation. Culture and antibiotic sensitivity was done in all cases; out of these the maximum cases were found to be sterile i.e.55%. The bacteria which have been isolated in the 45% cases in order of frequency were staphylococcus 20%, streptococcus12.5% and pneumococcus 12.5% respectively. Conclusion: Majority of the chronic dacryocystitis cases are caused by Staphylococcus, streptococcus and pneumococcus. Amoxyclav and 3rd generation cephalosporins can be used to treat chronic dacryocystitis. The epithelial hypertrophy and Non-specific chronic inflammation with fibrosis is indeed the most commonly reported histopathological finding in lacrimal sac wall biopsy specimens. Microbiological analysis and antimicrobial susceptibility pattern is mandatory for the selection of a specific antimicrobial therapy and to the control of further resistance development of bacterial strains.

IL13 activates autophagy to regulate secretion in airway epithelial cells

ABSTRACTCytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

Pathology of Mycoplasma gallisepticum infection in naturally infected layer birds

Sixty five commercial layer farms with lesions suggestive of respiratory problem were investigated during the study period from May 2014 to April 2015 to evaluate the pathology of Mycoplasma gallisepticum infection in laying chicken. Mycoplasma gallisepticum was confirmed through PCR in 36 out of 65 farms. The positive farms were examined for gross and histopathological changes in tissues like trachea, lungs, air sacs and liver. The highest incidence of MG infection occurred during the winter season. Grossly, MG affected birds showed catarrhal inflammation of trachea and bronchi, slight pneumonic changes in lungs, cloudiness, air sacculitis and caseous exudate in thoracic air sacs, congestion and haemorrhages in lungs, caseous mass in both thoracic and abdominal air sacs. Histopathologically, focal destruction of superficial cells, goblet cell formation, hyperplasia of mucosal epithelium, submucosal infiltration of lymphocytes, macrophages along with haemorrhagic areas were observed in the trachea. Lungs revealed interstitial haemorrhages, parabronchiolar oedema, mucosal hyperplasia of secondary bronchiole and interstitial pneumonia characterized by thickening of interstitium. Air sacs showed epithelial destruction and increased thickening due to proliferation of submucosal connective tissue with congested blood vessels and neovascularization. This study proved that MG infection occurs severely among laying chicken causing more economic loss to the farmers.

TGFβ signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.

The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor β receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFβ signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFβ restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFβ inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFβ in regulating goblet cell differentiation.

Role of Cyclooxygenase-2 in Exacerbation of Allergen-Induced Airway Remodeling by Multiwalled Carbon Nanotubes

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

Intraluminal pH and Goblet Cell Density in Barrett’s Esophagus

IntroductionGoblet cells in Barrett’s esophagus (BE) vary in their density within the Barrett’s segment. Exposure of Barrett’s epithelium to bile acids is a major stimulant for goblet cell formation. The dissociation of bile acids into forms that penetrate Barrett’s epithelium is known to be pH dependent. We hypothesized that variations in the esophageal luminal pH environment explains the variability in goblet cell density. The aim of this study was to correlate esophageal luminal pH with goblet cell density in patients with BE. MethodsA customized six-sensor pH catheter was positioned with the most distal sensor in the stomach and the remaining sensors located 1 cm below and 1, 3, 5, and 8 cm above the upper border of the lower esophageal sphincter in five normal subjects and six patients with long-segment BE. The luminal pH was measured by each sensor for 24-h and expressed as median pH. Patients with BE had four quadrant biopsies at levels corresponding to the location of the pH sensors. Goblet cell density was graded from 0 to 3 based on the number per high-power field. ResultsIn normal subjects, the median pH values recorded in the sensors within the lower esophageal sphincter (LES) and esophageal body were all above 5. In patients with BE, the median pH recorded by the sensor within the LES was 2.8 and increased progressively to 4.7 in the sensor at 8 cm above the LES. Goblet cell density was significantly lower in the distal Barrett’s segment exposed to a median pH of 2.2 and increased in the proximal Barrett’s segment exposed to a median pH of 4.4 (p = 0.003). ConclusionPatients with BE have a goblet cell gradient that correlates directly with an esophageal luminal pH gradient. This suggests that goblet cell differentiation is pH dependent and likely due to the effect of pH on bile acid dissociation.

Hypoxia and lineage specification of cell line-derived colorectal cancer stem cells

Hypoxia is an important regulator of normal and cancer stem cell (CSC) differentiation. Colorectal CSCs from SW1222, LS180, and CCK81 colorectal cancer-derived cell lines are able to differentiate into complex 3D lumen-containing structures in normoxia, whereas in hypoxia, they form undifferentiated dense colonies that have reduced expression of the enterocyte differentiation marker CDX1, lack goblet cell formation, and have increased expression of BMI1 and activated Notch1. Hypoxia increases the clonogenicity of CSCs, which is cumulative as each round of hypoxia enriches for more CSCs. The hypoxic phenotype is reversible, because cells from hypoxic-dense colonies are able to reform differentiated structures when regrown in normoxia. We show that CDX1 is able to stimulate the generation of lumens even in hypoxia and has a negative feedback on BMI1 expression. Knockdown of CDX1 reduces lumen formation but does not affect goblet cell formation, suggesting that enterocytes and goblet cells form from different progenitor cells. Notch inhibition by dibenzazepine (DBZ) allowed CSCs to form goblet cells in both normoxia and hypoxia. Finally, we show that Hif1α, but not CA9, is an important mediator of the effects of hypoxia on the clonogenicity and differentiation of CSCs. In summary, hypoxia maintains the stem-like phenotype of colorectal cell line-derived CSCs and prevents differentiation of enterocytes and goblet cells by regulating CDX1 and Notch1, suggesting that this regulation is an important component of how hypoxia controls the switch between stemness and differentiation in CSCs.

A Divergent Substrate-Binding Loop within the Pro-oncogenic Protein Anterior Gradient-2 Forms a Docking Site for Reptin

Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin–AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2–Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site , and for measuring the effects of the Reptin–AGR2 complex in cancer cell growth.

Regulation of mucin expression in respiratory diseases

Respiratory diseases such as asthma and COPD (chronic obstructive pulmonary disease) are characterized by increased numbers of goblet cells and excessive mucus production, which contribute to the underlying disease pathology. Mucins form a major component of the mucus contributing to its viscoelastic properties, and in the airways the mucins MUC5AC and MUC5B are found at increased levels in both asthmatic and COPD subjects. A diverse range of stimuli have been shown to regulate MUC5AC expression and cause increases in the number of mucus-producing goblet cells. Perhaps the best characterized of these mediators is the cytokine IL (interleukin)-13, which causes increases in MUC5AC-expressing goblet cells in the airways. Several transcription factors have been linked with goblet cell formation and mucus production and include STAT6 (signal transducer and activator of transcription 6), FOXA2 (forkhead box A2) and the SPDEF [SAM (sterile alpha motif) domain-containing prostate-derived Ets factor]. In mouse airways, goblet cells are normally rare or absent, but increase rapidly in number in response to certain stimuli. The origins of these goblet cells are not well understood, although Clara cells and ciliated cells have been implicated as goblet cell progenitors. An understanding of the origin and processes regulating goblet cell formation in human airway epithelial cells has important implications for the identification of therapeutic targets to treat respiratory diseases.

Regulation of Neuregulin 1β1–Induced MUC5AC and MUC5B Expression in Human Airway Epithelium

Excessive mucus production has been linked to many of the pathologic features of respiratory diseases, including obstruction of the airways, decline in lung function, increased rates of mortality, and increased infections. The mucins, MUC5AC and MUC5B, contribute to the viscoelastic properties of mucus, and are found at elevated levels in the airways of individuals with chronic respiratory diseases. The T helper type 2 cell cytokine, IL-13, is known to regulate MUC5AC expression in goblet cells of the airways, although much less is known about the regulation of MUC5B expression. In a study to further understand the mediators of MUC5AC and MUC5B expression, neuregulin (NRG) 1beta1 was identified as novel regulator of goblet cell formation in primary cultures of human bronchial epithelial cells (HBECs). NRG1beta1 increased expression of MUCAC and MUC5B proteins in a time- and dose-dependent fashion in HBEC cultures. NRG1beta1-induced expression of MU5AC and MUC5B was shown to involve v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB) and ErbB3 receptors, but not ErbB4 receptors. Treatment of HBECs with inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, and phosphatidylinositol 3-kinase indicated that these kinases were involved in NRG1beta1-induced MUC5AC and MUC5B expression. Additionally, NRG1beta1 was shown to induce the phosphorylation of the ErbB2 receptor, AKT, and extracellular signal-regulated kinase 1/2. NRG1beta1 protein was found increased in the airways of antigen-challenged mice, together with increases in MUC5AC and MUC5B message. Together, these data indicate that NRG1beta1 is a novel mediator of MUC5AC and MUC5B expression in HBECs, and may represent a novel therapeutic target for mucus hypersecretion in respiratory diseases.

Expression of Resistin-like Molecule Beta in Barrett’s Esophagus: A Novel Biomarker for Metaplastic Epithelium

The formation of goblet cells characterizes the intestinal metaplasia of Barrett's esophagus (BE). Hematoxylin-eosin (HE) staining may fail to show intestinal metaplasia in BE, and PAS-Alcian Blue may present difficulties of interpretation due to its more heterogeneous staining. Recent evidence indicates that expression of resistin-like molecule beta (RELMbeta), a goblet cell-specific protein, is uniquely restricted to intestinal epithelium. However, it still remains largely unknown whether RELMbeta can be served as a biomarker for metaplastic epithelium of BE. In this study, 104 biopsy specimens of the distal esophagus from 88 suspected BE patients were collected, including 56 suspected intestinal metaplasia, 26 gastric type mucosa, and 22 squamous epithelium. We evaluated the RELMbeta expression in these biopsy specimens, and compared with those of CDX-2 immunostaining and PAS-Alcian Blue staining (pH 2.5). Of the suspected intestinal metaplasia specimens, 46 presented intestinal-type goblet cells and were immunostaining positive for RELMbeta and CDX-2, the remaining ten possessed only goblet cell mimickers and were not reactive with RELMbeta and CDX-2. Of the gastric-type mucosa specimens, none reacted with either RELMbeta or CDX-2. Moreover, the squamous epithelium was not reactive with RELMbeta and CDX-2. Acid mucin was present in goblet cells in all cases of BE and columnar cells in ten gastric specimens. In addition, the reactivity of RELMbeta was enhanced in six BE specimens with dysplasia. These results provide evidence that RELMbeta protein may be a novel biomarker to distinguish the intestinal-type goblet cells and goblet cell mimickers, and useful in the correct diagnosis of BE.

Notch Inhibits Expression of the Krüppel-Like Factor 4 Tumor Suppressor in the Intestinal Epithelium

The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor-suppressive role for KLF4 is shown by the recent finding that Klf4 haploinsufficiency in Apc(Min/+) mice promotes intestinal tumorigenesis. Studies also show that KLF4 is required for the terminal differentiation of goblet cells in the mouse intestine. The Notch signaling pathway suppresses goblet cell formation and is up-regulated in intestinal tumors. Here, we investigated the relationship between Notch signaling and KLF4 expression in intestinal epithelial cells. The rate of proliferation of HT29 human colon cancer cells was reduced when treated with the gamma-secretase inhibitor dibenzazepine to inhibit Notch signaling or small interfering RNA directed against Notch. KLF4 levels were increased in dibenzazepine-treated or Notch small interfering RNA-treated cells. Conversely, overexpression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate. Treatment of Apc(Min/+) mice with dibenzazepine resulted in a 50% reduction in the number of intestinal adenomas compared with the vehicle-treated group (P < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from dibenzazepine-treated Apc(Min/+) mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki-67 staining when compared with those from vehicle-treated mice. Results of these studies indicate that Notch signaling suppresses KLF4 expression in intestinal tumors and colorectal cancer cells. Inhibition of Notch signaling increases KLF4 expression and goblet cell differentiation and reduces proliferation and tumor formation. KLF4 is therefore a potential mediator for the antitumor effect of Notch inhibitors such as dibenzazepine.